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MICROBIOLOGY 


MARSHALL 


MICROBIOLOGY 


7 


A TEXT-BOOK OF 

MICROORGANISMS GENERAL AND APPLIED 


CONTRIBUTORS 

F. T. Bioletti, Berkeley, California. J. G. Lipman, New Brunswick, New Jersey. 

R. E. Buchanan, Ames, Iowa. W. J. MacNeal, New York, New York. 

W. V. Cruess, Berkeley, California. E. F. McCampbell, Columbus, Ohio. 

M. Dorset, Washington, D. C. E. B. Phelps, Washington, D. C. 

S. F. Edwards, Lansing, Michigan. O. Rahn, Elbing, Germany. 

E. Fidlar, London, Ontario. L. F. Rettger, New Haven, Connecticut. 
W. D. Frost, Madison, Wisconsin. M. H. Reynolds, University Farm, St. Paul, 
A. Guilliermond, Lyons, France. Minnesota. 

F. C. Harrison, Macdonald College, Que., Canada. W. G. Sackett, Fort Collins, Colorado. 
E. G. Hastings, Madison, Wisconsin. W. A. Stocking, Ithaca, New York. 
H. W. Hill, London, Ontario. C. Thorn, Washington, D. C. 

Arao Itano, Amherst, Massachusetts. J. L. Todd, Montreal, Quebec. 

W. E. King, St. Paul, Minnesota. Z. Northrup Wyant, East Lansing, Michigan. 


EDITED BY 

CHARLES E. MARSHALL 

Amherst, Massachusetts 

PROFESSOR OF MICROBIOLOGY AND DIRECTOR OF GRADUATE SCHOOL 
MASSACHUSETTS AGRICULTURAL COLLEGE 


THIRD EDITION REVISED AND ENLARGED 
WITH 200 ILLUSTRATIONS 


PHILADELPHIA 

P. BLAKISTON'S SON & CO, 

1012 WALNUT STREET 


COPYRIGHT, 1921, BY P. BLAKISTON'S SON & Co. 


THE MAPLE PRESS TTOKK P A 


CONTRIBUTORS 


BIOLETTI, FREDERIC T., M. S. 

Professor of Viticulture and Enology, Viticulturist of Experiment Station, 

University of California, Berkeley. 
BUCHANAN, R. E., B. S., M. S., PH. D. 

Professor of Bacteriology, Bacteriologist of Experiment Station, and Dean 

of the Graduate College, Iowa State College, Ames. 
CRUESS, W. V. 

Assistant Professor of Fruit Products, Agricultural Experiment Station, 

University of California, Berkeley. 
DORSET, M., B. S., M. D, 

Chief of Biochemic Division, U. S. Bureau of Animal Industry, Washington, 

D. C. 
EDWARDS, S. F., B. S., M. S. 

Formerly Professor of Bacteriology, Ontario Agricultural College, Guelph, 

Canada. Director of The Edwards Laboratories, Lansing, Michigan. 
FIDLAR, EDWARD, B. A., M. B. 

Formerly Chief of Division of Pathology, Institute of Public Health; 

Pathologist of London Asylum and of Victoria Hospital; Professor of 

Pathology, W. U. Medical Faculty; Bacteriologist of London Board of 

Health, London, Ontario. Captain, C. A. M. C. 
FROST, W. D., PH. D., D. P. H. 

Professor of Agricultural Bacteriology, University of Wisconsin, Madison. 

GUILLIERMOND, A., DOCTEUR ES SCIENCES. 

Professor of Botany, University of Lyon, France. 
HARRISON, F. C., D. Sc., F. R. S. C. 

Principal and Professor of Bacteriology, Macdonald College (Faculty of Agri- 
culture, McGill University), Macdonald College, Que., Canada. 
HASTINGS, E. G., M. S. 

Professor of Agricultural Bacteriology, Bacteriologist of Experiment Station, 

University of Wisconsin, Madison. 
HILL, H. W., M. B., M. D., D. P. H. 

Formerly Executive Secretary, Minnesota Public Health Association, St. 

Paul; Director of Institute of Public Health of Western University, 

London, Ontario, Canada. 
ITANO, ARAO, B. S., PH. D. 

Associate Professor of Microbiology, Massachusetts Agricultural College, 

Amherst. 


VI CONTRIBUTORS 

KING, WALTER E., M. A., M. D. 

Formerly Professor of Bacteriology and Bacteriologist of Experiment Station, 

Kansas Agricultural College, Manhattan; Assistant Director of Research 

Laboratory, Parke, Davis & Co., Detroit, Michigan. Laboratory Director, 

Beebe Laboratories, Inc., St. Paul, Minnesota. 
LIPMAN, JACOB G., PH. D. 

Dean of Agriculture, Rutgers College; Director of Experiment Station, New 

Brunswick, New Jersey. 
MACNEAL, WARD J., PH. D., M. D. 

Professor of Bacteriology and Director of the Laboratories, New York Post- 

Graduate Medical School and Hospital, New York. 
McCAMPBELL, EUGENE F., PH. D., M. D. 

Professor of Preventive Medicine, Dean of the Medical College, Ohio State 

University. 
PHELPS, EARLE B., B. S. 

Professor of Chemistry, Hygienic Laboratory, U. S. Public Health Service, 

Washington, D. C. 
RAHN, OTTO, PH. D. 

Formerly Assistant Professor of Bacteriology, Illinois University, Urbana. 

Now Elbing, Germany. 
RETTGER, L. F., PH. D. 

Professor of Bacteriology and Hygiene (in Sheffield Scientific School), 

Yale University, New Haven, Connecticut. 
REYNOLDS, M. H., B. S., M. D., D. V. M. 

Professor of Veterinary Medicine and Surgery, Agricultural College, Univer- 
sity of Minnesota; Experiment Station, University Farm, St. Paul. 
SACKETT, WALTER G., B. S., Ph. D. 

Bacteriologist, Colorado Experiment Station, Colorado Agricultural College, 

Fort Collins. 
STOCKING, W. A., M. S. A. 

Professor of Dairy Industiy, Cornell University, Ithaca, New York; Dairy 

Bacteriologist of the Experiment Station. 
THOM, CHARLES, PH. D. 

Mycologist, Bureau of Chemistry, U. S. Department of Agriculture, Wash- 
ington, D. C. 
TODD, J. L., B. A., M. D., D. Sc. 

Associate Professor of Parasitology, McGill University, Montreal. 
WYANT, ZAE NORTHRUP, M. S. 

Research Associate in Bacteriology, Michigan Agricultural Experiment 

Station, East Lansing. 


INTRODUCTION TO THE THIRD EDITION 


The kindly reception of Microbiology, which has been progressive, 
makes a revision a pleasurable task. 

There has been little need of change in the basic facts presented, 
but there is always room for a clarification of thought and improvement 
in arrangement. As time has passed it has been found desirable, also, 
to emphasize and extend some of the chapters. 

Teaching has demonstrated that, in most instances, the chapters 
dealing with biological products follow more naturally and logically 
the chapter on immunity. Since the chapters on diseases are more of a 
reference character, they have been placed at the end. 

The war has made more prominent food contamination, preservation 
and decomposition. For this reason all chapters considering food have 
been brought together in a single division and greater attention has 
been given the subject by rewriting, insertions and enlarging the scope. 
Dairy microbiology has not been included in the division of food be- 
cause it has such a distinctive field of its own. 

The editor has a deep feeling of indebtedness to the contributors who 
have been so kindly disposed, ready and helpful in this revision, and 
to Miss Marion F. Dondale, for her immeasurable assistance. 

CHARLES E. MARSHALL, EDITOR. 
AMHERST, MASSACHUSETTS. 


vn 


INTRODUCTION TO THE SECOND 

EDITION 


The continued and growing demand for "Microbiology" has caused 
the contributors to undertake a thorough revision. In this they have 
been guided by the recent developments in this branch of science, 
and also by a desire to adjust and rearrange in the light of constructive 
suggestions and criticisms. 

The primary purpose of this text-book is to place in the hands of 
college students an elementary technical treatise of the subject matter 
included. No effort has been made to review or cite literature, for to 
do either would expand the volume beyond useful limits. To provide 
an introductory text-book mainly for recitations, or for a supplement 
to lecture or laboratory courses, is about all that can be satisfactorily 
comprehended in a single project. 

The cytological aspect of microbiology has seemed to us to deserve 
some emphasis, for it has become quite definite and has been suggest- 
ively indicating much of real value in connection with the active life 
processes of the cell and microbic activities in agriculture, medicine 
and wherever microbiology is applicable. 

The significance of "Intestinal Microbiology" has required a short 
chapter for its proper presentation. 

It has also been found desirable to treat the microbial diseases of 
insects, a growing subject, in a distinct chapter. 

The study of microorganisms flounders in a fog of unsettled ideas 
for a proper designation. Whether it should be called Protistology, 
Microbiology, Bacteriology, Mycology, or something else must be left 
for the future to determine. 

CHARLES E. MARSHALL, EDITOR. 
AMHERST, MASSACHUSETTS. 

ix 


INTRODUCTION TO THE FIRST EDITION 


By a process of adaptation and growth, the branch of science com- 
monly recognized as "Bacteriology" has for many years included, 
besides the bacterial forms, those microorganisms yielding to the same 
laboratory methods of study and investigation. This is a policy or 
purpose instituted by Pasteur. It is also the result of investigations 
and added knowledge, more definite arrangements of available facts, 
and the highly specialized training required for the work. In short, 
technic together with the economic relations of the subject-matter 
has no little influence in placing limitations. In the light of such cir- 
cumstances, it appears more pertinent to designate this text-book 
as "Microbiology" perhaps not the best term, but one much in accord 
with French usage. 

Agriculture, Domestic Science and certain other courses in scientific 
schools and colleges call for the treatment of the subject in such a man- 
ner as to make it basic to the interpretation of such subjects as air 
impurities, water supplies, sewage disposal, soils, dairying, fermenta- 
tion industries, food preservation and decomposition, manufacture 
of biological products, transmission of disease, susceptibility and im- 
munity, sanitation, and control of infectious or contagious diseases. 
A strong effort has been made to provide the fundamental and guiding 
principles of the subject and to show just how these principles fit into 
the subjects of a more or less strictly professional or practical nature. 
Here the instructional work of the microbiologist stops in most educa- 
tional institutions and the instruction of the practical or professional 
man begins. 

Because of the extreme massiveness and diversity of the subjects, 
Agriculture and Domestic Science and Industrial Vocations in general, 
a comprehensive consideration of the subject is demanded. Elimina- 
tion of many features not only becomes difficult but really precarious, 
because so many avenues are open to the student that pertinency cannot 
always be foreseen or determined. It is well to remember, too, that 

xi 


Xll INTRODUCTION TO THE FIRST EDITION 

such aggregate subjects as Agriculture and Domestic Science, unlike 
Engineering and Medicine, because of their youth, have not developed 
to that stage in their educational history where practice and the science 
upon which practice should be founded are amalgamated. The practi- 
cal man in Agriculture, and Applied Sciences generally, too frequently 
is so extremely traditional in his practice that he utterly fails to separate 
the true from the false, or, in other words, does not exercise his dis- 
criminative powers at all, but depends entirely upon so-called haphazard 
methods and self-willed processes. This factor operates against the 
proper development and logical study of any branch of science in its 
relation to the farmer, or manufacturer. 

The plan of a text-book in Microbiology which seeks to furnish 
basic principles, to train the mind in logical development and adjust- 
ment, and to prepare the student to undertake an intelligent study of 
strictly professional or practical subjects, must assume a definite and 
systematic arrangement. With this in mind, the text has been divided 
into three distinct parts: Morphological and Cultural, or that which 
deals with forms and methods of handling; Physiological, or that which 
deals strictly with functions, the key to the applied; Applied, or that 
which reaches into the application of the facts developed to the problems 
met in the study of professional or practical affairs. 

In a text-book, the product of several hands, there is the most serious 
difficulty in obtaining unity of thought and expression without repeti- 
tion; besides, that very conspicuous weakness of emphasizing some fea- 
tures unduly while other features of importance are scarcely mentioned, 
confronts us. A most earnest attempt has been made to overcome 
these faults as far as possible, but a complete mastery of them cannot 
be expected in the first product. However, what is lacked in unity 
and continuity of expression and in balance we sincerely hope will be 
made up, in part at least, by the selection and the value of the material 
contributed. 

Laboratory features of microbiology have been eliminated wher- 
ever it has been practicable. Should any demonstrations be added 
or needed, we have felt that they may be easily supplied by the instruc- 
tor, who, of course, will be governed by local facilities and conditions. 
Although no space has been given to laboratory exercises, it should not 
be gathered that the authors of this book are any the less earnest in 
urging a well-organized laboratory course to supplement the general 


INTRODUCTION TO THE FIRST EDITION Xlll 

instruction as an essential factor to a working appreciation of the 
subject. 

In matters of spelling, new words, and phrases, conservatism has 
controlled. Arbitrary decisions and selections have been forced in 
several instances to secure clearness, consistency and definiteness. 
It is painfully evident to anyone attempting to bring system out of 
the confusion and chaos existing in many fields of microbiological 
action that some rearrangement ought to be undertaken. As usual, 
however, this will be very slow on account of the many almost insur- 
mountable difficulties. 

We need and invite helpful suggestions and criticisms at all times,, 
for a valuable text-book of the nature of this is one of slow growth and 
development and not of "sport evolution." The editor is certain that 
each contributor will welcome suggestions and, further, will be in far 
better position to judge his own contribution after the material appears 
in book form and has been submitted to students for which it is designed. 

No one better than the editor realizes fully the sympathetic part 
played by the contributors. If any merit attaches to this book as it 
finds its place in microbiological instruction, such merit should be 
recognized as due the contributors whose unselfish aims have made it 
possible. 

CHARLES E. MARSHALL, EDITOR. 
AMHERST, MASSACHUSETTS. 


CONTENTS 


TITLE PAGE iii 

CONTRIBUTORS v 

INTRODUCTIONS (Editor) vii 

CONTENTS (Editor) xv 

HISTORICAL REVIEW (Harrison) i 

PART I. MORPHOLOGY AND CULTURE OF MICROORGANISMS 

GENERAL (Editor). OUTLINE OF PLANT GROUPS (Thorn) 
OUTLINE OF PROTOZOAL GROUPS (Todd) 

* 

CHAPTER I. ELEMENTS OF MICROBIAL CYTOLOGY (Guilliermond) 15 

Cells and energids. Structure of the cell, Nuclear structure (general structure of 
the nucleus, centriole, value of the nucleus, forms of nuclei, theory of binuclearity), 
cytoplasm (appearance of protoplasm, chondriosomes, vacuoles, reserve products), 
membrane, locomotion. Reproduction, Various processes, nuclear division (mito- 
sis, amitosis), sexual changes. 

CHAPTER II. MOLDS (Thorn) CYTOLOGY (Guilliermond) 36 

Fungi in general, Bacteria. Phycomycetes, Ascomycetes, Basidiomycetes, Imper- 
fect fungi. Cytology of molds, General structure of molds, cytoplasm, nuclei, 
metachromatic corpuscles and reserve products, cell wall. Molds, Cosmopolitan 
saprophytes, molds of fermentation, parasitic molds. Consideration of groups, 
Mucor, Thamnidium, Penicillium, Aspergillus, Monascus, Cladosporium, Alter- 
naria and Fusarium, Oidium, Monilia, Dematium, Saprolegniaceae. 

CHAPTER III. YEASTS (Bioletti) CYTOLOGY (Guilliermond) . 61 

Morphology of certain types, Definition and bases of classification. Cytology, 
General structure of yeasts, cytological phenomena during multiplication, variation 
in the cellular structure during development, cytological phenomena of the sporula- 
tion and germination of ascospores. The principal yeasts of importance to fermenta- 
tion industries, True yeasts, pseudo-yeasts. Culture of yeasts. 

CHAPTER IV. BACTERIA (Frost) CYTOLOGY (Guilliermond). 79 

Forms of lower bacteria, Fundamental form types, gradations, involution forms. 
Size. Motility, Brownian movement, vital movement, organs of locomotion, 
character of movement, rate. Reproduction, Vegetative multiplication, spore 
formation. Cell grouping, Cell aggregates among the micrococci, the bacilli, the 
spirilla, Zooglcea. Cytology of bacteria, General consideration of cytoplasm and 
nucleus, minute consideration of cytoplasm and nucleus, life cycle of bacteria 
(Editor), reserve products, general structure of cell wall, minute structure of cell wall, 
capsules, general consideration of flagella, minute consideration of flagella. Higher 
bacteria, The larger spirochaetes, trichobacteria, the sulphur bacteria. Classi- 
fication. Relationship of bacteria. Cultivation of bacteria. 

CHAPTER V. FILTRABLE MICROORGANISMS (Dorset) 119 

A brief general discussion of the available knowledge of filtrable microorganisms. 

XV 


XVI CONTENTS 

CHAPTER VI. PROTOZOA (Todd) 123 

Introduction. Structure of protozoa. Activities of protozoa, Locomotion, re- 
production, developmental cycle, encystment. Parasitism. Discussion of classifi- 
cation. Technic. 

PART II. PHYSIOLOGY OP MICROORGANISMS 

DIVISION I 
INTRODUCTION 145 

CHAPTER I. UNIT OF BIOLOGICAL ACTIVITY (Marshall and Itano) 147 

The mechanism of cells. 

CHAPTER II. A STUDY OF PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 

(Marshall and Itano) I5.S 

Introduction, Energy. Solutions. Electrical conductivity, iopization and 
dissociation, "True reaction," theory of H ion concentration. Surface tension. 
Adsorption. Brownian motion. Diffusion, osmosis, dialysis, permeability. 
Colloids and crystalloids. 

CHAPTER III. CHEMICAL STUDIES OF THE CONTENTS OF MICROBIAL CELLS (Marshall 

and Itano) 186 

Analyses, Moisture, proteins and other nitrogenous substances, carbohydrates, 
fats, ash elements, enzymes, toxins, vitamines. 

DIVISION II. NUTRITION AND METABOLISM (Rahn) 

INTRODUCTION (Revised by Marshall; a few paragraphs on protozoal nutrition by 
Todd) 195 

CHAPTER I. ENERGY REQUIREMENTS IN CELLULAR NUTRITION 199 

CHAPTER II. MECHANISM OF METABOLISM 203 

General theory of metabolism, Metabolism, katabolism, anabolism. Intra- and 
extra-cellular fermentation. Decomposition of insoluble food, properties of en- 
zymes, enzymes of fermentation, Classification of enzymes. Hydrolytic enzymes, 
enzymes of carbohydrates, enzymes of fats, enzymes of proteins, coagulating en- 
zymes. Zymases. Oxidizing enzymes. Reducing enzymes. Enzymic theory 
of katabolism. Enzymic theory of anabolism. General enzymic considerations. 

CHAPTER III. FOOD OF MICROORGANISMS 221 

Moisture requirement. Amount of food required. Food for growth, Sources of 
carbon, nitrogen, hydrogen, oxygen, minerals. Food for energy (oxygen relations). 

CHAPTER IV. PRODUCTS OF MICROBIAL ACTIVITIES 230 

General considerations. The chemical equations of fermentations. Products 
from nitrogen-free compounds, Sugars, starch, cellulose, acids, alcohols, fats. 
Products from nitrogenous compounds, Protein bodies, ptomaines, urea, uric 
acid, hippuric acid. Products from mineral compounds. Oxidations, reductions. 
Unknown products of physiological significance, Pigments, aromatic sub- 
stances, enzymes, toxins. Physical products of metabolism, Production of heat, 
production of light. 

CHAPTER V. PHYSIOLOGICAL VARIATIONS ASSOCIATED WITH METABOLISM AND 

NUTRITION 253 

Factors influencing the type of decomposition. 

CHAPTER VI. NUTRITION OF MICROORGANISMS AND THE ROTATION OF ELEMENTS IN 

NATURE 258 

Carbon cycle. Nitrogen cycle. Sulphur cycle. Phosphorus cycle. 

i 


CONTENTS XV11 

DIVISION III, PHYSICAL INFLUENCES (Rahn) 

CHAPTER I. WATER AS A PHYSICAL FACTOR 263 

Osmotic pressure. Plasmolysis (salt and sugar solutions, colloidal solutions). 
Desiccation. 

CHAPTER II. INFLUENCE OF TEMPERATURE 269 

Optimum temperature. Minimum temperature. Maximum temperature. 
Biological significance of the cardinal points of temperature. End-point of fer- 
mentation. Freezing. Thermal death-point. Resistance of spores. 

CHAPTER III. INFLUENCE OF LIGHT AND OTHER RAYS 278 

Phototaxis. X-rays. Radium rays. 

CHAPTER IV. INFLUENCE OF ELECTRICITY 282 

CHAPTER V. INFLUENCE OF MECHANICAL AGENCIES 283 

Pressure. Gravity. Agitation. 

DIVISION IV. CHEMICAL INFLUENCES (Rahn) 

CHAPTER I. STIMULATION OF GROWTH 286 

Chemotropism and chemotaxis. 

CHAPTER II. INHIBITION OF GROWTH 288 

Poisons, germicides, disinfectants, antiseptics, preservatives. Mode of action. 
Factors influencing disinfection. Classification of disinfectants. 

DIVISION V. MUTUAL INFLUENCES 
SYMBIOSIS. METABIOSIS. ANTIBIOSIS 297 

PART III. APPLIED MICROBIOLOGY 
DIVISION I. MICROBIOLOGY OF AIR (Buchanan) 

CHAPTER I. THE MICROORGANISMS OF THE AIR AND THEIR DISTRIBUTION. . . . 303 
Microorganisms present in the air. Occurrence in the air. How microorganisms 
enter the air. Conditions for subsidence of bacteria. Determination of the number 
of bacteria in the air. Number of bacteria in the air. Species of organisms in the 
air. 

CHAPTER II. MICROBIAL AIR INFLUENCE IN FERMENTATION, DISEASES, ETC. . . 308 
Air as a carrier of contagion. Organisms of the air and fermentation. Freeing air 
from bacteria. 

DIVISION II. MICROBIOLOGY OF WATER AND SEWAGE 

CPIAPTER I. MICROORGANISMS IN WATER (Harrison) 310 

Classes of bacteria found in water, Natural water bacteria, soil bacteria from surface 
washings, intestinal bacteria usually of sewage origin. The number of bacteria in 
rain, snow, hail, etc., and in water from wells, up-land, surface waters, rivers, and 
lakes. Causes affecting the increase and decrease of the number of bacteria in water, 
Temperature, light, food supply, oxidation, vegetation and protozoa, dilution, sedi- 
mentation, other causes. Interpretation of the bacteriological analysis of water, 
Quantitative standards, qualitative standards. Sedimentation, filtration and purifi- 
cation of water, Sedimentation and filtration, coagulating basins and filtration, 
porous filters, purification by ozone, purification by heat, purification by chemicals. 
Location and construction of wells. 


XV111 CONTENTS 

CHAPTER II. MICROBIOLOGY OF SEWAGE (Phelps) 330 

Bacterial flora of sewage. Types of sewage bacteria, Putrefactive and anaerobic 
bacteria (the liquefaction of protein, the fermentation of cellulose, the saponification 
of fats, the fermentation of urea, the reduction of sulphates and nitrates), oxidizing 
bacteria (the production of nitrates and nitrites, other oxidizing reactions), patho- 
genic bacteria (prevalence and longevity, life in septic tanks and filters). The culti- 
vation of sewage bacteria, Filters, anaerobic tanks. The destruction of sewage 
bacteria, By biological processes, by chemical processes. 

DIVISION III. MICROBIOLOGY OF SOIL (Lipman) 

CHAPTER I. MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 345 

Introduction. The soil as a culture medium. Moisture relations, The amount 
and distribution of rain fall, range of soil moisture, effect of drouth and excessive 
moisture. Colloidal nature of the soil. Aeration, Mechanical composition of 
soils, aerobic and anaerobic activities, rate of oxidation of carbon, hydrogen and 
nitrogen, the mineralization of organic matter. Temperature, Influence of cli- 
mate and season, early and late soils, production and assimilation of plant food. 
Reaction. Range of soil acidity, causes of soil acidity, soil reaction and hydrogen- 
ion concentration, change of reaction produced by microorganisms in the medium, 
effect of reaction on number and species. Food supply, Organic matter, the 
mineral portion of the soil. Biological factors, Fungi, algae, protozoa, higher 
plants, bacteria (numbers and distribution, bacteria in productive and unproduc- 
tive soils, distribution at different depths, seasonal variations of bacterial numbers 
and activities, morphological and physiological groups). Methods of study, 
Methods for counting bacteria, quantitative relations, qualitative reaction, trans- 
formation reactions, rate of oxidation of carbon, rate of oxidation of nitrogen, addi- 
tion of nitrogen, reactions concerning calcium, magnesium, sulphur, phosphorus. 

CHAPTER II. DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 375 

Carbohydrates, Origin, decomposition of cellulose, the production of methane and 
hydrogen, oxidation of methane, hydrogen, and carbon monoxide, the cleavage and 
fermentation of sugars, starches, and gums. Fats and waxes, Origin and decompo- 
sition. Organic acids, Sources, transformation and accumulation. Protein 
bodies, Amount and quality, carbon-nitrogen ratio. Transformation of nitrogen 
compounds, Ammonification, nitrification, denitrification. Analytical and syn- 
thetical reactions, Amount of bacterial substance in the soil, availability of bacterial 
matter, transformation of peptone, ammonia, and nitrate nitrogen. 

CHAPTER III. FIXATION OF ATMOSPHERIC NITROGEN. (Methods of Soil Inoculation, 

by Edwards.) 400 

The source of nitrogen in soils, Early theories, chemical and biological relations. 
Non-symbiotic fixation of nitrogen, Historical, anaerobic species, aerobic species, 
energy relations. Symbiotic fixation, Historical, modes of entrance and devel- 
opment, resistance, immunity, and physiological efficiency, mechanism of fixation, 
variations and specialization, relation to environment. Soil inoculation, Methods 
of soil inoculation, Inoculation with legume earth, inoculation with pure cultures, 
etc. (Edwards.) 

CHAPTER IV. CHANGES IN ORGANIC CONSTITUENTS 417 

Weathering process, Origin and formation of soil, influence of biological factors. 
Lime and magnesia, Removal and regeneration of carbonates, lime as a base, effect 
of calcium, magnesium compounds upon bacterial activities. Phosphorous, Avail- 
ability of phosphates, relation of phosphorus to decay and nitrogen-fixation. Sul- 
phur, Sulphur compounds in the soil, sulphur-phosphate composts, sulphur bac- 
teria, sulphofication, sulphate reduction. Potassium, The transformation of 
potassium compounds in the soil. Other mineral constituents, Iron, aluminum, 
manganese, and copper. Antagonism. Variability in soil fertility investigations. 


CONTENTS XIX 


DIVISION IV. MICROBIOLOGY OF MILK AND MILK PRODUCTS 

CHAPTER I. THE RELATION OF MICROORGANISMS TO MILK. (Stocking.) (The Acid- 
forming Bacteria, by Hastings.) 428 

Character of milk. Absorbed taints and odors. Changes due to microorganisms. 
Microbial content of milk, Common milk, special milks, certified milk. 
Sources of microorganisms in milk, Interior of cow's udder (healthy udders, 
diseased udders), exterior of cow's body, atmosphere of stable and milk house, the 
milker, utensils, water supply. Methods of preventing contamination of milk, 
Individual cows, care of the cow's body, avoid dust in atmosphere, dairy utensils, 
the milker. Groups or types of microorganisms found in milk, and their sources, 
General significance of acid-forming bacteria, groups of acid-forming bacteria (char- 
acteristics of the Bad. lactis acidi group, characteristics of the B. coli-aerogenes 
group, characteristics of the Bact. bulgaricus group, characteristics of the coccus 
group) (Hastings), bacteria having no perceptible effect upon milk, the casein-di- 
gesting or peptonizing bacteria, pathogenic organisms. Factors influencing the 
developing of microorganisms in milk, Initial contamination, straining, aera- 
tion, centrifugal separation, temperature, pasteurization, the use of chemicals. 
The normal development of microorganisms in milk, Germicidal period, period 
from end of germicidal action to time of curdling, period from time of curdling until 
acidity is neutralized, final decomposition changes. Abnormal fermentations in 
milk, Gassy fermentation, sweet curdling fermentation, ropy or slimy fermenta- 
tion, bitter fermentation, alcoholic fermentation, other fermentations. The com- 
mercial significance of microorganisms in milk, -Relation of dirt contamination to 
germ content. Milk as a carrier of disease-producing organisms, (acid forms, 
neutral forms, injurious organisms, epidemic diseases, non-epidemic diseases). 
Bacteriological analysis of milk. Bacteriological milk standards. The value of 
bacteriological milk standards and analyses. 

CHAPTER II. THE RELATIONS OF MICROORGANISMS TO BUTTER (Hastings) 47 

Types of butter, Sweet cream butter, sour cream butter. The flavor of butter, 
Control of butter flavor, kinds and numbers of bacteria in cream, spontaneous ripen- 
ing of cream, use of cultures in butter making, commercial cultures, use of pure cul- 
tures in raw cream, use of pure cultures in pasteurized cream, pure cultures in oleo- 
margarine and renovated butter, abnormal flavors of butter. Decomposition 
processes in butter. Pathogenic bacteria in butter. 

CHAPTER III. RELATION OF MICROORGANISMS TO CHEESE (Hastings) 486 

General. Types of cheese, Acid-curd cheeses, rennet-curd cheeses. Conditions af- 
fecting the making of cheese, Quality of milk, tests for the quality of milk, ripening 
of milk, curdling of milk, manipulation of the curd, ripening of cheese (theories of 
cheese ripening, present knowledge of causal factors, causes of proteolysis, preven- 
tion of putrefaction, other groups of bacteria in cheese, flavor production in cheese). 
Abnormal cheeses, Gassy cheese, miscellaneous abnormalities of cheese (bitter 
cheese, colored cheese, putrid cheese, moldy cheese). Specific kinds of cheese, 
Cheddar cheese, Emmenthaler cheese, Roquefort cheese, Gorgonzola cheese, Stilton 
cheese, Camembert cheese. 

CHAPTER IV. RELATION OF MICROORGANISMS TO SOME SPECIAL DAIRY PRODUCTS 

(Stocking) 504 

General. Condensed milk, Sweetened condensed milk, unsweetened condensed 
or evaporated milk, concentrated milk, powdered milk. Canned butter, and 
cheese. Special milk drinks made by the action of microorganisms, Kumyss, 
kefir, leben, yoghurt, artificial buttermilk. Ice cream. 


XX CONTENTS 

DIVISION V. MICROBIOLOGY OF FOODS 

CHAPTER I. DESICCATION, EVAPORATION, AND DRYING OF POODS (Buchanan) . . .516 
Agencies that bring about changes in dried foods. Factors which inhibit growth of 
microorganisms in food. Methods of drying, Carbohydrate foods, as fruits, 
macaroni, vermicelli, copra, syrups, molasses, jellies, jams; fats, as cotton seed, 
olive, and other oils, etc.; protein foods, as jerked meat, dried beef, dried fish, pem- 
mican, beef extract, gelatin, somatose, milk, eggs, etc. 

CHAPTER II. HEAT IN THE PRESERVATION OF FOOD PRODUCTS (Edwards) 524 

Historical r6sum. Economic importance, From the standpoint of health and 
dietetics, and from the standpoint of commerce. Alteration of foods, Physical 
changes (appearance, mechanical disintegration), chemical changes (appearance, 
chemical change, palatability and digestibility), biological changes (vital disorganiza- 
tion, normal flora and fauna). Pasteurization, Economic consideration, specific 
application (beer, fruit juices, milk and cream, condensed milk). Processing or 
sterilization, Economic considerations, specific application (meat, fish, vegetables, 
and fruits). Controlling factors in successful canning, Cleanliness, soundness of 
raw material, receptacle, water supply, degree of heat required. Home canning. 
Spoilage, Microbiological, detection of spoiled goods. Disposal of factory refuse. 

CHAPTER III. THE PRESERVATION OF FOOD BY COLD (MacNeal) 542 

Introduction. The effects of refrigeration upon foods in general, Changes during 
chilling, changes during storage, changes after storage. Refrigeration of certain 
foods, Meat, fish, poultry, eggs, milk, butter, fruits and vegetables. Legal con- 
trol of the cold-storage industry. 

CHAPTER IV. PRESERVATION OF FOOD BY CHEMICALS (MacNeal) 550 

The effects of preservatives upon foods in general, The process of curing, the period 
of storage, the after-storage changes. The chemical preservation of certain foods, 
Meats, fish, dairy products, prepared vegetables, and fruits. The nutritive value 
of preserved foods. The effects of food preservatives, Substances which preserve 
by their physical action, substances which preserve by their chemical action, inor- 
ganic food preservatives, organic food preservatives, substances added to foods to 
improve the apparent quality. The legal control of the preservation of foods by 
chemicals. 

CHAPTER V. MICROBIOLOGY OF FERMENTED FOODS 559 

Compressed yeast, yeast as food (Cruess). Bread (Cruess). Vegetables 
(Cruess). Olive pickling and canning (Cruess). Silage (Cruess). Malt syrups 
(Cruess). Tobacco (Cruess). Starch (Bioletti). Sugar (Bioletti). Tea (Biol- 
.etti). 

CHAPTER VI. MICROBIAL FOOD POISONING (MacNeal) 581 

General considerations. Infections of food-producing animals transmissible to 
man. Human infections transmitted in food. Food poisoning due to the growth 
of saprophytic bacteria in the food, Poisonous meat, sausage, fish, shell fish, milk, 
cream, cheese, and vegetable food. Specific diseases due to food poisoning, 
Botulism, and Bacillus botulinus, ergotism, pellagra. The chemical nature of food 
poisons. 

CHAPTER VII. MICROORGANISMS OF THE DIGESTIVE TRACT (MacNeal) 593 

Introduction. Microorganisms of certain portions of the alimentary canal, Mi- 
croorganisms of the mouth, microorganisms of the stomach, microorganisms of the 
intestines, microorganisms of the feces. General method of study, Collection of 
material. 


CONTENTS XXI 

DIVISION VI. MICROBIOLOGY OF ALCOHOLIC FERMENTATION AND DERIVED 

PRODUCTS (Bioletti) 

CHAPTER I. WINE .603 

Grape juice and wine as culture media. The microorganisms found on grapes, 
Molds, yeasts, pseudo-yeasts, bacteria. The microorganisms found in wine, 
Aerobic organisms (mycodermae, acetic bacteria), anaerobic organisms (slime- 
forming bacteria, propionic and lactic bacteria, mannitic bacteria, butyric bac- 
teria). Control of the microorganisms, Before fermentation, during fermenta- 
tion, after fermentation. Prohibition and wine. 

CHAPTER II. BEER 622 

Raw materials and microorganisms of brewing, Grains employed, yeasts of beer, 
kinds of beer. Process of brewing, Outline, malting (production of enzymes), 
work of enzymes and bacteria, fermentation (work of yeast), after treatment. 
Diseases of beer. 

CHAPTER III. MISCELLANEOUS ALCOHOLIC BEVERAGES AND PRODUCTS . , 628 

Cider and perry. Fermented beverages of various fruits. Hydromel or mead. 
Miscellaneous fermented beverages, Mexican pulque, sake, pombe, ginger beer.- 
Distilled alcohol, Introduction (uses and sources of alcohol), Methods (prep- 
aration of the sugar solution, fermentation). 

CHAPTER IV. MANUFACTURE OF VINEGAR 636 

Acetic fermentation, Nature and origin of vinegar, vinegar bacteria. Processes of 
manufacture, Raw materials, fermentation, starters and pure cultures, apparatus, 
domestic method, Orleans method, Pasteur method, Rapid methods, rotating 
barrels, function of the film, after treatment. Diseases. 

DIVISION VII. MICROBIOLOGY OF SPECIAL INDUSTRIES 

CHAPTER I. SPECIAL INDUSTRIAL FERMENTED PRODUCTS . 649 

Acetone and acetic acid (Cruess). Lactic acid (Cruess). Citric acid (Cruess). 
White lead (Cruess). Leather (Cruess). Indigo (Bioletti). Retting (Bioletti). 


DIVISION VIII. MICROBIOLOGY OF THE DISEASES OF MAN AND DOMESTIC 

ANIMALS 

CHAPTER I. METHODS AND CHANNELS OF INFECTION (McCampbell) t 659 

Infection defined. Microorganisms of diseases considered and classified, Patho- 
genic bacteria, pathogenic protozoa, ultra-microscopic microorganisms or viruses, 
the distribution of pathogenic microbic agents in nature. The occurrence of patho- 
genic microbic agents upon and in the bodies of healthy animals and man. The 
manner in which infectious agents enter the body and their sources, Air-borne infec- 
tions, dust infection, droplet infections, water-borne infections, infections from 
soil, infection from food, animal carriers of infection, human carriers of infection, 
contact infection. The routes by which infectious microorganisms enter the body. 
Variation in infection. The factors which influence the results of an infection, 
Virulence, number, avenue, resistance. The exact cause of infections, Soluble tox- 
ins, endotoxins, toxic bacterial proteins, other possible exact causes. The methods 
by which infectious microorganisms are disseminated. The methods by which in- 
fectious microorganisms are eliminated from the body. The effect of infectious 
microorganisms upon the body, The period of incubation, local reactions, general 
reactions (metabolism, blood-forming organs, parenchymatous tissues, epithelial and 
endothelial tissues, erythrocytes and leucocytes, antibody formation). 


XX11 CONTENTS 

CHAPTER II. IMMUNITY AND SUSCEPTIBILITY (McCampbell) 684 

General, Definition, hypersusceptibility or anaphylaxis, predisposition and non- 
inheritance of infectious diseases. Immunity, Natural immunity and susceptibility 
(racial immunity and susceptibility, familial immunity and susceptibility, individual 
immunity and susceptibility), factors of natural immunity (the protection afforded 
the body by the surfaces, skin and cutaneous orifices, subcutaneous tissue, the ex- 
posed mucous membranes of the body, nasal cavity, mouth, lungs, stomach, intes- 
tines, genito-urinary tract, conjunctiva, the protective nature of inflammatory 
processes, natural antitoxins, natural antibacterial substances, normal hemolysins, 
normal agglutinins, normal precipitins), acquired immunity (active immunity, pas- 
sive immunity). The origin and occurrence of antibodies, Antitoxins (the mech- 
anism of the neutralization of toxin by antitoxin, units of antitoxin), lysins and 
bactericidal substances (the structure of lysins, deviation of complement, the deflec- 
tion of the complement as a test for antibodies), cytotoxins and cytolysins, opsonins 
and phagocytosis (opsonic index, hemoopsonins), agglutinins (normal agglutinins, the 
production of agglutinins, the distribution of agglutinins in the blood, inherited 
agglutinins, the substances concerned in agglutination, structure of agglutinins and 
agglutinogens, agglutinoids, the stages of agglutination, hemoagglutinins), precip- 
itins (normal precipitins, mechanism of the formation of precipitins, autoprecipitins 
and isoprecipitins, the phenomena of specific inhibition, antiprecipitins, the precip- 
itinogen, precipitate, coprecipitins, the forensic use of precipitins). The theories of 
immunity, Noxious retention theory, exhaustion theory, Ehrlich's side-chain 
theory, phagocytic theory. 

CHAPTER III. MANUFACTURE OF VACCINES (King) 724 

Introduction. Actively immunizing substances (vaccines), Attenuated viruses, 
smallpox vaccine, blackleg vaccine, blackleg aggressin, blackleg filtrate, rabies 
vaccine, Dorset-Niles hog cholera serum, anthrax vaccine, tuberculosis vaccine. 
Bacterial vaccines (bacterins), Typhoid fever, pneumonia, influenza-pneumonia, 
canine distemper, Asiatic cholera, bubonic plague. Sensitized vaccine. Toxin- 
antitoxin mixture. 

CHAPTER IV. THE MANUFACTURE OF ANTISERA AND OTHER BIOLOGICAL PRODUCTS 

RELATED TO SPECIFIC INFECTIOUS DISEASES (King) 740 

Antitoxic sera, Diphtheria antitoxin, tetanus antitoxin, perfringens antitoxin. 
Antimicrobial sera, Antimeningococcic, antistreptococcic, antigonococcic, anti- 
pneumococcic, Dorset-Niles (antihog cholera), antirabic, antidysenteric, preserva- 
tion of antisera. Tuberculins, Koch's old, other tuberculins. Mallein. Suspen- 
sions for the agglutination tests. Substances used for diagnostic tests, Luetin, 
antigens, Schick test. 

CHAPTER V. CONTROL OF INFECTIOUS DISEASES (Hill) 754 

Principles. Practice. Public health methods as revised and promulgated by the 
Institute of Public Health, London, Canada, Householder's responsibility to 
board of health, physician's responsibility to board of health, penalties, definitions, 
rules for release of cases from isolation, placarding of house, quarantine periods for 
contacts, observation versus quarantine, regulations regarding visitors, in case of 
death. Disinfection. Carriage of infection by biological agents. 

-CHAPTER VI. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS (various authors) 775 
Diseases caused by molds and yeasts, Pneumomycosis, aspergillosis, secondary 
infections (Thorn), thrush (Thorn), dermatomy coses, barber's itch, etc. (Thom), 
favus (Thom), actinomycosis (Reynolds), mycetoma (Fidlar), mycotic lymphangitis 
(Reynolds). Diseases caused by bacteria, Botryomycosis (Reynolds), gonor- 
rhoea (Fidlar), epidemic cerebro-spinal meningitis (Fidlar), infectious mastitis (Rey- 
nolds), Malta fever (Fidlar), staphylococcic infections (Fidlar), streptococcic 
infections (Fidlar), pneumonia (Fidlar), anthrax (Harrison), bacillary white diar- 
rhaea of young chicks (Rettger), chicken cholera (Harrison), chronic bacterial en- 
teritis (Reynolds), 'contagious abortion (MacNeal), diphtheria (Fidlar), dysentery 


CONTENTS xxiii 

(Fidlar), fowl diphtheria (Harrison), glanders (Reynolds), influenza (Fidlar), whoop- 
ing cough (Fidlar), haemorrhagic septicaemia (Reynolds), leprosy (Fidlar), plague 
(Fidlar), swine erysipelas (Dorset), tuberculosis (Reynolds), foot rot of sheep 
(Dorset), malignant oedema (Fidlar), symptomatic anthrax (Reynolds), tetanus 
(Fidlar), typhoid fever (Fidlar), Asiatic cholera (Fidlar). Microbial diseases as yet 
unclassified, Scarlet fever, measles, German measles, Duke's disease, smallpox, 
chickenpox, mumps (Hill), canine distemper (Dorset), cattle plague (Dorset), 
contagious bovine pleuro-pneumonia (Dorset), cowpox, horsepox and sheeppox 
(King), dengue (Dorset), foot-and-mouth disease (Dorset), fowl plague (Dorset), 
hog cholera (Dorset), horse sickness (Dorset), infantile paralysis (Dorset), pella- 
gra (MacNeal), rabies (MacXeal), swamp fever (Reynolds), typhus fever (Dorset), 
yellow fever (Dorset), Diseases caused by protozoa (Todd), Rhizopoda: amoe- 
bic dysentery, entero-hepatitis of turkeys; flagellata and Leishmania: kala-azar, 
infantile kala-azar, Delhi boil; trypanosoma: sleeping sickness, human trypano- 
somiasis of South America, trypanosomiases of animals; sporozoa; coccidia; 
coccidiosis of rabbits, avian coccidiosis; haemosporidia: malaria, red water. East 
Coast fever, oroya fever, anaplasmosis; sarcosporidia; haplosporidia; myxosporidia; 
microsporidia; infusoria: balantidium enteritis; parasites of uncertain position: 
relapsing fever, syphilis, yaws or frambcesia, other spirochaetal diseases. 

DIVISION IX. MICROBIAL DISEASES OF INSECTS (Wyant) 

INTRODUCTION. Bacterial disease of June Beetle larvae, Lachnoslerna spp. Flacherie 
(silk worm). "Japanese gipsy-moth, disease."- Bacterial disease of locusts. Bacil- 
lary septicaemia of caterpillars, Arctia caja. Graphitosis. American foul brood.- 
Septicaemia of the cockchafer, Melolontha vulgaris. European foul brood. Bac- 
terial septicaemia of larvae of the Lamellicornce. Bacterial disease of the gut- 

-, epithelium cf the lug-worm, Arenicola ecaudata. Pseudograsserie of the gipsy- 
moth caterpillar. Sacbrood of bees. Wilt disease or flacherie of the gipsy-moth 
caterpillar, Porthetria dispar. Pebrine. Nosema-disease of bees. Miscellaneous 
insect diseases, Entomophthoracese (Thorn), Other microbial diseases (Wyant). 
General pathology and immunity studies 905 

DIVISION X. MICROBIAL DISEASES OF PLANTS (Sackett) 

INTRODUCTION 949 

CHAPTER I. BLIGHTS 95 1 

Stem blight of alfalfa. Bacteriosis of beans. Blight of lettuce. Blight of mulberry. 
Blade blight of oats. Stem blight of field and garden peas. Pear blight. 
Streak disease of sweet peas and clovers. Tomato blight. Walnut blight. 

CHAPTER II. GALLS AND TUMORS 966 

Crown gall. Olive knot. "Fingers and toes" of cabbages (Todd). Tuberculosis 
of sugar beets. 

CHAPTER III. LEAF SPOTS 973 

Citrous canker. Angular leaf-spot of cucumbers. Leaf-spot of the larkspur- 
Bacterial spot of plum and peach. Disease of sugar beet .and nasturtium leaves. 

CHAPTER IV ROTS 

Black rot of cabbage. Wakker's hyacinth disease. Basal stem rot of potatoes. 
Bud rot of cocoanut. Brown rot of orchids. Rot of cauliflower. Soft rot of calla 
lily. Soft rot of carrot and other vegetables. Soft rot of hyacinth. Soft rot of 
muskmelon. Soft rot of the sugar beet. 

CHAPTER V. WILTS 

Wilt of cucurbits. Wilt of sweet corn. Wilt of tomato, egg plant, Irish potato, and 
tobacco. Additional bacterial diseases. 

INDEX OF CONTRIBUTORS -993 

INDEX OF SUBJECTS 995 


LIST OF ILLUSTRATIONS 


Frontispiece 

1. Jansen's Microscope 2 

2. Kingdom of the Protista, diagrammatic . ff . . 12 

3. Cells of Saccharomyces cerevisics . . . . ' 16 

4. Cells made up of energids 16 

5. Diffuse nuclei of bacteria 17 

6. Nuclei in Cyanophycece 17 

7. Chromidia in protozoa 18 

8. Micro- and macro-nucleus in an infusorian 19 

9. Division of micro-nucleus and chondriosomes 19 

10. Formation of chloroplasts 20 

11. Mitochondria developing into amyloplasts 21 

12. Chloroplasts of different forms 21 

13. Metachromatic corpuscles 23 

14. Illustrating cyst and thread membranous walls 24 

15. Organs of locomotion in bacteria 25 

16. Division of Spongomonas uvella and Monas termo 26 

17. Transverse section illustrating trichocysts and cilia attachments 26 

18. Schizogony in Amceba polypodia 27 

19. Sporogony in Saccharomyces cerevisia, B. mycoides and Leucocytozoon lovali. 27 

20. Karyo kinesis in Acanthocystis aculeata and Coleosporium senecionis .... 29 

21. Protomitosis in Amoeba mucicola, Amceba froschi, Euglena splendens, and 

Amceba diplomitotica 31 

22. Mesomitosis in Pelomyxa palustris, Urospora lagidis, and Galactima succosa. 33 

23. Conjugation in Schizo Saccharomyces octosporus 34 

24. Nuclei in mycelium of Thamnidium elegans and Mucor circinelloides. ... 41 

25. Fragments of mycelia of molds with dividing nuclei 41 

26. Filaments of molds showing chondrium 43 

27. Nucleus of Mucor in various stages of division 43 

28. Metachromatic corpuscles in Dematium 44 

29. Metachromatic corpuscles in asci 44 

30. Metachromatic corpuscles in conidia 45 

31. Metachromatic corpuscles in cell of perithecium of Pestularia vesiculosa . . 46 

32. Mucor, general 49 

33. Mncor, zygospore 49 

34. Penicillium expansum. 52 

35. Aspergillus glaucus 55 

36. Aspergillnsfumigatus, A. nidulans 55 

37. Cladosporium herbamm 57 

38. Spores of Alternaria 57 

39. Fusarium 57 

40. Oldium lactis 58 

41. M onilia Candida 59 

42. Manilla sitophila, oidia in chains 59 

43. Yeast cell 62 

xxv 


XXVI LIST OF ILLUSTEATIONS 

44. Spore-bearing yeast cells 63 

45. Saccharomyces cerevisice showing vacuoles and metachromatic corpuscles 

stained 64 

46. Saccharomyces cerevisics showing cells with nuclei, nuclear division and 

glycogenic vacuoles with grains 64 

47. Saccharomyces cerevisice showing cells stained by a special method re- 

vealing a chondrium consisting of granular- and rod-mitochondria. . . 64 

48. Saccharomyces cerevisice, with both nucleus and metachromatic granules . 65 

49. Saccharomyces ellipsoideus cells with nucleus 66 

50. Copulation and sporulation in Schizosaccharomyces octosporus 68 

51. Various stages of nuclear division during sporulation in Schizosaccharo- 

myces octosporus 68 

52. Cellular fusion in Schizosaccharomyces pombe 69 

53. Heterogamous copulation in Zygosaccharomyces chevalieri 70 

54. Sporulation in Saccharomyces ludwigii 71 

55. Germination of ascospores in Saccharomyces ludwigii ., . . ^ . 72 

56. Wine and beer yeasts 74 

57. Wild and pseudo-yeasts 77 

58. Types of micrococci 79 

59. Types of bacilli 79 

60. Types of spirilla 80 

6 1. Involution forms 80 

62. The division of bacterial cells 83 

63. The formation of spores 85 

64. Location of spores in bacterial cells 85 

65. Spore germination 86 

66. Division forms of micrococci 87 

67. Division forms of bacilli. 88 

68. Threads of B act. anthracis 88 

69. Plasmolytic changes 89 

70. Karyokinetic appearances in Bad. gammari . . . . 91 

71. B. megatherium in process of division 92 

72. Diffuse nucleus in Chromatium okenii and Beggiatoa alba 93 

73. B. butschlii in division 95 

74. B. sporonema in spore formation with vestiges of ancestral sexuality . . 96 

75. B. radicosus with nuclear appearances 96 

76. B. flexilis in division of cell and formation of spores 98 

77. Retrogression of original nucleus and formation of diffuse nucleus in var- 

ious bacteria 98 

78. Life cycle of Azotobacter 100 

79. Differentiation of metachromatic corpuscles in various bacteria by means 

of stains 102 

80. Structure of bacterial membrane in section 103 

81. Capsules (Bact. pneumonic?) 104 

82. Distribution of nuclear substance and various flagella 105 

83. Monotrichous bacteria (Msp. comma) 105 

84. Monotrichous bacteria (Ps. pyocyanea) 105 

85. Lophotrichous bacteria (Ps. syncyanea) . 105 

86. Lophotrichous bacteria (Sp. rubrum) 105 

87. Peritrichous bacteria (B. typhosus) 105 

88. Crenothrix polyspora 109 

89. Spirophyllumferrugineum,Gallionella}erruginea,Leptothrixochracea... . no 

90. Pasteur-Chamberland or Berkefeld filtering apparatus 120 

91. Amceba vespertilio 124 

92. Paramecium caudatum dividing without mitosis 127 

93. Stages in division of Amoeba poly podia 128 

94. Multiplication of Coccidium schubergi 129 


LIST OF ILLUSTRATIONS XXvii 

95. Herpetomonas musca-domestica 134 

96. Trypanosoma tincce and Trypansoma perccB 135 

97. Trichomonas eberthi 136 

98. Lamblia intestinalis 137 

99. Development of sporozoits in Laverania malaria 138 

100. Solutions, diagrammatic 157 

101. Movement of electric current and ionization 159 

102. Apparatus employed in determination of H-Ion concentration 166 

103. Illustrating surface forces 169 

104. Illustrating surface pull 170 

105. Particle in Brownian motion 172 

106. Plasmolysis in cells 177 

107. An arrangement of dispersoids 181 

108. Comparison of particles of different size 182 

109. Ultramicroscope 183 

no. Illustrating cell activities 196 

in. Amoeba proteus 197 

112. Influence of oxygen on microorganisms 229 

113. Crystals of bacteriopurpurin 247 

114. Carbon cycle '....' 259 

115. Nitrogen cycle 260 

116. Sulphur cycle 261 

117. Action of light on bacteria 278 

1 1 8. Action of light on molds 279 

119. Action of light on mold colonies 280 

120. Chemotaxis 286 

121. Curve of disinfection 289 

122. Influence of filtered water on typhoid fever and Asiatic cholera 315 

123. Section of sand filter 323 

124. Unglazed porcelain filters 325 

125. 126, 127. Location of wells on farm 327 

128. Construction of model well 328 

129. Trickling filter, sand filter, dosing tank, septic tank 341 

130. Septic tank 342 

131. Non-symbiotic nitrogen-fixing organism (B. pastciirianns) 402 

132. Non-symbiotic nitrogen-fixing organism (A zotobacter vinelandi] 403 

133. Ps. radicicola 407 

134. Section through root tubercle 408 

i-SS* T 36, 137. Influence of Ps. radicicola 411,412,413 

138. Section of cow's udder 434 

139. Bacterial colonies in dust from udder 437 

140. Bacterial colonies from cow's hair 438 

141. Bacterial colonies from dust of stable 439 

142. Small-top milk pails 442 

143. Ropy cream 464 

144. Ropy cream organisms 465 

145. Chart of Rochester milk supply 469 

146. Gassy cheese 488 

147. Cheese from lactic starter 489 

148. Influence of lactic organisms on casein degradation 495 

149. Swiss cheese 500 

150. Kefir grain 509 

151. Chart. Effect of storage on bacterial content of ice cream 514 

152. Chart. Influence of temperature on sterilizing time 537 

153. Chart. Influence of number of spores on sterilizing time 537 

154. Chart. Influence of speed of rotation on heat penetration. 538 

155. Tubes for feces examination 602 


XXV111 LIST OF ILLUSTRATIONS 

156. Bacteria of slimy wine 610 

157. Bacteria of wine diseases 6n 

158. Vinegar bacteria 638 

159. Vinegar barrel 642 

160. Rapid process vinegar apparatus 645 

161. Oidium albicans 776 

162. Oidium albicans. (Kohle and Wassermann.) 776 

163. Trichophyton tonsurans 777 

164. 165. Actinomyces bovis 779, 780 

166. Gonococci 785 

167. Bad. anthracis, thread formation 803 

168. Bact. anthracis, spores 803 

169. Organisms of anthrax in capillaries 804 

170. Bact. diphtheria 813 

171. Wesbrook's types of Bact. diphtheria 814 

172. Bact. mallei 821 

173. Bact. pestis 831 

174. Bact. tuberculosis, branching forms 836 

175. Bact. tuberculosis, from sputum 836 

176. Bact. tuberculosis, in culture 837 

177. B. tetani, with spores. 843 

178. B. typhosus 848 

179. Ms p. comma 852 

1 80. M sp. comma colonies in gelatin 853 

181. Kidneys in hog cholera, hemorrhagic points 86 1 

182. Negri bodies 872 

183. Amoeba coll 877 

184. Leishmania donovani 880 

185. Structure of trypanosome 882 

1 86. Trypanosoma gambiense 883 

187. Glossina palpalis 884 

1 88. Colonization in Trypanosoma lewisi 887 

189. Malarial parasite in human and mosquito cycles 891 

190. Longitudinal section of Anopheles 893 

191. Babesia bigemina 895 

192. Ornithodoros moubata 901 

193. Spirochceta duttoni 902 

194. Treponema pallidum ; . . . 903 

195. Ps. medicaginis 952 

196. Pear blight 958 

197. Walnuts affected by bacteriosis 964 

198. Crown gall 966 

199. Roots of cabbage plant affected with "stump-root." 970 

200. Plasmodiophora brassica. . 971 

Colored Plate 
The Malarial parasites 891, 892 


HISTORY OF MICROBIOLOGY* 


Geronimo Fracastorio, of Verona, was born in 1484, studied medicine 
in Padua, and published a work in Venice in 1546, which contained the 
first statement of the true nature of contagion, infection, or disease 
organisms, and of the modes of transmission of infectious disease. He 
divided diseases into those which infect by immediate contact, through 
intermediate agents, and at a distance through the air. Organisms 
which cause disease, called Seminaria conlagionum, he supposed to be 
of the nature of viscous or glutinous matter, similar to the colloidal 
states of substances described by modern physical chemists. These 
particles, too small to be seen, were capable of reproduction in ap- 
propriate media, and became pathogenic through the action of animal 
heat. Thus Fracastorius, in the middle of the sixteenth century, gave 
us an outline of morbid processes in terms of microbiology. 

Athanasius Kircher, in 1659, demonstrated the presence of " minute 
living worms in putrid meat, milk, vinegar, etc.;" but he did not 
describe their form and character, and it is doubtful whether he ever 
saw microorganisms. 

In the year 1683 Antonius van Leeuwenhoek, a Dutch naturalist and 
a maker of lenses, communicated to the English Royal Society the re- 
sults of observations which he had made with a simple microscope of 
his own construction, magnifying from 100 to 150 times. He found in 
water, saliva, dental tartar, etc., what he termed "animalcula." He 
described what he saw, and by his drawings showed both rod-like and 
spiral forms, both of which, he said, had motility. In all probability, 
the two species he saw were those now recognized as Bacillus buccalis 
maximus and Spirillum spuligenum. Leeuwenhoek's observations 
were purely objective and in striking contrast with the speculative 
views of M. A. Plenciz, a Viennese physician, who in 1762 published a 
germ theory of infectious diseases. Plenciz maintained that there 
was a special organism by which each infectious disease was produced, 

* Prepared by F. C. Harrison. 


2 HISTORY OF MICROBIOLOGY 

that microorganisms were capable of reproduction outside of the body, 
and that they might be conveyed from place to place by the air. 

The important role that the compound microscope has played in 
microbiology calls for something regarding the invention of this in- 
strument an invention which antedates Leeuwenhoek's discovery by 
nearly 100 years. 

The first compound microscope was made by Hans Jansen and his 
son Zaccharias, in 1590, at Middelburg, in Holland. The instrument 
was composed of two lenses mounted in tubes of iron; a representation 
of it, made from the original and still kept at Middelburg, is shown 
in Fig. i. From that date the microscope gradually improved. In 
1844 the immersion lens was introduced by Dolland. In 1870 Abbe 
brought out the substage condenser, which still bears his name. Apo- 
chromatic lenses and many minor improvements were introduced by 
the firm of Zeiss about 1880. 


V 


a fib 


FIG. i. Longitudinal section of a compound microscope made by Zaccharias 
Jansen (1590). a, Microscope tube; &, objective tube; c, ocular. 

In 1786 O. F. Mliller (a Dane) first attempted to classify, according 
to theLinnean system, the various organisms previously discovered, and 
characterized four or five genera among them, the genus Vibrio, in 
which, under the terms bacillus, lineola, and spirillum, we recognize 
forms that correspond with our "bacteria." 

From the middle of the eighteenth century until well on into the 
nineteenth, the history of bacteriology is largely the story of a con- 
troversy between those who believed that minute living organisms, such 
as those above referred to, were produced from inanimate substances, 
and that their formation was spontaneous. Philosophers, poets, and 
common people of the most enlightened nations accepted this doctrine 
down to the eighteenth century. The hypothesis regarding this forma- 
tion was known as that of " spontaneous generation," "heterogenesis," 
and " abiogenesis." The opponents of this theory denied the possibility 
of a transition from a lifeless to a living condition, and contended that 
all life came from preexisting life a theory aphoristically summed 
up in the phrase "omne vivum ex vivo." Such was the doctrine of 
Biogenesis life only from life. 


HISTORY OF MICROBIOLOGY 3 

In 1668, Francisco Redi, an Italian, distinguished alike as scholar, 
poet, physician, and naturalist, expressed the idea that life in matter is 
always produced through the agency of preexisting living matter; but 
the beginnings of the real controversy date from the publication of 
Needham's experiments in 1745. The English divine boiled some meat 
extract in a flask, made the flask air-tight, and left it for some days. 
When the flask was opened, he found in it what he termed "infusoria." 
He naturally concluded that all life had been killed by boiling; and, 
as the entrance of fresh life from the outside was prevented by the 
closing of the flask, he considered that the living infusoria must have 
originated spontaneously from the inanimate constituents of the broth. 

Twenty years later Abbe Spallanzani alleged that the development 
of the infusoria "in an infusion maintained at boiling-point for three- 
quarters of an hour was possible only, provided air, which had not been 
previously exposed to the influence of fire, had been admitted." Ob- 
jections were made to these experiments and the controversy went 
merrily on. Gradually experimental evidence accumulated resulting 
largely from the work of Franz Schulze, and the discovery by Schroeder 
and Dusch in 1853, that putrescible fluids will not decay after boiling, if 
protected from the bacteria of the air by means of a cotton-wool 
filter or plug; and the epoch-making experiments of Pasteur in 1860, 
with the now well-known Pasteur flask, showed conclusively that the 
hypothesis of spontaneous generation, or abiogenesis, could not be 
proved. 

Liebig, the celebrated German chemist, strenuously opposed the 
theories of Pasteur; his authority and the brilliancy of his expositions 
influenced the scientific world during the period 1840-60. To Liebig, 
fermentation was a purely chemical phenomenon unassociated with any 
vital process; and he treated Pasteur's results with disdain. "Those 
who pretend to explain the putrefaction of animal substance by the 
presence of microorganisms," he wrote, "reason very much like a child 
who would explain the rapidity of the Rhine by attributing it to the 
violent motions imparted to it in the direction of Bingen by the numer- 
ous wheels of the mills of Mayence." Again and again Liebig formally 
denied the correctness of Pasteur's assertions; finally Pasteur challenged 
him to appear before the Academic Commission to which they would 
submit their respective results. Liebig, however, did not accept the 
challenge; the victory was with the French savant. 


4 HISTORY OF MICROBIOLOGY 

In 1841 Fuchs investigated some blue and yellow milk. He exam- 
ined it with the microscope and discovered the presence of organisms. 
He succeeded in cultivating the "blue milk" microbe in mallow slime, 
and re-developed the blue color in milk by introducing some of his 
culture. The organisms obtained were sent to Ehrenberg, who named 
them Bacterium syncyaneum, now known as B. cyanogenus, Ps. syn- 
cyanea and B. synxanthus, a name which is still retained in the 
literature. 

Since 1860 the master mind of Louis Pasteur has dominated the 
realm of microbiology. His epoch-making discoveries were largely due 
to his intuitive vision, his skill in device and in the adaptation of means 

i 

to ends, his prodigious industry, and the enthusiasm and love with which 
he inspired his associates. Trained as a chemist, his first appointment 
was to a professorship of chemistry, and his earliest research dealt with 
problems in molecular chemistry and physics. On his being elected 
Dean of the Faculty of Sciences at Lille, he commenced to study fer- 
mentation. His work in this field was soon followed by important 
results: the discovery of the organisms which produce lactic and butyric 
fermentation, and of anaerobic life, or life which flourishes without 
free oxygen. He devised an improved method of making vinegar, and 
demonstrated the presence of the acetic organism which he named 
Mycoderma aceti. Later he studied the diseases of wine, and dis- 
covered that bitterness or greasiness was due to a special ferment, and 
suggested the heating of wines in closed bottles to a temperature of 
60, in order to kill the injurious microorganisms. This process, since 
called pasteurization, is now largely used, and makes it possible for 
manufacturers and merchants to keep and export wine without losing 
its flavor or bouquet. It is interesting in this connection to note that 
a French confectioner named Appert published, in 1811, his method of 
preserving fruits, vegetables, and liquors by heating and sealing,, and 
hence may be looked upon as the founder of the packing and canning 
industry. 

In 1864-65 the silk districts of that region of France, known as the 
Midi, suffered such serious losses that the yield of cocoons fell from 
twenty-six million kilograms to four million, which entailed a loss of 
twenty million dollars and caused widespread distress and poverty. 
An epidemic had broken out among the silk-worms the dread 
disease known as Pebrine. Pasteur was induced to make an in- 


HISTORY OF MICROBIOLOGY 5 

vestigation as to the best means of combating the epidemic; and, after 
several years of study, he found the organism causing the disease, 
suggested remedies, and brought back wealth to the ruined com- 
munities, but at the cost to himself of impaired health and partial 
paralysis. 

Pasteur's results were very suggestive; and one outcome of his work 
was that between 1870 and 1880 several important discoveries were 
made by other investigators. Prior to the dates mentioned, the 
mortality from blood poisoning, gangrene, and other infections follow- 
ing operations was extremely high. Surgeons regarded such a result 
as inevitable, and many agreed with the saying of Velpeau, that "the 
prick of a pin is the open door to death;" but, in 1860, Joseph Lister, 
an Edinburgh surgeon, began to study the possible role of microbes in 
the infection of wounds. By sterilizing his instruments, sponges, liga- 
tures, etc., and using antiseptics, he was able to obtain such a high 
percentage of recoveries that in two years he saved thirty-four patients 
out of forty a percentage unheard of up to that time. Hence the 
origin of the antiseptic and aseptic methods of surgery is traceable 
to Lister's efforts. Lister's methods, suggested by the ideas of Pas- 
teur, have rendered possible the marvelous surgery of the present day, 
banished hospital gangrene, and robbed confinement of its terrors. 

To Lister must also be given the honor of devising the first practical 
way of obtaining a pure culture of bacteria by means of high dilutions. 
By using this method, Lister obtained some idea of the different fer- 
mentations of milk, such as souring, curdling, etc. He also confirmed 
the conclusion of Robert Hall (1874), that milk could be obtained 
from the animal in a sterile condition, thus proving that the souring 
of milk was caused by organisms from some external source. 

In 1872, F. Cohn's System of Classification, based on morphological 
characters, appeared. He distinguished six genera micrococcus, bac- 
terium, bacillus, vibrio, spirillum, and spirochaete; four years later this 
investigator made the important discovery of endospores (spores formed 
within cells), and noticed that organisms in this state were more re- 
sistant to heat than the rods from which they were derived. This fact 
was observed in the well-known "hay bacillus." 

In 1871, Weigert succeeded in staining bacteria with picro-carmine; 
but it was not until 1876 that he used the aniline colors, or dyes, for this 
purpose, and thus opened up a new field which was exploited with such 


6 HISTORY OF MICROBIOLOGY 

beautiful results by Ehrlich, Koch, Gram, and others. The staining 
of microorganisms rendered it possible to obtain pictures of them by 
photographic methods; the art of photomicrography developed thus 
rapidly. 

In 1879, Miquel discovered bacteria which grew or developed at tem- 
peratures between 65* and 75. He isolated them first from the waters 
of the Seine, and subsequently from dust, manure, and other substances. 
Later researches have shown that these thermophilic organisms play im- 
portant roles in various fermentations. 

The ninth decade of the last century was prolific in important bac- 
teriological events. Discovery followed discovery in rapid succession. 
In 1880, Laveran, a French military surgeon, discovered the protozoon of 
malaria; in 1881 Robert Koch introduced the poured gelatin and agar 
plate, which made it possible to obtain pure cultures without difficulty. 
Investigators were quick to take advantage of this method and notable 
results followed. Eberth and Gaffky discovered the bacillus of typhoid 
fever, and succeeded in growing it in culture media. In 1882, Loefrler 
and Schiitz discovered the bacterium which causes glanders; and in the 
following year Koch isolated the vibrio of Asiatic cholera from the in- 
testines of cholera patients. In 1883 Klebs described the diphtheria 
bacterium; and, in 1884, Loeffier grew the organism in pure culture. 

In 1884, Koch published his results on the etiology of tuberculosis, 
in a paper which will remain as a classical masterpiece of bacteriological 
research, owing to the difficulty of the task and the thoroughness of the 
work. Not only did Koch show the tubercle bacterium by appropriate 
staining methods, but he succeeded in obtaining pure cultures of it and 
in producing tuberculosis by inoculation with his isolated cultures. 

In 1885, Nicolaier observed the tetanus bacillus in pus produced by 
inoculating mice and rabbits with soil; later, in 1889, Kitasato isolated 
this organism, and showed that the cause of the failure in earlier 
attempts to isolate it were due to the fact that it could grow only in the 
absence of free oxygen. The specific infecting agents in pneumonia 
were discovered by Friedlander and Fraenkel about this time, as were 
also several organisms associated with inflammation and suppuration, 
such as the Streptococcus pyogenes and the Staphylococcus pyogenes, 
discovered by Rosenbach, and the green pus germ (Pseudomonas 
pyocyanea] by Gessard. 

*A11 temperatures are stated in Centigrade scale, unless otherwise indicated. 


HISTORY OF MICROBIOLOGY 7 

While these discoveries were taking place, largely in Germany, Pas- 
teur had been engrossed with his prophylactic studies. In 1880, he dis- 
covered a method of vaccination against fowl cholera; and in 1881 he 
published his method of vaccination against anthrax. On a farm at 
Pouilly le Fort, sixty sheep were placed at Pasteur's disposal; ten of 
these received no treatment, and twenty-five were vaccinated. Some 
days afterward the latter were inoculated with virulent anthrax, and also 
twenty-five which had received no vaccine. The twenty-five non- 
vaccinated sheep died, and the twenty-five vaccinated ones remained 
healthy and in the same state as the ten control animals. This con- 
vincing experiment was followed by others; and, in the twenty-five 
years immediately following the introduction of the method, more* 
than ten million animals were vaccinated in France alone, with ex- 
cellent results. In 1885, as the result of much animal experimentation, 
Pasteur related to the Academy of Sciences his discovery of a method 
of vaccination against, rabies, or hydrophobia; and six months after 
the successful treatment of the first case, 350 persons bitten by rabid 
dogs were vaccinated. An institute for the preparation of vaccines 
was built by public subscription and named the Pasteur Institute; and 
since that date more than thirty similar establishments have been 
founded in different parts of the world. 

This eighth decade, so pregnant with discoveries of the utmost im- 
portance to medicine and surgery, was also notable for its discoveries in 
agricultural bacteriology. The honor of having been the first to work 
out the causal relation between a specific .microbe and a plant disease 
belongs to Burrill, who discovered the organism of Fire or Pear Blight; 
and in 1883 to 1888 Wakker discovered the bacillus which produces the 
"yellows" of the hyacinth, a disease of considerable economic im- 
portance in Holland. To Beyerinck, Hellriegel, and Wilfarth we owe 
our earlier knowledge of the development and morphology of the 
nitrogen-fixing organism which produces the nodules or tubercles on 
the roots of legumes. In 1888 Winogradsky isolated from soils nitrify- 
ing microbes which grew in a medium devoid of all traces of organic 
matter. During this period, Hansen's investigations along the line of 
the fermentation industry were most important. He devised methods 
for securing pure cultures of yeasts starting from a single cell, showed 
that yeasts produced diseases in beer, and established the method of 


HISTORY OF MICROBIOLOGY 

identifying yeasts by observing their microscopic appearance, the for- 
mation of ascospores, and the production of films. 

The tenth decade of the nineteenth century was almost as prolific in 
discovery as the ninth. In 1890 Behring discovered the antitoxin for 
diphtheria, as a result of the pioneer work on toxins by Roux and 
Yersin. Five years later, this serum came into general use as a cura- 
tive agent; and the efficiency of the treatment is shown by a comparison 
of the death rate from diphtheria before and after the introduction 
of the antitoxin. The average annual death rate from diphtheria in 
eight large cities, during the period 1885-94, was 9.74 per 10,000 of 
the population before the use of antitoxin; and during the antitoxin 
period of 1895-1904 it was 4.29. 

The subsequent researches on the constitution of toxins and anti- 
toxins by Ehrlich, Metchnikoff, Madsen, and others have been pro- 
ductive of a better understanding of the problems of immunity. 

In 1892 Pfeiffer discovered the organism of influenza or grippe; and 
in 1894 Yersin and Kitasato independently discovered the bacterium of 
bubonic plague. 

The now well-known serum diagnosis of typhoid fever, whereby 
living and motile typhoid bacilli are clumped and lose their motility 
when placed in the diluted serum of a patient suffering from the 
fever, was due to the work of Gruber and Durham, and the exploitation 
of the method by Widal dates from 1896. 

In 1898, Shiga discovered the bacterium of dysentery, and the pos- 
sible cause of pleuro-pneumonia in cattle was found by Nocard. This 
latter organism was so minute as to be at the extreme limit of micro- 
scopic definition, and suggested that other well-known diseases, such as 
foot-and-mouth disease, are probably caused by ultra-microscopic 
organisms. 

This year, Ronald Ross worked out the relation between man, the 
mosquito, and the malarial parasite a discovery which at once sug- 
gested the best means of controlling the disease. 

In 1905, Schaudinn definitely established the causal agent of syphi- 
lis, a spirochaete-shaped organism, which he named Treponemapallidum, 
and which had escaped earlier discovery on account of its being refractory 
to the ordinary staining methods. 

In the last decade, our knowledge of certain communicable diseases 
has been extended considerably. Preventive and prophylactic measures 


HISTORY OF MICROBIOLOGY Q 

have been studied extensively and carried out on a scale never before 
contemplated, and probably made possible only by war conditions. A 
few of these may be mentioned as examples of the progress made: 
the Dakin-Carrel treatment of septic wounds, the immunization of 
troops against typhoid, tetanus and pneumonia; the increasing use, 
improvement in manufacture and efficacy of protective and curative 
sera and vaccines; the importance of the carrier in many infections, 
and the means whereby he is dealt with, as instanced in the case of 
infection with the meningococcus; the discovery of filtrable viruses as, 
to quote the most recent (1919), the inciting agent of mumps. 

No one can deny that the progress of microbiology in the last fifty 
years has been wonderful, and in the last few years extraordinary, but 
much still remains unknown and new problems appear from time to 
time. The etiology of certain diseases yet remain undiscovered. The 
cause of the disease known as influenza which carried off so many in 
the fall of 1918 remains as yet unknown although some reports of 
alleged discoveries have been made. Trench fever is another example 
of a problem suddenly appearing and necessitating instant solution. 
'in the last few years a group of pleomorphic organisms have 
been discovered, which are associated with typhus, Rocky 
Mountain fever and trench fever. These organisms are carried by 
insects but have not yet been cultivated." 

So also with other fields of research. Great progress has been made 
in water and food microbiology; more attention is being paid to parasi- 
tology; soil organisms and especially soil protozoa are receiving more 
study and our technique has advanced with great strides. 

In short the work of the microbiologist has become of increasing 
interest and importance in all lines of work. 

The record of past achievement is an inspiration; and the knowledge 
that each discovery is the result of persistent and concentrated effort, 
may give us of the present day firmer faith and greater strength for 
work in the broad and inviting field outlined in this text book. 


PART I 

THE MORPHOLOGY AND CULTURE OF MICRO- 

ORGANISMS 


GENERAL* 

Microbiology is concerned with organisms which range between 
well defined plant life on the one hand, and well defined animal life 
on the other. These living forms are in the main unicellular in 
structure. A gradation exists from the plant world into this mi- 
crobe-world and also from the animal world. No sharp lines can 
be established because Nature seems to blend from one type into 
another leaving no particularly characteristic barrier, although man, 
for his own convenience, strives to construct Nature with very 
definite lines of demarcation. Haeckel was so impressed with the 
organisms which lie between the animal and plant world that he 
found it undesirable to attempt to classify them in the one or the 
other kingdom. Accordingly, he believed it of sufficient importance 
to give a specific name, Protista, to the microorganisms included in 
this specific kingdom. This relationship is clearly set forth by an 
illustration furnished by Minchinf (Fig. 2). 

Morphology has been paramount in classification in the past, yet, 
at first, bacteria were called animals and later plants. With the ad- 
vancement and importance of physiology, it becomes necessary to 


* Editor. 

t Minchin, E. A.: An Introduction to the Study of the Protozoa. 

II 


12 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


consider physical, chemical, nutritive or digestive and general physiolo- 
gical processes along with morphological characters. When these are 
considered there is a marked resemblance of microorganisms, even 
molds and yeasts, to animal life. Assignment to either animal or plant 
life is precarious and unnecessary, for in making such an attempt the 
scientist really does nothing more than prescribe for Nature restrictions 
rather than follow Nature as she exists. 


FIG. 2. Graphic representation of the relation of the animal and vegetable 
kingdoms to the kingdom of Protista (Protistenrcicli}. The Protozoa are represented 
by the portion of the triangle representing the animal kingdom which lies within 
the circle representing the Protista. (After Minchin.} 


From the organization of microbiology by Pasteur, the technic of 
the subject together with, in large part as well, its economic bearing 
seems to be the applied determining factor in bounding the field. The 
subject of microbiology is following at present the course of all scien- 
tific branches it is undergoing division for purposes of intensification 
demanded by practice and by the limitations of man's capacity. 


OUTLINE OF PLANT GROUPS 


OUTLINE OF PLANT GROUPS* 

The following is a diagram of plant groups, showing one scheme of 
placing the bacteria, yeasts, and molds in relation to other groups. 
Only a few of the sub-groups can be shown in such a scheme. 


Plants 


Schizophyta 
(fission-plants) 


/ Schizomycetes (fission-fungi), bacteria. 

I Schizophyceae (f ission-algas) , blue-green algae. 

f Chlorophyceag green algae. 
Alg33 \ Phaeophyceae brown algae. 

( Rhodophyceae red algae. 
Characeae. 

Myxomycetes. 
Actinomycetes 


Thallophyta 


Fungi 


Phycomycetes 


Chytridineae. 
Zygomycetes 

Oomycetes 


(Mucors). 
Saprolegniacese. 

(water fungi). 
Peronosporaceae. 

(downy mildews). 


Ascomycetes 


Imperfect Fungi, 
Conidia only 

Basidiomycetes 


Hemiasci (Monascus). 

Protoascinese (Saccharo- 

myces, Yeasts). 
Protodiscineae. 
Euasci Discomycetes. 

Plectascineas (Aspergil- 
lus, certain Penicillia) 
Pyrenomycetineae. 
f Penicillium, Fusarium, Alternaria.f 
I Oidium, Cladosporium, and others. 
Rusts. 
Smuts. 
Mushrooms. 


Bryophyta (mosses and liverworts). 
Pteridophyta (ferns, etc.) 
Spermatophyta (seed plants). 

t Ascomycetous species occur among these genera but such species are rarely met in bacteriol- 
ogical work; many of the common species of Aspergillus lack the ascigerous form, hence are 
classified by their conidial forms only. 

OUTLINE OF PROTOZOAL GROUPS f 

U AN OUTLINE CLASSIFICATION OF THE PROTOZOA," embracing only parasitic 
and more especially the forms pathogenic for man and domestic animals. For 
discussion of classification see p. 133. 


Protozoa Rhizopoda 


I 


Entamdba buccalis 

Entamceba coll 
Entamceba ' Entam&ba hlslolytlca 

Ent amoeba mehagrldis 
Plasmodiophora {Plasmodiophora brasslca. 


'Charles Thorn, 
t J. L. Todd. 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


Protozoa 


Flagellata 


Sporozoa 


Infusoria 


Parasites 
position 


of uncertain 


Leish mania 


Crithidia 


Trypanosoma 


Leishmania donovani 
Leishmania tropica 
Leishmania infantum 
Trypanosoma gambiense 
Trypanosoma rhodesiense 
Trypanosoma cruzi 
Trypanosoma brucei 
Trypanosoma equinum 
Trypanosoma evansi 
Trypanosoma lewisi 
Trypanosoma equiperdum 


Trypanoplasma 

Cercomonas 

Trichomonas 

Monas 

Plagiomonas 

Lamblia \Lambha intestinalis 

Gregarina 

Coccidium 


Trichomonas intestinalis 
Trichomonas vaginalis 


Haemosporidia 


Plasmodium 


Babesia 


I Eimeria cuniculi (Coccidium stiedce) 
[ Eimeria avium 

Plasmodium vivax 
Plasmodium malaria 
Plasmodium falciparum 
Proteosoma 
Haemoproteus 
Haemogregarina 
Hepatozoon 

Babesia bovis (bigemina} 
Babesia canis 
Babesia parva 
Bartonella 
Anaplasma 

Sarcosporidia { Sarcocystis { Sarcocystis miescheriana 
Haplosporidia { Rhinosporidium { Rhinos poridium kinealyi 
Myxosporidia { Myxobolus { Myxobolus pfeifferi 
Microsporidia { Nosema { Nosema bombycis 
Balantidium{ Balantidium coll 
Toxoplasma 
Histoplasma 
Chlamydozoa 
Rickettsia 
Ultramicroscopic viruses 

Spirochceta recurrently 
Spirochceta \ Spiroch&ta mncenll 

( Spirochata gallinarum 

r~ { Treponema pallidum 

Treponema \ 
i ( Treponema pertenue 


CHAPTER I* 

ELEMENTS OF MICROBIAL CYTOLOGY 

CELLS AND ENERGIDS 

The microorganisms are confined to cells, such as algae, molds, 
bacteria, yeasts, and protozoa, or cytoplasmic masses with a nucleus 
associated with each (Fig. 3). Some are, however, made up of rows 
of cells, such as threads of Cladothrix, occasionally capable of branching 
out, like the mycelium of a mold (Fig. 4, A). There are also some cells 
which have a special structure. In each cell are enclosed several 
nuclei. If certain amoebae are examined, for example, Pelomyxa pa- 
lustris (Fig. 4, 5), inside of what appears to be a cell there are found 
many nuclei. Such cells have not the anatomical value of true cells, 
but seem to represent as many cells as there are nuclei. Each of 
these nuclei with the cytoplasm which surrounds it, equivalent to a 
cell, may be called specifically an energid. Some algae and fungi are 
made up of threads of cells enclosing several nuclei; each cell in- 
cluded in a thread consequently represents a group of organized ele- 
ments, the union of several energids in the same anatomical unit (Fig. 
4, A). 

STRUCTURE or THE CELL 

A typical cell is constituted of three essential elements: the nucleus; 
the cytoplasm; and the cell-membrane. 

The general characteristics of these three elements, and, follow- 
ing this, the study of cell reproduction, may now be systematically 
presented. 

THE NUCLEAR STRUCTURE. General Structure of the Nucleus- -The 
nucleus frequently takes in microorganisms the typical form which it 
assumes in the higher organisms, namely, that of a spherical vesicle 
limited by a membrane, enclosing a hyaline substance called the 
nuclear-fluid, or nudeoplasm (Fig. 22, A, a, B, a). In this nuclear 

*By A. Guilliermond. 

15 


1 6 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

fluid are found : the nucleolus, a spherical corpuscle made up of pyrinin 
to which the chromatin, a characteristic substance of the nucleus, fre- 
quently attaches itself; the chromatic network, the thread of which is 
made up of limn, a very slightly chromophilic substance, enclosing 
some grains, the grains of chromatin, which possess a special affinity 
for basic stains. The chromatin or nuclein is the most important 
substance of the nucleus. 

Centriole. In intimate contact with the exterior of the nucleus and 
sometimes inside is usually found a small body called the centrosome, 
or, if the dense chromatin alone is considered, the centriole (Fig. 21, 
B, a). It is a small chromophilic grain which is often surrounded by a 
clear zone of protoplasm called archoplasm. 


m 
* * 


B 


FIG. 3. FIG. 4. 

FIG. 3. Cells of Saccharomyces cerevisia. 

FIG. 4. Cells made up of several energids. A, A portion of the mycelium of a 
mold, Aspergillus ochraceus. (After Dangeard.} B, Cell of an amoeba, Pelomyxa 
palustris. (After Doflein). 

Value of the Nucleus. The nucleus is an organ indispensable to 
cellular life. It directs for the most part the physiological functions 
of the cell. It plays an active part in nutrition as is indicated by the 
fact that the greater part of the products of nutrition or of reserve 
spreads itself around the nuclear membrane. Finally, it assumes an 
important role in cellular division and in sexual phenomena. 

The experiments of Balbiani which have been repeated by other 
authors show that the cell cannot function without its nucleus. By 
cutting an infusorial cell in two portions, one of which contains the 
nucleus and the other only its cytoplasm, Balbiani found that the 
nucleated part was able to resist the wound which it had received 
and regenerate the cytoplasm which was lacking; whereas the enucleated 
portion soon perished. 


ELEMENTS OF MICROBIAL CYTOLOGY 


It does not seem probable, therefore, that cells can exist without 
their nuclei. Nevertheless, to the present time it has not been possible 
to find conclusive proof of the presence of a true nucleus in bacteria. 
The presence in their cells, however, of a great num- 
ber of small chromatin grains like the chromatin ma- ? 
terial of nuclei, and their evolution during the forma- 
tion of spores, force the observer to admit that these 
represent grains of nuclear substance, and that bac- 
teria have a kind of diffuse nucleus, which is scattered 
in the form of small grains (Fig. 5) in the cytoplasm 
of the cell. 


- 


1 


FIG. 5 Dif- 
fuse nuclei of 
bacteria. A, B. 
wiycoides. (After 
Forms oj Nuclei in Microorganisms. The nucleus Guilliermond.} B, 

of primitive microorganisms is far simpler than in Thiothrixten- 
the higher forms, where it becomes fairly complex. Sweilengrebel.} 
Consequently in the Cyanophycece or blue-green algae, 
the lowest of all algae, the nucleus is in a very primitive state. It is 
large, not separated from the cytoplasm by a membrane, and is made 
up simply of a nuclear fluid and a chromatic network. The cyto- 
^_ plasm is confined to a thin cortical layer 

and the nucleus nearly fills the cell (Fig. 6). 
In other microorganisms the nucleus is 
much more complex. Yet frequently this 
nucleus is found in a primitive state quite 
different from typical nuclei of higher 
organisms. In some amoebae, the nucleus 
is formed simply of a poorly defined mem- 
brane filled with nuclear fluid, and a large 
body of chromatin resembling a nucleolus 
called the karyosome or centriole-nudeolus 
(Fig. 22), because it acts both as a cen- 
triole and as a nucleolus. In the center of 


C 


D 


FIG. 6. Nuclei of Cyano- 
phycecB. A, Thread of Rivu- 

laria bullata with nuclei in . ,. 

process of division. B,-D, :he karyosome is frequently seen a more 

Fragments of threads of Colo- intensely chromophilic corpuscle corre- 

thrix puhinata showing nuclear ,. . , /T -,. 

division. spending to the centriole (Fig. 21, B, a). 

Many protozoa and some algae have a 

centriole-nucleolus, but it is wholly enclosed in the nuclear fluid. 

The chromatin appears as little grains or as a network (Fig. 21, A, a). 

In the higher microorganisms (protozoa and fungi) the nucleus 


1 8 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

begins to take the form of typical nuclei. The centriole detaches 
itself from the karyosome which becomes a true nucleolus, and may 
remain either wholly intranuclear (Fig. 20, A, a, 22, A, a), or become 
entirely extranuclear (Fig. 20, B, a, 22, B, a). 

Theory of Binudearity of Cells and Chromidia. In the infusoria, the 
nuclear structure divides into two nuclei (Fig. 8); a large one, the 
macronucleus or vegetative nucleus, which functions during the vegetative 
life of the cell, and a small one lodged in a hollow of the macronucleus, 
the reproductive nucleus or micronucleus. At fertilization, the macro- 
nucleus is disorganized and its place taken by the micronucleus which 
reproduces by division both a micronucleus and a macronucleus. 
Certain flagellates have likewise two nuclei, a large vegetative and re- 
productive nucleus, and a small micro- 
n or kinetonucleus which controls the for- 
mat i n f the flagellum. 

Starting from these facts, a few in- 
B vestigators have tried to demonstrate 

Fig. 7 .-Chromidia in pro- that a11 Cells have two nuclei ' Recent 
tozoa. A, The cycle of the mi- evidence reveals that there are in the 

uf^^^r^S. cytoplasm of most protozoa small chro- 
maba histolytka. (After Hart- mophilic granules, like the chromatin 
chromidia"' Nnclfuf ' chr ' material, which are supposed to emigrate 

from the nucleus during certain phases 

of development, and which are likened to the nuclear substance 
(Fig. 7). These granules are called chromidia, and all the granules 
scattered in the cytoplasm are designated as the chromidial structure 
or chromidium. Chromidia have been found in the cells of higher 
organisms. There is a theory that this chromidial system repre- 
sents a second nucleus, the vegetative nucleus, scattered in the cyto- 
plasm, and that the entire cell is provided with two nuclei, one of 
which has passed unseen up to this time because of its diffuse form. 
This theory is much doubted to-day, and it seems probable that the 
chromidium is simply a reserve material for the cell, or corresponds 
to formations which will be described later as mitochondria. 

CYTOPLASM.- -Appearance and Properties of Cytoplasm Cytoplasm 
may be denned for our purposes as a semi-fluid substance, granular in 
appearance, and reacting with an acid stain. It has three essential 
physiological properties, nutrition, motility, and sensibility. Cyto- 


ELEMENTS OF MICROBIAL CYTOLOGY 19 

plasm appears to be composed largely of protein substances and of 
diverse lipoid substances in a state of colloidal- solution. It varies 
widely according to circumstances, consequently it may be useless to 
search for any definite structure. In many microorganisms, as for 
example the protozoa, there is on the periphery of the cell a hyalin zone 
which is called the ectoplasm to distinguish it from the rest of the 
cytoplasm, the endoplasm (Fig. 17). 

Chondriosomes. Recent research has demonstrated special func- 
tioning bodies in the cytoplasm, the mitochondria, which seem to be 
the constructive elements of cytoplasm. They are a part of its struc- 
ture, and are supposed to play an important physiological role in the 
cell. These structures, visible in the living organism, but stained 


/ 

%', 


r'* 


j 

A' 


t 


\ 
1 


^ 

> 


. 


I 
i 


ch 


n \ 

\ f 


? 

e . , 


-B 


" I 'I , 


I 

f 

mu"' - 

K * \u\\ui' 

\ /* - i\y 

$Ky A * 

FIG. 8. Glaucoma piriformis, FIG. 9. Division of micronu- 

infusorian with (N) ' macronu- cleus and of the chondriosomes 

cleus, (n) micronucleus, (ch) in Carchesium polypinum, infu- 

mitochondria, (vp) pulsating sorian. (After Faure-Fremiet.) 
vacuole. (After Faure-Fre- 
miet.) 

only by a special process, are sometimes in the form of small isolated 
granules (granular mitochondria, Fig. 8, B), or of small threads (thread- 
mitochondria} or sometimes of rods much like certain bacilli (rod- 
mitochondria, Fig. 8, A). These forms frequently change from one to 
the other. The granular mitochondrium is able to elongate itself into 
a rod which is itself capable of dividing up into thread-mitochondria. 
All the mitochondria of one cell are called the chondrium. These 
structures seem to be made up of lipoidal substance and phosphates of 
albumin. 

The mitochondria cannot generate themselves directly from the 
cytoplasm, but are formed always from preexisting mitochondria by 
division. They apparently transmit themselves, after having divided, 
from the egg to the adult individual, and from the adult individual 
to the egg (Fig. 9). 


20 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

Physiologically, mitochondria are organs of elaboration. In 
them, through some unknown physico-chemical phenomena, most of 
the products of cell activity may be formed. The product, whatever 
may be its specific nature, has its origin in a granular mitochondrium 
or in a rod-mitochondrium. Each product is surrounded by a 
mitochondrial exterior surface inside of which it develops slowly; the 
exterior surface remains until the product has reached its state of 
maturity. 

It has been known for some time that there exist in higher plants 
corpuscular elements called plastids or leuco plastids, which also possess 
a synthetic function. Some, the chloroplastids, make the chlorophyl 


' 


& 


A 

FIG. 10. Formation of chloroplasts in the young leaf of barley. A, Very young 
cells in which appear rod-mitochondria. B, Older cells in which the rod-mitochondria 
are transforming themselves into chloroplasts. C, Cells in which the chloroplasts 
are definitely constituted. 

which, by using rays of light as energy, forms starch; others, the 
amylo plastids, confine themselves to forming starch from the excess 
sugars found in the cells; still others, the chromoplastids, constitute the 
pigment bodies of plants (xanthophyl, carotins). It has been recently 
shown that plastids are nothing but mitochondria which have under- 
gone greater differentiation and specialization than those which, at the 
expense of ordinary mitochrondria derived from the egg, have increased 
in size (Figs. 10, n). 

Mitochondria have been found in most protozoa and fungi. In the 
latter they take part in the formation of reserve products, especially 
the met a chromatic corpuscles of which more will be said later. 

Mitochondria are most highly developed in algae where they give 
origin to chloroplastids as in higher plants. On the other hand 3 in 


ELEMENTS OF MICROBIAL CYTOLOGY 


21 


the lower forms, no mitochrondria seem to exist, but the chloroplastids 
take on certain special characteristics. Instead of small scattered 
corpuscles is found one, or occasionally several, large chloroplastids 
filling most of the cell. They are in various shapes ribbons, spirals, 
nets, etoilated bodies (Fig. 12), etc. but all appear to be made up of 
a mitochondrial substance. Their physiological role is much more 
general than in the chloroplastids of higher plants. They produce 
not only the chlorophyl, but other pigment bodies, the starch or para- 
mylum, metachromatic corpuscles, and globules of fat. Conse- 


0-X- 


chr 


FIG. IT. FIG. 12. 

FIG. ii. A cell from the root of a bean in which the rod-mitochondria (cli) 
form in the course of their development amyloplasts from which (p) spring grains 
of starch (a). 

FIG. 12. A, Euglena viridis with its star-like chloroplasts (chl.) at the center 
of the organism, the pyrenoid body (Py) surrounded by grains of paramylum (Par), 
eye-spot (o), contractile vacuole (v), flagellum (/), nucleus (). (After Dangeard.) 
B, Micro glena pitnctifera, with two elongated chromatophores arranged longitudinally. 
(A fter Stein.} 

quently the complex chloroplastids of the algae with their general 
function have been considered as a special form of chondrium which, 
instead of being scattered in the cytoplasm as a number of small 
structures, finds itself gathered in very compact masses. 
. The Cyanophycea are the only microorganisms in which the chon- 
drium has not been found. In the Cyanophycece the chlorophyl and the 
blue pigment (phycocyanin) associated with it are diffused throughout 
the cytoplasmic area surrounding the nucleus. The very primitive 
structure of the algse explains to some extent this absence of an im- 
portant structure of the cell. 


22 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

Vacuoles- -There is always in the cytoplasm one (or several) rather 
bulky vesicle filled supposedly with an aqueous solution of mineral 
salts called a vacuole. Vacuoles play an important part in the ab- 
sorption of liquids by the cell. Owing to the mineral salts dissolved 
in the vacuole-nuid, the concentration of which is ordinarily higher 
than that of the surrounding medium, the vacuoles become the center 
of osmotic forces which consequently cause a part of the ambient 
liquid to penetrate the cell and determine its turgescence. 

Very curious vacuoles are found in many protozoa, namely, the 
pulsating vacuoles (Figs. 8, 12). They are small vacuoles which expand 
and contract rhythmically, and which are considered as excretory and 
respiratory organs. The water that has entered the cell gathers in this 
vacuole and is expelled as it contracts. Probably in crossing the body 
this water yields its oxygen to the cytoplasm in order to charge itself 
with carbonic acid and the products of metabolism. 

Reserve Products. --The cytoplasm encloses some structures differ- 
entiable by means of certain stains or chemical reagents as granulations, 
but which are not constituent elements of cytoplasm; they come 
from a secretion of the cytoplasm, and only under certain conditions. 
These grains may be found either in the cytoplasmic substance itself, 
or in the vacuoles included in the cytoplasm. Most of these granules 
are reserve products which appear when nutrition is deficient. Among 
the reserve products most common in microorganisms are the granules 
called metachromatic corpuscles (Fig. 13, A). These bodies, which 
are the object of a special study in connection with molds and yeasts, 
are made up of a substance the nature of which is still unknown, and 
are found in nearly all fungi, in most algae and bacteria, and in many 
protozoa. 

Glycogen and paraglycogen are equally well distributed in micro- 
organisms (fungi, protozoa). Among algse, glycogen is found only 
in the Cyanophycea, but it is elsewhere replaced by starch or para- 
mylum (Fig. n), common products of chlorophyllic assimilation. 

There are also the protein substances, such as crystalloids of 
mucorin scattered in the Mucorina, or the globules of fat common 
in all cells (Fig. 13, B). 

Most of these substances seem to result from the activity of the 
chondrium structure. Recent investigation shows that the meta- 
chromatic corpuscles have their rise among the mitochondria. It 


ELEMENTS OF MICROBIAL CYTOLOGY 


has long been known, on the other hand, that the starch and paramylum 
are always formed in the chloroplastids. 

MEMBRANE. The cell is usually enveloped in a more or less heavy 
membrane, secreted by the cytoplasm, which acts as a protective 
organ for the cell. 

The presence of the membrane is not, however, indispensable; 
many protozoa do not have it, and are consequently naked cells. 
Motility in many microorganisms is closely associated with the mem- 
brane, for the movement of cytoplasm and the flexibility of the mem- 


*' 

*'* 

* 


-- cm 


f A 


FIG. 13. A, Metachromatic corpuscles (cm), in Sarcosporidia, Sarcocysth tenella. 
(After Erdmann.} B, Fat globules (g) in Trypanosoma rotatorium. (Ajter Doflein.} 

brane are essential factors. Cells as a rule have a membrane of 
different degrees of thickness and composition. It may be albuminoid 
or chitinous (Infusoria), or it may be made up of carbohydrates, as 
cellulose, pectose, and callose (algae, fungi). Bacteria always have a 
membrane, but its nature has not yet been definitely determined. 
Often the cell membrane is able to thicken noticeably, and thus protect 
the cell from influences of environment; the cell may then be regarded as 
transformed into a cyst which passes into a state of sluggish existence. 
Encystment is frequent with protozoa, and is produced when the 
environment becomes unfavorable (Fig. 14, A). 

The external layer of the membrane frequently undergoes modi- 
fications, transforming itself into a mucilaginous or gelatinous sub- 


24 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

stance as we see in many CyanophycetB,"m bacteria surrounded by 
capsules, and in zooglea. The membrane then becomes extremely 

thick (Fig. 14, B). 

LOCOMOTIVE STRUCTURE. Most algae and fungi cannot move. 
Many bacteria and all protozoa have more or less perfected locomotive 

structure. 

The Cyanophyeea and many bacteria, although without loco- 
motive organs, present nevertheless oscillatory movements which seem 
due to a general movement of the cytoplasm translated exteriorly 
because of the flexibility of their membrane. With these exceptions, 
movement is effected by means of a locomotive structure. 

This structure is found in its simplest 
form in the pseudopodia of the amceba. 
The naked cell of the amceba pushes out 
pseudopods, simple expansions of the ecto- 
plasm arising at any part of the body, 
which take various shapes, and reenter the 
body without leaving the least trace of their 
existence. It is a result of motility of the 
cytoplasm, one of its essential properties, 
shown here exteriorly because of the absence 
of a cellular membrane. 
geard.) B, Thread of nostoc The locomotive structure is more com- 

la U gin o U u n s d case by " thkk mUd " P 1 ^ other protozoa; the pseudopod 

is replaced by contractile appendages- 
flagetta, or mbratile cilia. 

The flagellum is a contractile appendage of definite shape and 
position which draws the body after it by means of waving movements. 
It is found on bacteria and flagellates. 

The organ of locomotion of bacteria is still little known (Fig. 15). 
It consists of a certain number of contractile appendages placed at 
one end of the cell, or at both, or sometimes distributed over the whole 
body. These appendages, which may be called vibrating appendages, 
have the characteristics of flagella. Their existence, for a long time 
doubted, is now well established. 

The locomotive structure of the Flagellata is much better known. 
It is characterized by one or more flagella inserted in the anterior 
extremity of the cell. In case of more, one frequently folds back 


ELEMENTS OF MICROBIAL CYTOLOGY 25 

toward the posterior end. In the lateral region of the cell it unites 
with a contractile membrane, the undulating membrane, running in 
spiral form along the length of the body, of which it is the free end. 
Flagella are made up of one or more elastic fibers, surrounded by a 
thin cytoplasmic sheath. 

The vibrating cilia are also contractile appendages, differing from 
the flagella only in their smaller size. They cover the whole body 
of the cell, as in the case of infusoria, enabling them to move about 
very easily in liquids. This interpretation is not concurred in by all 
investigators. 

Certain facts lead us to believe that flagella are only transformed 
pseudopods in which the cytoplasmic structure has changed and at the 
same time the kind of movement. Thread- 
like pseudopods are found with a rapid 
rhythmic movement which may serve as 
intermediate forms. Be that as it may, the A 
method of forming these organs is of special 
interest. Apparently they are formed under 

the influence and at the expense of the cen- FIG. 15. Organs of loco - 
f- r i o l e motion in bacteria. A, B. 

subtilis. (After Fischer} 
In the Flagellata the flagellum is always B, Microspira comma. 

inserted in the centriole or in a similar organ (After Fischer and Migula.} 

. C, Spirillum ruorum. 

which appears to issue from the centriole. 

It is not rare to find in cellular division some cells in which the nucleus 
is dividing with a centriole at each of its poles. Each serves as a point 
of insertion for a flagellum (Fig. 16, A, D, E). 

According to recent works, the flagellum is formed in general 
in one of two somewhat different methods. 

In the first case, the centriole divides itself by an elongation, followed 
by a contraction into two centrioles which remain united to each 
other by means of a fine thread, the centrodesmose. The centrodesmose 
then elongates and is transformed into a flagellum. 

In the second case, the centriole divides itself a first time just as 
in the preceding case, but the centriole farthest from the nucleus im- 
mediately undergoes a second division, thus making three centrioles. 
The one nearest the nucleus remains a centriole during nuclear division. 
The centriole situated somewhat farther from the nucleus becomes the 
point of insertion for the flagellum, and is called the blepharoplast or basal 


26 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

grain. The centriole is united to the blepharoplast by a centrodesmose t 
the rhizoplast, which is often absorbed. Finally, the last centriole 
situated beyond the blepharoplast about equally .distant, also unites 
with this cell-organ by a centrodesmose and, by approaching the 
extremity of the cell, causes the elongation of the centrodesmose which 
transforms itself into a flagellum. 

In the infusoria the vibratile cilia insert themselves in the ectoplasm 
and pass through the cuticle to reach the exterior. At the point of 


tr 


FIG. 16. FIG. 17. 


end 


FIG. 16. A, Spongomonas uvella. The nucleus is undergoing mitotic division. 
Two centrioles, each at the base of a flagellum, are located at the two extremes of 
the spindle. (After Hartmann and Chagas.) 

B, Monas termo. The cell lies in repose; a centriole (a) lies at the base of the 
flagellum; in (C) there are two centrioles, in (D) the two centrioles occupy the two 
poles of the nucleus during the process of mitosis; in (E) exists the final nuclear 
division. (After Martin.} 

FIG. 17. Fragments of the peripheral portion of Prorodon teres (infusorian) 
with vibratile cilia and their basal corpuscles, (ect) Ectoplasm; (end) endoplasm; 
(tr) trichocysts. (After Maier and Gurwitch.) 


insertion of each of these cilia is a small chromatic corpuscle or basal 
grain, a trichocyst, also supposed to arise from a repeated division of 
the centriole (Fig. 17). 

The centriole which, as we shall see later, seems to be a motor 
organ associated with the internal cytoplasmic movements during 
cellular division, appears also to be connected with the external move- 
ment of the cell. 


ELEMENTS OF MICROBIAL CYTOLOGY 


REPRODUCTION OF THE CELL 

VARIOUS PROCESSES OF REPRODUCTION. Reproduction of microbes 
is affected by various processes; the cell may reproduce itself by trans- 
verse or longitudinal fission, binary division, schizogony (bacteria, 
flagellata, molds, Figs. 6, A; 18; 20, A). This is by far the most fre- 
quent. It sometimes, however, divides itself by budding, gemmula- 
tion (Yeast, Fig. 3); that is, by the formation of a small protuberance 
which separates itself from the mother cell as a small daughter cell 
which, once free, grows slowly to maturity. 

Finally, a last process and a very frequent one is the formation of 
internal spores, or sporogony (Fig. 19). The nucleus undergoes a 


FIG. 1 8. Schizogony in Amoeba 
polypodia with amitotic division 
of the nucleus. (After Schnlze 
and Lange.} 


FIG. 19. Sporogony. A, Formation 

of spores in Saccharomyces cerevisice. B, 

Formation of spores in B. mycoides. (After 

Guilliermond.) C. Formation of spores in 

Lencocytozoon lovati. (After Fantham.) 


certain number of divisions, and the cytoplasm divides itself inside the 
cell in as many small cells as there are nuclei. These cells become 
spores and are set free by a rupture in the wall of the mother cell. 
Sometimes all the cytoplasm of the mother cell divides into spores, and 
sometimes only a part of the cytoplasm is used, the rest epiplasm 
serving as nourishment to the spores during their growth. 

Whatever the means by which the cell reproduces itself, cyto- 
plasmic changes and nuclear changes take place at the same time. 
The most important of the cytoplasmic changes is the distribution 
of the chondrium structure between two daughter cells, often preced- 
ing the division of this cytoplasmic structure (Fig. 9). 


28 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

The nuclear phenomena are much more important, and better 
known. The nucleus divides in order to furnish each daughter cell 
with a nucleus containing the same amount of chromatin. 

NUCLEAR DIVISION. Nuclear division may occur in one of two 
ways, one very complex, (i) the indirect mode, karyokinesis or mitosis; 
the other very simple, (2) the direct mode, or amitosis. 

Indirect Division, Karyokinesis, or Mitosis. We shall begin with 
the indirect mode which is by far the more common, using as an example 
a Heliozoon, the Acanthocystis aculeata (Fig. 20, A). The nucleus of 
this protozoon at rest contains a large karyosome of a spongy structure, 
and a chromatic network. Outside the karyosome in the nuclear 
vesicle is a centriole surrounded by a hyaline zone, the archoplasm 
(Fig. 20, A, a). 

Mitosis may be divided into four steps or phases. 

The first phase or prophase begins by the emigration of the centriole 
from the nucleus outside of which it surrounds itself by cytoplasmic 
irradiations, making a star-like body, called the aster (Fig. 20, A, b). 
Following this, the karyosome dissolves in the nucleoplasm, supposedly 
conveying material to the chromatic network which enriches itself 
noticeably in chromatin. The chromatic network then relaxes, thickens 
and transforms itself into a more or less spiral cluster, the spireme 
(Fig. 20, A , c) . At the same time the centriole divides into two centrioles, 
each surrounded by an aster (Fig. 20, A, c). Soon these centrioles place 
themselves at the two opposite poles of the nucleus (Fig. 20, A, d), while 
the spireme breaks itself up into a definite number of chromatic sec- 
tions, the chromosomes. While this is taking place, the nuclear mem- 
brane dissolves itself into a series of cytoplasmic fibrils, the achromatic 
spindle, resistant to nuclear stains. They appear in the middle of 
the nucleus and converge at each end to the centrioles (Fig. 20, A, d, 
c). The chromosomes group themselves in the center of the spindle 
as the equatorial plate (Fig. 20, A, e), the formation of which completes 
the prophase. Each of the chromosomes is attached to one of the 
fibrils which make up the achromatic spindle. 

The second phase or metaphase consists of the longitudinal di- 
vision of the chromosomes each of which divides itself into two equal 
chromosomes. 

In the third phase or anaphase the chromosomes equally divided 


ELEMENTS OF MICROBIAL CYTOLOGY 


2 9 


move to the two poles where they make two polar plates. The cen- 

trioles located here seem to have some attraction for the chromosomes. 

Finally comes the telo phase or phase of reconstitution of the two 

nuclei which terminates the process. In this phase, the chromosomes 


X 


wm^***%^^N^, 


^ 

3 


& 


f? 

b 


. 

y 


5 


FIG. 20. Karyokinesis (metamitosis) . A, 'Acanthocystis aculeata; (a) nucleus 
in state of repose with an intranuclear centriole; (6) (prophase) the centriole moves 
to the periphery and out of the nucleus and forms an aster (After Hertwig) ; (c) the 
division of the centriole and spireme; (d) the formation of the equatorial plates and 
the achromatic spindle; (e) equatorial plates; (/) anaphase; (g) telophase. (After 
Schaudinn.) B, In Coleosporium senecionis (Uredineae) . (a] Nucleus at rest with 
its centriole extranuclear; (&) formation of chromosomes; (c) equatorial plate; (d) 
metaphase; (e) anaphase; (/) (g) (i] telophase. (After Madame Moreau.) 

form a spiral chromatic cluster making a spireme at each of the poles 
(dispireme stage, Fig. 20, Ajg); each of the spiremes is then surrounded 


30 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

by a nuclear membrane in which is included the centriole. Thus the 
two nuclei are formed in which a nucleolus soon appears. Mean- 
while the cell has elongated, become constricted in the center, and 
finally broken into two cells (Fig. 20, B, f, g, i). The achromatic 
spindle completely disappears. 

This method of division represents the typical method of karyo- 
kinesis, that which is observed in higher organisms with the single 
difference that the centriole is intranuclear, whereas in the cells of 
higher organisms it is ordinarily outside the nucleus in contact with the 
nuclear membrane. An analogous mitosis is found in the Uredinea 
(Fig. 20, B, a-i), except that the centriole is here found to be extra- 
nuclear (Fig. 20, B, a), the asters are lacking, and the nucleolus persists 
to the end of mitosis expelled in the cytoplasm. The physiological 
significance of the nucleolus in this case is not known. This method of 
division is seen in certain molds and higher protozoa, and is called 
metamitosis or perfect mitosis. 

Summing up, mitosis is a process functioning to make an absolutely 
equal division of the chromatin between the two nuclei. This dis- 
tribution is performed by the breaking up of a spireme into a definite 
number of chromosomes, a number varying according to the species 
but always constant for any single species, and then by a longitudinal 
division of the latter. The centrioles seem to play an important role 
in this phenomenon, in directing it, and in attracting the chromosomes 
once divided toward the poles of the cell where the nuclei are formed. 

It is not necessary to conclude that the processes of mitosis are 
as complex as in other microorganisms. Relatively simple in the 
lower forms, mitosis becomes complicated as it climbs the ladder, 
gaining the characteristics of metamitosis only in the most advanced 
forms. 

The simplest case is found in the Cyanophycece (Fig. 6). Here 
cellular division begins by the outline of the transverse partition 
which appears in the form of a peripheral ring. At the same time 
the chromatic network takes a definite arrangement; its filaments 
arrange themselves parallel to the longitudinal axis of the cell, thus 
giving this division the appearance of a mitotic division. The outline 
of the partition extends little by little toward the middle of the cell, 
leaving open only a small spherical space in its center to which the 
fibers of the network then contract, and the nucleus takes the form of 


ELEMENTS OF MICROBIAL CYTOLOGY 


B 


T 

* 

c 


^ 


> 


v 


''"iiL\{H 

,<r.i, v// 


/ 


FIG. 21. Protomitosis. yl, In Amoeba mucicola. (a) Nucleus at rest; (b) 
beginning of prophase; (c) division karyosome; (d) division of centriole; (e) (/) 
equatorial plate; (g) metaphase; (i) (k) telophase. (After Chatton.} B, In Amoeba 
froschi. (After Nagler.} C, In Euglena splendens. (After Dangeard.) D, In 
Amoeba diplomitotica. (After Beaurepaire Arago.) 


32 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

a dumb-bell. Soon the partition stops completely, the filaments of 
the contracted part of the nucleus break up and the two daughter cells 
appear separated by a partition. The two nuclei whose filaments have 
been sectioned by the partition are not slow in recovering their in- 
tegrity (Fig. 6, b). 

We find in the Amoeba mucicola (Fig. 21, A) a much more char- 
acteristic mitosis, though more primitive. The nucleus of this amoeba 
when at rest is made up of a nuclear fluid surrounded by a membrane 
in which are a large karyosome and some small grains of chromatin 
localized on the periphery (Fig. 21, A). In the center of the karyosome 
is a small chromophilic centriole. The prophase begins by the elonga- 
tion of the karyosome to a rod-shaped body (Fig. 21, A, b) which then 
transforms itself into a dumb-bell (Fig. 21, A, c). The centriole 
also elongates and becomes constricted in the center (Fig. 21, A, d). 
At the same time an achromatic spindle appears all about the con- 
stricted region of the karyosome in the middle of which the grains of 
chromatin arrange themselves peripherally to form an equatorial 
plate, but there is no differentiation of this chromatin into two 
chromosomes (Fig. 21, A, c, d). In the metaphase the karyosome 
and the centriole divide into two polar masses (Fig. 21, A, e, /), the 
equatorial plate separates into two plates which, in the anaphase, 
emigrate to the poles (Fig. 21, A, g) drawn by the centrioles. In the 
telophase the spindle elongates, disappears, and the two nuclei are 
formed at the poles (Fig. 21, A, i, k). The nuclear membrane exists 
during the entire phenomenon. 

In other microorganisms (Amoeba, Flagellata, Euglence) is found a 
similar mitosis except that the chromatin distributed in the resting 
nucleus as a network or as rod-shaped bodies forms an equatorial plate 
made up of true chromosomes (Fig. 21, B, C). 

Another form of mitosis, promitosis, is characterized, by the fact 
that the centriole is included in the karyosome, by the persistence of 
the nuclear membrane, and by the simultaneous division in the meta- 
phase of the karyosome and of the chromatin gathered in an equatorial 
plate. 

Between promitosis and metamitosis are a series of intermediate 
forms. In the Pelomyxa palustris, for exam'ple, the centriole while 
remaining intranuclear is able to separate itself from the karyosome 
(Fig. 22, A, a). The prophase here begins with the usual division of 


ELEMENTS OF MICROBIAL CYTOLOGY 


33 


the centriole (Fig. 22, A, b, c), and the two resulting centriole- threads 
pass to the extremities of the achromatic spindle, while the karyosome 
cooperates in the formation of the chromosomes (Fig. 22, A, d, e). 

In other cases (various fungi, Gregarin&j etc.), the centriole be- 
comes extranuclear, and the karyosome acts as a true nucleolus (Fig. 


* 

. 


B 


* 


I 


FIG. 22. Mesomitosis. A, In Pelomyxa palustris. (a) Nucleus at rest; (b) 
(e) division of centriole; (/) (g) equatorial plate; (h) anaphase. (After Bott.} 
B, In Urospora lagidis (Gregarina). (a) Nucleus with extranuclear centriole and 
aster; (b} the centriole is divided and the spireme is formed; (c) spireme; (d) equa- 
torial plate; (e) anaphase. (After Brasil.) C, In the ascus of Galactima succosa 
(Ascomycete). (a) Equatorial plate; (b) anaphase; (c) telophase. 

22). Sometimes it dissolves at the beginning of mitosis, seeming to 
aid the development of the chromatin of the spireme, and sometimes 
it persists during the entire process and is expelled in the cytoplasm 
at the end of the phenomenon without any known function. The 

3 


34 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

name of mesomitosis has been given to all the mitoses which distinguish 
themselves from promitosis by the persistence of a nuclear membrane 
throughout the phenomenon. 

Direct Division or A mitosis --This consists simply of an elongation 
of the nucleus followed by a median constriction, then by a rupture of 
this constricted part without an equal division of the chromatin be- 
tween the two nuclei which often are not the same size. It is a simple 
breaking up of the nucleus. Amitosis, then, does not necessarily in- 
sure the equal distribution of chromatin between the two nuclei. 
This rare process is found in higher organisms only in old cells that are 
degenerating, or in diseased cells. Although for a long time it was 
thought to be a primitive phenomenon, it is now considered to be 
degenerative. We see, however, in certain Amoeba and Mycetozoa 
the karyosome enclosing all the chromatin divides itself into two equal 


FIG. 23. Conjugation in Schizosaccharomyces octosporus. (a) Two gametes in 

the process of fusion; (6) (c) nuclear fusion. 


bodies, showing the characteristics of a very primitive mitosis (Fig. 18). 
Amitosis seems to exist normally in yeasts and in certain molds. In 
the yeasts, for example, the nucleus divides by amitosis in the course 
of budding (Fig. 3), and mitosis is found only in the course of sporu- 
lation. 

SEXUAL CHANGES. In most microorganisms at certain times during 
their existence occur sexual changes, or fertilization, which seem to give 
them a new strength. It is followed by a period of very active re- 
production, whence the name of sexual reproduction given to these 
changes. This consists essentially in the fusion of two equal 
isogamous (is o gamy) or unequal, anisogamous (heterogamy) cells or 
gametes. In the latter case, the male is small and active, and the 
female large and passive. The fusion between the two cytoplasms 
and the two nuclei takes place at the same time (Fig. 23). 

If nuclear fusion were not compensated by an elimination of 
chromatin, the nucleus would increase in this substance at each fertili- 


ELEMENTS OF MICROBIAL CYTOLOGY 35 

zation. But this change is succeeded immediately in protozoa by a 
common process called chromatic reduction. The chromosomes 
in the course of the divisions which precede the formation of the 
gametes reduce themselves to half by a complex process which it 
would be superfluous to describe here. The same chromatic reduction 
takes place in the fungi and algse, but this does not always precede 
fertilization. It may follow it immediately as in the yeasts where it 
seems to produce itself during the nuclear divisions in the ascus. It 
may also occur during other stages of development. 


CHAPTER II* 

MOLDS 

FUNGI IN GENERAL 

Certain fungi commonly designated molds are constantly met in 
microbiological studies. They are found as contaminating colonies in 
microbial cultures. They are agents together with other microorgan- 
isms in the processes of fermentation 'and decay in the soil, and in the 
spoilage of food-stuffs. Certain of these forms approach the structure 
and habits of other microorganisms very closely. Members of these 
border groups have been sometimes described as bacteria, some- 
times as fungi; among them the Actinomycetes have been principally 
studied by bacteriologists, but recently Drechsler has succeeded in 
describing their fruiting forms in terms which leave no doubt that 
they are a fungous group. In describing microorganisms, cultural 
reactions have been largely used in characterizing the bacteria and 
related organisms; morphology has been the basis of nearly all descrip- 
tions of the mycelial fungi. 

With some exceptions, there is, among the cells of the true fungi, 
a differentiation of function into vegetative or assimilative cells and re- 
productive cells. The fungous body is usually composed of threads 
(technically called hyphce, singular, hypha). These hyphce usually 
branch in more or less complex manner forming networks or webs, 
collectively called mycelium. Hyphae may be one-celled or composed of 
many cells placed end to end as shown by the cross walls, called septa, 
seen in them. These threads grow either by the formation of new cells 
at the growing tips (called apical growth) or by the division of cells in 
the hypha (intercalary growth). The fungous cells rarely divide in 
three planes to produce solid masses of cells. Both vegetative and 
reproductive masses are formed in great variety from such hyphae. 
Often the thread-like character is almost or quite obliterated in the ripe 

* Prepared by Charles Thorn. A. Guilliermond has furnished the sections on " Cytology of 
Molds " 

36 


MOLDS 37 

masses, which may be fleshy, woody, carbonaceous, leathery and 
even horn-like in texture, as seen especially in the mushrooms, bracket- 
fungi, etc., but even in such cases the early stages show the structures 
to originate from masses of fungous threads. 

The formation of differentiated reproductive cells is, in general, 
characteristic of the fungi. The method of reproduction presents great 
variety. In the simplest forms, the reproductive cells are scarcely, if 
at all, distinguishable from the vegetative cells. In some species whole 
hyphae break up so that each cell forms the starting-point of a new 
colony. Other forms develop special branches bearing reproductive 
cells. From these it is but a step to the production of fruiting branches, 
characteristic in form, called conidiophores, bearing cells markedly 
specialized as reproductive by form and frequently also by color, 
called conidia. These conidia are entirely asexual in origin and capable 
of growing directly into new colonies, although in many cases they are 
provided with resistant walls which enable them to live for long periods 
if conditions are unfavorable to growth at once. In other species, 
specialized resting cells with resistant walls are formed to enable the 
plant to survive unfavorable conditions. These are called chlamydo- 
spores or sometimes cysts. The name gemma is sometimes applied to 
similar structures, preferably to such as grow at once. The same end is 
reached in still other groups by the formation of sclerotia which are 
hard masses or balls of thick-walled cells filled with concentrated food 
materials. These sclerotia are frequently distinctive of the species 
producing them by size and appearance. They vary from microscopic 
in size to masses weighing many pounds and may perhaps in cases be 
aborted fruits. They sometimes resemble such sexual fruiting bodies. 
Resting structures of either type, especially when large, commonly 
produce typical spore-bearing structures at once after germinating. 
Sexuality among the fungi has been difficult to demonstrate in some 
groups with complex fruit bodies. It is certainly suppressed, if not 
entirely wanting, in whole series of cosmopolitan forms and present 
only in rare species in other groups. 

The systems of classification used are largely based upon the types of 
sexual fruit bodies produced. Where such fruit bodies are not known, 
the method of formation of the asexual spores furnishes the most 
satisfactory basis for grouping. In classifying fungi, certain types of 
spore formation are found to be characteristic of particular groups. 


38 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

Since within these groups various accessory types of fruiting occur, so 
that some species show three or even more forms of spores, that type 
of spore formation which is regarded as characteristic of the group is 
known as the perfect stage. If sexual fruits are found, these constitute 
the perfect stage of the group; if no such fruit is found, the most 
characteristic asexual form is used. 

Between the typical forms are many gradations resulting in many 
families whose relationship to one or the other group is difficult to 
determine. Probably the ancestral history (phylogeny) of the fungi, 
if known, would show several or many lines of descent rather than one. 
Many thousands of species of fungi have been described, principally 
in Latin, German, French and English. The literature is widely 
scattered in monographs, reports and journals published in various 
languages. For the purposes of the bacteriological worker, a few 
representative groups with some of the significant species will be 
considered here. 

BACTERIA. In the scheme of plant grouping presented (page 13), 
which is only one of many attempts to show relationships, the bacteria 
are placed with a group of single-celled green or blue-green forms as 
Schizophyta or fission-plants because of reproduction only by the divi- 
sion of the cells. Recent work of Lohnis, Cort and others have opened 
possibilities of specialized spore-production in the bacteria. Such 
reproductive bodies, if proved present and fully described, would 
probably furnish a sound basis for a scheme of relationships of the 
bacteria. At present it is undecided whether they are specialized 
from higher forms by suppression of characters or represent a primitive 
morphology with highly specialized physiological relations. 

PHYCOMYCETES. The Phy corny cetes are called algal fungi because 
they resemble certain groups of green filamentous forms in many 
particulars. In this group two general types of sexual reproduction 
appear zygospore formation and oospore formation. The first, 
found in the Zygomycetes represented by the common mucors, consists 
of the fusion of terminal cells of branches of the mycelium similar in 
appearance but differentiated in sex. As a result of this fertilization 
large thick-walled resting cells are produced, called zygospores, from a 
Greek root meaning yoked (Fig. 33). In oospore formation, found 
in the Oomy cetes, the conjugating cells differ in appearance as well as 
in function. The oospore or egg-cell is large and is rich in food 


MOLDS 39 

materials; the antheridium is much smaller, penetrates and fertilizes 
the oospore, which afterward develops into a thick-walled resting spore. 
The very destructive downy mildews belong to this group. 

ASCOMYCETES. In this great group sexuality was denied until recent 
years, but has been proved in cases enough to establish a presumption of 
more general occurrence. The characteristic structure of the group is 
the ascus, a sac containing, when ripe, typically eight spores, some- 
times a less number by the failure of some to develop, sometimes a larger 
number, usually some multiple of eight. The ascus when sexuality is 
known is developed subsequent to fertilization, not directly from an 
egg cell. The group presents a great variety of fruiting masses pro- 
duced in connection with the asci. The simplest forms are loose webs 
of hyphae enmeshing a few asci; other forms show clubs, cups, flask 
forms, crusted areas, the type of mass in each case being characteristic 
of the family, genus and species represented. Only a few of many 
thousands of these forms are encountered in bacteriological work. 
One genus is, however, constantly found. The commonest species of 
AspergUlus produces bright yellow, globose fruiting bodies, called 
perithecia (singular, perithecium) , rilled with asci. These are borne upon 
the surface of the substratum and often give a yellow color to the colony 
by their abundance. Such perithecia consist of the ascogenous cells 
and the asci produced by them, about which a more or less completely 
closed sac or wall has been formed, by the development of the sterile 
cells adjacent to the fruiting ones. 

BASIDIOMYCETES. In the Basidiomycetts there is still further reduc- 
tion of the evidences of sexuality. 

In some sections of this group an essential sexuality has been corre- 
lated with the fusion of nuclei at stages of life history characteristic 
for the particular sections of the group. Such fusion seems to underlie 
the development of the typical faint body, although it has only been 
demonstrated in a small number of species. The typical structure is 
the basidium, a spore-bearing cell characteristically producing four 
protuberances called sterigmata (singular, sterigma), each bearing a 
.single spore. These basidia are grouped into many kinds of fruit bodies 
varying from occurrence here and there upon a loose web of hyphae to 
dense columnar areas covering the gills of the mushrooms or lining the 
cavities of the puffballs. Very few of these species are encountered in 
bacteriological studies. 


40 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

IMPERFECT FUNGI. A very large number of species are known 
which have never been seen to produce sexual fruits or fruits character- 
istic of the great groups. These are brought together and described as 
form-genera by their method of asexual spore formation. From the 
lack of the organs used in classifying the other groups, these are called 
the Imperfect Fungi and their grouping regarded only as temporary, 
a convenience for the identification of materials. These include many 
forms of economic importance, and many of the species most frequently 
met in bacteriological work. Sometimes one or a few species of a large 
group produce the perfect form while very many species cannot be 
induced to do so. Some of these species undoubtedly represent stages 
of perfect fungi whose perfect forms simply are not recognized as 
connected with these; others as in the more common species of Peni- 
cillium reproduce for an indefinite number of generations by conidia. 
Such species do not appear to need the perfect form and hence ap- 
parently have, in some cases, lost the power to produce it. Genera 
consisting entirely of such species are very properly retained as form- 
genera in the Imperfect group. 

As found in nature all these forms are parasitic, saprophytic, or 
capable of both modes of life. All depend more or less completely 
upon organic matter for nourishment. Great diversity exists, how- 
ever, in their adaptation to environment. Many of them are not only 
parasitic but so closely adapted to parasitizing particular host-species 
as not to be found elsewhere. Others attack several or many species, 
usually related. Even among saprophytes many species are found only 
upon particular forms of decaying animal or vegetable matter. The 
great economic importance of these parasitic and closely adapted sapro- 
phytic species has been recognized by the development in recent years 
of the literature of plant pathology (phytopathology). These cannot 
be considered in this work. 

CYTOLOGY OF MOLDS* 

GENERAL STRUCTURE OF MOLDS.- -Three kinds of cell-structure 
formation are found in molds: 

i. Some, belonging to the Phy corny cetes, show no cross- walls; they 
have a much branched, felted mycelium, but in the early stages there 

* Prepared by A. Guilliermond. 


MOLDS 


are no true transverse septa. Septa appear in many forms only when 
fruiting begins, but in the opinion of some they merely separate the 
living portions of the mycelium from those in which the cytoplasm is 
dead or degenerating. The cytoplasm in the unseptate mycelium forms 
one continuous mass; it contains a great many nuclei (Fig. 24, i and 

2). Each nucleus with the cyto- 
plasm surrounding it, according 
to Sachs, may be considered a 
physiological unit acting in a 
somewhat similar capacity as a 

' . '.* * V Ji^*^ 

,.- cell, or may be designated as an 


1 

. . 
2 


\ 


FIG. 24. FIG. 25. 

FIG. 24. i, Part of the mycelium of Thamnidium elegans (Mucor}. 2, Ex- 
tremity of a filament of Mucor circinelloides showing three swellings about to form 
sporangia. 3, A spore of the same mold. 4, Yeast forms from the same mold. 
(After Leger.) 

FIG. 25. i, Mycelial filament of Endomyces magnusii. 2, Extremity of a 
filament of the same mold in the process of growth, with a dividing nucleus. 3 and 
4, Filaments of Endomyces fibuliger. In 4, metachromatic corpuscles are seen 
in the vacuoles. 5, Filament on the way to increase, from the same mold, the 
nucleus dividing. 

energid. This view is not held by all observers, however. Con- 
sidered thus, the mycelium represents the collection of a great many 
indistinct cells which are not separated by walls. The Mucorinece, for 
example, belong to this structural type. 

2. Other fungi, especially among the Ascomycetes, have a septate 
mycelium, but one in which the transverse septa do not restrict cellular 


42 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

functions as true cells. It consists of compartments containing a 
variable number of nuclei called coenocytes (Fig. 25, i). Each compart- 
ment may be considered, not as a true cell, but as a colony of rudi- 
mentary cells, energids. 

3. Still other molds have a mycelium consisting of true cells with 
a single nucleus, as for example En domyces fibuliger (Fig. 25, 3 and 4) 
and Endomyces decipiens. 

There are, moreover, molds which show both these last two struc- 
tural types, with transitional forms between the two. For instance, 
in Endomyces magnusii, the mycelium, ordinarily consisting of areas, 
each containing many energids, can in some parts progress to a 
uninuclear cellular structure. 

The conidia or spores of many molds may have either one or 
many nuclei, according to the species. The spores of the Mucorinea, 
for example, always have many nuclei (Fig. 24, 3); on the contrary, 
the ascospores of the Ascomycetes, the conidia of Penicillium and 
Aspergillus, contain generally but a single nucleus. 

The yeast forms which result from the budding of the mycelium in 
some molds, most frequently have a single nucleus (Fig. 24, 4) ; how- 
ever, in some, Dematium, are sometimes found yeast-forms containing 
several nuclei. The yeast-forms of the Mucorinece, which are not other- 
wise very typical forms, are always multinuclear. 

To whichever of these three structural forms a mold belongs, it 
always represents some similar constitutional elements which we will 
now consider. 

CYTOPLASM.- -The cytoplasm is a semi-fluid mass, somewhat dense, 
sometimes homogeneous and containing a more or less considerable 
number of vacuoles. Certain methods of fixing and staining have 
recently made possible a demonstration, in the cytoplasm of the 
most diverse molds, of the presence of a chondrium, very clear and 
always splendidly exhibited. This consists mostly of fine rod- 
mitochondria, very long and flexible, generally lying parallel with 
the longitudinal axis of the cell (Fig. 26). Sometimes also it contains 
granular mitochondria. 

The cytoplasm also has reserve products, of which we shall speak 
later. 

NUCLEI. The nuclei show a differentiated structure which is 
sometimes difficult to demonstrate. They consist of a nuclear 


MOLDS 


43 


membrane, a hyaline nucleoplasm, a large nucleolus and a chromatic 
network. The last is sometimes indistinct, and it frequently happens 
that the nucleus appears to contain only a nucleolus; but a very 
careful examination always reveals the network (Fig. 25, 3 and 4). 

The division of the nucleus is not always easy to observe. To study 
it, one must examine the growing tips of the mycelium. In some cases 
this consists in an elongation of the nucleus which soon assumes the 


-. 


FIG. 26. 


FIG. 27. 


FIG. 26. Various molds fixed and stained by a special technic, showing 
their chondrium. i, Filament of Rhizopus nigricans (Mucor). 2-4, Filaments of 
Penicillium glaucum, 5 and 6, Fragments of the conidial organ of the same 
mold. 7, Filament of Endomyces magnusii. 8 and 9, Oidia of the same mold. 
In all these molds, chondrium is represented by long filaments, or sometimes 
by small grains. The filaments often show small vesicles at their crossing. 

FIG. 27. Nucleus of the Mucor (1-4), and various stages of its division (5-8). 
(After Moreau.} 

form of a very slender dumb-bell which breaks apart at the narrow 
portion. This is the extent of an amitotic or direct division (Fig. 25, 
2 and 5). 

Karyokinesis is usually seen only in the organs of fructification 
(asci, basidia, etc.) ; nevertheless, in the mycelium of the Basidiomycetes 
and Mucorinece, true metamitoses have been found. In the Mucorinea 
for_example (Fig. 27), the nucleus loses its membrane (1-4) and gives 


44 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


rise to a spindle ending in a centrosome at either extremity, while two 
chromosomes form the equatorial plate at the center (5). Each of 
the two chromosomes divides and the four resulting chromosomes are 

distributed between the two poles (6-8) where 
they form the two daughter nuclei (Moreau). 
METACHROMATIC CORPUSCLES AND RESERVE 
PRODUCTS.- -The vacuoles always contain a 
great many shining granules, showing Brownian 
motion and capable of being stained in the living 


I 


FIG. 28. 


FIG. 29. 


FIG. 28. Dematium species stained by a method permitting the differentiation 
of the metachromatic corpuscles, i, Filament. 7 and 9, Yeast forms. 9, Yeast 
form starting to bud from mycelium. The metachromatic corpuscles are situated 
in the vacuoles in the form of small grains joined in chains (6) or isolated. Many 
appear like large granules (9). n. Nucleus, v.c. Vacuole with metachromatic 
corpuscles. 

FIG. 29. Various stages of the development of the ascus in Aleuria cerea. 
i and 2, Young asci with their nucleus and many metachromatic corpuscles. 3, 
Fragments of an ascus after the second nuclear division. 4, Ascus, still young, 
in which the ascospores are surrounded by metachromatic corpuscles. 5, Older 
ascus in which most of the metachromatic corpuscles have been absorbed by the 
ascospores. 

state by neutral red and methylene blue. These bodies have staining 
qualities which permit them to be easily characterized. They are stained 
a violet-red by most of the basic dyes, aniline blue or violet. They also 
take on a very pronounced reddish tinge with hematoxylin (Fig. 28). 
By reason of this property of metachromatism, they have been called 
metachromatic corpuscles. These bodies, which are very common in the 
Protista, have been found in yeasts, bacteria, algae and protozoa. The 
chemical nature of the substance constituting them is still unknown, 


MOLDS 45 

but the name metachromatin is often used for it. 1 Some authors, among 
whom is Arthur Meyer, believe them to consist of a combination of 
nucleic acid, but this is a mere supposition. 

On the other hand, the role of the metachromatic corpuscles is now 
well known. It is evident that they are reserve substances. Their 
evolution proves it. Thus metachromatic corpuscles appear in great 
abundance in the young asci of the higher Ascomycetes (Fig. 29, i and 2), 
then accumulate in the cytoplasm of the epiplasm which is not 
utilized in the formation of the ascospores, gather all around the 
ascospores at the time of their forming (3-4), and are gradually 


FIG. 30. Conidial organ of Aspergillus niger with metachromatic corpuscles. 

absorbed by the latter in the course of their development (5). 
They therefore furnish nourishment for the ascospores and from this 
standpoint behave exactly like glycogen and the globules of fat 
which are usually coexistent with them in the cytoplasm. We 
shall see, moreover, that they undergo a similar evolution in the 
asci of yeasts. Likewise in the conidiophores of molds, notably in 
the fruiting heads of Aspergillus and Penicillium, the metachromatic 
corpuscles are produced in great abundance (Figs. 30 and 31), then 
gradually disappear as the conidia form (30, 3). Here again they 
serve as food for the conidia. 

1 Because of the priority and more exact signification, the names metachromatic corpuscles 
and metachromatin are preferable to the terms grains of volulin and volutin given by Arthur 
Meyer. 


46 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

Metachromatic corpuscles appear not only in the vacuoles, but also 
in the perivacuolar cytoplasm. There they spring up, to diffuse finally 
in the vacuole where they increase. It is difficult to observe their 
manner of forming in the mycelial filaments, but in the preparation for 
sporulation in some molds (asci of the higher Ascomycetes), it has 
recently been demonstrated that they start in the midst of the elements 
of the chondrium, which act as plastids similar to the plastids of the 
higher plants. They start in the interior of the granular-mitochondria 
or in the rod-mitochondria (Fig. 31). In the former case, a small cor- 


-s. 


FIG. 31. Formation of metachromatic corpuscles in a cell of the perithecium of 
Pestularia vesiculosa. The rod-mitochondria form, on their crossings, vesicles (c) 
consisting of a metachromatic corpuscle unstained by the special method which 
served to differentiate the chondrium. Some corpuscles (a), more highly developed, 
are found in the vacuoles still surrounded by their mitochondrial shell; others (c) 
at the completion of their development have worn through their mitochondrial 
covering. 

puscle appears in the substance of a mitochondrium, then develops 
gradually, while the mitochondrial membrane which envelops it grows 
thinner; is reduced to a small capping of the grain on one side; then 
disappears when the latter reaches maturity. It is noteworthy that the 
corpuscles emigrate with their plastid to the interior of the vacuoles 
during their development. 

When the corpuscles start in a rod-mitochondrium the process is 
much the same as in the granular-mitochondria; when several rod- 
mitochondria are involved, at their junction small corpuscles are 
seen to form; the parts of the rod-mitochondria which join are then 
absorbed and the corpuscles, enclosed in their mitochondrial membrane, 
once separated, undergo the same evolution as above. 

Thus the metachromatic corpuscles, like grains of starch in the 
higher plants, start in the midst of the mitochondria and develop 
gradually out of their mitochondrial matrix with the aid of the vacuolar 
substance. 


MOLDS 47 

In molds are found still other reserve products. One often sees 
globules of fat in the cytoplasm, which are easily stained by a black- 
brown by osmic acid; and glycogen which can be differentiated by iodine 
in iodide of potassium. The glycogen is contained in either the 
cytoplasm or the vacuoles. It is generally very abundant. 

These products (fat and glycogen) undergo the same evolution as 
the metachromatic corpuscles, and they also accumulate in the organs 
of fructification (asci, conidial organs) to serve in the nourishment of 
spores and conidia. 

CELL-WALL. The cell-wall of molds is quite distinct and often 
thick. It is sometimes cutinized. According to Mangin, it consists 
of callose and pectose with which is often associated a kind of cellulose. 

SPECIFIC CONSIDERATION OF MOLDS* 

A few species are found to grow very constantly in the same situa- 
tions as bacteria. These are associated with forms of decay, fermenta- 
tion, or disease, either as primary or secondary causes. They thus 
become important to the bacteriologist who studies them by the same 
methods as bacteria. These species belong to widely scattered groups 
of fungi, so that species found under the same conditions frequently 
differ greatly in appearance. The common term, molds, is applied 
collectively to these organisms, though no sharp limits can be set to 
the use of the term. Physiologically these species can be considered 
in three series: 

COSMOPOLITAN SAPROPHYTES. Certain species are capable of 
growing within very wide limits of temperature and of composition of 
substrata. Many of these have accompanied man everywhere and are 
constantly found upon every kind of putrescible matter, especially as 
the causes of fermentation or decay in food. Their spores (conidia) are 
produced in countless numbers, and are so light that they float in air 
currents and are carried by contact in every conceivable manner by 
animals and by man. The life cycle from spore to spore is frequently 
very short, often being completed in twenty-four hours or less. Many 
of these forms are propagated for an indefinite number of generations by 
asexual spores or conidia but produce sexual fruit when special conditions 
are furnished. Some of them have never been induced to develop a 

* Prepared by Charles Thorn. 


48 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

"perfect' f-orm. These species are the ' weeds' 1 of the bacterial 
culture-room, since they cannot be entirely eliminated and often times 
will survive conditions more severe than the bacteria themselves. 

MOLDS or FERMENTATION.- A few species have acquired special 
importance by their fermentative action. Certain of these forms are 
widely distributed and able to utilize other media and conditions. 
They differ from closely related species of the same genera in the ability 
to produce special enzymes or especially large amounts of such enzymes 
as bring about particular forms of fermentation. Certain of these 
species have been utilized in the manufacture of drinks, of citric acid, 
in cheese ripening, etc. Others are so adapted to growth under condi- 
tions of fermentation as to be found constantly in connection with such 
processes, in which their vigorous growth and fermenting power 
seriously interferes with control of results. 

PARASITIC MOLDS. Many species of molds have been described 
as the cause of diseases in man and domestic animals. A few of 
these forms have been isolated and studied. Some of them attack 
the lungs, others the kidneys, but the larger number appear 'as the 
cause of diseased areas (dermatomycoses) of the skin, hair follicles, 
and external ears, or swellings or malformations of the extremities. 
Of a large number of forms named from a microscopic determina- 
tion of their presence in particular lesions, only a few have been 
adequately characterized and shown to be primary agents in caus- 
ing the injuries observed. Aspergillus fumigatus, various species of 
Sporotrichum and Actinomyces, the scalp organisms of herpes and 
favus appear to be real pathogens. 

GENERIC CONSIDERATION OP GROUPS* 

THE MUCORS OR BLACK MOLDS. The mucors or black molds con- 
stitute a large group of species belonging to the Phycomycetes or algal 
fungi whose general characters are a unicellular mycelium, at least in the 
vegetative stage, and quite generally a well-developed form of sexual 

*The series of forms presented contains representatives of the most common groups as 
they occur in laboratory cultures, and such as have acquired importance to the worker in 
bacteriology by participation in processes regularly studied by the bacteriologist. For more 
complete discussion of the fungi, the student is referred to standard text-books of cryptogamic 
botany. For discussions of species, Lafar's Technical Mycology includes the groups found 
associated with the bacteria; for other groups, special botanical literature must be consulted. 


MOLDS 


49 


reproduction (Figs. 32 and 33). In the mucors, the mycelium is usually 
richly developed within and often also on the surface of the substratum; 
asexual reproduction is accomplished by spores borne as conidia or 
borne within sporangia; and sexual reproduction is accomplished by 
the conjugation of special branches from the mycelium forming zygo- 


FIG. 32. FIG. 33. 

FIG. 32. Mucorinecs. Mucor. From Tabula Botanicce, showing sporangia 
originating from mycelium, spores and spore germination, and the formation of 
zygospores in a heterothallic species (diagrammatic). (Reduced one-half.) (By 
permission of A . F. Blaskeslee.} 

FIG. 33. Mucorinecs. Mucor, Rhizopus. A, B, C, D, Formation of the zygo- 
spores from conjugating branches; E, section of D; F, mature zygospores in section; 
G, germination of zygospores; H, diagram of fruiting stolons of Rhizopus nigricans; 
K, section of sporangium during spore formation, highly magnified (From Tabula 
Botanicce.} (Reduced one-half.) (By permission of A. F. Blaskeslee.} 

spores (Figs. 32 and 33). The typical mucors produce sporangia as 
capsule-like dilations at the ends of erect fertile hyphae, each con- 
taining many spores. Septa are commonly developed in the mycelium 
when sporangia begin to appear. These fertile hyphae may be micro- 
scopic or attain a length of several centimeters. 

Important Species. Perhaps the commonest form is Rhizopus 
nigricans (syn. Mucor stolonifer), the black mold of bread, a cosmo- 

4 


50 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

politan species associated with the decay of many kinds of food stored 
in wet condition or in humid situations. Typical clusters of spor- 
angiophores are borne on stolons or runners, which are hyphae extending 
radially from the center of the colony and fastened to the substratum 
or to the support at intervals by root-like outgrowths. Abundant 
growth of this species is found only under very moist conditions or 
in substrata with high water content. Rhizopus is a very common 
contamination in laboratory cultures. 

Many species and races of Rhizopus have been described. These 
have been studied especially in connection with the fermentation 
industries of Japan and China. Rice, wheat, and soy-beans in various 
mixtures pass through an initial process of " Koji" preparation in which 
raw or cooked materials are exposed to the air for several days. These 
processes offer ideal conditions for the entrance of the mucors as 
contaminations among the organisms desired. 

There are many common species of the genus Mucor, very few of 
which are identifiable without critical study. The specific names as 
commonly cited often designate groups of species or varieties rather 
than sharply marked forms. Certain of these may be briefly considered. 

Mucor mucedo L. is a common form upon dung, characterized by 
heads (sporangia) upon long sporangiophores,* at first yellow then 
becoming dark brown or black and studded upon the surface with 
needles of lime. 

Mucor racemosus, Fresenius, is characterized by the production of 
chlamydospores or cysts in the mycelium within the substratum, as 
elliptical thick-walled cells. The sporangiophores typically branch to 
make racemes of sporangia. The racemose mucors are active agents in 
changing starch to sugar and in the production of traces at least of 
alcohol from sugars. 

Mucor rouxii (Calm.), Wehmer, (syn. Amylomyces rouxii) is the 
most important of a series of forms with sporangiophores branching 
sympodially which are active in changing starch to sugar and in produc- 
ing traces at least of alcohol. The mycelium of Mucor rouxii develops 
in fluid cultures as yeast-like cells and groups of cells. The typical 

* The term sporangiophore is composed of the word sporangium combined with the suffix 
phore, meaning bearer. In sympodial branching the first fruit is on the tip of the original hypha, 
the first branch arises below this fruit and is terminated by the second fruit. Each successive 
branch and fruit originates in similar manner. 


MOLDS 51 

mucor fruits are produced only under special cultural conditions. 
This organism is used in the amylo process of alcoholic fermentation. 

Fermentation activity has been described for numerous species of 
Mucor and Rhizopus. Among them are Mucor circinell aides, Van 
Tieghem, Mucor javanicus, Wehmer, Mucor plumbeus, Bonorden, 
Rhizopus oryzce, Went, Rhizopus javanicus. The fermenting power 
of mucors like that of yeasts varies greatly with the species or even 
with races used, approaching in some species the efficiency of the 
more active yeasts. 

THAMNIDIUM. Of related genera, Thamnidium differs from Mucor 
in the production of two kinds of sporangia. The terminal sporangium 
of a fruiting hypha resembles that of Mucor; the secondary or accessory 
sporangia which are borne upon side branches of the sporangiophores 
are smaller, lack the columella, and produce few to several spores 
within an outer wall. 

Thamnidium elegans, Link, produces primary and secondary spor- 
angia on different hyphse, together making white colonies. The fertile 
side branches are produced in whorls and bear whorls of branchlets 
from their centers which in turn produce sporangioles from the tips of 
short straight twigs or branchlets. 

PENICILLIUM. The extremely abundant green molds most fre- 
quently belong to the genus Penicillium, although some members of 
other groups may be confused with them at times. 

Characters. Colonies are composed of loosely woven hyphse, 
branched, septate, colorless, or bright colored. The fertile hyphae 
(conidiophores) are mostly erect, arising either from submerged hyphse, 
or as branches of aerial hyphae, septate, usually branched only in the 
fruiting portion. Conidial fructifications consist of more or less com- 
plex systems of branches and branchlets, the ultimate fertile cells each 
producing a chain of conidia (Fig. 34). The whole system is usually 
grouped near the end of the conidiophore, giving the appearance of 
one or more brooms 01 brushes (whence the name). Very few species 
are known to produce asci, hence these are rarely encountered. The 
conidial form continues for an indefinite number of generations, there- 
fore all the activities of the genus are associated with this form. The 
classification of the whole group, technically transferred by some 
workers to the ascomycetes on account of certain forms, becomes 
misleading, because it contains so few species producing asci. 


52 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

So far as evidence has accumulated nearly all the forms met are 
imperfect. 


w '1 ff P" I' r f I//1 ' 
1"! If ft 1 1 If// 


FIG. 34. Penicillium expansum, Link, a, 6, /, Branching and arrangement of 
branches of conidial fructification (Xgoo); c, d, e, conidiiferous cells and conidial 
chains (XQOO); g, h, j, k, I, sketches of fructifications (Xi4o); m, n, o, germination 
of conidia (XQOO); r, s, sketches from photographs showing in 5 loose aggregations 
of conidiphores beginning to develop into zonately arranged coremia, in r a 
coremium i mm. in height. (From Bui. 118, Bureau of Animal Industry, U. S. 
Dept. Agriculture.) 


Cultural Considerations. Among the numerous species and races 
are certain green forms which are widely distributed and almost om- 
nivorous in habit. Other species are closely restricted to particular 


MOLDS 53 

substrata. Starches and sugars appear to be especially favorable 
components of nutrient media for members of the group. The larger 
number of the species grows best at temperatures from 15 to 30; 
a very few of them reach their optimum at 37, but many species are 
entirely inhibited and some killed at blood-heat. Vegetative mycelium 
begins to be produced at temperatures very close to freezing, but 
colored conidia are produced slowly or not at all at low temperatures. 
The species of Penicillium thrive through a wide range of concentration 
of culture media, though perhaps the most characteristic growths are 
produced in media high in water content. The common species of 
this genus grow in all the standard bacteriological media. With few 
exceptions the species grow well in synthetic media composed of 
assimilable carbohydrates and inorganic salts. A few species require 
the presence of some one of the higher nitrogenous compounds, but 
many species refuse to produce typically colored fruit without some 
form of starch or sugar in addition to ordinary peptone and beef- 
extract. Very few species grow well in alkaline media, but most 
species are tolerant of organic acids at the concentrations found in 
fruits and vegetables. 

Some Common Species. Penicillium roqueforti, Thorn, is a green 
form constantly found in pure culture in Roquefort cheese, frequently 
also in ensilage. It is widely distributed and grows under many sets 
of conditions. 

Penicillium camemberti, Thorn, is the chief organic agent in ripening 
Camembert cheese. Cultures of this species are floccose or cottony, 
at first white, later gray-green. 

Penicillium expansum, Link, is a green form, always obtainable from 
apples decaying in storage, upon which it frequently produces large 
coremia or stalks bearing conidia in masses sometimes several millimeters 
in diameter. It is one of the most abundant species of the genus, 
widely distributed in different countries. In cultures, colonies produce 
a characteristic odor, suggestive of its common habitat, decaying apples. 

Penicillium brevicaule, Saccardo, (Scopulariopsis repens, Bainier) 
is a form with rough or spiny brown spores which has been used phy- 
siologically to detect the presence of arsenic by its ability to set free 
arsine from such substrata. A whole series of forms has since been 
found to possess this character correlated with characteristic spore 
formation. These species or races are common in the soil both in 


54 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

Europe and America and appear as frequent contaminations upon 
cheese and upon cured meats to both of which products they impart 
peculiarly penetrating ammoniacal flavors. One member of this group 
has been reported as present in war- wounds. 

Except species associated with particular processes or substrata, 
the identification of the green species of Penicillium requires special 
methods and greater care than is possible aside from special study of 
the group. 

ASPERGILLUS (AND STERIGMATOCYSTIS). The genus Aspergillus in- 
cludes numerous species which develop under widely different condi- 
tions. Many of these forms reach their typical development under 
drier conditions than Penicillium and Mucor, such as stored grain, her- 
barium specimens, dried flesh, or foods containing concentrated sugars, 
such as jams, jellies, etc. Some excite processes of fermentation, and 
a few are associated with diseases. 

Characters. The vegetative hyphae are creeping, submerged in the 
substratum or sometimes aerial also, branched, septate, usually color- 
less, and sometimes bright colored. Conidiophores or fertile hyphae 
arise by transformation of single hyphal cells into thick-walled and 
often characteristically shaped foot-cells from which the fertile stalks 
arise as perpendicular branches which are erect, unseptate, or few- 
septate, usually much larger in diameter than the vegetative hyphae, 
and gradually enlarged upward, ending in more or less abrupt dilations 
or heads which bear closely packed columnar sterigmata or conidiif erous 
cells over the whole or a large part of their surface (Fig. 35, b). Each of 
these cells bears, in one group of species, a single chain of conidia; in 
other species (called by some authorities Sterigmatocystis) all or part of 
these sterigmata bear several secondary sterigmata which bear the 
conidial chains. Part of the species produce also thin-walled perithecia 
as variously colored spherical bodies upon the surface of the substrata. 
These perithecia are filled with eight-spored asci (Fig. 35, e). Species 
in certain groups produce sclerotia instead of perithecia, but many 
species are not known to produce either perithecia or sclerotia. 

Important Species. Among the species constantly met with, 
Aspergillus niger is recognizable by its black or very dark brown spores 
and in some strains by black sclerotia. Several black-spored forms are 
described, but their separation is usually impossible by ordinary 
methods of culture. Aspergillus niger ferments sugar solutions with 


MOLDS 


55 


the production of oxalic acid in considerable quantity. Citric acid 
fermentation with Aspergillus niger has been successfully developed 
upon a factory basis by Currie in the United States. 

Of green forms, Aspergillus* glaucus, Link (Aspergillus herbariorum, 
Wiggers), and Aspergillus repens, De Bary, both produce abundant 
yellow perithecia. These abound upon herbarium specimens, hay, 
grain, concentrated foods, such as jellies, preserves, dried bread and 
dried meats upon which they produce green conidial areas which are 
later dotted with bright yellow perithecia. 


FIG. 35. FIG. 36. 

FIG. 35. Aspergillus glaucus. a, Conidiophore showing increased diameter 
over the vegetative cells at its base (Xi28); b, sterigmata (X45o); c, conidia, smooth 
thick. walled in this variety, other varieties are spiny (X45o); d, peri thecium(X 128); 
e, ascus containing ascospores (X45o). (Original.) 

FIG. 36. Aspergillus. (i) A. fumigatus, Fres; (2) A. nidulans. i and 2, Show 
the simple sterigmata of A . fumigatus and the secondary sterigmata of A. nidulans. 
The conidia of these species do not remain attached in ordinary fluid mounts. 
(Original.) 

Aspergillus fumigatus, Fresenius, is a green form characterized by 
short conidiophores enlarging gradually into heads and bearing a single 
set of sterigmata on the very apex, with chains of thin-walled green 
spores about 3^f m diameter. This sppcies produces a destructive 
disease of birds known as aspergillosis. The same species is sometimes 
reported as pathogenic to man. 

Aspergillus nidulans differs by having two sets of sterigmata but 
otherwise frequently closely resembles Aspergillus fumigatus. It is 

*Recent examination of a large number of Jlftnerican specimens shows that Aspergillus 
repens is the usual green form in this country. 

1"The unit of measurement is the micron GO or micro-millimeter (.001 mm. or J-Ssooo in.) 


56 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

widely distributed in soil. Although it has been reported as pathogenic 
the identification of this species in pathogenic lesions is not confirmed. 

Aspergillus oryzce and A. flavus form a closely intergrading series, 
certain members of which are used in the fermentation industries of 
Japan and China. A. oryzce as described by Wehmer is used in the 
fermentation of rice to produce Sake, an alcoholic drink. The diastatic 
enzyme of A. oryza grown upon rice converts the starch into sugars 
which are fermented by yeasts into alcohol. Other members of the 
series more closely approximating A. flavus are widely used in the 
fermentation of soy-beans. A mixture of cooked soy-beans and 
cracked roasted wheat is inoculated with A. flavus in special koji 
fermenting chambers. In three days the entire mass becomes fully 
overgrown with mycelium and covered with the ripe conidia of this 
form. The wheat and beans are partially penetrated by the mold 
hyphae. The mass is then transferred to brine strong enough to inhibit 
further growth except of a few yeasts. In a long period, several 
months to three years, the whole mass becomes digested to form 
soy-sauce, or shoyu. This product is the basis of meat sauces such as 
Worcestershire. 

Aspergillus wentii, Wehmer, characterized by its long conidiophores 
and coffee-colored heads of conidia, is found in the Soja preparation in 
Java. 

Of other forms constantly met, Aspergillus candidus has white or 
pale cream fruiting surfaces. A. terreus is avellaneous in color; Asper- 
gillus ochraceus, ocher or tan. 

Much confusion is still found in the literature of this genus, so that 
frequent references to the activities of particular species are difficult or 
impossible to verify. 

MONASCUS.- -The organism of red ensilage is widely distributed in the 
silos of America. Ensilage infected with Monascus forms into red balls 
or masses up to a foot in diameter held together by mycelium. The 
masses are red from coloring material partly in the mycelium and spore- 
masses and partly in the silage. The same or a nearly related form 
described as Monascus purpureus, Went, is used in producing red rice, 
A ng-quac, in China. The mycelium penetrates the^ice grains, produces 
a friable texture and gives the whole mass a purple red color. Ang- 
quac (or Ang-khak) is used to color Chinese sauces and reaches America 
especially upon Chinese soy-bean cheeses. 


MOLDS 


57 


CLADOSPORIUM (AND HoRMODENDRON).--The species of Cladospor- 
ium occur frequently in cultures of decaying vegetable matter, of milk 
and cream, or butter. The colonies liquefy gelatin. Both mycelium 
and spores are at first colorless, but later dark colored to almost black, 
with spores becoming two-celled in very old cultures. 

Cladosporium herbarum is the commonest species encountered.* 
Colonies in culture media differ so greatly in structure from those upon 
natural substrata as to make identification of species questionable. 
(Fig. 37). Much confusion is therefore found in the use of the names 
of species of Cladosporium and the related genus, Hormodendron, which 
is separated by some. 


FIG. 37; FIG. 38. FIG. 39. 

FIG. 37. Cladosporium herbarum, showing the forms of conidiophores and conidia 
which are very common upon laboratory culture media. (Original.} 

FIG. 38. Spores of Alternaria sp. (Original.) 

FIG. 39. Fusarium from decaying potato, a, Spores showing curvature and 
septa; b, germination of spores; c, development of spores in petri-dish culture; d, 
mass of spores as found in culture. (Original.) 

ALTERNARIA AND FUSARIUM.- -The frequent occurrence of species of 
Alternaria andFusamim in cultures demands that the generic characters 
be recognized. Both, as a rule, produce abundant growth with a tend- 
ency to over-run cultures of other forms (Figs. 38, 39). The spores of 
Alternaria are brown, Indian-club form or muriform (divided into 
several cells by longitudinal as well as cross walls), and are connected 
together into chains (Fig. 38). The spores of Fusarium are colorless, 
either straight, sickle-shaped, or crescent-shaped, divided into several 
cells by cross walls, are produced in chains or adhere into masses on the 
tips of the fertile branchlets. The morphology of colonies in culture 
varies widely from the descriptions of the same species under natural 
conditions. Species of Fusarium frequently produce bright colors in 

* This species has been shown to be a conidial form of Spaerella lulasnei Janczewski, but the 
bacteriological student will meet only the conidial stage. 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


the mycelium and substrata; colonies of Alternaria often become almost 
black. Identification of species in cultures is thus far impossible, 
except for the specialist. 

OIDIUM. Oidium (Oospora) lactis is universally found in cultures 
from milk and milk-products and occurs very frequently in decaying 
vegetables, manure, etc. Colonies of the species are colorless, have 


FIG. 40. Oidium lactis. a, b, Dichotomous branching of growing hyphse; c, d, 
g, simple chains of oidia breaking through substratum at dotted line x-y, dotted 
portions submerged; e,f, chains of oidia from a branching out-growth of a submerged 
cell; h, branching chain of oidia; k, I, m, n, o, p, s, types of germination of oidia under 
varying conditions; /, diagram of a portion of a colony showing habit of Oidium 
lactis as seen in culture media. (From Bull. 82, Bur. Animal Industry, U. S. Dept. 
Agr.) 

vegetative mycelium entirely submerged, become powdery-white with 
spores when mature, liquefy gelatin, and produce a strong character- 
istic odor (Fig. 40). Microscopically the species is recognized by dicho- 
tomous branching of the hyphae at the margin of the rapidly growing 
colonies, and by the spores or oidia which are abruptly cylindrical, 
varying with conditions in length and diameter and produced both 


MOLDS 


59 


above and below the surface of the substratum in long chains which 
break up readily. At times the whole mycelium appears to break up 
into oidia. Oidium lactis is a factor in the ripening of many kinds of 
cheese: Limburger, Harz, Camembert, Gorgonzola, etc., and in the 
deterioration of butter in storage. Its activity is associated with 
strong odor and taste. 


FIG. 41. A colony of Manilla Candida. (Photographed by Z. Northrup.) 


FIG. 42. Forms of oidia in chains. (Manilla sltophlla.} 

MONILIA. The generic name Monllla is very loosely used in the 
fermentation literature for certain forms in which the conspicuous 
development consists of chains of cells like strings of beads. There is a 
series of borderline forms between the true yeasts, the mycoderma 
group of yeasts and the true hyphal fungi. The name Manilla Candida 


60 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

was applied by Hansen to one of these which is found frequently in 
breweries. The genus Willia has been created for another series of 
these forms. Another widely distributed species, Manilla s^o/>Ma, forms 
loose salmon-pink masses of conidia on the surface and in the interior of 
bread, in cereals and other foods. In culture media Monilia sitophila 
fills culture tubes and dishes with loose fluffy salmon masses of conidia. 
This organism frequently overruns an incubator or a culture room in- 
fecting everything fermentable. 

DEMATIUM. One species of Dematium, Dematium pullulans, has 
been much studied. This is frequently found within decaying fruit as 
dark brown colonies. In culture, mycelium is sparingly produced, 
either colorless or colored, and conidia are borne in clusters and chains 
all along the hyphae submerged in the substratum. At first both myce- 
lium and conidia are colorless, later some or all of the cells develop 
heavy dark brown walls. Although not active as an agent of fermen- 
tation, it occurs very frequently in the fermentation industries some- 
times discoloring the fermenting products. The conidia bud out from 
the cells of the mycelium in a manner resembling the yeasts. Its 
occurrence with the yeasts has led to many careful descriptions of its 
several types of spore production and its biological activities. 

SAPROLEGNiACE/E.--This is an aquatic group of Phy corny cetes, which 
includes both saprophytes and parasites. Its commonest members 
grow as shimmering masses of cottony mycelium upon the bodies of 
flies or other insects in aquaria. Other members of the same group 
are parasitic, some attacking young fish and producing characteristic 
lesions. Both sexual and asexual spores (motile swarm spores) are 
abundantly found. 


CHAPTER III 
YEASTS* 

MORPHOLOGY OF CERTAIN TYPES 

DEFINITION AND BASES OF CLASSIFICATION. If the cloudy freshly 
expressed juice of grapes or other fruits be passed through a centrifuge, 
the sediment will be found to consist principally of amorphous particles 
of dirt and plant tissue. If the clear juice is now allowed to stand in a 
warm place for a few days it will ferment and the sediment thrown 
down by the centrifuge may be shown by the microscope to consist prin- 
cipally of unicellular microorganisms. 

These microscopic cells are called collectively ''yeast" and belong 
to various groups of fungi. Some of them are special vegetative forms 
of Phy corny cetes (Mucor), others of Ascomycetes (Saccharomyces, Asper- 
gillus), while others are unknown in any other form and are classed as 
Fungi imperfecti (Mycoderma, Torula). They are widely-distributed in 
nature and some of them occur on all exposed surfaces and particularly 
on moist organic substances containing sugar and acid. The true 
yeasts (Saccharomy cetes), which are of the greatest importance indus- 
trially, occur naturally on the raw material (S. ellipsoideus on grapes) 
or are known best in the cultivated condition (S. ceremsia of beer). 

The true yeasts occur in the form of spherical or more or less elon- 
gated cells varying in normal width from 2.5^1 to 12/1. The first classi- 
fications were based on shape and size alone but these vary and depend 
so much on cultural conditions that they are of little value in differen- 
tiating species or varieties. 

The range of variation in shape and size, especially of the spores, 
under given conditions of culture medium and temperature, is now used 
only in conjunction with the reactions brought about in various solu- 
tions to distinguish the various forms. 

The true yeasts are characterized by the formation of endospores 
and are classed with the Gymnoascea. Each cell seems capable, under 

* Prepared by F. T. Bioletti. A. Guilliermond has furnished the sections on the " Cytology 
of Yeasts." 

61 


62 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


favorable conditions, of developing into an ascus. Many unsuccessful 
attempts have been made to connect the true yeasts genetically with 
various forms of fungi such as Mucor, Ustilago and Dematium. At 
present they must be considered as distinct species. 

Some yeasts have a tendency during fermentation to remain at the 
bottom of the liquid; others form a thick foamy layer on top. These 
are known respectively as bottom and top yeasts. No sharp distinction 
can be made as there are intermediate forms. 

The vegetative reproduction in the genus Saccharomyces takes place 
by budding, in Schizosaccharomyces by fission. 


FIG. 43. Yeast cell. (Original.) 

The extreme temperatures for budding lie between i and 47, vary- 
ing with different species. The optimum temperature varies in the 
same way between 25 and 35. The rate of multiplication under favor- 
able conditions will range from one to several hours for the formation of 
a new cell. 

When young, vigorous, well-nourished cells are supplied with abun- 
dant air and moisture at a comparatively high temperature under con- 
ditions that discourage budding (lack of nutriment) they form endo- 
spores. These spores are usually about half the diameter of the mother 
cell and from one to eight or more may occur in each cell. They may 
be formed by cells before or after budding and may even change to asci 
and form new spores. They are generally spherical or slightly ellip- 
soidal, rarely kidney-shaped (S. marxianus) or furnished with a zonal 
ring (S. anomalus) (Fig. 43). 


YEASTS 


In nutrient solutions they swell, burst the mother cell, become free 
and germinate by budding, usually producing vegetative cells directly, 
though occasionally producing first a short promycelium (S. ludwigii). 

In Schizosaccharomyces octosporus the ascus is formed by the fusion 
of two cells. Sometimes in other species, two or more spores in one cell 
will fuse before germination. 

Staining with warm carbol-fuchsin and partial decolorization with 
weak acetic acid leaves the spores red and the cell colorless. 


FIG. 44. Spore-bearing cells. A, S. pasteurianus. (After Bioletti.} B, Sch. 
octosporus. (After Schionning.} C, S. anomalus. (After Kayser.} 


CYTOLOGY OF YEASTS* 

GENERAL STRUCTURE OF YEASTS. The structure of yeasts in no 
way differs from that of the other fungi, only it is seemingly more complex 
and consequently more difficult to interpret on account of the abundance 
of the stainable granulations which sometimes accumulate in the cells 
and occasionally hinder the differentiation of the nucleus. This explains 
why it has until recently remained a subject of controversy. It is now 
fairly well understood. 

* Prepared by A. Guilliermond. 


64 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

In order to understand clearly this structure, one must observe 
young cells taken from a culture at the beginning of development. 
For this purpose we use Saccharomyces cerevisice which, because of the 

relatively large size of its cells, lends itself better than 
any other yeast to a cytological study. Examined in 
the living state, highly magnified, the cells of this 
yeast show a dense and homogeneous cytoplasm with 
a group of small vacuoles or a single large vacuole at 
FIG. 45. Sac- the center. In the vacuoles and also in the perivacu- 

charomyces cere- o j ar cytoplasm, we can clearly distinguish a great 

msics. Young J 

cells examined in many small shining granules, of varying sizes, which 

the living state manifest Brownian motion. It is easy to stain them 

m a solution of . 

neutral red. The in the living state (Fig. 45) with a very dilute solu- 

vacuoles, stained t ion o f neu tral red or methylene blue. These are 

pale red, contain 

m e t a c hromatic only metachromatic corpuscles. 

corpuscles col- j n xe( j an( j stained preparations (Fig. 46, i-io) is 
ored dark red. . n - i 

seen in each cell a single, large nucleus, whose struc- 
ture is exactly like that which we have discussed in molds. This 
nucleus is surrounded by a membrane and contains a hyaline nucleo- 


^ 


-. 

^ 

FIG. 46. FIG. 47. 

FIG. 46. Saccharomyces cerevisice. i-io, Young cells with nucleus, showing its 
structure. 6-8, The same: division of the nucleus. 11-13, Cells after twenty-four 
hours' fermentation, with a very large glycogenic vacuole filled with lightly colored 
grains. 

FIG. 47. Saccharomyces cerevisice. Young cells fixed and stained by a special 
method revealing in the cytoplasm a chondrium consisting of rod mitochondria and 
granular mitochondria. 

plasm in which is easily seen a large nucleolus and some chromatin; 
this latter is scattered through the nucleus, sometimes found in the 
nucleoplasm in the form of a network, sometimes reduced to a num- 


YEASTS 65 

her of granules smaller than the nucleolus, and sometimes even found 
gathered on the circumference of the nuclear membrane. 

The cytoplasm is dense and homogeneous. A special technic has 
recently enabled the demonstration of a chondrium in the cytoplasm. 
This seems to consist both of granular mitochondria and of more or 
less elongated and flexible rod-mitochondria (Fig. 47). 

The vacuole shows in its interior numerous metachromatic corpus- 
cles of varying sizes (Fig. 48). As in molds, these corpuscles appear not 
only in the vacuole, but also in the perivacuolar cytoplasm; there they 
start, and are next diffused in the vacuole where they finish their growth, 
then dissolve when the need is felt. It is 
difficult in the case of yeasts to determine , 
their origin; nevertheless, observations ^ 
made of fungi with larger cells than we 

~r j 

have previously described, show that the 

metachromatic corpuscles start in the .I;-!. 

midst of mitochondrial elements, and it r" 

seems certain that after that the process 5 6 I 

is the same in yeasts. FIG. 48. Saccharomyces cere- 

Tn the rvtonla^m of vpasts a ho have visi<B > stained b y a method re- 
cytopiasm yea. ,s, ai o, nave vealing both ^ nuc i eus and 

been noted granulations, which can be the metachromatic corpuscles, 
stained with ferric haematoxylin, and which 

have been named basophile grains; but these formations, which are not 
well defined, seem to us to represent simply products from the altera- 
tion of the chondrium under the influence of imperfect fixing agents. 

The membrane of yeasts is quite thick and very distinct. Its 
chemical nature is still little known. According to some authors, it 
consists of a cellulose; others think that it contains only pectose. Ac- 
cording to Mangin, it is formed of callose. Finally, some authors have 
thought they discerned chitin. 

The structure we have just described is found in all the species 
(Fig. 49), only it is sometimes much less distinct because of the smallness 
of the cells. In the elongated yeasts, and in the cells composing the 
mycelial formation which are encountered under some conditions, 
especially in the films, the nucleus generally occupies the center of the 
cell; it is situated in a kind of matrix or bridge consisting of a very 
dense cytoplasm, while a vacuole filled with metachromatic corpuscles 
occupies each of the two extremities of the cell. 

5 


66 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

Summing up, the elements of which a yeast cell consists are a cyto- 
plasm with a chondrium, a nucleus with clearly differentiated structure, 
vacuoles containing numerous metachromatic corpuscles, a membrane 
of a nature not yet clearly denned. 

CYTOLOGICAL PHENOMENA DURING MULTIPLICATION. During the 
budding of the yeasts, cytoplasm enters the young bud with some chon- 
drium; then, when the bud has reached a certain size, the cytoplasm 

forms in it a little vacuole in which appear 
metachromatic corpuscles (Fig. 48, 2-7). 

In the course of these phenomena, the 
nucleus retains the position which it occupied 
in the mother cell before the appearance of 
the bud. Only when the bud is quite large 
does the nucleus begin to divide. It is elon- 
gated so that one end penetrates the bud; the 
nucleus then resembles an elongated dumb- 
bell with the larger head remaining in the 

'~ 5 a y harom y^ s mother cell and the other, smaller head, in the 
s. Young cells 

each with nucleus. bud (Fig. 46, 6, 7 and 8; Fig. 48, 2, 7; Fig. 49). 

Soon the part of the dumb-bell which is 

stretched out breaks near the neck of the bud, forming two nuclei of 
unequal size, at first tapering spherical in shape, and later rounded 
off: one is the nucleus of the mother cell and the other that of the 
bud. This division is therefore effected by the direct method; it is an 
amitosis. In the Schizosaccharomyces, where the cells do not multiply 
by budding as in other yeasts, but by a transverse partition, the 
nuclear division is effected by amitosis: the nucleus, situated in the 
center of the cell, elongates along the longitudinal axis of the cell and 
resembles a dumb-bell, ending by dividing in the middle, thus forming 
two nuclei of the same size. Soon a transverse septum appears be- 
tween the two nuclei and separates the two daughter cells. 

We have now to note the modifications which arise in the structure 
of the cells during the different phases of development and at the time 
of sporulation. 

VARIATION IN THE CELLULAR STRUCTURE DURING DEVELOPMENT. 
In the course of development, especially during fermentation, yeasts 
reveal cytological phenomena which render their structure more com- 
plex and more difficult to interpret. Let us take for example the study 


YEASTS 67 

of the S. cerevisia. After twelve hours of fermentation, the meta- 
chromatic corpuscles become more numerous. At the same time, the 
cytoplasm forms little vacuoles which contain no metachromatic cor- 
puscles, but only glycogen, easily detected by iodo-iodide of potassium. 
These are gradually fused into a single vacuole, which enlarges much 
and modifies materially the cell structure. The glycogenic vacuole, 
increasing, pushes back to the periphery of the cell the cytoplasm, the 
vacuoles with metachromatic corpuscles, and the nucleus whose chro- 
maticity increases and which becomes homogeneous in appearance 
(Fig. 46, n). After forty-eight hours, moreover, the cell is found to 
consist of an enormous vacuole filled with glycogen which occupies 
most of it, while the nucleus, the vacuoles with metachromatic cor- 
puscles and the cytoplasm are pushed back to one side of the cell, which 
is then transformed into a kind of glycogen sack (Fig. 46, 12 and 13; 
48, 6-8). At this time the glycogenic vacuole contains a great many 
small granulations (Fig. 46, 12-13), which easily fix some staining 
materials, especially ferric haematoxylin, and whose origin and signifi- 
cance have not been determined. 

Toward the end of fermentation, the glycogen gradually diminishes 
and the glycogenic vacuole is gradually reduced, then ends by dis- 
appearing. The cell after this resumes its original structure. 

In the course of these phenomena, the membrane apparently shows 
no modification. It is known, however, that under some conditions, 
yeasts secrete gelatinous substances which englobe their cells in a kind of 
jelly and so appear like zoogloea (Hansen). It is well to add, on the 
other hand, that many pathogenic yeasts, when living in the host, have 
the ability to protect their cells against the reaction of the organisms, 
by secreting a very thick capsule of gelatinous nature: each of their 
cells is then surrounded by a large capsule. 

CYTOLOGICAL PHENOMENA or THE SPORULATION AND GERMINATION 
OF ASCOSPORES. For a study of the sporulation, we will consider a 
representative of the species Schizosaccharomyces, the Sch. octosporus, 
in which these phenomena are easily observed and especially well 
understood. 

We know that in this yeast, as in some others, sporulation is pre- 
ceded by a sexual phenomenon consisting of an isogamous copulation. 
The ascus results from the fusion of two similar cells. The gametes are 
ordinary cells which have the structure which we have previously 


68 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


described, with one nucleus and one or more metachromatic vacuoles 
containing corpuscles (Fig. 50, a). Fusion takes place between the two 
cells which are nearest together. Each of these two cells sends out a 
tiny beak; the two little beaks thus formed anastomose and form a 

channel of copulation joining the two 
rv^ cells (Fig. 50, b, c, d). The septum 


L 


FIG. 50. Successive stages of 
copulation and sporulation in Schizo- 
saccharomyces octosporus. 


separating the two gametes in the 
middle of the channel is quickly 

/ 

h absorbed, and the two cells then 
have free communication. The cyto- 
plasm of the two cells draws together 
and mingles in the channel; there the 
two nuclei draw near to each other 
(Fig. 50, e) and fuse into a single 
nucleus (Fig. 50, /, g, ti). Next the 

zygote ends its fusion; instead of its original dumb-bell appearance, it 
assumes the form of an oval cell, then grows large (Fig. 50, i). Occa- 
sionally, however, it retains a vestige of the individuality of the two 
gametes, showing two swellings joined by a somewhat narrower middle 
portion (Fig. 50, /). 

During this time, the cell becomes filled with little vacuoles and 
assumes a more or less alveolar structure. 
These vacuoles contain a number of metachro- 
matic corpuscles. The nucleus which occupies 
the center of the zygote begins to divide. The 
ascus, containing sometimes four, sometimes 
eight ascospores (Fig. 50, j), will then undergo 
two or three successive divisions, as the case 
may be. These divisions are accomplished by 
karyokinesis or mitosis. In the stages preceding 
nuclear division, the nucleus is very large and 
shows a very clear structure with a nucleolus 
and a chromatic reticulum (Fig. 51, a). It 
soon elongates and assumes a special structure. 
Its membrane loses its clearness, and in the midst of the nucleoplasm 
an achromatic spindle appears, ending at each of its two poles in a 
very small centrosome and containing at its center a group of fine 
granulations representing the equatorial plate (Fig. 51, b and c). The 


FIG. 51. 
charomyces 


-Schizosac- 
octosporns. 


Various stages of the 
nuclear division during 
ion. 


YEASTS 69 

nucleolus always persists on one side of the spindle. At a subsequent 
stage the chromatic granulations or chromosomes are divided between 
the two poles of the spindle, the nucleoplasm is mixed with cytoplasm, 
then the spindle elongates, while the chromatic granulations form a 
homogeneous mass at the two poles (Fig. 51 d, e, g and h). The 
nucleolus is quickly absorbed, then the two nuclei are formed at the 
expense of the two chromatic masses (Fig. 51, /). To summarize, 
therefore, this division consists in mesomitoses of a primitive kind, 
which appear to take place in the interior of the nucleus, whose mem- 
brane is absorbed only at the end of the phenomenon. They show 
the characteristics of the mesomitoses which have been described in 
the asci of the higher Ascomycetes. 


\ 

- 9 
- -. 


A- 
3 , 

r 


,~ 

i- 2 e J r a 

FIG. 52. Successive stages of copulation and sporulation in Schizosaccharomyces 
pombe. 1-2, Cells just as sporulation is about to begin. 3-7, Union of the two 
gametes and nuclear fusion. 8, Ripe ascus. Cellular fusion being incomplete, 
the ascus retains the shape of the two cells joined by a channel of copulation. 

When these divisions are accomplished, the nuclei seem to be scat- 
tered in the cell (Fig. 50, i)\ they are soon surrounded by a thin layer of 
cytoplasm which is separated from the cytoplasm by a membrane; 
these are the ascospores. At first very small, these gradually increase 
at the expense of the cytoplasm which has not been used in their forma- 
tion in other words epiplasm then reach the point where they oc- 
cupy the whole of the ascus, after having absorbed this epiplasm (Fig. 
50, _/.) The metachromatic corpuscles scattered in the vacuoles of 
the epiplasm disappear during these phenomena, being absorbed by 
the ascospores. At no time during the development of the ascus can 
glycogen be seen any more than in plant cells, but this is replaced 
by an amyloid substance which is stained blue by iodo-iodide of potas- 
sium. This substance impregnates the membrane of the ascospores 
and disappears during their germination, utilized as a reserve product. 

In some Schizosaccharomyces or ordinary yeasts which bud (zygo- 


70 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

saccharomyces) the ascus comes from an egg which starts in a similar 
manner (Fig. 52.) In some species, this egg is formed by a hetero- 
gamous copulation between an adult cell (macrogamete) and a very 
young cell which has just separated from the mother cell (micro- 
gamete) (Fig. 53). On the contrary, in most species, the ascus results 
from the simple transformation of an ordinary cell without previous 
copulation. Whatever may be its origin, the ascus shows cytological 
phenomena quite similar to those which have just been described in 
Sch. octosporus, with mere differences of detail. Always in Sch t 


FIG. 53. Heterogamous copulation in Zygosaccharomyces chevalieri. 1-3, 
Gametes sending out a beak in anticipation of copulation. 4-7, Micro- and macro- 
gametes joined by their channel of copulation. 8, The partition separating the 
two gametes is absorbed. 9-18, The contents (nucleus and cytoplasm) of the micro- 
gamete enter the macrogamete and are fused with the contents of the latter. 
19-21, Ripe asci. 22-23, Freeing of the ascospores by rupture of the membrane of 
the ascus. 

octosporus are seen only a few metachromatic corpuscles in the ascus. 
In most of the other yeasts, on the contrary, the ascus contains a very 
large number of metachromatic corpuscles, and it is easier there to fol- 
low the evolution of these bodies which present interesting singularities 
clearly demonstrating their role as reserve substances. 

Let us observe, for example, the cytological phenomena which ap- 
pear during sporulation in Saccharomyces ludwigii. In this yeast, 
which shows no sexuality in the origin of the ascus, the cells which are 
preparing to sporulate assume a finely vacuolar structure (Fig. 54, 8 
and 9) and produce a large quantity of reserve products: metachromatic 
corpuscles, glycogen and fat globules. Metachromatic corpuscles spring 
up in some vacuoles, glycogen in others; as for the fat globules, they 


YEASTS 


are located in the cytoplasmic web. The nucleus is situated on one 
side of the cell, surrounded by a thin layer of very thick and homo- 
geneous cytoplasm which is to become the sporoplasm, at whose 
expense the ascospores are formed, the remainder that is to say the 
vacuolar cytoplasm being destined to compose the epiplasm or nourish- 
ing plasm. 

At a later stage, the metachromatic corpuscles undergo a kind of 
pulverization transforming them into small grains, and begin to dis- 


r 


. 


I '4 


= 


. 

i 

. 


FIG. 54. Sporulation in Saccharomyces ludwigii. Figs, i and 7 showing the 
evolution of the nucleus. Figs. 8-9, the metachromatic corpuscles, stained by a 
method permitting a differentiation, except in Fig. 8, are dissolving, and the sub- 
stance of the vacuole which contains them shows a diffuse metachromatic coloring 
(here gray) like the corpuscles. 


solve in the vacuoles surrounding them, the latter at this time taking, 
with aniline blue stains, a diffuse red coloring similar to that of the 
metachromatic corpuscles (Fig. 54, 9). At the same time, the nucleus 
undergoes two successive divisions, but these have not been discern- 
ible up to the present time, because of the density and the strong 
chromaticity of the sporoplasm surrounding the nucleus. They are 
manifested merely by the appearance of the two daughter cells which 
migrate to the two poles of the cell, carrying with them a part of the 
sporoplasm, which assumes the appearance of a dumb-bell and whose 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


slender part ends by breaking (Fig. 54, 2, 3 and 4). The cell, there- 
fore contains at this time at each of its poles a small mass of sporo- 
plasm having first one, then two, nuclei (Fig. 54, 5 and 10). After 
this, the sporoplasm condenses around each of these nuclei (Fig. 
54, 6), thus delimiting at each of the poles two small ascospores. 

During these phenomena, the metachromatic corpuscles congre- 
gate around the ascospores (Fig. 54, n and 12), then gradually dis- 
solve. The ascospores constantly increase in size at the expense of 
the epiplasm, which becomes disorganized and is reduced to a vacuo- 
lar liquid containing in suspension metachromatic corpuscles, fat 
globules and glycogen. They succeed in absorbing entirely the epi- 
plasm and in occupying the whole of the ascus (Fig. 54, 13 and 14). 
The metachromatic corpuscles, like the glycogen and the globules of 
fat, are then completely absorbed by the ascospores, which indicates 
clearly that they, as well as the latter substances, act as reserve prod- 
ucts. When the ascospores are ripe, they contain in their vacuoles 
metachromatic corpuscles (Fig. 54, 14). 


FIG. 55. Germination of ascospores in Saccharomyces ludwigii. i, Beginning 
of the fusion of the ascospores. 2, The ascospores are joined two by two by a 
channel of copulation, but their nuclei are not yet fused. 3, The nuclei are fused. 

4, At the left two ascospores, joined, have formed at the middle of the channel of 
copulation a bud which has ruptured the membrane of the ascus. At the right, the 
two ascospores, joined by a channel of copulation have not yet fused their nuclei. 

5, Formation of the bud at the expense of the two fused ascospores. Two other 
ascospores have not yet begun their fusion. 6, The bud formed at the channel of 
copulation is already established and separated from this channel by a transverse 
septum. 

In all yeasts, at the time of budding, the ascospores have the appear- 
ance and structure of plant cells. Their germination does not differ 
from ordinary plant multiplication. In some species, however, espe- 
cially in S. ludwigii t copulation, suppressed at the beginning of sporula- 


YEASTS 73 

tion, is replaced by a compensating phenomenon which intervenes at 
the germination and consists in the fusion of the ascospores two by two 
(Fig. 55). The ascospores anastomose at their extremities by a chan- 
nel of copulation which, as soon as the nuclear fusion is accomplished, 
becomes the seat of a budding. 

THE PRINCIPAL YEASTS OF IMPORTANCE TO FERMENTATION 

INDUSTRIES* 

TRUE YEASTS, SACCHAROMYCETES. The various yeasts used in 
brewing and some of those used in producing distilling material are 
grouped together as S. cerevisia. They are large and round or slightly 
oval. 

They are divided into three main groups the bottom yeasts which 
are used in the manufacture of German beer, and which, usually, are 
capable of producing only a moderate amount of alcohol; the top yeasts, 
used in English beers and compressed yeast, capable of producing more 
alcohol, and the distillery yeasts, which have great fermentative power 
and produce large amounts of alcohol. 

Many forms of these yeasts have been described in great detail by 
Hansen and others but the distinctions are based principally on physio- 
logical peculiarities such as the temperature and time limits of film and 
spore formation, and the character of the fermented liquids. The vari- 
ous forms seem to be fixed, and to retain their characteristics unchanged 
under almost all forms of treatment. 

The wine yeasts, S. ellipsoideus, seem to be even more diverse than 
the beer yeasts, but have been less thoroughly studied. They are some- 
what smaller than the latter and usually slightly more elongated. They 
form spores much more abundantly and easily than the beer yeasts 
and the cells in film formation are often much elongated. 

Their fermentative power is considerable, some of them being capa- 
ble of producing over 16 per cent by volume of alcohol. W. V. Cruess 
has obtained 21 per cent from a Burgundy wine yeast. They differ in 
the flavors and aromas which they produce in the fermented liquid, and 
especially in the rapidity with which they settle. Some yeasts, such 
as those of Champagne and Burgundy, form a compact sediment which 
settles quickly and leaves the liquid clear. Others remain suspended 
for a long time and settle with difficulty. 

* Prepared by F. T. Bioletti. 


74 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


Every region seems to have its own forms and the characteristics of 
the various forms seem to be as well fixed as those of beer yeasts. 

Wines are manufactured by the use of these yeasts. They are also 
employed in distilleries. In breweries they are considered disease yeasts 
and have a deleterious effect on the beer. 


B 


D 


FIG. 56. Wine and beer yeasts. A, S. ellipsoideus, young and vigorous; B, S. 
ellipsoideus, (i) old, (2) dead; C, S. cerevisioe, bottom yeast; D, S. cerevisice, top yeast. 
(Original.) 

S. pyriformis resembles in shape S. ellipsoideus, and in association 
with Bacterium vermiforme produces ginger beer. 

S. vordermanni is concerned in the manufacture of arrack. It fer- 
ments the sugar produced from rice by the molds, Mucor oryzce and Rhi- 
zopus oryzce. 

S. fragilis and other yeasts have been found in kefir and other fer- 
mented drinks made from milk. These yeasts working in conjunction 
with bacteria produce alcoholic acid beverages. 


YEASTS 75 

Many true yeasts are more or less injurious. They do not, like 
bacteria and pseudo-yeasts, cause serious diseases, capable of completely 
ruining the fermented product, but they may injure the quality more or 
less. Some yeasts are useful in certain cases and injurious in others. 
If beer yeasts become contaminated with wine yeast the resulting beer 
may be persistently turbid. If one attempts to ferment grapes 
with beer yeast, a wine with a disagreeable beer aroma and of poor 
keeping qualities is produced. 

S. pasteurianus occurs in several forms as an injurious yeast in brew- 
eries, causing bitterness and turbidity. Similar forms occur in wine but 
do little harm except in the absence of the true wine yeast. The cells of 
this species vary from oval to long ellipsoidal, often being much elon- 
gated and in film formation sometimes producing a branching mycelium. 
Spores are formed easily and abundantly. 

The apiculate yeast, S. apiculatus, is very abundant on grapes and 
most acid fruits. It is very variable and undoubtedly includes many 
varieties. The cells are small, vary in shape from oval to cylindrical, 
most of them having an apiculation at one or both ends, making them 
pear or lemon shaped. According to Lindner they form spores in drop 
cultures, one in a cell. Under favorable conditions this yeast increases 
with great rapidity, but is checked by 3 to 5 per cent of alcohol. It 
causes cloudiness in wine, interferes with the growth of the proper 
yeast and injures the flavor. 

Many yeasts, mostly small and some of them rose-colored, have 
been found on grapes and in wine, but they do not develop under 
ordinary conditions of wine making sufficiently to be harmful. 

Schizosaccharomyces pombe is a yeast found in pombe or millet beer, 
made by negroes in Africa. It is cylindrical and large, though variable 
in size. Both ends are rounded. It multiplies by forming a septum 
near one end, the smaller division then growing into a normal cell. 
From one to four spores are formed in a cell. These spores are often 
produced in the fermenting liquid. The fermentative power is high and 
a large percentage of alcohol may be formed. 

Several other species of this genus have been isolated from grapes 
and from Jamaica rum. 

PSEUDO YEASTS. Budding cells often occur in fermenting liquids 
which have all the characteristics of yeast except that of producing 
endospores. They are grouped together under the name of Torula. 


76 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

They are usually small, spherical or slightly elongated. Some species 
produce a little alcohol and some none. They seldom occur in suf- 
ficient quantities to be harmful and one form is accredited with pro- 
ducing the special flavor of some English beers. 

The forms included under Mycoderma resemble yeast in shape 
but produce little or no alcohol, are strongly aerobic and do not 
produce endospores. Their most noticeable characteristic is that they 
grow only on the surface of the liquid, where they produce a thick film. 
They cause complete combustion of the alcohol and other organic 
matters, making beer and wine vapid and finally spoiling them. 

CULTURE OF YEASTS 

PURE CULTURES. Yeast can be properly studied only in pure cultures. The 
media used are either the liquids in which the yeasts are to be used such as wort, cider, 
grape juice, or a special medium devised for a special investigation. An example of 
the latter is Laurent's medium: 

Ammonium sulphate, 4 . 71 g. 

Potassium phosphate, o . 75 g. 

Magnesium sulphate, o. 10 g. 
Water, i L. 

To this is to be added any carbohydrate to be studied. Media may be made 
solid by the addition of gelatin or agar. 

Pure cultures can be made, rarely, by inoculation from a naturally pure source, 
such as the sporangium of a Mucor. 

Physiological Separation. The first attempts at purifying mixed cultures were by 
means of physiological differences. Pasteur freed yeast from bacteria by growing it 
in a medium containing 2 per cent, of tartaric acid. Effront used fluorides in the same 
way. These methods may be made more effective by repeated transfers of the 
culture. Each transfer will contain a larger proportion of the form most suited to 
the conditions, until finally a pure culture may be obtained. The principle of these 
methods is of great use in practical fermentation, but is of little use in rigidly separat- 
ing forms. Methods of general application for the latter purpose must be such that 
a single cell can be isolated in a sterile medium and a culture propagated from 
this single cell. 

Separation by Dilution in Liquid Media. A mixed culture is diluted with steri- 
lized water until on the average every two drops contain one cell. A large number 
of flasks of a sterilized nutrient medium is then inoculated from the dilution, one 
drop in each flask. If the dilution has been properly made, about half of the flasks 
will remain sterile and half will show growth. Many or most of the latter will 
contain pure cultures. 

Separation by Dilution in Solid Media. If we dip a sterilized platinum wire into 
a mixed culture and then draw it repeatedly over the surface of a solid culture medium 


YEASTS 


77 


such as a slice of sterilized potato or a layer of nutrient gelatin in a petri dish we will 
get a series of streak cultures. The first of these will develop a strong growth of mixed 
forms. The last will show more and more isolated colonies until some of them will 
show only a few, some of which may be pure cultures. 


A 


B 


6 
/ 


D 


FIG. 57. Wild and pseudo yeasts. A, S. pombe. (After Lindner). B, Torulce. 
(After ^ Pasteur.} C, Mucor, (i) spores; (2) germinating spores and mycelium. D, 
S. apiculatus. E, Mycoderma vim. (After Bioletti.} 

The most useful method of separation and one which is applicable to most cases 
is that of plate cultures, first used by Koch and improved by others. In this method a 
drop of the mixed culture is thoroughly distributed in 10 to 20 c.c. of liquefied 
nutrient gelatin or agar. A drop of this mixture is then diluted in the same way in 
another portion of the same medium. This process is continued until the requisite 


78 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

degree of dilution is obtained. The various portions of nutrient gelatin are then 
poured, with precautions against outside infection, on glass plates or more conven- 
iently into petri dishes. On cooling and solidifying, the gelatin imprisons every cell, 
each of which on growing gives rise to a colony. It has been found that in practice 
a small percentage of these colonies may arise from two adhering cells and thus fail 
to be pure culture. 

Hansen's modification of the method is intended to obviate this uncertainty. By 
making the dilutions in the way described for liquid media, a drop of gelatin contain- 
ing only one cell is obtained, placed on a cover-glass over a culture slide and, by direct 
observation, the presence of a single cell verified. The development and multiplica- 
tion of this cell can be watched. 

DIFFERENTIATION OF YEASTS. With magnifications of 300 to 500, yeast cells 
can be examined conveniently. Contamination with bacteria and molds of special 
form can be detected, but otherwise a simple microscopic examination is of little 
value in determining the purity of a culture. Some information regarding the 
health, nutrition and vitality of the yeast may be obtained and the form of the spores 
is of some value in distinguishing species. Yeast cells vary in size as much as in 
form but under standard conditions each variety will show a certain normal range of 
dimensions. 

If a young, vigorous yeast, in a favorable liquid culture medium, is allowed to 
remain at rest at a suitable temperature with full access to air and protection from 
contamination, a growth of cells on the surface will usually take place. This growth 
may extend over the whole surface (Him formation] or may be restricted to the edges 
(ring formation) . This growth occurs at once with a few species (S. membrancefaciens) - 
or at the end of several days (S. ellipsoideus II] or may require several weeks. 
The time and optimum temperature of film formation have been used as descriptive 
characters. 

All the morphological and cultural characteristics of yeast are insufficient for 
diagnostic purposes and must be supplemented by the physiological characteristics 
such as their action on various sugars and other carbohydrates. 


CHAPTER IV 
BACTERIA* 

The bacteria naturally fall into quite distinct groups or orders 
the true bacteria and the sulphur bacteria. 

A portion of the true or Eubacteria together with the sulphur forms, 
are designated as the higher bacteria. The forms usually spoken of 
as bacteria belong to the group of lower bacteria, and when the 
word "bacteria" alone is used reference is usually made to the lower 
bacteria. These constitute a group of microorganisms quite distinct 
and characteristic, while the higher bacteria form links, as it were, 
between the lower bacteria and other closely related microorganisms. 
The morphology of the two groups will need to be discussed 
separately. * 

FORMS OF LOWER BACTERIA* 

FUNDAMENTAL FORM TYPES. The forms of bacteria are exceed- 
ingly simple. They are either spheres, straight rods, or bent rods 
(spiral). In the spherical form they are known as cocci, or micrococci 
(sing, coccus or micro coccus) . The straight rods are bacilli (sing. 
bacillus) and the bent rods are spirilla (sing, spirillum). 


.. 

. . ;. " 

v * 

as 

FIG. 58. Types of micrococci. (After Williams.) 


FIG. 59. Types of bacilli. (After Williams.) 
Prepared by W. D. Frost, with cytology by A. Guilliermond. 

79 


So 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


FIG. 60. Types of spirilla. (After Williams.} 

GRADATIONS. The difference between these fundamental form 
types is frequently very slight. It becomes a very difficult matter, 
for instance, to distinguish at times between the micrococcus and the 
bacillus. There is a number of bacteria, and among them the well- 
known example of B. prodigiosus, which are described at. one time by one 
investigator as micrococci and at another time, or, by another inves- 
tigator, as bacilli. The pneumonia germ is also another illustration 
of an organism that occupies a dual position. Migula has suggested 
a method of differentiating these which will be discussed under a 
later head. The bacilli pass almost imperceptibly into the spirilla. 
The cholera bacillus of Koch is in reality a spirillum. 


FIG. 61. Involution forms. Here are illustrated unusual forms of B. subtilis, 
water bacteria, Bact. aceti, Bact. pasteiirianum, bacteroids in root nodules, Bact. 
tuberculosis, Bact. diphtherias. (After Fischer from Frost and McCampbell.) 


BACTERIA 8 1 

INVOLUTION FORMS. *- -The forms of bacteria are quite constant under 
normal conditions, but very frequently they show abnormal or bizarre 
shapes. These are known as involution forms (Fig. 61). It is some- 
times suggested that these involution forms represent another stage in 
the developmental history of the organism, and upon this supposition 
certain bacteria which very regularly show these involution forms have 
been classified as belonging to a different suborder from that in which 
the lower bacteria are placed. The ordinary view of the involution 
forms is, however, that they are degeneration forms, that they cor- 
respond, in other words, to the halt and maimed in society and are to 
be accounted for by the fact that they are deformed by their own by- 
products. In fact, it is quite probable that they are autogenic. In- 
volution forms are very likely to occur in artificial culture and are much 
more common with some species than with others. (See page 100.) 

SIZE* 

The bacteria were formerly spoken of as the smallest of living things, 
but since the recognition of the ultramicroscopic organisms it is neces- 
sary to be somewhat more specific in characterizing their dimensions. 
The unit of measurement in microscopy is the micron (/*), or micro- 
millimeter. This is .001 mm. or approximately 1/25000 of an inch. 
Applying this unit to the bacteria we find that the micrococci and the 
short diameter of the bacilli and spirilla average about i^u. The micro- 
cocci vary in diameter from a small fraction of a micron to three or four 
microns in diameter. The bacilli are sometimes very small, as the 
influenza bacterium with a width of 0.2^ and a length of 0.5^, and 
sometimes very large as, for example, the Bact. anthracis with a width 
of I.2/A and a length of 5.20/4. The spirilla average about i.o/z in 
diameter but may be as long as 30^-40^. 

MOTILITY* 

When bacteria are viewed under the microscope in a living condition 
many of them are seen to move. This movement may be one of two 
kinds. In some cases it is progressive, the individuals move about from 
one part of the field of the microscope to another and change their rela- 
tive positions. In other cases the movement is vibratory, the bacteria 
move back and forth and rotate but do not progress or change their 
relative positions to any extent. This latter form of movement is 
known as brownian movement, because it was first described by Brown. 

Prepared by W. D. Frost. 


82 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

BROWNIAN MOVEMENT.- -This movement is probably caused by the 
impact of the molecules of the suspending medium and for this reason 
is sometimes called molecular movement. It is not characteristic of 
bacteria, or indeed of life, but is shared by many small microscopical 
objects when suspended in a fluid medium. Most beautiful examples 
of brownian movement can be seen by suspending granules of India 
ink or carmine and examining them under the microscope. This 
brownian movement is to be sharply differentiated from vital movement 
which is possessed by some bacteria. 

VITAL MOVEMENT. As already indicated, bacteria have the power 
of independent movement due to inherent vital power. ' Only a few of 
the micrococci are motile, while many of the bacilli and spirilla are. This 
movement is a change of position and is caused by certain protoplasmic 
processes which these bacteria possess, known as cilia (sing, cilium) or 
flagella (sing, flagellum}. The fact of motility or non-mo tility of an 
organism is of considerable value to the systematist. It is determined 
by examination in a hanging drop. At times, however, it varies so little 
from the brownian movement that it is difficult to tell whether a par- 
ticular organism or culture does or does not possess vital movement. 
An opinion can be more definitely formed at times if some chemical 
producing an anaesthetizing effect on the bacteria is introduced into 
the examining medium. In case the organism is actually motile its 
movement will be altered by the anaesthetic but in case it is merely a 
brownian movement there will be no change. 

ORGANS OF LOCOMOTION. The protoplasmic threads referred to as 
the organs of locomotion are known as flagella, or cilia. The difference 
between the cilium and flagellum is the fact that a cilium has a simple 
curve while a flagellum has a compound curve, like a whip lash. Most 
of the bacteria possess flagella rather than cilia. The size, arrange- 
ment, etc., of these flagella are constant and characteristic of a par- 
ticular organism. Their structure and arrangement, therefore, will be 
discussed later. 

CHARACTER OF MOVEMENT. Different bacteria exhibit different 
kinds of movement. Some dart forward with great rapidity, others 
move slowly; some move in straight lines, others wobble, but any 
particular character is quite constant and many of the bacteria may 
be recognized by their peculiar movements. 

RATE. The rate at which the bacteria travel when they possess 
vital movement varies greatly. Some of them move very fast, others 


BACTERIA 83 

very slowly. Many of them appear to move with wonderful rapidity. 
Van Leeuwenhoek, when he first saw these moving bacteria, said that 
they traveled with such great rapidity that they tore through one 
another, but it must be borne in mind that under the high powers of 
the microscope the rate of movement is magnified to the same extent 
as the object, and that in reality the rate of movement is not excessive. 
When compared to their size, the rate of movement is probably little 
greater than that of a trotting horse and considerably less than that 
of a speeding automobile or a railroad train. 

REPRODUCTION* 

Reproduction among the bacteria is largely asexual and takes place 
ordinarily by what is known as binary fission. In addition to this a 


QOQDODOQ 

FIG. 62. The division of bacterial cells (diagrammatic). (After Novy.} 

number of bacteria go into a resting stage, or produce spores. The 
spore formation is not, however, a method of multiplication, because 
usually only a single spore is formed in a cell, but serves to tide the 
organism through unfavorable conditions. 

VEGETATIVE MULTIPLICATION. This is accomplished by means of 
binary fission (Fig. 62). When a bacterium has reached maturity, fis- 
sion begins. Division begins by an invagination of the protoplasm 
in the middle of the cell, which proceeds until the cell protoplasm is 
completely separated. The cell wall then grows in and finally splits 
forming the two ends of the new cells. These new cell walls are formed 
at right angles with the long axis of the cell in the case of the bacilli 
and spirilla, except in rare instances. In the case of micrococci, the 
throwing of the cell wall across one diameter is quite as economical 
as any other and may therefore proceed in any direction. Migula 
makes a considerable point of the fact that bacilli and spirilla elon- 
gate before division and micrococci divide before they elongate; this 

Preparedly W. D. Frost. 


84 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

would be the criterion which he would use to separate these two form 
types. A generation among the bacteria is from one division of the 
cell to another. This is sometimes very short, in fact, only twenty to 
thirty minutes. Many of the bacteria after half-an-hour's time have 
grown from newly formed cells to maturity and are ready to divide 
again. This makes it possible for bacteria to multiply with very great 
rapidity, and if we know the length of the generation in a particular 
bacterium it would be easy enough to estimate the rate of multiplica- 
tion, at least theoretically. It would be only a matter of geometrical 
progression. It is of course quite impossible for the bacteria to main- 
tain their theoretical rate of growth for any length of time, but, prac- 
tically, they grow with enormous rapidity, as is shown in cultures and 
by the changes which they bring about in nature, such as the produc- 
tion of fermentation and the generation of toxin. Four periods in the 
life history have been described. A latent or lag period, which is the 
time elapsing between the seeding and the time at which the maximum 
rate of growth begins; the logarithmic period or the time when the rate 
of growth is at its maximum; a stationary period when the increase 
becomes less and less and finally ceases; and the period of decline when 
the organisms begin to die. 

SPORE FORMATION. A considerable number of bacteria form spores 
within the cell. Because they are formed within the cell they are 
spoken of as endospores. Endospores are formed by the bacilli and the 
spirilla, but not by the micrococci. Their chief value to the cell is their 
ability to resist unusual conditions, and to enable the individuals of a 
species to pass through unfavorable conditions which to the ordinary 
vegetative form of the cell would prove disastrous. At the maturity 
of the cell, spore formation may begin. It is an open question whether 
spore formation occurs as a regular 'stage in the life history of an 
organism, or is produced only under the stimulus of unfavorable en- 
vironmental conditions. Both theories have their advocates. The 
first evidence of spore formation in the cell is a granulation of the 
protoplasm of the cell. As spore formation proceeds the granules 
become larger and collect at one portion of the cell. These granules 
then fuse to form the spore, which soon surrounds itself with a spore 
wall. At times the spore is smaller than the mother cell and is formed 
without changing the shape of the cell. At other times it is larger 
than the mother cell and causes a bulging of the latter. The position 


BACTERIA 


of the spore in the cell varies (Fig. 64). In some species it is equatorial, 
in others it is polar, and in still others it has an intermediate position 
between equatorial and polar. When the spore is larger than the 
mother cell and is situated equatorially it causes the cell to bulge with 
the formation of a barrel-shaped organism, a clostridium. If the 
spore is situated at the poles and is larger than the mother cell, a 
capitate or drum-stick bacillus is produced. When the spore is smaller 
than the mother cell and the cells form in chains, there is frequently a 
tendency for the spores to be formed in opposite ends of contiguous cells 
of the chain so that they appear in pairs. The reason for this is not 
understood. When the spore has reached maturity, the mother cell 
disintegrates and finally disappears, leaving the endospore free. 

The endospores possess remarkable powers of resistance due to the 
concentrated character of the protoplasm, or to the character of the 


j 


FIG. 63. 


FIG. 64. 


FIG. 63. The formation of spores. (After Fischer from Frost and McCampbell.) 
FIG. 64. Spores and their location in bacterial cells. (After Frost and McCampbell.} 

spore wall. The resistance here may be due to the structure of the wall 
itself or to the chemical substances which it contains. It is readily con- 
ceivable that the presence of certain fatty acids, or higher alcohols, 
might give the spore its remarkable resistance. These spores are very 
resistant to desiccation; they have been preserved in a dried condition 
for many years. They are also very resistant to the action of heat; 
some forms are known to withstand a temperature of boiling water for 
as long a time even as sixteen hours. They are resistant also to chem- 
icals and the action of sunlight, although in some cases, as pointed 
out by Marshall Ward, the very chemical substances which furnish 
them the powers of resistance toward environmental factors may be 
broken up under the influence of sunlight, forming poisons so that the 
spore is killed more readily than the vegetative cell would be. 


86 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

When these spores are brought under favorable conditions of 
moisture, temperature, and food supply, they germinate. There are 
several types of germination (Fig. 65). In some cases the spore wall 
ruptures at the pole and the young cell emerges so that its long axis is 
in the same direction as the long axis of the spore. In another type 
the spore ruptures equatorially and the young cell emerges with its 
long axis at right angles to the long axis of the spore. In still another 
type the spore swells and the young cell absorbs the wall of the spore. 

In the lower bacteria only a single spore is formed in a cell. 
In the case of the higher bacteria, however, a number of spores may be 
formed at the distal end of the filament. These are spoken of as 
conidia, and possess properties similar to those of the endospores. 


b 


n 
u 


FIG. 65. Spore germination, a, Direct conversion of a spore into a bacillus 
without the shedding of a spore- wall (B. leptosporus); b, polar germination of Bad. 
anthracis; c, equatorial germination of B. subtilis; d, same of B. megatherium; e } same 
with "horse-shoe" presentation. (After Novy.) 

In some cultures of bacteria, as for example in the micrococci, 
certain cells seem to be larger and different from the other cells. In a 
streptococcus filament, certain cells suggest to the observer the joint 
spores of the algae and have therefore been spoken of as arthrospores or 
joint spores. There is, however, no evidence of an experimental 
nature, which warrants the belief that these cells are in reality spores, 
and it must be said that at the present time the presence of arthro- 
spores among the bacteria is purely hypothetical. 

CELL GROUPING* 

Bacteria rarely occur singly but usually in groups. These cell 
aggregates are frequently very constant and quite characteristic of the 
organism possessing them. They are of sufficient definiteness and 
constancy to be used by the systematists in characterizing large groups. 

^Prepared by W. D. Frost, 


BACTERIA 87 

CELL AGGREGATES AMONG THE MICROCOCCI. The grouping of 
micrococci depends upon the plane of division and also upon the cohe- 
sion of the cells. Since it is quite as economical for the micrococcus to 
divide in one direction as another, it is possible for a number of different 
cell groupings to occur. Whatever the direction of the dividing walls, 
it is usually quite constant; if a particular species of micrococci has its 
planes of division parallel, there will be formed chains of micrococci. 
In some cases the cohesion is slight and only two cells remain attached 
to each other, forming what are ordinarily known as diplococci. There 
is a considerable number of very well-known bacteria that are diplo- 
cocci (Fig. 66). If the cohesion is stronger, we have chains of micro- 
cocci or rosaries formed which are known as streptococci. Well-known 
and very important bacteria are grouped in this way. In other micro- 
cocci the cell wall is not formed continuously in parallel planes but in 


QQ 


FIG. 66. Division forms of micrococci. a, Diplococcus, perfect form with 
flattened opposed surface (gonococcus) , lanceolate form (pneumococcus] ; b, strepto- 
coccus; c, consecutive fission yielding a tetrad; d, sarcina form resulting from division 
of tetrad c; e, staphylococcus. (After Novy.} 

planes which alternate at right angles to each other. In this way cell 
aggregates occupying two dimensions of space are formed. These are 
known as tetracocci, or merismopedia. Still again, the planes of division 
may proceed at right angles to each other in three dimensions of space. 
In this case packets are formed which are known as packet cocci, or 
sarcincz. Another group of the micrococci occurs, known as the staphy- 
lococci, so called because they are arranged in irregular bunches, like a 
bunch of grapes. This arrangement may be due to the fact that these 
micrococci divide in many different planes, or because during the course 
of their growth their arrangement is changed. 

CELL AGGREGATES AMONG THE BACILLI. In the case of the bacilli, 
one diameter is usually considerably shorter than the other, so that 
nature almost invariably throws the new cell wall across the bacilli 
at right angles to their long axis (Fig. 67). There is, therefore, only 
one arrangement or cell grouping possible, and that is end to end, so 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 

that streptobacilli are formed. When arranged in pairs, the designa- 
tion is diplobacilli. The length of the chains appears to depend not 
only upon the cohesion of the bacilli but also upon the shape of the 


FIG. 67. Division forms of bacilli, a, Single; b, pairs; c, in threads. (After Novy.) 

end; those which have square ends frequently have very long chains, 
while those with rounded ends have short chains or occur singly. 

A unique growth-form or cell aggregate is that due to the post fission 
movement of the cell as described by Hill in cultures of Bact. diph- 


f/ 


III I /"/ 

!'iii 4* 

tiff 

iii ' "I/ >' 

?/ ; ,"// // 

II 

Ili/L 

"'ii tiiii/i/ 

'//// /;////// 


FIG. 68. Threads of Bact. anthracis. (After Migula.) 

theriae. On fission the two daughter cells are not completely separated 
but remain attached at one place. This leads to a movement similar 
to the closing of a jack knife. In this way the two sister cells are 
brought to rest at an obtuse, a right or an acute angle to each other. 
They may be even brought parallel. 


BACTERIA 89 

CELL AGGREGATES AMONG THE SPIRILLA.- -The same kind of 
arrangement is maintained among the spirilla. 

ZOOGLCEA. Some of the bacteria secrete a mucilaginous substance 
which causes the cohesion of the cells frequently in considerable number. 
This aggregate of cells may assume some characteristic appearance and 
a great many attempts have been made by systematists to make use 
of this in differentiating species. These zooglceic masses usually 
assume the forms of pellicles, but their value as diagnostic features is not 
great. The formation of zooglcea is very frequently only a stage in 
the life history of an organism. 

THE CYTOLOGY OF BACTERIA 

: The typical cell, such as that of a higher plant or animal, is made 
up of cytoplasm surrounded by a cell wall. The cytoplasm contains a 
nucleus. There are also frequently present other evidences of struc- 
ture in the cytoplasm, such as nucleolus, polar bodies, etc. In addition 
to these there may be appendages, such as the cilia or flagella. In 
the case of bacterial cells, we find most of these structures present, 
such as cell wall, cytoplasm, and appendages. 

GENERAL CONSIDERATION OF CYTOPLASM AND NUCLEUS.* The 
cytoplasm of the bacterial cell is similar to the cytoplasm of other cells 
except that chemical analyses seem to show that it contains a higher 


a. 


FIG. 69. Plasmolytic changes. (After A. Fischer.) a, Cholera vibrio; b, typhoid 

bacillus; c, Spirillum undula. (From Novy.} 

percentage of nitrogen. As viewed under the microscope, in either an 
unstained or stained condition, it appears as a homogeneous mass 
filling the entire cell and rarely showing any evidence of structure. 
Ordinary stains, such as are used in animal and plant histology, fail 
to reveal the presence of a nucleus, the whole cell being usually uni- 
formly stained with those stains generally characterized as nuclear 
stains. When these stains are applied to some bacteria, particularly 
at certain stages of their growth, certain parts stain more readily than 
others, and we get either what is known as a bi-polar stain or polar 

Prepared by W. D. Frost. 


QO MORPHOLOGY AND CULTURE OF MICROORGANISMS 

granules. In the first case, the ends of bacilli are stained more deeply 
than the center so that the cells appear very much as diplococci. This 
bi-polar stain is characteristic of such organisms as the bacterium of 
chicken cholera or the bacterium of bubonic plague. The polar 
granules are frequently seen in the diphtheria bacterium and may 
be located at the poles and also at the center. In this germ and in 
some others it is possible, by special staining, to give the granules a dif- 
ferent color from the rest of the organism. In this case these bodies are 
spoken of as metachromatic granules which are considered later under 
" Reserve Products." The presence of these granules might possibly 
be explained upon the theory that the cells are plasmolyzed (Fig. 69). 
As a result of plasmolysis the protoplasm of the cell is drawn away 
from the cell wall and concentrated in areas which would very well 
explain the appearances. And it seems likely also that the methods 
employed in staining might lead to plasmolysis, but the metachromatic 
granules can hardly be explained upon this supposition. 

The cytoplasm of the bacterial cell is slightly refractive. It is 
colorless except in a few cases in which the green coloring matter, like 
chlorophyl, is present, as, for instance, Bad. viride and Bact. chlorinum. 
In the purple sulphur bacteria, the coloring matter bacteriopurpurin 
is present. The bacterial cytoplasm contains vacuoles at times. 

MINUTE CONSIDERATIONS OF CYTOPLASM AND NUCLEUS.* The 
question of the cytology of bacteria has long excited the curiosity 
of biologists. It is indeed of great importance from many points 
of view. In the first place, we are interested to know whether 
bacteria are ordinary cells having a nucleus; or whether, as some 
maintain, they lack entirely a nuclear element and are an exception 
to the rule elsewhere established. Moreover, the cytologic study 
of bacteria may furnish useful knowledge concerning the phylogeny 
and taxonomy of these organisms, a matter not yet solved. Finally, 
we may hope that it will throw light upon some problems of a physio- 
logical or pathological nature. 

Unfortunately this study is very delicate, because of the extreme 
minuteness of the bacterial cells, so that in spite of the large number of 
researches which it has incited in the last twenty-five years, it is to this 
day a matter of controversy. 

At present three theories are held by authors relative to the inter- 
pretation of the general structure of bacteria. We will examine these 

Prepared by A. Guilliermond. 


BACTERIA 


three theories one by one, endeavoring to determine which one, in our 
opinion, seems most probable. 

One of these theories claims that bacteria are cells of very primitive 
organization lacking nucleus and consisting simply of cytoplasm with 
vacuoles. The cytoplasm contains many stainable granulations, but 
these represent products of nutrition. Such an opinion scarcely accords 
with our knowledge of the constitution of the other Protista, in all of 
which the existence of a typical nucleus, or at least of chromatic 
elements replacing the nucleus, has been established. This view has 
not, therefore, had many supporters. 

Another theory maintains that bac- 
teria have a typical nucleus and are in 
no way structurally different from ordi- 
nary cells. This opinion was suggested 
by Arthur Meyer, who claims to have 
succeeded in differentiating, in a great 
many bacteria, granules which fix nu- 
clear stains, and of which one or often 
several appear in a cell. These granules 
he would consider nuclei. It seems to 
be established, however, that the ma- 
jority of the elements noted by Meyer FlG 70 Bacterium gammari 
are not nuclei, but reserve products and a filamentous bacterium from 

,. ,, the intestine of Bryodrilus. (After 

common among the Protista and known vtjdowsky.) 

as metachromatic corpuscles. 

Vejdowsky's efforts have resulted in much weightier proofs in favor 
of the existence of a true nucleus. In the Bacterium gammari, a 
species discovered by him in the sections of a little fresh water crus- 
tacean, Gammarus zschokkei, Vejdowsky has been able to demonstrate 
in each cell a typical nucleus which is always present. This nucleus 
appears very clearly; it consists of a colorless nucleoplasm surrounded 
by a membrane and containing karyosomes (Fig. 70). The author had 
the good fortune to ascertain in several cases karyokinetic representa- 
tions of the division of this nucleus (a, b, c). In short, the presence 
of this nucleus is indisputable. 

The same author discovered a similar structure in a filamentous 
bacterium found in the digestive tract of an Annelida (Bryodrilus 
ehlersi) (Fig. 70, d). 


Q2 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

These conclusions are positive, but the species observed by Vej- 
dowsky are not well-defined bacteria, and may be thought to belong 
to the molds rather than to the bacteria. It has also been said, 
not without reason, that Bad. gammari might be a yeast of the genus 
Schizosacchromyces and that the filamentous bacterium studied by 
Vejdowski seems to resemble a filamentous mold. 

However this may be, one of Vejdowsky's pupils, Mencl, has en- 
deavored to apply these conclusions to other bacteria, which are well- 
defined, notably B. megatherium, but has only succeeded in bringing 
forth proofs which are much less convincing of the existence of a nucleus. 
The author strived to discover a nucleus, but this organ ,is not constant 
and does not show the structure of a true nucleus. 

Both Kruis and Rayman have discovered a nucleus in different 
bacteria (B. myco'ides, radicosus, etc.). This nucleus appears only in 
very young cells; it is not found in older cells, and seems (like the nucleus 

noted by Mencl) to represent merely the 

*, . [2tJ] t t <%^ incipient transverse septum which fixes 

I , 2 stains well at the beginning of its forma- 

** u, O ..., tion and in some ways resembles a nucleus. 

3 4 The studies of Penau, who also endea- 

FIG.^ 71. Bacillus megathc- vored to prove the existence of a typical 

rium. (After Penau.} i > 

nucleus in bacteria, were no more success- 
ful. In B. megatherium, he describes the following phases. In the 
youngest cells he observes a stage where the cytoplasm is very dense 
and uniformly stained, without a trace of differentiation. Immediately 
succeeding is a phase where the cytoplasm becomes less chromatic and is 
filled with vacuoles. At this point the author finds in each cell a tiny 
granule (Fig. 71, i), homogeneous and easily stained, situated at one of 
the poles of the cell, very near the membrane. This granule he con- 
siders to be a nucleus. Moreover, in the cytoplasmic web he observes 
a series of stainable granules connected by slender trabeculae, thus 
forming a kind of network which he likens to mitochondrial and chro- 
midial formations. At the time of sporulation, Penau finds an in- 
crease in the size of the nucleus (Fig. 71, 2 and 3) which changes to 
a large granule; this is soon surrounded by a membrane and becomes 
the spore (4), which is therefore formed mostly of chromatin. 

The same author discovers a very different structure in Bact. 
anthracis. Here, after a stage of undifferentiated structure which 


BACTERIA 93 

characterizes the youngest cells, follows a phase where the cytoplasm 
becomes alveolar. At this time, at one of the poles of each cell, appears 
a very large homogeneous granule which Penau regards as a nucleus. 
This nucleus, however, has only an ephemeral existence and quickly 
undergoes a cytolysis during which it disintegrates. The disintegra- 
tion products then impregnate the trabeculae of the cytoplasm and the 
nucleus becomes diffuse. In a last phase which corresponds to sporo- 
genesis, the chromatin which impregnates the cytoplasm is partly con- 
densed at one of the poles, where it forms first a mass of grains, then a 
large granule which changes to a spore. 

Nothing is less conclusive than these results, since the author cannot 
discover an homologous structure in the different species which he 
studies, and since the nucleus which he describes is only a transitory 
organ not showing the distinguishing characteristics of a nucleus. 

To prove the existence of a nucleus in bacteria, it is necessary to 
show a nucleus with a differentiated structure, the constant presence 
of the nucleus, and to follow the division of this organ during the cellular 
separation. So far no one has apparently been able to differentiate 
such an organ in well-defined bacteria. We must conclude, therefore, 
that with the exception of the results obtained by Vejdowsky, all ob- 
servations so far gathered in favor of the existence of a typical nucleus 
in bacteria are by no means convincing. 

The third theory asserts the existence of a diffuse nucleus in bacteria. 
It was first suggested by Weigert and more carefully formulated by 
Blitschli. This author describes in a certain number of Sulpho-bacteria 
of large size, Beggiatoa, Chromatium, a kind of central body occupying 


FIG. 72. i. Chromatium okenii. 2. Beggiatoa alba. These two bacteria have 
a central body containing chromatic grains and considered by Biitschli as the 
equivalent of a nucleus. (After Biitschli.) 

nearly the whole volume of the cell and consisting of an alveolar cyto- 
plasm of highly stainable web, containing within its knots numerous 
chromatic granulations (Fig. 72). The remainder of the cell consists 


94 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

of a thin cytoplasmic layer, less easily stainable, surrounding the 
central body. Biitschli compares this structure with the one which 
has been demonstrated in the Cyanophycea, and claims that the central 
body represents the equivalent of a nucleus. It would be a sort of large 
nucleus occupying most of the cell, not bounded by a membrane, and 
scarcely distinct from the cytoplasm. This structure has recently been 
verified in Chromatium okenii by Dangeard. The Sulpha-bacteria, 
however, are organisms morphologically entirely distinct from ordinary 
bacteria, and are apparently directly related to the Cyanophycecz. 
Such a structure is not found in other bacteria, in which it is impossible 
to demonstrate a central body and in which, one must admit, the 
nucleus is still more diffuse. 

To Schaudinn we are indebted for the most exact observations in 
favor of the theory of the diffuse nucleus. He had the good fortune 
to discover in the intestine of the cockroach, Periplaneta orientalis, a 
bacillus of very large size which he named B. biitschlii. It is the largest 
bacillus known at present (4^ wide), and lends itself readily, therefore, 
to cytological studies. His minute observations have shown that 
there is no nucleus, the cells enclosing a finely alveolar cytoplasm, 
whose net contains many small grains which take nuclear stains 
(Fig. 73, 1-6). 

At the time of sporulation the chromatic grains increase in size 
(Fig. 73, 7-9), then gather at the center of the cell in a kind of axial 
wreath (Fig. 73, 10). The two extremities of this wreath quickly swell 
with an accumulation of chromatic grains and form two granular 
masses, one at either pole. These two masses form the beginning of 
the two spores, for each cell forms two spores (Fig. 73, n and 12). 
The grains which compose these two rudiments then condense to form 
two large homogeneous granules (Fig. 73, 13) which strongly resemble 
nuclei and which Schaudinn considers to be such. Around these two 
granules is soon condensed a thin cytoplasmic zone which in turn is 
separated from the surrounding cytoplasm by a membrane (Fig. 73, 
13). Henceforth the spores cannot be stained by ordinary means 
because of the thickness of their membrane which prevents the pene- 
tration of stains (Fig. 73, 14). The granules of the wreath, which 
join the two rudiments of spores, gradually disappear as well as 
the cytoplasm, while the spores increase in size. Then the sporangium 
ends by breaking and setting free the two spores. Germination con- 


BACTERIA 


95 


sists simply of a swelling of the spore, then the formation of a small rod 
which issues from the spore and forms a septum for itself (Fig. 73, 15 
and 1 6). As soon as the spore germinates, the nucleus ceases to exist 
as a morphologic entity; it is scattered in the cytoplasm in the form of 
little grains. 


13 14 


FIG. 73. Bacillus butschlii. 1-16, Vegetative cells and their division. 7-9, Begin- 
ning of sporulation: the cells about to sporulate are partitioned off crosswise; then 
the septum thus formed is absorbed, at which time sporulation begins. Schaudinn 
considers this partitioning off followed by fusion of the two daughter cells as a rudi- 
mentary sexuality. 10-13, Formation of the beginnings of the two spores, at the 
poles of the cell. 14, Ripe spores. 15-16, Germination of the spore. (After 
Schaudinn.) 

In another bacillus smaller in size (B. sporonema), Schaudinn has 
found an analogous structure only at the time of sporulation; he does 
not prove the formation of an axial filament but only the condensation 
of a portion of the chromatic grains into a large granule which forms the 
beginning of the spore (Fig. 74). 

By the fact that in these two bacilli the beginning of the spores 
appears as a granule equivalent in some respects to a nucleus and 
resulting from the condensation of a portion of the stainable grains, 
Schaudinn is led to believe that these grains are composed of chromatin 
and represent a kind of diffuse nucleus. 


9 6 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


These results have been confirmed by our studies of a large number 
of endospore bacilli (B. megatherium, radicosus, mycoides, aster ospor us, 
alvei). Upon examination at the very outset of their development, 
these bacteria present a homogeneous appearance and are uniformly 


1 

. / 


FIG. 74. Bacillus sporonema. i, Cell about to sporulate. 2, This cell grows 
narrow at the center, as if it were going to be divided (Schaudinn regards this pinch- 
ing together which afterward disappears (5), as the vestige of an ancestral sexuality 
like that of B. biitschlii). 3-5, Formation of the beginning of the spore. (After 
Schaiidinn.) 

stained with no great differentiation, explicable by the density of the 
cytoplasm or by a special condition of the membrane. At this stage 
the cells are in the process of active divisions, after which the transverse 
septa are formed as follows: On the side walls of the bacillus appear 
two small granules which take some stains (Fig. 75, i). These soon 


FIG. 75. i-io, Bacillus radicosus. i, Beginning of development. 2-3, Cells 
at the end of eight hours; 4-6, sporulation. 9-10, Cells in which the chromatic 
grains are located in the middle in a mass slightly resembling a nucleus. 11-12, 
Spirillum volutans. 

disintegrate at the center of the cell to form a thin band marking out 
the two daughter cells and forming the beginning of the transverse 
septum. This strongly resembles a nucleus and has apparently been 
considered as such by a number of authors (Rayman and Krius, Mencl). 
Toward the eighth hour of development, the cells show clearly their 


BACTERIA 97 

structure which is changed in appearance; the cytoplasm vacuolizes and 
ends by displaying a fine alveolar structure. The web contains in its 
knots small, highly stainable granules (Fig. 75, 2 and 3). In some 
cases (cultures on special media for example), there is noticeable a 
localization of these granules at the center of each cell, forming a 
granular region which recalls somewhat the appearance of a large 
nucleus and which is separated into two portions at the time of the 
cellular division as if it were indeed a true nucleus (Fig. 75, 7 and 10). 

These granules fix the nuclear stains, and it seems permissible to 
consider them chromatic in nature. 

At the time of sporulation there forms at one of the poles of the 
cell a small oval mass, easily stained, which is like a nucleus in appear- 
ance (Fig. 75, 4 and 5). This results from the condensation of part of 
the chromatic granules of the cytoplasm, gradually grows larger, and 
changes to a spore. When the spore has reached a certain size, it is 
surrounded by a membrane which prevents the penetration of ordinary 
stains (Fig. 75, 6); it appears then like a large colorless sphere in the 
stained cytoplasm of the cell (Fig. 75, 6). 

At no stage of the development have we observed the least trace of 
a nucleus. May there be a nucleus which our present technic would 
not enable us to differentiate? That has seemed to us scarcely probable, 
for if this nucleus existed, it would certainly be visible in a species 
as large as B. biitschlii and would not have escaped Schaudinn. The 
most reasonable hypothesis, the one which we have adopted, is to 
consider like Schaudinn that bacteria contain chromatin more or less 
mingled with cytoplasm, differentiated in the case of small grains and 
condensing at the time of sporulation to form the spore which would 
consist principally of chromatin. The cells of bacteria would accordingly 
have a very primitive structure. 

Granted the clearly demonstrated existence of this particular struc- 
ture in the Cyanophyceas, there is no reason for not admitting that the 
nucleus, very rudimentary in the Cyanophycece, might be even more so 
in bacteria, being reduced to a diffuse nucleus consisting of chromatic 
grains scattered in the cytoplasm. 

These observations have, moreover, received a series of new con- 
firmations by the labors of a great many authors (Swellengrebel, 
Ruzicka, Ambrez, etc.) and especially by the later researches of Dobell. 
The latter investigator discovered, in the intestines of frogs and toads, 

7 


9 8 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


a large bacillus (2^ wide) almost as large as B. butschlii, and named it, 
B. flexilis. This species shows exactly the same cytological charac- 
teristics as B. butschlii (Fig. 76). 

Through a study of a number of different bacteria found in the in- 
testine of toads, frogs and lizards, Dobell has endeavored to show that 
this diffuse nucleus is not original, but derived from the retrogression 
of a more highly differentiated nucleus. 

Thus in various micrococci he was able to show in each cell the 
existence of a central stainable granule, dividing by constriction at the 
time of cellular division, and which he regards as a nucleus (Fig. 77, 


12 


FIG. 77. 


FIG. 76. Bacillus flexilis. i, Beginning of the division of a cell about to sporu- 
late (vestige of sexuality). 2, Disappearance of the incipient division. 3, Forma- 
tion of the chromatic axial filament. 4, Formation of the beginning of two spores. 
5, Ripe spores. (After Dobell.) 

FIG. 77. Various bacteria, showing the successive types of the retrogression 
of the original nucleus and its transformation to a diffuse nucleus. (After Dobell.) 

1-5). In other cocco-bacillary species of bacteria characterized by 
spherical shape capable of elongation, Dobell discovers a similar nucleus 
in the spherical cells. When the cell lengthens and assumes the ap- 
pearance of a bacillus, this nucleus changes to a spiral axial filament 
(Fig. 77, 5 and 6). 

In various bacilli the same author demonstrates a filament which is 
ever present (Fig. 77, 7-11). The spore results from the condensation, 
at one of the poles, in the shape of a large chromatic granule, of part 
of the grains which compose this filament (Fig. 77, 12 and 13). An 
interesting variation of this structure is found in B. saccobrinchi. 


BACTERIA 99 

In this bacillus is noticed first an initial stage where the nucleus is 
represented by an axial filament quite similar to that otB.spirogyra 
(Fig. 77, 14). In the course of development, however, this filament 
resolves itself into a great many grains which scatter through the 
cell (Fig. 77, 15 and 16). The nucleus then becomes diffuse. Part of 
this diffuse nucleus next condenses at the time of sporulation into a 
large chromatic grain which forms the beginning of the spore. Finally, 
in other bacilli, Dobell finds in the whole development no more than a 
diffuse nucleus, that is, the structure described by Schaudinn and by 
Guilliermond. 

In the group of spirilla, Dobell notices these three types of structure: 
In some species he finds present a spherical body resembling a nucleus ; 
other species show a zigzag or a spiral filament; still others have a 
diffuse nucleus. 

From these observations, Dobell feels authorized to conclude that 
bacteria are organisms originally containing a nucleus, but in which the 
nucleus, as a result of parasitism, has undergone a series of retrogres- 
sions which have ended by making it diffuse. 

This opinion would have the advantage of reconciling opposed 
theories. It would explain how some authors have been able to dis- 
cern a true nucleus in various forms. 

Another more weighty reasoning which might also explain these 
contradictions is the fact that under the name of bacteria are gathered 
forms perhaps very different, some of which seem to belong to the 
Sulpho-bacteria and others might be considered as molds. 

Although we have just mentioned numerous works, the conclusion, 
to my mind, would be that while some bacteria may contain a more or 
less rudimentary nucleus whose existence is nowhere else precisely 
demonstrated, so far, in the great majority of the species, nothing more 
has been found than a diffuse nucleus consisting only of grains of chro- 
matin scattered through the cytoplasm. 

Life Cycle of Bacteria* .--The life-cycle of bacteria will prove a very 
important factor in the study of their morphology, their cultivation, 
their cultural characteristics and their classification, if its development 
takes place along the line so definitely advanced by Lohnis and Smith f. 
The variation in the appearance of a species of bacteria has long been 

* Prepared by the Editor. 

f Lohnis, F. and Smith, N. R.: Jour. Agr. Research, VI, 18, 675. 1916. 


IOO 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


recognized; cultivation has been fraught with difficulties which have at 
times been in some way associated with the change in form or in a sense 
connected with "involution" alterations; cultural characteristics have 
likewise been subject to variations which have depended upon the 
so-called vigor of the organism; and classification of bacteria may be 
materially affected since some of the cycles approach closely those of 
protozoa. 

Perhaps the most significant changes upon which the life-cycle of 
bacteria is based may be those represented by Jones,* and Lohnis and 
Smith in the life of A zotobacter-types. The polymorphous character of the 


FIG. 78. Change of Azotobacter from the normal cells (I) to arthrospores (II) 
and involution forms (III) to be lost in symplastic stage (IV) and recovering cell- 
form in V. Diagrammatic from Lohnis and Smith. 


Azotobacter group has been a matter of intense interest for a long period. 
Lohnis and Smith have not only endeavored to follow the variations 
through a consistent historical developmental cycle but have attempted 
to organize their observations and have them in accord with past 
observations. 

The organism may be assumed to exist in the form of a distinct cell 
and at other times in an amorphous condition called by the authors, the 
symplastic stage. In the usual cell-form the organism may multiply 
by fission as is the case with all bacteria, may produce endospores 

*Jones, D. H. : Cent. f. Bact. ; Trans. Royal Society of Canada, 1913. 


BACTERIA 101 

as is a common mode of reproduction, or arthrospores, when the entire 
organism appears to transmute to a resting stage or spore, or, the organ- 
ism may pass to the amorphous or symplastic condition. There is 
also a possibility of a union or " conjunction" of cells suggesting the 
functioning of gametocytes. 

In passing into the symplastic stage the cells passing through involu- 
tion forms appear to form clumps and lose completely their individual- 
ity of form and contents in a general mass of disorganized protoplasmic 
debris. Presumably scattered throughout this mass exists what may 
be recognized in protozoal forms, yeast cells, et cetera, nuclear centers, 
for out of this more or less homogeneous unvarying background of 
protoplasmic substance appear many lines resulting in modified forms 
which pass on to forms similar to the original cellular forms from which 
this amorphous mass was at first derived. 

The form of Azotobacter upon which this life-cycle theory is based 
may not be, of course, conclusive; however, Jones has confirmed many 
of the findings of Lohnis and Smith in the case of Azotobacter but is 
not ready to subscribe to all of their interpretations. Jones * claims, too, 
that so far as other species of bacteria are concerned in this theory 
of life-cycle, he has been unable to confirm Lohnis and Smith who 
assert that in the forty-eight species studied, they find practically the 
same developmental cycle. 

This subject is of so wide importance that it deserves much atten- 
tion and study. 

RESERVE PRODUCTS, f Besides the grains of chromatin which we 
have just been considering in bacteria are found other granulations 
which do not show the characteristics of chromatin and which act as 
products of nutrition. These granulations are characterized by the 
reddish color which they assume with most of the aniline blue or violet 
dyes, as well as with haematoxylin. These bodies, which are common 
to the majority of the Protista, are metachromatic corpuscles. 

They are found in larger or smaller numbers according to the species, 
the age of the cells, and the medium in which they are living. Some 
bacteria contain few metachromatic corpuscles (B. radicosus, megathe- 
rium, mycoides}; others produce many (B. alvei, asterosporus, Sp. 
volutans, Bact. tuberculosis and diphtheria). The metachromatic 

* Jones, D. H.: Jour, of Bact., Vol. V, p. 325. 
f Prepared by A. Guilliermond. 


IO2 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


corpuscles appear at the beginning of development in the form of very 
small grains, which generally increase gradually in size during de- 
velopment, and finally are absorbed in the very old cells. They are 
sometimes distributed through the whole cell (Spirillum volutans) as 
grains of chromatin (Fig. 79, 8 and 9), but most often they tend to 
gather at the two poles of the cell, or line up all along the bacillus 
(Fig. 79, i to 4, 6, 10, u). In some species (B. alvei, asterosporus, 
Bad. tuberculosis and diphtheria), these corpuscles grow bigger until 
they attain relatively large dimensions, surpassing the bacillus in size. 

Thus they cause a series of swellings all 
along the bacillus, which in consequence 
appears somewhat like a necklace (Fig. 
79, n). They then give the illusion of 
spores; one can easily understand the 
error of some authors who have confused 
them with spores, notably in the case of 
the Bact. tuberculosis. 

In B. asterosporus, the metachromatic 
FIG. 79. Various bacteria -, n ,, 

stained by a method which corpuscles usually appear in the youngest 

differentiates only the meta- cells, singly and in the shape of a small 

chromatic corpuscles. 1-4, 1 111 -i T 

Bacillus radicosus. 5-6, Bacii- central granule closely resembling a nu- 

lus asterosporus. 7, The same, cleus and which A. Meyer seems to have 

The cells have formed their -, , /T,. N 

spore and the metachromatic taken for such ( Fl S- 79, 5)- 

corpuscles outside of the spores During sporulation, the metachromatic 

have not yet been absorbed by . . j r , , 

it. 8-9, Spirillum volutans. corpuscles exist just outside of the spore 

lo-n, Bacillus alvei. (Fig. 79, 7), then are finally absorbed by it. 

They therefore act like reserve products. 

Moreover, in the cells of bacteria other reserve products, notably 
globules of fat and of glycogen, have been found. 

BACTERIAL CELL WALL. General Structure* All the bacteria have 
cell walls and it is these that give definite form to the cell. These walls 
are rigid and elastic and are probably made up of two layers, the outer one 
of which is able to deliquesce and form capsules, or perhaps zooglcea. 
The inner part retains the elasticity and gives the form to the bacteria. 
These cell walls are readily permeable to water and it is through 
them that all of the nourishment of the cell is obtained; that is, 
there are no openings for the entrance of food or the discharge of 

* Prepared by W. D. Frost. 


BACTERIA 


103 


by-products, but the intake and output goes on through the cell wall 
which is entire. 

Minute Structure of Cell Wall.* -In some species of large size, 
the membrane can be distinguished when strongly magnified, and 
appears with a double contour. Usually it is scarcely visible, and can 
be observed only when the contents of the cell has been contracted by 
plasmolysis or by a suitable reagent. It is sometimes thin, some- 
times more or less thick. In the latter case, it is often possible to 
recognize two layers, an inner or cuticular layer, very thin and trans- 
parent; and the other external, not so well defined and thicker, jelly- 
like in appearance. This latter or gelatinous layer seems to result 
from a special differentiation of the peripheral zones of the inner layer. 
The outer layer ordinarily resists staining reagents and appears as a 
kind of transparent zone about the colored elements. It can acquire 
a relatively great thickness, and the formations described as capsules 
are only an exaggeration of this gelatinous layer. 

Schaudinn has been able to observe quite care- 
fully the construction of the cuticular layer in 
B. butschlii. According to him, the membrane 
seen in profile would appear to consist of a 
series of disks alternately clear and cloudy (Fig. 
80, A and B). Seen from the front, it would 
give the impression of a network whose meshes 
are more refringent and stain more highly (C). 
It is laid on a peripheral zone of cytoplasm, a 
kind of ectoplasm with closer network, and is 
clearly differentiated from the rest of the cyto- structure of the mem- 
plasm. The spore is provided with a double brane and of the ecto- 
, j i p ., i r derm in Bacillus 

membrane and has at one of its poles a sort of bMsc hUL C, Membrane 

micropyle through which germination is effected of the same bacillus, 
/-r,. j ^\ front view. (After 

(Fig. 73, 15 and 1 6). Schaudinn.) 

The chemical composition of the membrane 

is little known. According to some authors, this membrane consists 
of cellulose; according to others, it contains a lipoid substance; 
finally, by many authors it is supposed to be composed principally 
of nitrogenous compounds. Let us remark further that chitin has 
supposedly been detected therein. 

* Prepared by A. Guilliermond. 


104 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

Capsules* A considerable number of the bacteria regularly, or 
under certain conditions, form what are known as capsules (Fig. 81). 
These are mucilaginous envelopes which in width frequently exceed 
that of the organism itself. In microscopical preparations of bacteria 
it is important to differentiate these from artifacts, since by ordinary 
staining methods the capsules are not colored but appear as colorless 
areas surrounding the bacteria. If, due to shrinkage of the bacteria, 
or other material on the preparation, clear spaces are formed, it is 
readily seen that these might be confused with the real capsule. It is 


:;^^^B|/ : V->; -"' .-'.:&' 


FIG. 81. Capsules. Bad. pneumonia (Friedlander). (After Weichselbaum from 

Frost and McCampbell.) 

possible to stain the capsules by special methods; these must be used in 
order to determine positively the existence of the capsules. The 
bacteria which grow in the bodies of animals frequently contain these 
capsules but fail to show them when grown upon artificial culture media. 
It is difficult, therefore, to determine whether or not an organism has a 
capsule by mere examination of cultures. Some culture media, how- 
ever, do cause a formation of capsules in the case of capsulated bacteria. 
These are blood serum, sometimes, and milk, usually. Beautiful cap- 
sules can be obtained by growing such bacteria as the Bact. pneumonia, 
Bact. capsulatum, and Bact. Welchii in milk cultures. Strept. mesen- 
teroides is a bacterium which grows in the syrup of the sugar refineries 
and forms abundant capsules. This organism changes the char- 

* Prepared by W. D. Frost. 


BACTERIA 


105 


acter of the syrup, and its entrance and growth is frequently the cause 
of serious loss. 

FLAGELLA. General Consideration of Flagella* The flagella are 
very narrow thread-like structures. It is not known how narrow since 


A. / 


FIG. 82. FIG. 83. FIG. 84. 

FIG. 82. Chromatium okenii; 2, Bacterium lineola; 3, 4 and 5, sulpho-bactena; 
7, Ophidomonasjenensis; 8, and 9, Spirillum undula; 10, Cladothrix dichotoma. (After 
Biitschlifrom Guilliermond review, Bull. Inst. Past.} 

FIG. 83. Micros pira comma. Monotrichous bacteria. (After Migula from 
Schmidt and Weiss.} 

FIG. 84. Pseiidomonas pyocyanea. Monotrichous bacteria. (After Migula from 
Schmidt and Weiss.} 

they cannot usually be seen without staining and they can only be 
stained by precipitating some chemical which may add considerably to 
their width. They are frequently longer than the organism which 


\ 


FIG. 85. FIG. 86. FIG. 87. 

FIG. 85. Pseiidomonas syncyanea. Lophotrichous bacteria. (After Migula from 
Schmidt and Weiss.} 

FIG. 86. Spirillum rubrum. Lophotrichous bacteria. (After Migula from 
Schmidt and Weiss.} 

FIG. 87. Bacillus typhos us. Peritrichous bacteria. (After Migula from Schmidt 
and Weiss, and Frost and McCampbell.} 

possesses them and sometimes many times that length. B. sympto- 
matici anthracis found in the soil has a flagellum sixty times its own 
length. The arrangement of the flagella on the bacteria is quite constant 

* Prepared by W. D. Frost. 


106 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

and is used by some authors to differentiate genera. Very few of the 
micrococci are provided with flagella, as was indicated above, and in 
the bacilli and spirilla they may be arranged at the poles singly or in 
brushes, or they may be arranged on the entire periphery of the cells. 
When bacteria are provided with a single flagellum at one pole, the 
arrangement is said to be monotrichous (Figs. 82, 83 and 84). When they 
are arranged in brushes, the arrangement is spoken of as lophotrichous 
(Figs. 85 and 86) and when they are arranged on the entire periphery, 
the arrangement is said to be peritrichous (Fig. 87). It frequently 
happens that in the case of the monotrichous and lophotrichous the 
flagella occur at both ends of the organism. This is explained by the 
fact that the organism is just undergoing binary fission and that the 
second group is on the newly forming cell. It is worth while in this 
connection to call attention to the fact that the flagella on one end are 
new, while those on the other end may be thousands of generations old. 

Minute Consideration of Flagella.* The question of the cilia or 
flagella of bacteria is not yet entirely decided. The absence of cilia 
in large bacteria capable of motion gives the idea that these are not the 
only organs of motion, and that contraction of the protoplasm certainly 
plays the most important role in the phenomena of motility. More- 
over, the nature of cilia has been debated. Van Tieghem and Biitschli, 
taking their stand primarily on the difficulty of staining cilia by the 
reagents which rapidly color protoplasm, have considered these cilia 
to be simply prolongations of the membrane, lacking all contractibility 
and locomotive power. According to Van Tieghem, when two cells 
formed by the division of the same element separate, the common por- 
tion of the transverse septum, instead of dividing neatly in two, can 
stretch out into a filament which breaks at a greater or less distance from 
each of the two daughter cells. This prolongation composes the 
vibratile cilium. 

This theory, however, does not explain the existence in certain 
bacteria of clusters of cilia at the two poles, or of cilia distributed over 
the whole surface of the membrane. Other authors, as for example 
A. Fischer, consider the cilia true prolongations of the protoplasm 
issuing through tiny apertures in the membrane. This view at present 
tends more and more to predominate, and the existence of flagella on 
bacteria appears to be demonstrated. 

Prepared by A. Guilliermond. 


BACTERIA I0y 

Another interesting peculiarity, moreover, has recently been estab- 
lished independently by Swellengrebel and by Dangeard. According 
to these authorities, in some species (Chromatium okenii and Spirillum 
wlutans) the cilia have connection with one of the chromatic grains of 
the diffuse nucleus. There is a chromatic filament starting from the 
base of the cilium and ending in connection with a chromatic grain, 
similar to the organisms with flagella in which the flagellum is in 
relation to a basal chromatic grain (blepharoplast) . 

THE HIGHER BACTERIA* 

The so-called higher bacteria include some of the spiral forms, at 
least the larger spirochaetes, the thread or trichobacteria, and the 
sulphur or thiobacteria. 

The spirochaetes and trichobacteria contain so many forms of 
interest that their form and structure needs special consideration. 

THE LARGER SPIROCHAETES. Spirochaetes differ so much among 
themselves that it seems necessary to divide them into two groups. 
The members of one of these groups, the small spirochaetes, are prac- 
tically identical with the true bacteria, and naturally fall in the family of 
the Spirilliacea. Members of this group, however, so gradually approach 
the other group, the large spirochaetes, that it is difficult to draw a line 
of separation between the two, yet the large spirochaetes resemble in 
so many essential details the trypanosomes that they are usually placed 
as a coordinate genus with them under the flagellates a sub-class of 
the Protozoa. The larger spirochaetes are described as follows:. 

Form and Size. In form the spirochaetes are long, very thin and 
flexible spirals. Their length is usually not less than twenty times their 
breadth. Some forms are as long as 500 /z. It seems probable that 
some of them are flattened and hence in form are more like a spirally 
bent ribbon than rod. 

Motility. These organisms move very rapidly under normal con- 
ditions. The character of the movement may be of three kinds: 
(i) Lashing, eel or snake like; (2) undulatory, compared to the flapping 
of a sail in the wind; (3) rotation, similar to a cork-screw when pushed 
into a cork. 

Reproduction. Multiplication is by means of binary fission. If 
these forms are to be considered as bacteria, the division would be 
expected to be by means of transverse partition walls. A number of 

* Prepared by W. D. Frost. 


108 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

workers, however, have described a process of longitudinal division. 
Forked forms also which are frequently seen are held to indicate longi- 
tudinal divisions. Some observers have claimed that conjugation 
occurs among the spirochaetes. If this is true their relation to the 
Protozoa would be quite likely, but accounts of this phenomenon are 
inconclusive. Several observers have described " rolled up " specimens, 
oval and ovoid forms, which have been assumed to be cysts. The 
spirochaetes break up into granules or short segments and such speci- 
mens are sometimes spoken of as "monili form." It is not definitely 
known whether these coccoid forms are simply degenerative forms or 
the equivalent of bacterial spores. 

Sheaths. A definite sheath has been described for some forms 
and the irregularity in the disposition of this around the cell may 
account for the structures that have been taken for undulating 
membranes. 

Cell Aggregates. There is apparently no definite cell grouping but 
tangled masses of these organisms have been described in several 
species. 

THE TRiCHOBACTERiA.--The trichobacteria (Chlamydobacteriacece) 
are thread or filamentous forms. The cells are cylindrical and similar 
in form and may or may not vary in size in different parts of the fila- 
ment. The individual cells are capable of independent existence, but 
when growing in the filament give evidence of differentiation in func- 
tion. Sometimes these filaments are attached to the substratum or 
some object in it; at other tunes they are free. In case of the sessile 
forms the cells at the attached end (base) are smaller than those at the 
apex. In other members of the group the ends of the thread are swollen 
or become club-shaped (Fig. 88). In some forms cell division takes 
place. in three directions of space, thus forming a thread of massed cells. 
Branching. The filaments are usually unbranched, but some 
forms show true branching, such as is found among the plants fungi 
and algae. Some again exhibit what is called false branching. This 
is due to a misplaced cell, which grows parallel or at an angle to the 
parent thread and suggests branching. 

Reproduction. The cells throughout the filament may divide to 
form spores, but the apical cells of the thread are frequently set apart 
for the purpose of reproduction, and by a process of division form 
spores or conidia. The conidia are usually round and without any 


BACTERIA 


ICQ 


resting stage may produce new threads of cells. Sometimes spores 
germinate while still in the old thread (Fig. 88), giving a tangled 
mass of cells or whorls of new threads at intervals on the old. The 
conidia may be either motile or non-motile. The motility of these 
conidia when it exists is due to flagella. 

Sheath. The threads of cells are sometimes surrounded by sheaths 
of varying thickness. This sheath is a thickened and hardened mem- 


FIG. 88. Crenolhrix polyspora Cohn, Brunnenfaden. 

and Weiss.) 


(After Migula from Schmidt 


brane, and forms a tube in which the different cells of the bacteria are 
contained. This sheath is homologous to a capsule. In -it are fre- 
quently deposited characteristic by-products of the cell. In Creno- 
thrix (an iron bacterium), for example, we have iron oxides. 

Among the iron bacteria are several interesting forms. Crenotkrix 
polyspora is one of the best known. Its general morphology is shown 
in Fig. 88. The attached, sessile, threads are shown at a. The 
tufts of short threads, radiating from the larger threads, are 


no 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


formed by the germination of conidia while they are still in the parent 
threads. The large threads, b, c, d, and e, show more details. In e a 
uniform thread is shown with the separate vegetative cells; in d these 
have broken up into conidia. The flaring form of the threads are shown 
in c and b where the conidia are formed in large numbers. These 
figures also show the sheath which is indicated by the double line in 6 
and by the extension of the lines beyond the cell contents. 

Chlamydothrix ochracea Migula is composed of filamentous, cylindri- 
cal, colorless threads. The sheath is at first thin and colorless but later 
becomes thicker, yellow or brown due to encrustations of iron oxide. 
Multiplication is by means of cell division and swarm cells. These 
latter may sometimes germinate in the sheath, giving the 1 appearance of 
branching (Fig. 89, c). 


P'iG. 89. A, Spirophyllum ferrugineum; B, Gallionella ferruginea; C, Leptothrix 

ochracea. X about 1080. (After Harder.} 

Gallionella ferruginea Ehr., in its typical form, consists of spiral 
threads coiled together in double or quadruple coils like a rope. The 
threads are cylindrical but comparatively thin. Individual cells have 
not been distinguished in the threads (Fig. 89, B). 

Spirophyllum ferrugineum Ellis is very similar to and associated 
with the above. It differs principally in the shape of the threads 
which are flat or ribbon-like. The threads are always twisted but may 
occur singly or be coiled into ropes (Fig. 89, A). 


BACTERIA III 

All of these iron bacteria have the power of changing certain 
soluble salts of iron into insoluble forms and thus precipitate them from 
solution. Growing in the pipes of a city water supply their deposits 
choke up the pipes and hence they are frequently referred to as "water 
pests." As a result of researches in recent years these iron bacteria are 
now regarded as important geological agents and to them is ascribed a 
large share in the deposition of iron ores. 

Other thread bacteria of considerable importance are the acti- 
nomycetacece. Some of them are common in the soil and recently 
have been given special study. Others cause disease and a well known 
form, Actinomyces boms Hartz, is the cause of lumpy jaw in cattle. 

The actinomycetes are mold-like organisms and often show true 
branching. They reproduce vegetatively or by means of conidia. 
They are without sulphur granules, not colored with bacteriopurpurin 
and the sheaths, if present, are not impregnated with iron. The struc- 
ture of Actinomyces boms is shown in Fig. 165, p. 780, while the charac- 
teristic radiating clubbed ends of the filaments, as these organisms grow 
in the tissues of cattle, are shown in Fig. 164, p. 779. 

THE SULPHUR BACTERIA. The sulphur bacteria are filamentous 
forms which may reach a length of many microns. They are cylin- 
drical or perhaps sometimes flat. They may be either attached or 
actively motile. The movement when present is due not to flagella, 
but to an undulatory motion like that of the spirochaetes or Oscillaria 
among the algae. As they move forward they rotate on their own axis 
and swing their free ends. 

Spore formation is unknown in some forms where multiplication is 
accomplished by the breaking up of the threads in short segments. 
In the case of the sessile forms conidia are produced at the end of the 
thread and are motile (Thiothrix nivea). The sulphur bacteria contain 
at certain stages strongly refractile sulphur granules in their bodies. 

CLASSIFICATION* 

The classification of bacteria was early recognized by Mueller as a 
matter of difficulty, since he says: "The difficulties that beset the in- 
vestigation of these microscopic animals are complex; the sure and 
definite determination (of species) requires so much time, so much of 
acumen of eye and judgment, so much of perseverance and patience, 
that there is hardly anything else so difficult." 

Prepared by W. D. Frost. 


112 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

A considerable number of systems for the classification of the bac- 
teria have been proposed. One of the most widely used at the present 
time is that devised by Migula. His system is based on the principle, 
universally followed by botanists and zoologists, of using morphological 
characters only to distinguish genera. There has been, however, a 
growing conviction among bacteriologists that it is necessary to take 
physiological characters into consideration in determining even the 
major groups of bacteria in any system of classification. This revolu- 
tionary doctrine was presented in an extreme form by Orla Jensen who 
used the metabolic processes of the bacteria as the chief criteria for 
establishing not only genera but families and orders ' as well. A 
Committee of the Society of American Bacteriologists have recently 
reported on the Families and Genera of Bacteria*. This system makes 
use of both morphological and physiological characters and promises to 
be an important step towards a natural system of classification. Mi- 
gula's system and that of the Committee of the Society of American 
Bacteriologists, in skeleton form, follow: 

MIGULA'S CLASSIFICATION 

ORDERS OF THE SCHIZOMYCETES 
Cells contain sulphur. Colorless or pigmented rose, 

violet or red by bacteriopurpurin never green.. THIOBACTERIA 
Cells free from sulphur and bacteriopurpurin, 

colorless or faintly colored EUBACTERIA 

FAMILIES OF EUBACTERIA 
Cells globose in a free state, not elongating in any 

direction before division into i, 2 or 3 planes.. . . COCCACE^E 
Cells cylindrical, longer or shorter, and only divid- 
ing in one plane, and elongating to twice the 
normal length before division 

1. Cells straight, rod-shaped, without sheath, 

non-motile or motile by means of flagella . . . B ACTERIACE/E 

2. Cells crooked, without sheath SPIRILLACE.E 

3. Cells inclosed in a sheath CHLAMYDOBACTERIACE/E 

GENERA OF THE COCCACE^: 
Cells without organs of locomotion 

1. Division in one plane Streptococcus 

2. Division in two planes Micrococcus 

3. Division in three planes Sarcina 

Cells with organs of locomotion 

1. Division in two planes Planococcus 

2. Division in three planes Planosarcina 

*Jour. Bact. II, p. 505, 1917. 


BACTERIA 113 

GENERA OF THE BACTERIACEJE 

Cells without organs of locomotion Bacterium 

Cells with organs of locoomtion 

1. Flagella distributed over the whole body. . . .Bacillus 

2. Flagella polar Pseudomonas 

GENERA OF THE SPIRILLACE.E 

Cells rigid not snakelike or flexuous 

1. Cells without organs of locomotion Spirosoma 

2. Cells with organs of locomotion 

(a) With one, very rarely two or three polar 

flagella Microspira 

(b) Cells with polar flagella in tufts of five 

to twenty Spirillum 

Cells flexuous Spirochaeta 

GENERA OF THE CHLAMYDOBACTERIACE^E 

Cell contents without granules of sulphur 

1. Cell threads unbranched 

(a] Cell division always only in one plane. . Chlamydothrix 
(&) Cell division in three planes previous to 

conidia formation 

i. Cells surrounded by a very 
delicate, scarcely visible, sheath 

(marine) Phragmidiothrix 

ii. Sheath clearly visible (in fresh 

water) Crenothrix 

2. Cell threads branched (pseudobranches) Sphaerothrix 

FAMILIES OF THE THIOBACTERTA 

Filamentous bacteria which do not contain bac- 

teriopurpurin. Cells contain sulphur granules . .BEGGIATOACE^E 

Cells contain bacteriopurpurin, sulphur granules 

may also be included RHODOBACTERIACEvE 

GENERA OF THE BEGGIATOACE.E 

Cells non-motile, threads attached to some object. .Thiothrix 
Moves by means of an undulating membrane Beggiatoa 

GENERA OF THE RHODOBACTERIACE.E 

This family includes twelve genera as follows: Thiocystis, Thiocapsa, Thiosarcina, 
Lamprocystis, Thiopedia, Amcebobacter, Thiothece, Thiodictyon, Thiopoly- 
coccus, Chromatium, Rhodochromatium and Thiospirillum. 

8 


114 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

THE FAMILIES AND GENERA OF THE BACTERIA 

Report of the Committee of the Society of American Bacteriologists. C.-E. A. 

Winslow ct al. (Artificial key) 

ORDERS OF THE SCHIZOMYCETES 

Cells united during the vegetative stage into a 

pseudoplasmodium MYXOBACTERIALES 

Cells not forming a pseudoplasmodium 

Cells free or united in elongated filaments, often 
with a well denned sheath. Conidia fre- 
quently formed. Free sulphur, iron or 
bacteriopurpurin often present. 
Cells typically containing granules of sulphur or 

bacteriopurpurin or both THIOBACTERIALES 

Suilphur and bacteriopurpurin absent; iron often 

present CHLAMYDOBACTERIALES 

Cells ne\~er in sheathed filaments. Conidia only 
in mycelial Mycobacteriaceae. Flagella often 
present. Free iron, sulphur, or bactiopurpurin 
never present .EUBACTERIALES 

FAMILIES OF THE EUBACTERIALES 

Cells spiral with polar flagella IV. SPIRILLACE^E 

Not as above 

Cells spherical; rarely, if ever, motile; spores 
never produced; never securing growth energy 

from nitrogen or ammonia V. COCCACEJi 

Not as above 

Cells short rod-shaped with a single, rarely two, 
polar flagellum; usually forming green or 

yellow pigment III. PSEUDOMONADACE^ 

Not wholly as above 

Spores formed VIII. BACILLACE^ 

Spores never formed 

Metabolism simple, securing growth energy 
from carbon, hydrogen, or their simple 

compounds; flagella, if present, polar I. NITROBACTERIACE^ 

Metabolism complex, dependent upon more 
complex carbohydrate and protein sub- 
stances; flagella, if present, peritrichic. 
Cells clubbed, fusiform, filamentous, 
branching or mycelial; those not distinctly 
so are either acid-fast or show barred 

irregular staining IT. MYCOBACTERIACE^ 

Not as above 

Gram positive; non-motile VI. LACTOBACILLACE^ 

Gram negative; often motile VI. BACTERIACE,E 


BACTERIA 115 

GENERA OF THE EUBACTERIALES 

I. NITROBACTERIACE.E 

Fixing nitrogen or oxidizing its compounds 
Fixing free nitrogen 

Cells large; in soil 7. Azotobacter 

Rods minute; in roots of leguminous 

plants 8. Rhizobium 

Oxidizing nitrogen compounds 

Oxidizing ammonia 5. Nitrosomonas 

Oxidizing nitrites 6. Nitrobacter 

Not as above 

Oxidizing hydrogen i. Hydrogenomonas 

Oxidizing carbon compounds 

Oxidizing alcohol; branching forms 

common 4. Mycoderma 

Not as above, using simpler carbon 
compounds 

Oxidizing CO 3. Carboxydomonas 

Oxidizing CH 4 2. Methanomonas 

II. MYCOBACTERIACE^; 

Slender rods staining with difficulty and 

acid fast 3. Mycobacterium 

Not as above 

Mycelium and conidia formed 

With aerial hyphae and conidia; usually 

saprophytic soil organisms 2. Nocardia 

Hyphae and conidia not aerial; usually 

parasitic in animals i. Actinomyces 

Not as above; cells rod-like, usually somewhat 
curved, clubbed, fusiform, or even 
branched, but never mycelial 
Thick, long threads, fragmenting into 

short thick rods 6. Leptotrichia 

Not as above 

Cells usually elongate and fusiform, 
filaments, if formed not branch- 
ing; stains somewhat irregularly. .5. Fusiformis 
Cells slightly curved, clubbed, or in 
old cultures even branching; not 
filamentous; showing definite bar- 
red staining 4. Corynebacterium 

III. PSEUDOMONADACE^E 

Generic characters mainly those of family. . i. Pseudomonas 


Il6 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

IV. SPIRILLACE^: 

Flagellum single (rarely 2 or 3) i. Vibrio 

Flagella tufted (5 to 20) 2. Spirillum 

V. COCCACE.E 

Abundant red-pigmented growth on agar. . 7. Rhodococcus 
Not as above 
Gram negative 

Normally in pairs of flattened cells; 
growth on plain agar scanty, never 

bright yellow i. Neisseria 

Normally in plates, packets, or irregu- 
lar masses; growth on plain agar 
abundant, pigment definitely 
yellow 

Cells in regular packets 6. Sarcina 

Cells not in regular packets 5. Micrococcus 

Gram positive (exceptions rare and not 

easily confused with above genera) 
Cells normally in chains, sometimes in 
pairs (especially in acid environment) 
never in large irregular masses. 
Gelatin rarely liquefied. Growth on 
plain agar usually translucent, never 

heavy, never yellow or orange 2. Streptococcus 

Cells normally in groups and masses; 
(occasionally in plates in Albo- 
coccus) chains short and irregular, 
if present. Gelatin often lique- 
fied. Agar growth abundant, 

white to orange 

Pigment orange (rarely lacking); 

gelatin often liquefied actively.. . .3. Staphylococcus 
Whitish to porcelain white; liquefac- 
tion less vigorous 4. Albococcus 

VI. BACTERIACE^E 

Plant pathogens 2. Erwinia 

Not as above; saprophytes or in animal 

habitats (intestines, tissues, etc.) 
Usually motile and exhibiting active 
fermentative powers; typically para- 
sitic in intestines of man and higher 
animals; growing well on ordinary 
media. . i. Bacterium 


BACTERIA Iiy 


Not wholly as above 

Growing only in presence of hemo- 
globin, ascitic fluid or serum 4. Hemophilus 

Growth on media scanty, but less 
sensitive than the above; short rods 
with tendency to bipolar stain 3. Pasteurella 

VII. LACTOBACILLACE^: 

Generic characters mainly those of family. . i. Lactobacillus 

VIII. BACILLACE^: 

Aerobic, usually saprophytic; cells not 

greatly enlarged (if at all) at sporulation. i. Bacillus 

Anaerobic, often saprophytic; cells fre- 
quently enlarged at sporulation 2. Clostridium 

NOMENCLATURE 

It is most important that each kind of bacterium should have a 
definite name. The name should be a binomial and not a trinomial. 
It is also very desirable that all bacteriologists should adhere to the rules 
that govern botanists in these matters. Probably the most important 
points to remember are: To use Latin names for all groups; to recognize 
only one valid designation for each organism or group and that the 
oldest (with certain limitations); to designate orders with the ending 
ales, families with the ending aceae, sub-families with oideae, tribes with 
eae, and sub-tribes with inae\ to use generic names as substantives and 
write them with a capital letter; to designate all species by the name of 
the genus and a specific name or epithet, usually of the nature of an 
adjective (the two names forming a binomial or binary name). 

RELATIONSHIP or BACTERIA* 

There has been a great deal of discussion as to whether bacteria 
are plants or animals. They were first described as animalcula and 
to the popular mind they are usually animals or "bugs." It is diffi- 
cult to determine their exact relation philogenetically. These diffi- 
culties are so great that some scientists, as Haeckel, would create a 
new kingdom, call it Protista, and put in it some of the lower plants 
and animals which are difficult to classify, together with the bacteria. 
The bacteria are undoubtedly more closely related to the blue-green algae 
than to any other forms of life. They resemble these organisms in form, 
method of reproduction, and absence of definite nucleus. It is quite 

* Prepared by W. D. Frost. 


Il8 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

impossible to decide, furthermore, whether some forms, such as Bact. viride 
and Bad. chlorinum, are blue-green algae or bacteria. On the other 
hand, there are some points of resemblance between the bacteria and 
the protozoa. Spore formation, similar to that among the bacteria, 
occurs among some of the protozoa. Another point of resemblance is 
the possession of flagella. Some of the flagellates quite closely resemble 
the bacteria in many ways, and the Spiroch<zt(B y which are usually 
believed to be bacteria, have been classed as flagellates by eminent 
protozoologists. 

Physiologically the bacteria are quite closely related to the fungi, 
and are frequently classed with them under the term Schizomycetes. 

ARTIFICIAL CULTIVATION OF BACTERIA* 

The introduction of methods of artificial cultivation marks the beginning of the 
science of microbiology. These methods were developed by Pasteur and Koch and 
are depended upon by the microbiologist of to-day as the foundation for most of his 
work. It has been the aim of investigation to discover a more general culture 
medium. So far it has been impossible to do this, but beef broth, made after a 
formula suggested by LoefSer many years ago, forms the basis of nearly all of our 
culture media. This beef broth, or nutrient bouillon, is made by extracting meat 
free from fat in water, adding a small per cent of peptone, correcting the chemical 
reaction, clarifying and sterilizing. To this broth various substances are added 
for special purposes; gelatin and agar, in order to solidify the media, and various 
sugars and other chemical substances for the purpose of determining the physiological 
characteristics of various bacteria. One of the difficulties with the present methods 
of the artificial cultivation of bacteria is the inconstancy of the composition of the 
media, due to the fact that the extract of beef, the peptone, and other ingredients, 
cannot be obtained chemically pure. If it should prove possible to use synthetic 
substances, such as the polypeptids, it would mark a great step in advance, but it is 
probably quite impossible to devise a single medium upon which all bacteria will 
grow. Some bacteria, such as those which produce nitrification, refuse to grow on 
ordinary media containing organic material. The cultivation of bacteria in pure 
culture is dependent upon isolation, and the method of isolation suggested by Robert 
Koch in 1880, and known as the plate culture method, has given eminent satis- 
faction. This method is dependent upon the use of -liquefiable solid media, such 
as gelatin or agar. 

'Prepared by W. D. Frost. 


CHAPTER V 

FILTRABLE MICROORGANISMS* 

The terms "filtrable microorganisms' 3 and 'filtrable viruses' 1 
are used to designate a group of disease-producing microorganisms that 
are characterized by their ability to pass through ordinary "bacteria- 
proof" filters. In the past it has been customary to speak of these filter 
passers as invisible or ultramicroscopic because of the fact that, besides 
being filtrable, they were, with the single exception of the virus of 
bovine pleuro-pneumonia, invisible under the microscope and incapable 
of multiplying in vitro on any of the usual culture media. Recent 
discoveries indicate that the terms " in visible" and "ultramicroscopic' 3 
are incorrect, at least with respect to some members of the group. 
So long as clear, filtered fluids that gave no visible evidence of life were 
capable of setting up infectious disease in men or in animals there was 
some reason for the use of those terms and even for Beijerinck's fanciful 
conception of a "living fluid contagion." However, the brilliant 
researches of Noguchi have offered a technique by means of which some 
of these hitherto invisible viruses have been cultivated outside of the 
animal body and made visible under the microscope. While some 
members of this group may indeed be of ultramicroscopic size, there is 
reason to believe that many of them will eventually be rendered visible 
through improvements in bacteriological technique. 

The characteristics of the filtrable viruses may be best understood 
by consideration of a typical example, the virus of foot-and-mouth 
disease. In this disease vesicles form in the mouths and on the feet 
of infected cattle. The virus is known to be present in the lymph 
which forms in these vesicles because this lymph will produce typical 
attacks of foot-and-mouth disease when inoculated into susceptible 
animals. If now this infectious lymph be diluted with water and passed 
through a Berkefeld filter the resulting filtrate will be found to be free 
from all visible microorganisms and in addition the usual culture tests 
will give negative results. Notwithstanding this apparent sterility, 

* Prepared by M. Dorset. 

119 


I2O 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


a 


FIG. 90. Apparatus for fractional filtration, designed for use with Pasteur- 
Chamberland or Berkefeld filters, a, Glass mantle surrounding filter; b, Chamber- 
land filter; c, paraffin joint; d and e, rubber stoppers;/, double side-arm suction flask; 
g, pinchcock controlling outlet from suction flask; h, outlet tube surrounded by glass 
shield and attached to lower end of suction flask by means of short rubber tubing; 
i, glass shield fused to and surrounding outlet tube as a protection against contamina- 
tion when the filtrates are drawn off; j, glass inlet tube plugged with cotton, for ad- 
mitting air into suction flask; k, pinchcock governing the admission of air into flask; 
I, vacuum gauge; m, stopcock connected with vacuum pump. (U. S. Dept. of Agri- 
culture^ Bureau of Animal Industry, Bull. 113.) 


FILTRABLE MICROORGANISMS 121 

however, the filtrate will produce disease in cattle in the same manner 
as the imfiltered lymph. It is known that the symptoms produced 
by the filtrate are caused by a living organism and not by a toxin, 
because by successive nitrations and inoculations the disease can be 
transmitted through a long series of animals, thus indicating clearly 
that there exists in the filtered lymph a living organism which is capable 
of reproduction. Another proof that the virulence of the filtered lymph 
is caused by the presence of living corpuscular elements, and that it is 
not a mere solution of a toxin, is found in the failure of the virus to 
pass through filters of finer grain than the Berkefeld as, for example, 
the Kitasato filter. The microorganism of foot-and-mouth disease has 
not been cultivated nor made visible. Among other diseases produced 
by filtrable viruses which as yet remain invisible, are hog cholera, 
rinderpest, swamp fever, fowl plague and South African horse sickness. 

The invisibility of this group of microorganisms may depend upon 
either their minute size or their peculiar structure. The most powerful 
microscopes will not enable us to discern with distinctness objects which 
are less than o.i/j in diameter. We know of bacteria which in size 
approach this limit quite closely (M. progrediens, 0.15^ in diameter) 
and there is no reason for believing that the size of organisms is limited 
by our ability to see them. As already stated, invisibility may also 
result from a peculiarity of structure, such as complete transparency 
and failure to stain with the reagents ordinarily used for this purpose. 

The ability of microorganisms to pass through filters is dependent 
upon a variety of factors. The size and plasticity of the organism, 
the fineness of the pores, and the thickness of the walls of the filter as 
well as the conditions under which the filtration is performed, will all 
influence the result. 

The failure of the filtrable microorganisms to develop under artificial 
conditions is to be attributed to their strict parasitism and to our in- 
ability to imitate exactly in the laboratory the conditions which exist 
in the animal body. The method of Noguchi, referred to above, and 
which has done so much to advance our knowledge of the filtrable 
viruses, was first used to cultivate Treponema pallidum. The culture 
medium is placed in long narrow test tubes and consists of a piece of 
fresh sterile rabbit's kidney placed in the bottom of the tube over which 
is poured sterile unheated and unfiltered ascitic fluid or a mixture of 
ascitic fluid and agar. The surface of this medium is covered with a 


122 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

layer of sterile paraffin oil to exclude oxygen. The material from which 
cultures are to be made is introduced into the bottom of the tube by 
means of capillary pipettes. * 

While the filtrable microorganisms possess certain qualities in com- 
mon, in some respects they differ widely from one another. Some will 
pass only through the coarsest of bacteria-proof niters, while others pass 
readily through the densest niters, thus indicating wide differences in 
size or in structure. Some are very susceptible to the action of germici- 
dal agents, whereas others are more resistant than the ordinary bacteria. 
Some produce disease in only one species of animal, while others show 
little or no limitation in this respect. The diseases produced by these 
microorganisms likewise differ markedly, some being comparatively 
benign and local in character, whereas others appear as the most pro- 
found septicaemias. Some are extremely contagious, while others can 
be transferred from one animal to another only by means of an inter- 
mediate host. In fact these invisible microorganisms seern to differ 
among themselves quite as widely as do those which are visible to us. 
The existence of a filtrable microorganism is determined as follows: 
The infectious agent must pass through a bacteria-proof filter, which 
is free from imperfections as shown by tests with visible organisms of 
small size. Pressure exceeding one atmosphere should not be employed 
during filtration. The time of filtration should not exceed one hour. 
The filtrate should remain free from all visible bacteria as shown by 
microscopic examination and cultural tests. The filtrate should 
possess the specific disease-producing qualities of the unfiltered material. 
Animals infected with the filtrate should yield material which, after 
filtration, will in its turn possess the attributes of the original unfiltered 
material. Recent suggestive developments have thrown some light on 
the possible nature of filtrable viruses. The reader is referred to the 
work of Flexner and Noguchi since 1912, published in the Journal 
of Experimental Medicine; he is also requested to read the article by 
Lohnis and Smith already mentioned on page 99. 

* For details of this method see J. Exp. Med., 1911, et seq. 


CHAPTER VI 

PROTOZOA* 

INTRODUCTION 

Many of the diseases which are known to be due to an infecting 
agent are caused by bacteria; but others are caused by protozoa. 

The bacteria belong to the vegetable kingdom. The protozoa are 
unicellular animals; they are extremely numerous and are very widely 
distributed in nature. They occur in water, soil and in the bodies of 
most animals. 

From a zoological point of view, the protozoa constitute an impor- 
tant sub-kingdom. It is sometimes difficult to say whether a minute 
organism is a plant or an animal. For this reason, primitive unicellular 
organisms are sometimes classified by themselves, as Protista (pages n, 
117), a kingdom which thus includes not only primitive organisms which 
have not yet been definitely established in either group but also certain 
unicellular animals and plants. It appears preferable, however, to 
determine as far as possible the genetic relationship of various or- 
ganisms and, by the study of their physiology and modes of develop- 
ment, to differentiate between those which are plant-like and those 
which are animal-like in character. The protozoa are thus included 
in the animal kingdom and have been defined as "unicellular animals." 
They are to be distinguished, on the one hand from primitive forms such 
as bacteria which, lacking differentiation of nucleus and cytoplasm, 
do not conform to the type of structure of true cells, and on the other 
hand, from primitive unicellular organisms of plant-like character such 
as^algae and fungi. | 

Many protozoa live in fresh water. Others live in the sea; chalk is 
formed from the skeletons of myriads of protozoa which once lived in 
the ocean. While a large proportion of the protozoa are free-living, 
others are parasitic on animals and plants. Some of the parasitic 
protozoa are practically harmless and do no apparent injury to the 

Prepared by J. L. Todd. 
t See page 13, 

123 


124 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


hosts which support them; others produce severe diseases. Before 
mentioning those especially which cause disease (see page 876) it will 
be well to consider the protozoa as a class and to discuss the characters 
which all have in common. 

STRUCTURE OF THE PROTOZOA 

Most protozoa are so small as to be visible only by the aid of the 
microscope but certain species are visible to the naked eye as individuals, 


IIP*/ 

& "'5Br : . 

;' I 


&; '*-'; vr "WVX- v 

*T*? .",* ""* ^O^^V 

.:- 1 pi - 

" ' ' ~ > ~-'}-tfJ?-' ^" ' 

~"^ - ' 

- . ' v-KJ^Cv 

. ' ' S^;S 

.-,-, a&Sr,- ^ 


FIG. 91. Amoeba vespertilio. (After Doflein.) 

or as agglomerated masses of individuals. For example, the Sarco- 
sporidia, which occur in the muscles of mice and other animals, can 
easily be seen without a microscope, and the huge plasmodial masses 
of Mycetozoa, which are sometimes seen on rotting wood or in tan 
pits, may measure many centimeters in breadth. 

Like all living things, the protozoa are composed of protoplasm (page 
1 8) and its products. Protoplasm is a complex mixture of various sub- 
stances in a colloidal condition. When studied by appropriate methods, 


PROTOZOA 125 

the protoplasm of a cell appears to be alveolar or foam-like in structure. 
This is because the protoplasm is emulsoidal in character being com- 
posed of a mixture of many more or less non-miscible substances, 
some of which are fluid in character, others more of the nature of 
solids. In such a mixture, the more viscid materials form tiny 
globules, and each of these is surrounded by a layer of softer material 
(Fig. 91). Consequently, cytoplasm is alveolar in structure; it has an 
appearance similar to that produced by the myriads of bubbles in a 
mass of foam. The walls of the outer layer of alveoli, or of alveoli 
which surround a resistant structure within the cell, are perpendicular 
to the surface against which they lie but the outline of the alveoli, 
which are not in contact with a firm structure, is more nearly circular. 
An exactly similar arrangement of the alveoli may be seen in a mass of 
soapsuds contained in a bottle; wherever the bubbles touch an un- 
yielding surface, their outline becomes rectangular. 

Recent studies in colloidal chemistry and in the microscopic dissection 
of cells have furnished valuable contributions to the knowledge of the 
chemical and physical properties of protoplasm. The view has been 
advanced that protoplasm consists largely of material in a state known 
in colloidal chemistry as a gel, some portions being firm and viscid 
and others very soft in character. Procedures which convert such 
material into a sol or fluid state are said to cause the protoplasm to 
quickly disintegrate. Certain portions of the cell such as the limiting 
membrane, the nuclear membrane and the nucleolus are of firmer 
consistence than other portions, and some cells contain globules and 
granules of various types. 

The protoplasm of a protozoon may be divided into two main 
portions: the cytoplasm and the nucleus (Chapter I). The cytoplasm, 
as a whole, may be divided, more or less easily, into a clearer, denser, 
more resistant outer layer the ectoplasm; and a more fluid, granular, 
internal portion the endoplasm. Denser, more resistant fibers some- 
times run through the cytoplasm and, like a skeleton, serve to fix the 
shape of the organism in which they exist. 

The nucleus, in its simplest form, is a structure which is differ- 
entiated from the remainder of the cell by being more refractile and 
by being colored more deeply in specimens which have been stained 
by dyes. It stains deeply because it contains a substance called chro- 
matin. The chromatin usually occurs in granules which may vary 


126 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

considerably in size and which are supported upon a linin framework 
that does not stain by ordinary methods. The interstices of the 
nucleus are filled with nuclear sap. A limiting nuclear membrane 
may be present, but it is not an essential part of the nucleus. The 
nuclear material may be all gathered together in a single mass, or it 
may be distributed in small granules termed chromidia so that, at the 
first glance, no nucleus seems to be present. Such chromidia may be 
said to constitute a distributed nucleus, although the term nucleus is 
usually applied to a well differentiated cell structure. 

The nucleus (page 15) is to be regarded as the most important unit 
in the structure of the cell and is apparently essential for the con- 
tinued existence of the latter. If cells are divided portions contain- 
ing no nucleus invariably die while portions containing the nucleus 
may continue to live and eventually recover from the injury. The 
role of the nucleus is not fully understood but it seems certain that it 
is a controlling center for the cell's activities. It is concerned in the 
nutrition of the cell, frequently nuclear structures have to do with the 
motility of cells and the chromatin serves as a medium for the 
hereditary transmission of specific characteristics. Its functions, 
therefore, are at least three-fold since it is active in trophic, kinetic 
and reproductive capacities. Usually, all these functions are subserved 
by a single nucleus; sometimes, however, as in the flagellates and 
many ciliates they are divided between two nuclei (page 18). 

ACTIVITIES or THE PROTOZOA 

The higher animals or Metazoa are composed of a great number 
of cells. A protozoon consists of a single cell. In the former the 
various functions of the body are each carried out by a special type 
of cell; for example, movement is performed by the muscle cells, 
digestion is provided for by the cells of the alimentary tract, and urine 
is excreted by the kidney cells. A protozoon being a unicellular 
animal, these various functions must be performed within the single 
cell of which it consists. Consequently certain parts of its protoplasm 
are especially differentiated and function in a manner similar 
to the organs of multicellular animals. Such differentiated parts are 
termed organellce and by means of these the protozoa move about, 
feed, and excrete waste products in many respects like the higher 
animals. 


PROTOZOA 


127 


n.- 


The activities of a protozoon may be considered under LOCOMOTION, 

METABOLISM* and REPRODUCTION. 

LOCOMOTION. The protozoa have several different modes of mov- 
ing themselves about. Some of them move by the formation of 
temporary processes or pseudopodia; in 
this method of progression, the protoplasm 
flows out, in finger-like processes, from the 
body of the organism and, as the protoplasm 
flows into these processes, the whole organ- 
ism progresses, literally, by flowing along. 
Some of the gregarines move about by 
means of a flowing of the protoplasm which 
always takes place in one direction; it is 
probable that the control of the direction 
of the flow in these parasites is effected by 
the contraction of myonemes. These are 
contractile fibers, which usually lie near the 
surface of the organism possessing them. 
Through their contraction, the form of the CVr- 
body of the parasite may be altered and, in 
this way, motion may be produced. Cilia 
are small hair-like processes, which may 
occur either in definite areas or in large 
numbers over the whole surface of a proto- 
zoon. They produce motion by waving 
and, acting together, make a strong simul- 
taneous stroke in one common direction. FJG g2 ._ Paramecium 
The movement of all the cilia of an organ- caudatum: division showing 

ism is, however, usually not synchronous . the macronucleus (N) divid- 

J J mg without mitosis, the mi- 

but proceeds in waves across the surface cronucleus O) dividing mi- 

of its body so that the appearance is simi- totlcall y- c- 1 .. Old, and c -f-, 

J new. contractile vacuoles. 

lar to that produced when a breeze passes (Minchin, after Butschli and 

across a field of grain. Flagella are larger 

than cilia; they are whip-like processes Wandtaflen, No. LXV.V 
which have a lashing movement. They 

are usually few in number and are often placed at the ends of the or- 
ganism. Undulating membranes consist either of a thin fold of the sur- 
face layer or of rows of fused cilia and form either fin-like organs ex- 

* (See p. 195.) 


cu- 


128 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


tending along the surface of the organisms or special organs for the 
intake of food. 

REPRODUCTION 

The protozoa reproduce in many different ways and several of these 
ways may occur in a single organism. For this reason, their repro- 
ductive power is very great; in power of repeating their like, they fall 
just short of the bacteria. The union of a male and a female form does 


/'*%, - 

'-.*: 

^ . -. : 

$ :. 5 v. .' 

- :: ' : J..A- '' - \ 

^SS^^P 


FIG. 93. Stages in the division of Amoeba poly podia. (After F. E. Schulze and Lange 

from Doflein.} 

not always precede multiplication; sexual union and reproduction, 
though now combined in many animals, may have been originally two 
entirely distinct phenomena and, in the protozoa, though sexual union 
may be concerned with the production of new individuals, it is often 
especially associated with the regeneration of the protoplasm of the 
parasites taking part in it. 

The simplest of the methods of reproduction is simple binary divi- 
sion, in which the organism divides into two equal parts. A modifica- 
tion of this process is gemmulation, in which a small protozoon buds off 


PROTOZOA 


129 


from a larger parent; sometimes many buds are formed rapidly, one 
after the other, until the parent protozoon disappears in a swarm of 
daughter cells. When a protozoon divides at a single division to pro- 
duce a large number of daughter cells simultaneously, the process is 


FIG. 94. Coccidium schubergi. A-C, asexual multiplication; D-K, sexual multi- 
plication; D, microgametes; E, macrogamete; F, G, fertilization; H, 7, K, division 
and spore production. (After Schaiidinn, from Doflein.} 


called schizogony and the young parasites are called merozoites, if a 
sexual fertilization has not immediately preceded the act of division; 
if such a division, in which the parent organism disappears, takes place 
after a fertilizing act, the process is called sporogony and the young 
parasites are sporozoites. 


130 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

In protozoa, as in metazoa, the essential process in fertilization is the 
union of two nuclei of opposite sex. In dividing, cells may go through 
a process called mitosis during which the chromatin of the nucleus is 
grouped into more or less rod-shaped masses which are called chromo- 
somes. The number of chromosomes which are formed during mitosis 
is constant and characteristic for each species. In the reproductive 
areas, during the two divisions just preceding the maturity of cells 
which are to become ova or spermatozoa, the number of chromosomes is 
reduced to exactly one-half of the number which are formed during the 
division of cells outside of the reproductive areas of the same animals. 
The process by which the number of chromosomes is reduced to one-half 
is termed chromatic reduction, and the fragments of chromatin which in 
the female are unused and which are extruded from the cell during the 
process are called polar bodies. While reduction in the number of 
chromosomes has been shown to occur prior to fertilization in a number 
of the protozoa, in many species a more primitive process consisting of 
the mere extrusion of masses of chromatin irrespective of the number of 
chromosomes is found to occur. It is evident that the chromatin is, 
at least usually, reduced in amount preparatory to the sexual process. 

Although in certain of the protozoa nuclear division is accomplished 
by a process of mitosis similar to that which occurs in multicellular 
animals, in many it is affected by a much more primitive process. 
The nucleus may be resolved into scattered granules of chromatin- 
chromidia which may subsequently become reconstructed into a num- 
ber of nuclei. The nucleus may divide by direct division, that is, by sim- 
ple constriction into two approximately equal parts. Between this form 
of division and the classical mitosis there is every possible transition. 
The centrioles or centrosomes are frequently intranuclear in the 
protozoa. In the case of primitive nuclei without definite nuclear mem- 
brane a division simulating mitosis is termed promitosis. In other 
forms in which there is a nuclear membrane but in which the centrioles 
remain intranuclear throughout division, the process is called meso- 
mitosis. The nuclear membrane often persists throughout division 
and the chromosomes are in many forms very minute or are not 
definitely formed. 

The fertilizing processes which occur in the protozoa may be grouped 
under three heads: Copulation, Conjugation and Self-fertilization. In 
copulation two whole cells unite. The cells taking part in this union 


PROTOZOA 131 

are called gametes and there are the male or micro gametes, and the 
female or macro gametes. The cells which produce the gametes are 
called gametocytes. The product of the union is called a copula or 
zygote. If the uniting cells be equal in size the copulation is isogamous; 
if they be unequal, the copulation is said to be anisogamous. Aniso- 
gamous copulation, the union of two unequal cells, is most typically 
seen in the fertilization of a large macrogamete by a small microgamete. 
Copulation is the most common fertilizing process among the patho- 
genic protozoa. Conjugation, the second method of fertilization, only 
occurs among the ciliata. In it, two adult individuals place themselves 
in apposition. The nucleus of each cell first reduces and then divides 
into two halves, one male, the other female. Each organism retains 
its female half nucleus, while an exchange of the male half nuclei is 
effected. Processes of self-fertilization, such as autogamy and partheno- 
genesis, are included under the third heading. In autogamy the nucleus 
of a single cell divides into two parts. Each of these may undergo 
further division, during which the chromosomes are reduced or there 
may be a simple extrusion of a portion of the chromatin. The two 
resulting, reduced nuclei then unite, in the same cell, to form a new 
nucleus. Parthenogenesis is the development of new individuals from a 
female cell without a preceding fertilization; this process possibly occurs 
in many protozoa, and through it perhaps may be explained the reap- 
pearance of malaria in patients who once suffered from that disease 
and were thought to have recovered. 

The LIFE CYCLE of a protozoon consists of the changes through 
which it passes in the period intervening between each fertilizing act. 
In many of the pathogenic protozoa, an alternation of generations 
occurs; that is, cycles of development in which an asexual method of re- 
production occurs, alternate with cycles of development in which re- 
production is effected by sexual methods. The developmental cycles 
are commonly punctuated by binary or multiple division, by encyst- 
ment, and by transference to a second host as a necessary factor for the 
completion of the life cycle. An alternation of generations occurs 
in the life cycle of one of the most important of the pathogenic protozoa, 
the parasite which produces malaria (Fig. 189). While it is in the body 
of its mammalian host, man, it multiplies through multiple fission or 
schizogony; the sexual, or propagative phase of its development 
occurs within the body of its invertebrate host, a mosquito. The 


132 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

host in which the adult, sexual stages of the parasite occur, in this 
instance the mosquito, is said to be the definitive host; hosts harboring 
the parasite while it is in other stages are called intermediate hosts. 

ENCYSTMENT. Under unfavorable conditions, such as dry surround- 
ings, many protozoa are able to surround themselves by a resistant 
cyst and to enter upon a resting stage of indefinite length. The cyst 
protects them from harmful influences and, surrounded by it, they 
remain in a resting state until favorable circumstances come about once 
more. The power of forming resistant cysts plays an important part 
in the life history of many parasitic protozoa; it is especially so with 
those protozoa which have become so specialized that multiplication 
or continuous existence independent of their appropriate host has 
become impossible for them. It is often through the formation of 
cysts that an infection by a protozoon is spread, and, as in the coccidia 
(page 889), the presence of such a stage is often absolutely essential 
in the life history of a parasite. 

PARASITISM 

A parasite is an organism which is, at some time, directly dependent 
upon another, usually, a larger organism. 

Although the word parasite is often used as though it referred only 
to organisms belonging to the animal kingdom, parasites may be 
either animal or vegetable; bacteria and fungi, which live at the 
expense of other living beings, are parasites just as the disease-pro- 
ducing protozoa and the biting insects which transmit them are 
parasites. 

Most parasites are simple organisms, low in the scale of life. They 
nourish themselves without exertion, at the expense of their hosts, and 
as might be expected, their unemployed organs, such as the sensory 
locomotory and seizing appendages, by means of which food is usually 
obtained, gradually disappear; degeneration always occurs in an 
organism which assumes a parasitic mode of life. 

Organisms, such as the malarial parasite, which are wholly de- 
pendent for existence upon their hosts, are called obligatory parasites; 
those which are not, such as the infusoria usually found in the stomach 
of herbivorous animals, are facultative parasites. Facultative parasites 
often feed upon organic material provided by the host, and not upon 


PROTOZOA 133 

the host itself; but they are capable of living indefinitely apart from 
the host. 

If an organism is attached to a host, and neither harms nor benefits 
it, such an organism and its host are said to be commensals. For 
example, the spirochsetes found about the teeth of many persons are 
usually harmless ; they are commensals of their host. When the host of an 
obligatory parasite dies, the parasite often perishes also. Consequently, 
it is contrary to the interest of such a parasite to destroy its host; yet 
parasites often do harm their hosts. The harm done by a parasite to its 
host is the disease which that parasite causes. Disease is recognized by 
symptoms. The nature of the symptoms depends directly upon the 
nature of the harm done by the parasite. The symptoms are the result 
of interference by the parasite with tissues, or the functions of tissues, 
in the host. The pathogenic protozoa may injure their hosts in at least 
three ways: They may feed upon, and destroy cells; they may produce 
poisonous toxins; and their presence may do damage by mechanically 
obstructing some of the functions of its host. All three of these ways 
are well exemplified by the action of the malarial parasite in man 
(page 892). 

DISCUSSION OF THE CLASSLFJ CATION* 

i 

The following grouping of the Protozoa gives a general idea of the 
position, in zoological sequence, of the individual parasites which are 
spoken of in the subsequent pages. The Protozoa are here grouped 
in four classes: the RHIZOPODA, the FLAGELLATA, the SPOROZOA, and 
the INFUSORIA; and these classes are divided directly into genera. This 
is by no means a complete classification of the protozoan families. 
Many orders, families and genera are unmentioned because they are 
parasitic neither in man nor in animals; and of the organisms mentioned, 
only those which are constantly causes of disease are described. 

The form of a protozoon may vary greatly at different stages of its 
development; for example, the adult herpetomonas is an active organism 
moving by means of a flagellum, quite unlike its spherical form which 
is without a flagellum. Consequently, the whole life history of a proto- 
zoon must be known before it can be classified with absolute certainty. 
The whole of the life history is known for only a few protozoa; and, 

(See p. 13.) 


bl 


134 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

though the organisms mentioned in this classification are placed in 
the position usually given to them, it must be understood that this 
classification is not final, and that the discovery of new stages in the 
life history of some of these protozea may make it necessary to remove 
them from the classes in which they have been placed. For example, 

before its flagellate stage was known, 
Leishmania donovani was classified with 
the sporozoa; now it is grouped with the 
herpetomonads. 

The characteristics of , the different 
genera and of the unimportant parasites 
are very briefly mentioned in the follow- 
ing paragraphs; the important parasites 
are treated more fully in the pages indi- 
cated by the references given, in brackets. 
The RHIZOPODA include the simplest 
forms of animal life. A rhizopod, such 
as an amoeba, consists of a single cell, 
without a protective covering, and with- 
out permanent organs of locomotion; it 
moves about and captures its food 
through the agency of its pseudopodia. 
Very few of the rhizopods are parasitic; 
most of those which are parasitic, belong 
to the genus Entamoeba. Different 
species of parasitic amoebae may occur 
in the alimentary canals of various ani- 
mals. Certain of these produce serious 
diseases (page 876). 

The FLAGELLATA are distinguished 
by possessing one or more flagella; 
they often have, also, a fin-like, un- 
dulating membrane extending along the surface of their body. 
Many possess two nuclei, a larger trophonucleus which has to do 
with nutrition and a smaller kinetonucleus which is intimately 
connected with the organs of locomotion. This group has been 
termed the Binudeata by certain systematists. Most flagellates are 
free-living. Comparatively few species are parasitic, but some of 
these cause very serious diseases (page 879). 


FIG. 95. Herpetomonas 
musca-domestica (Burnett). A, 
Motile individual with two flag- 
ella; B, cyst; , nucleus; bl, 
kinetonucleus. (After Pro- 
wazekfrom Minchin.) 


PROTOZOA 


135 


A Herpetomonas is an elongated organism which possesses trophonu- 
cleus and kinetonucleus. The latter is situated near the flagellar or 
anterior end of the parasite, and from it arises a terminal flagellum. 
A Herpetomonas has no undulating membrane. A Crithidia is an organ- 
ism like a Herpetomonas, but possessing an undulating membrane. 
A Trypanosoma is an elongated parasite which has a trophonucleus, 
a kinetonucleus usually situated near its aflagellar extremity and an 


FIG. 96. A^ Trypanosoma tinea of the tench; note the very broad and undulat- 
ing membrane in this species; #., C., T. percce of the perch, slender and stout forms. 
(After Minchin, X 2000.) 

undulating membrane along the border of which the flagellum extends 
to terminate in a whip-like appendage. Species of Herpetomonas, 
Crithidia and Trypanosoma are frequently found in the intestines of 
insects. One species of Herpetomonas is a frequent and harmless para- 
site in the intestine of the house fly. Many serious diseases are caused 
by trypanosomes. The genus Trypanoplasma includes organisms 
which have a flagellum at either end, as well as an undulating mem- 
brane. They are parasitic in the blood of fishes. The genera Cerco- 
monas, Nonas, and Plagiomonas include small, unimportant flagellate 


136 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


organisms which have been found, occasionally in the human intestine 
and vagina, and in necrotic material from the lungs. Trichomonas 
is a pear-shaped organism which has four flagella attached to its blunt 
end, and an undulating membrane extending from the origin of the 
flagella at the anterior end posteriorly over the surface of its body. 


FIG. 97. Trichomonas eberthi, from the intestine of the common fowl; ///., 
anterior flagella, three in number; P.fl., posterior flagellum, forming the edge of the 
undulating membrane; chr. I., "chromatinic line," forming the base of the undulating 
membrane; chr.b., "chromatinic blocks;" bl., blepharoplast from which all four 
flagella arise; m., mouth opening; N., nucleus; ax., axostyle. (From Minchin, after 
Martin and Robertson.) 

One of the four flagella is usually directed backwards and extends along 
the border of the undulating membrane. One species is sometimes 
found in the human bladder. Other species are common, usually 
harmless, parasites in the intestines of pigs, frogs and other animals. 
The most important species of the genus Lamblia is Lamblia intestinalis. 
It also is a pear-shaped organism. It has several flagella and is dis- 
tinguished by possessing a depressed sucker, by which it attaches itself 


PROTOZOA 


137 


to the intestinal epithelium of the animal in which it lives. It is a cause 
of diarrhoea in man, and also of a fatal disease of the intestines in 
rabbits; but it is almost invariably found in the duodenum and first 
portion of the small intestine of normal laboratory animals such as 
mice, rats, and rabbits. 


FIG. 98. Lamblia intestinalis. A, Ventral view; N., one of the two nuclei; ax.i 
axostyles;/. 1 , ft. 2 , fl. z , fl-*, the four pairs of flagella; s., sucker-like depressed area on 
the ventral surface; x., bodies of unknown function. (After Wenyon (277) from 
Minchin.) 

The SPOROZOA are parasitic protozoa which multiply by the produc- 
tion of spores at some stage of their life cycle. There are very many 
sporozoa and so, for convenience of classification, they are subdivided 
into seven orders. The Gregarincz have a^very distinctive shape; the 
single cell, of which they are composed, is divided into two or more 
divisions. The first of these divisions is furnished with hooks or other 
structures through which the parasite attaches itself to its host. None of 
the gregarines are parasitic on mammals; worms are the hosts for some 
of them. The Coccidia are usually parasitic within certain cells of their 


138 


MORPHOLOGY AND CULTURE OF MICROORGANISMS 


host, for example, Coccidium stieda (Eimeria cuniculi] (page 889) enters 
the epithelium of the small intestine and of the bile ducts of the 


B 


1 


E 


D 


te 


\ 


FIG. 99. Sporozoits in the oocyst of Laverania malaria. A, Formation of 
nuclear points which serve as the foci from which the sporozoits develop; B, a more 
definite shaping of protoplasm and nuclei; C, Z), mature sporozoits in the oocyst 
arranged about centers from which they radiate; E, a portion of one enlarged. 
(After Grassi, from Doflein.} 

rabbit, while Eimeria avium enters and destroys the cells lining the 
intestines of the birds which it infects (page 889). The H&mosporidia 
live, for a part of their life cycle, within the red cells of the blood of 


PROTOZOA 139 

vertebrate animals. They are a very important order. The genus 
Plasmodium causes malaria in man (page 890) ; while Proteosoma and 
H&moproteus are malarial parasites of birds (page 890) . The Hcemogre- 
garina are usually harmless parasites of reptiles and batrachians 
(frogs) ; a part of their life is passed within the red cells of their host, 
but they have a slowly moving stage, somewhat resembling a gregar- 
ine, which occurs free in the blood. Hepatozob'n perniciosum is the 
best known of a group of haemogregarine-like parasites which are 
parasitic, often within the white cells of the blood, in dogs, in rats, and 
in other rodents; so far as is known, they do not cause disease. The 
genus Babesia (page 894) includes parasites which cause important 
diseases in cattle, sheep, horses and dogs. Similar parasites have 
been found in the blood of monkeys, of dogs, of rats and other rodents. 
The Sarcosporidia are tube-like in shape and filled with spores. They 
are found within the cells of the voluntary muscles. TheHaplosporidia 
are a group of very small sporozoa of which little is known. Some of 
them are parasitic in fish; one of them, Rhino sporidium kinealyi, has 
been found in a tumor of the nose of a native of India. The Myxo- 
sporidia (page 899) are recognized by the peculiar form of their spores; 
each spore has one or more capsules each furnished with a coiled fila- 
ment or thread which is extruded under certain conditions and probably 
serves to anchor the spore to a surface upon which further development 
may occur. Members of this order are parasitic in various tissues of 
fishes and they often produce disease in their hosts. The spores of the 
Microsporidia (page 899) are exceedingly small; a member of this 
order is the cause of pebrine in silk- worms (page 937). 

The INFUSORIA (page 899) are a large class. Most of them are not 
parasitic. They are the most highly developed of the protozoa and 
their bodies are more or less covered with cilia, by which they move 
themselves through the liquids in which they live. 

Lastly, under the heading Parasites of Uncertain Position, are 
grouped a number of organisms which cannot be classified because 
so little is known of them at present. The spirochaetiform organisms, 
Histoplasma capsulatum (page 900), the Chlamydozoa (page 900), the 
Rickettsias, and the Ultramicroscopic viruses (page 119) are all asso- 
ciated with important diseases in men and in animals. 

The SPIROCH^T^E (page 900), as their name signifies, are thread-like 
organisms, which seem to be coiled in a spiral. It is probable that the 


140 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

curves of certain spirochaetes lie in one plane and, consequently, that 
their bodies are really waved and not spiral. These organisms have 
no organized nucleus. The chromatin is distributed throughout their 
bodies. 

Those parasites which are important enough to require special con- 
sideration are described (page 876) in the order in which they are men- 
tioned in the classification (page 13). Whenever it is possible to do so, 
a single species is taken as the type of each genus and that species, with 
the disease it produces, is described; if the remaining species of the 
genus are mentioned, they are spoken of only to indicate how they 
differ from the description of the type. 

r 


TECHNIC* 

The methods employed in studying the pathogenic protozoa are very similar to 
those used in bacteriology. Microscopes, with the highest magnifications, are 
essential for successful work. 

It is of great importance in the study of protozoa to examine them in the living 
condition. In no other way can their mode of locomotion be determined and 
frequently their contour is quite different in living and in fixed preparations. 
A small amount of the material in which they occur may be placed beneath a cover- 
glass on a clean slide and examined immediately with the microscope by ordinary 
daylight. In case large organisms are examined in rather thin fluid it is well to 
prevent their being crushed by interposing several minute globules of paraffin 
between slide and cover-glass. This is readily accomplished by touching paraffin 
with a hot needle and transferring it thus melted to several points on the slide before 
the preparation is made. When very minute forms are to be studied it is necessary 
to utilize what is known as the dark field illumination. This brings out very minute 
organisms and particles which, being transparent, are invisible to ordinary trans- 
mitted light. The dark field apparatus consists of a strong source of light such as a 
small arc lamp, a special condenser which deflects the light so that objects in the 
microscopic field are illuminated by light directed from the sides, causing them to 
appear bright on a dark background. Another method of obtaining a dark field is 
to mix on a slide a small drop of the material to be examined with an equal-sized 
drop of India ink, or better of saturated aqueous solution of nigrosin, and then to 
smear this mixture across the surface of the slide. It is then dried and examined at 

For more detailed instructions for the study of protozoa see Fantham, Stephens and 
Theobald, The Animal Parasites of Man, William Wood & Company, New York; Castellani 
and Chalmers, Manual of Tropical Medicine, Bailliere, Tindall & Cox, London; Stitt, Practical 
Bacteriology, Blood Work, Parasitology, Blakiston, Philadelphia; Brumpt, Precis de Parasit- 
<logie, Masson, Paris; Langeron, Precis de Microscopie, Masson, Paris; Doflein, Lehrbuch der 
Protozoenkunde, Gustav Fischer, Jena; and Prowazek, Der mikroskopischen Technik der 
Protistenuntersuchung, Leipzig. 


PROTOZOA 141 

once by the oil immersion lens. Only ordinary daylight is required for this method 
but it does not serve in the study of the motility of organisms. 

By special apparatus it is possible after obtaining a certain amount of skill to 
dissect many forms of protozoa. In this way knowledge is obtained of the physical 
properties of various portions of their bodies and it is also possible to inject various 
chemicals into their substance. This method of study is made possible by the me- 
chanical devices utilized by Barber to whose work the reader is referred.* 

In order to make stained preparations the material may be either smeared in a 
thin film upon clean slides or sectioned after appropriate treatment. In each case 
the material requires fixation. For the preparation of stained smears the Giemsa 
method is widely used. This is briefly as follows: 

1. Make thin smears of material on a clean and dry slide. 

2. Fix immediately by covering the smear with pure methyl alcohol which should 
be allowed to act for ten to twenty minutes. 

3. Dry by waving slide to and fro. 

4. Stain for four to twenty-four hours, according to the depth of stain desired, 
in a solution made by an addition of one drop of Giemsa stain to i c.c. of distilled 
water. 

5. Rinse with distilled water. 

6. Dry and mount in immersion oil or any acid-free balsam. 

It is frequently desirable to keep stained smears unmounted as they apparently 
retain their color for a longer period of time. They may be studied with the oil 
immersion lens but the oil should at once be rinsed off with xylol, for if left upon the 
preparation an insoluble substance is formed which produces a clouded appearance. 
All stained preparations should be stored away from the light when not in use. For 
the above method it is important to have all glassware perfectly clean and without 
trace of acid. The stain must be used immediately after preparation. Certain 
materials may be smeared very readily with the platinum loop ordinarily used in 
bacteriology. A very practical method for making blood smears is to gather a 
minute drop of freshly drawn blood from a small needle-prick of the skin on one 
edge of the end of a slide. The latter is placed in contact with the surface of another 
slide and being held at an angle of 45 degrees is pushed steadily lengthwise across its 
surface. By increasing or decreasing this angle a thicker or thinner film may be 
made. Certain investigators prefer to use what is termed the wet method for the 
fixation of smears. In this case the smear is dropped face down immediately and 
before drying into a fixative composed of two parts of a solution of saturated HgCU 
in distilled water and one part of absolute alcohol. The technic employed in the 
staining of sections is then followed and the smear is not allowed to dry at any step 
in the procedure. 

The preparation of stained sections requires a considerable amount of technical 
skill. Tissue is first fixed to render its structure permanent. It is then dehydrated 
in alcohol of increasing strengths, next placed in chloroform or some other clearing 
reagent and it is then imbedded in paraffin, after which it may be sectioned. Foi 

* Barber: University of Kansas, Science Bulletin 1907-4-3; also Journal of Infectious 
Diseases, 1911, 8, 248, and 1911, 9. H7- 


142 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

the details of sectioning and the staining of sections the reader is referred to Mallory 
and Wright's Pathological Technic, W. B. Saunders and Co., and to Lee's Vade 
mecum. 

The cultivation of free-living protozoa is usually accomplished by keeping a 
supply of the medium in which they live on hand. Hay infusion prepared by boiling 
a quantity of chopped hay in water is an easy and valuable method of preparing 
culture media. For the cultivation of amoebae, the following media is widely em- 
ployed. It should be noted, however, that the amoebae which have been cultivated 
are regarded as free-living forms and the attempts to cultivate parasitic amoebae 
have thus far been unsuccessful. 

MEDIUM OF MUSGRAVE AND CLEGG 

Agar 20 to 30 g. 

Liebig's extract of beef 3 to . 5 g. 

Common salt 3 to . 5 g. 

Water 1,000 c.c. 

This medium is designed to provide for slow bacterial growth in order to provide 
food for amcebae. On a richer medium the latter are overwhelmed by the rapid 
growth of bacteria. 

For the cultivation of trypanosomes, leishmania and other flagellates the so- 
called triple N media is employed. This is prepared as follows: 

NlCOLLE, NOVY, MACNEAL MEDIUM 

Water 900 c.c. 

Salt 6 g. 

Agar 16 g. 

Dissolve, distribute in tubes, sterilize and add to the medium in each tube after 
liquefying and cooling to 4o-5oC. one-third its volume of rabbit blood obtained by 
cardiac puncture. Slope the tubes for twelve hours, incubate at 37 for five days 
to prove the sterility of the medium and then keep them at the ordinary temperature 
of the laboratory for a few days before sowing them. (The tubes should be sealed to 
prevent evaporation.) 

The malaria organisms have been made to continue development outside the body 
by the following method devised by Bass. 

Bass's Method. The blood in 10- to 2o-c.c. quantities is taken from the patient's 
vein and received in a centrifuge tube which contains )-fo c.c. of 50 per cent, glucose 
solution. A glass rod, or piece of tubing, extending to the bottom of the centrifuge 
tube is used to defibrinate the blood. After centrifugalizing there should be at least 
i inch of serum above the cell sediment. The parasites develop in the upper cell 
layer about J^Q to %Q m ch from the top. All of the parasites contained in deeper 
lying red cells die. To observe the development, red cells from this upper % 
portion are drawn up with a capillary bulb pipette. 


PROTOZOA 143 

Should the cultivation of more than one generation be desired, the leucocyte 
upper layer must be carefully pipetted off, as the leucocytes immediately destroy the 
merozoites. Only the parasites within red cells escape phagocytosis. Sexual 
parasites are much more resistant, and the authors think they observed partheno- 
genesis The temperature should be from 40 to 41 and strict anaerobic conditions 
observed. /Estivo-autumnal organisms are more resistant than benign tertian ones. 
Dextrose seems to be an essential for the development of the parasites. 

Noguchi first cultivated spirochaetes; others have extended and modified his 
work. If a few drops of blood containing spirochaetes is dropped into sterile ascitic 
fluid, hydrocele fluid, or blood plasma, to which a piece of sterile tissue, such as 
rabbit's kidney, is added, the spirochaetes multiply. The test tube should contain 
about 15 ccm. of culture fluid. It is advantageous to cover the fluid with sterile 
paraffin oil. 


PART II 

PHYSIOLOGY OF MICROORGANISMS 


DIVISION I 

INTRODUCTION* 

Microbial physiology seeks to understand the material or concrete 
processes and functions of protoplasmic activity which integrate in the 
phenomenon of life. They are embodied in some form of an organism. 
The many assembled and harmonious forces involved in a unit of life, 
while they may be resolved in a degree elementally, are dependent upon 
the structure, composition, and energy values of the life-form, and also 
upon its environment; likewise the reverse is true. The concomitant 
relations of forces to the life-form and life-form to the forces are more or 
less hidden at present, yet they are slowly becoming apparent. 

Owing to the multiplicity and variety of forces operating in physio- 
logical functioning, it is patent that physiology is complexly composite 
in nature and must resort to the elemental branches of science as 
physics, chemistry and morphology for its understanding and exposi- 
tion. Shrouded along with demonstrated knowledge is the mysterious 
veil of life which makes of it a reality, subject to the ready onslaught 
of scientific attack and to a spirit which halts approach. 

Besides the basis of facts found in physics, chemistry and morphol- 
ogy which contribute freely to the structure, there are the immediate 
matters of cytology, anatomy both gross and histological, and environ- 
mental conditions which lead into fields of essential technic, before 
physiology can be truly grasped or successfully studied. When the 


* Prepared by Charles E. Marshall and Arao Itano. 
10 145 


146 PHYSIOLOGY OF MICROORGANISMS 

study is attempted in an elementary manner it consists in recognizing 
observational functions without attempting to explain or understand 
the mechanism behind. Such attempts belong to the first steps in 
primary and secondary education. The limitations or boundaries of 
physiological knowledge are those established by the human mind in 
laying hold of and utilizing the nature and the facts of the elemental 
studies upon which physiology rests and its ability to translate them in 
the life-mechanism at work. 


CHAPTER I 

THE UNIT OF BIOLOGICAL ACTIVITY* 

Whether a cell acts in the capacity of an individual entity or alone, 
as in the case of S. cerevisice, or in its intimate association with 
other cells possessing an individual entity and having an intercellular 
relationship as yeasts and acetic bacteria, or even in close dependent 
relationship with other cells in forming a multicellular entity as in the 
case of metazoa and metaphytes, its self -functioning processes or its 
performances are confined within the limits of the cell, are the operating 
mechanism of the cell and are independent of other cells, notwithstand- 
ing the controlling influences of environmental conditions upon its 
activities. The self-contained cell, which is a cellular entity, may not 
be subject as a rule to the more immediate influence of other cells, yet 
the associative influence exists in most cases whether near or remote 
and is more or less essential to the life of the cell. There is, in other 
words, a biological interdependence in most living forms. It follows 
that the cell has therefore a distinct life of its own within its sphere 
of activity and in addition an equally important office to perform in its 
association with other cells; in both cases, it functions only together 
with its environment. 

The cell is at the mercy of environmental conditions. S. ceremsia 
is master of itself until the factors of food, moisture, respiration, 
temperature and reaction are demanded for the sustenance of life; it 
then becomes the menial servant of each, for each and every factor is 
essential to its existence. In turn, food, required gases, temperature, 
and other environmental factors, such as may be needed for cell life, 
may have their source in the activities of other cells. Yeast cells 
produce alcohol for the acetic bacteria as certain organs of the body 
produce hormones for the activity of other organs. One cell, therefore, 
through external factors may become dependent upon another and 
probably is in the case of most cells. 

Unless the microorganisms which seemingly live directly upon the 
very simple elements of nature are excluded, cellular life is so intricately 

* Prepared by Charles E. Marshall and Arao Itano. 

147 


148 PHYSIOLOGY OF MICROORGANISMS 

bound up with other cellular life that it ceases without such association, 
whether it is regarded from the standpoint of individual entities, the 
protophytes and the protozoa, or the standpoint of the complex entities, 
the metaphytes and metazoa. Virchow made the cell the working unit 
system of life, but it was done in the sense that the "House of Roths- 
child 1 has become a unit in the financial world. Pfluger, Verworn, 
Ehrlich and Vaughan, however, resolve the cell or the "House" into 
the ultimate coordinated agencies within, molecular complexes, which 
are responsible for the inception and continuing of all the activities. 
There are cells, it is true, of many kinds and different degrees of struc- 
ture and organization, accordingly a more elemental unit must be 
sought which is essential to establish harmony or unity in life's ultimate 
phenomena or reactions. Vaughan, who is the last of the above to write 
from his own investigations, says: "The cell is not the unit of life; life 
is molecular. Life is function, not form." Again he says: "Cells 
consist of a chemical unity made of giant molecules." Moore states 
that "the unit of the biologists is the living cell," but he himself 
approaches it from the standpoint of molecular structure. He would 
impugn the attitude and circumscribe the field of the biologist by the 
limits of morphology whereas, in fact, the biologist interprets organic 
life by means of the various ultimate elements included so far as this is 
possible and endeavors to unify all forces and structures in an inti- 
mate unity. 

Physiology in taking cognizance of the cell itself and its environment 
is reduced to its simplest and lowest terms in the cell possessing an 
individual entity, for a large part of this physiology is found represented 
in its most rudimentary and elemental forms and consequently quite 
easily studied. 

Nutrition as it is illustrated in E. sub tills is easily approached as 
compared with that of man. It is not difficult to reproduce, change, 
and control nutritional conditions in a unicellular organism as compared 
with the multicellular organisms. Methods which enable the investi- 
gator to reproduce, manipulate and supervise microorganisms enable 
him to attack problems excluded from the category of the multicellular 
physiologist. However, the physiologist of complex forms, as the 
human, not only has his problems rendered more intricate by the organ- 
isms he studies but he also has them multiplied because of the many fold 
combinations of cells. Bayliss is substantially correct when he says, 


THE UNIT OF BIOLOGICAL ACTIVITY 149 

'The physiology of unicellular organisms, although of considerable 
importance, is not to be regarded as a general physiology," because the 
physiology of microorganisms leads only to the point where one phase 
rule must give way to another phase rule the physiology, in fact, of 
any restricted number of organisms harmoniously related can never 
cover the field of general physiology. Let us examine this more 
particularly for the purpose of securing the most effective attitude in 
the study of microbial physiology. 

The differences which separate unicellular physiology from the 
specific human physiology are worthy of consideration. Neither the 
one nor the other can be considered general or comparative physiology 
but both have values which are of interchangeable advantage. The 
unicellular organism enters upon its nutritional career as a free cell 
found in an environ of food which, in its specific manner, it must 
prepare for absorption and assimilation. The human first brings its 
food into a canal, a tube, the alimentary canal, lined with specialized 
cells which contribute to the preparation, the absorption and assimila- 
tion of the food. Enzymes are secreted by the unicellular form into the 
food medium undergoing preparation for absorption, just as enzymes 
are secreted by the cells of the walls of the stomach and intestines of 
the human species. The same purpose is apparent in each case. The 
food is absorbed through the cell-wall or directly into the protoplasm in 
the unicellular organism, while in man the cells lining the alimentary 
tract operate in much the same manner. In the human the distribution 
takes place by means of a carrier system, the circulation, in order to 
reach the distant points w r hile with the single-celled organisms the process 
is one of diffusion. Enzymes are present in both organisms engaged in 
the process of converting food material into protoplasm and the 
production of waste. Aside from the distributive method, the nutritive 
processes are much the same in both. It now remains to conclude 
which is the more simple to study, the more accessible for study, and 
the more adaptable to study. In this the single-celled organism has 
many advantages. This does not constitute, however, general or 
comparative physiology for either one of these assumes a large number 
and a varied number of living forms into which creep specific differences. 
The similarity paralleled in nutrition could be carried into other 
functions but this is not the purpose. It is desirable, mainly, to em- 


150 PHYSIOLOGY OF MICROORGANISMS 

phasize the possible extension of the simple and even rudimentary 
basic facts to the field of general physiology. 

The complicated structures of the metazoa and metaphytes can 
scarcely be compared with microorganisms. It is difficult to speculate 
intelligently on the development of shape and structures in biological 
forms in the light of present knowledge, yet proceeding from simple 
forms to the more complex, there is constantly confronting the mind 
the possibility on the part of nature to adjust form and structure to the 
growing and expanding demands of protoplasm. The struggle on the 
part of a microorganism to secure oxygen or to get away from it, the 
action of light and darkness on the growth of molds and other factors 
signify as much to a simple single-celled organism as the prehensile 
tendencies of an insect or an ape. It is something sought by the use of 
different structures and of different agents. There are so many 
indications of incipient developments in the unicellular organism that 
much time and space could be given to speculative possibilities. Only 
a suggestion is required, however, to start the thinking student to 
fruitful reflection. The morphologist approaches the subject of the 
place of microorganisms in nature through the channels of " degenerated 
forms" from "higher forms" or " types of simple forms in process of 
evolution." This view does not harmonize with the physiological 
approach in which functioning supercedes form unless degeneration 
and evolution is made a matter of functioning instead of form. 

A cell as a free unit and a cell as a unit but tied into a community 
of cells awaken interest. The free cell might be likened to primitive 
man, a Jack-of-all-trades, but a cell interwoven into a multicellular 
organism is that of a Jack-of-all-trades, an individual, and a man in a 
social organization, a specialist. This seems to be apparently true 
except where the cells simply form an aggregation or a colony in which 
all cells function alike. There is, too, the tendency of the single free 
cell to specialize as those of the alcoholic group, the lactic group and 
many others. To say that they are in the process of functioning to- 
gether in an organism as the yeast and acetic organisms is wholly 
chimerical yet very suggestive in nature's slow evolutions. Few facts 
can be brought forward to disentangle definitely such conceptions. 

As a working basis to-day it is necessary, because of restricted 
human advancement, to consider the cell as the unit of life although its 
compositional structure when understood may reveal the unit of life 
for to-morrow. 


THE UNIT OF BIOLOGICAL ACTIVITY 151 

THE MECHANISM OF CELLS 

In the early stages of this book Guilliermond has revealed the struc- 
tures of microbial cells and has indicated in his treatment the probable 
relations of these structures to functioning. The immediately fore- 
going section carries the suggested creative powers of the various cell- 
structures to molecular foci which seem to be the centers in which vital 
changes are occurring. It now remains to set forth the mechanism of 
the cell, functioning as a whole, which can be done only by approaching 
it through the channels of its activities. 

A living organism is conspicuous as a mechanism because it has the 
power of self-maintenance in the presence of a suitable environment, 
it can reproduce its like when conditions are favorable, it frequently has 
the freedom of motion, and possesses many reacting responses to stimuli 
or expressions of dynamic existence. These are characteristics which 
belong peculiarly to cell-life. 

With food at hand and other favorable conditions the cell becomes 
an automaton. The material needed in growth and the energy required 
for operation are. obtained without assistance and regulated to meet the 
exact demands of the cell. If food is plentiful, growth and reproduction 
reach out to conserve it; if food is scarce, the cell accommodates itself to 
its limitations by reduced activity or a resting stage. This great power 
of adjustability while having boundaries, is as useful to the life of the 
cell as it is wonderful. Likewise, if solid food alone is available, the 
food is reduced to a liquid form or changed that it may be incorporated 
in this bit of protoplasm, the cell. When there, it is transmuted into 
the living substance or is converted from dead matter to living matter. 
In performing this work, the food consumed has had to provide the 
energy as well as the material which rejuvenates the protoplasm, for 
this constant rejuvenating process in the case of protoplasm seems 
essential to life as well as to the constant supply of energy to make it 
possible. 

There are agents, or properties, or substances of the cell which make 
this possible, it is true, and there are structures of the cell which are 
involved in it, but these are necessarily subordinate to the innate proto- 
plasmic forces which give them birth and which in some manner create 
and supervise them. These detailed features are the subjects of physi- 
ological study. 


152 PHYSIOLOGY OF MICROORGANISMS 

Unicellular organisms are concerned with what may be called 
respiration. Oxygen, in its free forms, is needed in most instances for 
body-processes and carbon dioxide is given out. The amount required 
varies with different organisms and is not determined by the amount 
present in the air. Other gases are also used. Nitrogen is taken up 
by the microorganisms which grow on the roots of legumes; marsh-gas, 
carbon monoxide, carbon dioxide, hydrogen and other gases are claimed 
to be utilized. Each species of microorganisms seems to possess greater 
or less specificity in its gas-relations. No species can extend its influ- 
ence over all amounts of a single gas or to all gases. From this situation 
it is a forced conclusion that all cells do not function alike, accordingly 
must have different mechanisms although the capacity for life appears 
common to all. 

Some organisms emit light or manifest radiant energy, other organ- 
isms produce pigments which vary with different species, still others 
elaborate deadly poisons, called toxins, and other substances of patholog- 
ical significance. Briefly, microorganisms give rise to many different 
products whether they take the form of secretions, excretions, energy 
manifestations, or are referable to action upon environmental media. 
Enumeration is not the purpose here. These very numerous products 
indicate the many-sided activities of microbial life. If the substances 
which enter a mechanical device are the same, the products passing out 
are the same. In this case, the food substances and the conditions 
controlling the microorganisms may be identical yet the issuing products 
are different. It follows, therefore, that the mechanism of cehVmust 
be very different, yet as mentioned heretofore the life-mechanism is 
common and the same so far as determinable. 

Many unicellular forms have a cell-wall and when no cell-wall is 
present there is likely to be a distinctive and denser layer of cytoplasm 
on the outer zone. Some organisms when placed in a dense salt 
solution will lose a considerable amount of their water-content and 
shrink while others will not; in other words, some microorganisms have 
the power of withstanding dense brine while others do not. Osterhout 
has repeatedly demonstrated that where two salts are together in a 
medium acting as a substratum for microorganisms there may exist 
an antagonistic or favorable action of one of the salts upon the absorp- 
tion of the other salt by the cell but that cells vary in these responses. 

It is a common knowledge that cells have a selective action which 


THE UNIT OF BIOLOGICAL ACTIVITY 153 

differs with different microorganisms. The capacity to utilize certain 
foods in the presence of others, to acquire certain ingredients, and to 
leave others behind makes for great variability in the functioning of 
microbial cells. 

Spores of some microorganisms are exceedingly resistant, with- 
standing over twenty hours of heat at 100. Spores of other species 
are destroyed in from one to five minutes at 100 under the same con- 
ditions. Wide differences are found among vegetative cells likewise. 
The molecular stability which embodies the life of the cell against heat 
must be as variable as the dissolution of chemical substances under 
the influence of heat. Life then can be associated with many kinds of 
molecular mechanisms although for each species there is a more or 
less constancy. 

In the process of selecting, digesting and assimilating their food, 
certain microorganisms develop an unusual amount of heat so much that 
many microorganisms cannot live in its presence, yet these heat- 
producers (thermophile microorganisms) are dependent upon this 
excessive heat for their proper growth and functioning. 

So far evidences of varied processes existing in different species 
and strains have been set forth in the products and energy resulting 
from growth and development, or, more briefly, in the processes of 
metabolism. These clearly convey the very noticeable variation which 
must exist in the mechanisms which are responsible for such differences. 

There is another approach which will contribute force to what has 
already been said concerning the mechanisms of living cells. 

Chemists have repeatedly shown that cells differ in their chemical 
compositions. When chemists begin their destructive laboratory 
manipulations for the purpose of ascertaining what is present in a cell, 
they may not be working with the real substances or bodies making the 
living protoplasm. However, the bodies which they do study and 
which are fairly constant in the same kind of cells or in the same species 
are doubtless products which in one form or another enter into the 
complexity of protoplasm. The chemical substances studied in nervous 
tissue differ from those of muscles; the chemical substances as products 
of certain species of microorganisms differ from the products of other 
species. Some of these 'substances are definitely known to differ and 
others, although they cannot be satisfactorily determined, are suspected 
to differ in different tissues and different species of microorganisms. 


154 PHYSIOLOGY OF MICROORGANISMS 

A specific and more definite consideration of this subject will appear 
later. Our purpose here is to point out simply that the material making 
up the molecular mechanism of the cell must be exceedingly delicate 
and complex because of its products, and the mechanism of one cell 
must differ very significantly from that of another also because of its 
products, which as cell-contents are determinable; and further, the 
mechanism must be highly responsive because of its ready reactions to 
various influencing agents. All of these evidences seem to accord 
with the interpretation advanced in the previous chapter the cell 
consists of chemical foci and physical forces harmoniously constructed 
into an operating mechanism, the protoplasm, which is arranged effec- 
tively in a cellular laboratory, the cell. 

Literature freely consulted and recommended for extended study: 

BAYLISS, The Principles of General Physiology. 

CHILD, Individuality in Organisms. 

CZAPEK, Chemical Phenomena in Life. 

HENDERSON, The Fitness of the Environment. 

MOORE, The Origin and Nature of Life. 

VAUGHAN, Protein Split Products in Relation to Immunity and Disease. 

VERWORN, General Physiology. 


CHAPTER II 

A STUDY OF PHYSICAL FORCES INVOLVED IN BIOLOGICAL 

ACTIVITIES* 

INTRODUCTION 

It is becoming more and more evident that besides the chemical 
components and forces which constitute the mechanism and activities 
of the cell and which will be treated in the next chapter, there are 
certain physical forces and conditions as important and very depen- 
dently related which are operative. In the physical or chemical ap- 
proach to physiology the writers do not assume the role of being 
chemists or physicists and they write somewhat hesitatingly and reluc- 
tantly upon such matters, yet the stream separating the various 
branches of science must be spanned. The purpose, as writers, will be, 
therefore, to bring the elementary gist of the physical laws and phenom- 
ena, which bear upon microbial physiology, to the attention of the 
student for memory-helpfulness and suggestiveness. Should greater 
knowledge or more extensive reading be desired the student is asked to 
consult the literature appended at the end of this chapter. 

ENERGY 

Energy may be most effectively presented in the general law 
through which it defines itself and governs applications, and in the 
specific expressions in applications which provide the detailed back- 
ground. Energy is work or capacity to perform work. This is found 
in all living units. Work is going on. Whether this energy is desig- 
nated as mechanical, thermal, electrical, chemical makes no particular 
differences for the form is only one aspect of it. 

Energy may be transformed from thermal into electrical or chemical 
into thermal; in fact "all forms of energy are convertible. The total 
energy of any substance or system cannot be altered by the mutual 
actions of its parts." Furthermore, energy is practically indestructible. 

* Prepared by Charles E. Marshall and Arao Itano. 

155 


156 PHYSIOLOGY OF MICROORGANISMS 

This is true whether energy exist as potential energy in the form of 
rest or as kinetic energy in the active form. "In every modification 


of a material system, not affected by forces foreign to a system, the 
sum of its potential and kinetic energies remains constant." This 
statement represents the great law of Conservation of Energy. To 
determine the total energy of a body is difficult for it usually contains 
potential and kinetic energy which may not find expression in measur- 
able units. It follows that " the total intrinsic energy of a body or 
system of bodies is never known. When bodies mutually react it is 
only the difference of the energy of each body in two states which is 
considered. If a body has less energy in its actual than in its standard 
state the expression for its energy is negative." The functionings 
of a living organism may well be said to be the energizing powers of 
such organism. These powers are maintained by the food consumed, 
part of which enters into the synthesis of new protoplasm or in renewal 
processes, and part furnishes the energy for the work performed. 
Whether the energy goes to constructive and reconstructive purposes in 
the maintenance of the mechanism or whether to the manifest liberation 
of energy as work, it is desirable to remember that the ramifications of 
either route are multitudinous, intricate and mostly concealed. The 
principle of the Conservation of Living Forces states " that the differ- 
ence of the force-functions (or work) at the beginning and at the end 
of the motion of a system is equal to the difference of the vires vivce 
(kinetic energies) at the beginning and the end of the motion." This 
is readily seen to represent only measurable and definite performance 
and does not in any sense represent potential possibilities. 

If living energy is functional energy or functioning then an under- 
standing of it with any degree of comprehensiveness means that it is 
to be found in the study of the many functional processes of the body 
in detail. To this attention is directed. 

SOLUTIONS 

When any substance is placed in or is associated with another 
substance in such a manner as to enable both to establish a uniform 
ionic, molecular and particulate mixture, such a combination is called 
a solution. In a true solution, this combination, in which the ionic or 
molecular components are intimately merged or blended and are 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 157 

uniformly distributed or dispersed, is considered a homogeneous system. 
It is also a one-phase system because its components are not mechanically 
separable or physically different. On the other hand, colloidal solutions 
(see colloids and crystalloids) consisting of components which are 
physically different and mechanically separable form a heterogeneous 
system. Such a system or solution is therefore either diphasic or 
polyphasic. 

A true solution, composed of two substances, may have one sub- 
stance regarded as that dissolved and the other as the dissolving agent. 
If table salt is dissolved in water, the salt would be regarded as the 
substance dissolved in water as the dissolving agent. In this case, salt 
would be the solute and water the solvent. However, just the converse 
may be equally true: water dissolved in salt. Colloidal solutions 
are somewhat different because they consist of fine or very small par- 
ticles in suspension or emulsion. In such solutions, therefore, the 
suspended particles in solution (called suspensoids] or emulsified 
particles in solution (called emulsoids) are the substances distributed or 
dispersed in a medium or menstruum. The particles are called the 
disperse phase and the medium or menstruum the dispersion means. 
Together they constitute a heterogeneous system, dispersoids, also a 
polyphasic system. Some colloids approximate very closely ionic and 
molecular solution. 

A diagram (Fig. 100) will aid in understanding. 

Solutions 


True Colloidal 


i 

Ionic Ionic Purely colloidal Suspensions 

and molecular and not easy and emulsions 

molecular of easily 

mechanical separated 

separation mechanically 
Gaseous Liquid Solid 


Gaseous Liquid Solid 

FIG. 100. 


158 PHYSIOLOGY OF MICROORGANISMS 

From the diagram it will also be noted that, whether in true solution or 
colloidal solution, solutions are not confined to the action of water on 
some salt but extend in like manner to gaseous, liquid and solid alike, 
as gas in gas, solid in gas, gas in solid, liquid in gas, liquid in solid, etc. 

The solute may exist in the form of a substance which becomes 
electrically dissociated into its ions, as sodium chloride is dissociated 
into its ions, sodium and chlorine, in water, when it is called an elec- 
trolyte. The solute may be resolved only into its molecules in water, as 
sugar, when it is called a non-electrolyte. Such substances are desig- 
nated usually as crystalloids, but this is not uniformly so. Again, 
instead of using the term solute, in its place there is used the term 
disperse phase. This is made of small particles greater in size, as a rule, 
than molecules, and only in suspension. The solution takes on the 
differentiated form as represented by heterogeneous, polyphasic, and 
colloidal characters. The so-called solid solution signifies that one 
solid substance may become distributed throughout another solid 
substance as in the case of "crystals which are uniformly composed 
by two crystalline substances which present similarity in crystalline 
form as well as of chemical composition" or as coloring matter in 
mineral salts. 

The significance of the possibilities of solutions should be grasped 
to understand the changes taking place in protoplasm through meta- 
bolism, egestion and ingestion. 

ELECTRICAL CONDUCTIVITY, IONIZATION AND DISSOCIATION 

When a salt as sodium chloride (NaCl) is dissolved in water it 
undergoes dissociation or breaks up into atoms sodium (Na) and 
chlorine (Cl). Each atom is made up of electrons some of which 
constitute the center and are positively charged with electricity while 
some are on the outside and are negatively charged with electricity. 
These latter are more mobile, are not held so tenaciously, and are called 
the valence electrons because they establish the valency of the atom. 
These electrons could be regarded as the units of electric charge. If 
sodium (Na) and chlorine (Cl) unite in the formation of sodium chloride 
(NaCl) it appears to be by electric attraction. This may be due to the 
transfer of valence electrons from one to the other or attributable 
to the rearrangement of valence electrons within the atom thus creating 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 159 


the attraction. The two atoms, however, remain electrically neutral. 
Any free atom or combination of atoms which carries a charge of electricity 
is called an ion. When sodium chloride (NaCl) is resolved into the two 
atoms sodium (Na) and chlorine (Cl) in solution and each contains an 
electric charge, the process is called ionization or electric dissociation. 
The sodium chloride (NaCl) or like substance is called an electrolyte. 
The positive ions are known as cations and the negative as anions. 


NaCl 


Catkod 


- Anode 


FIG. 1 01. Movement of Electric Current and Ionization. 

In the dissociation of sodium chloride (NaCl) in water a negative 
unit of electricity in the form of a valence electron goes with the chlorine 
(Cl) and a positive unit of electricity in the form of a valence electron 
is thus established with the sodium (Na) . These electrons are further 
attached to molecules of water together with which are formed the 
electrically charged ions. The loss of the negative valence electron 
by the sodium (Na) when it passes with the chlorine atom (Cl) leaves 
the sodium atom (Na) positive or, in other words, the withdrawal of the 
negative electron from the sodium (Na) disestablishes the neutrality 
of the sodium (Na) by making it positive and that of the chlorine (Cl) 
by making it negative. The result creates a flow of electricity because 
there is a difference in the potential of the two atoms. The potential 
determines the flow of electricity in much the same manner as pressure 
determines the flow of a liquid. 

Such resistance as is offered by a solution to a current of electricity 
may be measured by the Wheatstone Bridge. 


l6o PHYSIOLOGY OF MICROORGANISMS 

When an electric current is introduced into a solution of sodium 
chloride (NaCl), the ions carry the electricity, sodium (Na) carrying 
the positive charge and chlorine (Cl) the negative charge. The positive 
or sodium ions gather at the electrode or pole or cathode, the pole by 
which the current leaves the solution; the chlorine ions which have the 
negative charge pass against the current to the anode, the pole by which 
the current enters the solution. Those gathered at the cathode are 
the cations, those at the anode, the anions. 

The electric relations existing between sodium (Na) and chlorine 
(Cl) in sodium chloride (NaCl) also exist in hydrochloric acid (HC1) 
in which hydrogen and chlorine bear the same relationships as sodium 
(Na) and chlorine (Cl) in sodium chloride (NaCl). 

The strength of the acid is measured by displaceable hydrogen atoms. 
In nitric acid (HNO 3 ) one hydrogen atom (H) is displaceable, in acetic 
acid (CH 3 COOH) only one hydrogen atom (H) is displaceable, for the 
hydrogen atoms of the methyl group (CH 3 ) are not displaceable. 

If an acid is neutralized it is the hydrogen which has been substi- 
tuted by some other cation. 

Since it is the hydrogen (H) which determines the strength of an acid 
and this hydrogen (H) has definite values in every acid then it must be 
possible to establish a standard of measurement by taking a gram- 
molecular solution as of hydrochloric acid (HC1) which is called a normal 
(N) solution. On the other hand, alkali solutions may be established 
of standard values against the acid standards. In these the hydroxyl 
(OH) ions act as the neutralizing agents against hydrogen (H) ions 
of the acids in such proportion as to form water. 

"True Reaction" ("True Acidity," "True Alkalinity," "True 
Neutrality").*- -The "true acidity" of an acid solution is brought about 
by the dissociated (hydrogen) ions; therefore the acidity is proportional 
to the concentration of the dissociated hydrogen ions, and not to the 
total gram molecules of acid present. For example, if one-tenth normal 
hydrochloric acid is taken, approximately only 91 per cent, of the total 
amount of acid becomes dissociated. The "true acidity," i.e., the 
hydrogen ion concentration, of this solution is only 91 per cent, of the 
one- tenth normal hydrochloric acid, or ninety-one thousandths normal. 
The dissociation of weak acid is still less. For instance, in a solution of 

* Bull. 167, Mass. Agr. Exp. Sta. by Arao Itano. 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES l6l 

one- tenth normal acetic acid only i . 3 per cent, approximately of the 
total acid is dissociated, and the hydrogen ion concentration of this 
solution is therefore thirteen ten-thousandths normal. The 'true 
acidity" of one- tenth normal hydrochloric acid is also about seventy 
times greater than that of one-tenth normal acetic acid, although both 
solutions contain the same amount of acid. 

The same holds true with the electrically dissociated base in which 
the metallic and hydroxyl ions are dissociated. The " true alkalinity' 
of such a solution is not determined by the total amount of base 
present, but exclusively by the concentration of dissociated hydroxyl 
ions. For example, in a one- tenth normal solution of the strong base, 
sodium hydroxide, about 84 per cent, of the total amount of the base is 
dissociated, and in the case of a weak base, such as ammonium hy- 
droxide, approximately 1.4 per cent, of the total amount of the base. 
The "true alkalinity' of these solutions, therefore, is eighty-four 
thousandths normal and fourteen thousandths normal, respectively. 
Thus, regarding the alkalinity as in the case of acidity, we may say in 
conclusion that "true alkalinity" of a solution is proportional to the 
concentration of hydroxyl ions. 

From the above discussion, "true neutrality" of a solution may be 
stated as follows : it is a solution in which the same amount of H and OH 
ions are present. For example, a "true neutral solution," viz., pure 
water, contains as many hydrogen ions as hydroxyl ions. It can be 
expressed as follows: 

+ 
H 2 O <=> H + OH 

in which CT-^ CTT, C indicating the concentration. 


Again, a solution may not necessarily be neutral, although it con- 
tains equivalent quantities of acid and alkali. For example, if a 
solution which contains hydrochloric acid and sodium hydroxide is 
taken, it can be expressed in the following manner: 

+ + - + - 

H_ Cl + Na OH Na Cl + HOH 

hydrochloric sodium hydroxide salt water 

acid 

This solution is neutral only when it contains just as many hydrogen 
as hydroxyl ions, or when both the acid and alkali are equally 

dissociated. 
11 


l62 PHYSIOLOGY OF MICROORGANISMS 

It is understood, therefore, that the "true acidity, alkalinity and 
neutrality' are not determined by the amount of such substances 
present, but entirely by the H and OH ion concentration. 

Theory of H Ion Concentration-^^ announcement of the theory of 
electric dissociation by Svante Arrhenius, in 1887, marked a new era in 
physical chemistry. It was F. Kohlrausch and A. Heydweiller who 
demonstrated that even the purest water is a conductor of electricity, and 
accordingly prepared a distilled water of the least specific conductance. 
They measured the specific conductance by means of electric conduc- 
tivity. Later, other methods for the estimation of dissociation were 
established, and the results obtained by Kohlrausch were confirmed. 
Now it is proved that a very small portion of the water molecule is 
dissociated into two electrically charged parts (or ions), as follows: 


H 2 O * H + OH 

Its dissociation takes place according to the law of mass action in 
accordance with the following equation:- 

(H)(OH) _ 


in which K denotes the ionization constant; that is to say, the product of 
the hydrogen and hydroxyl ion concentration, divided by the concentra- 
tion of the undissociated water molecule, should be constant. 

The concentration of water is generally constant. Therefore it may 
be expressed as follows :- 

(H).(OH) = Kw (2) 

in which Kw denoted K.H 2 O, or ionization constant of water. 

Equation (2) is another form of equation (i). 

This ionization constant of water has been determined by several 
noted physical chemists, and found to be io~ 14 at 22; that is, 

NOTE. (H) and (OH) express the concentration. 

(H).(OH) = Kw or 

Kw = io~ 14 (3) 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 163 

Since pure water is a neutral solution it contains the same number of 
dissociated hydrogen and hydroxyl ions. Therefore equation (3) can be 
expressed as follows: 

io~ 7 X io~ 7 = io~ 14 (4) 

That is, a pure water contains of each io~ 7 dissociated hydrogen and 
hydroxyl ions, or .000000 1 gram ions per litre, which is, in a general 

N 

term, one ten-millionth normal The acidity, alkalinity 

10,000,000 

and neutrality, therefore, are expressed in terms of hydrogen ion 
concentration in the following manner : 

Acid reaction (H) > io~ 7 
Alkaline reaction (H) < io~ 7 
Neutral reaction (H) = io~ 7 

That is, in an acid solution there are more than pram 

10,000,000 

molecule of dissociated hydrogen; in an alkaline solution, less; and in a 

neutral solution, just - gram molecule. Thus the reaction is 

10,000,000 { 

usually expressed in terms of hydrogen ion concentration unless it is 
indicated otherwise. 

From the above discussions it is readily seen that if the ionization 
constant is known, and the hydrogen ion concentration is determined 
experimentally, then the hydroxyl ion concentration can be calculated. 
The determination of hydrogen ion concentration is accomplished by 
the use of the gas cell, of which the principle is based upon the potential 
of the chain. This chain as described in physical chemistry, consists 
of 

Hg-HgC] | n/ioKCl | cone. KC1 | solution | Pt H 2 

calomel electrode concentrate (unknown) platinum elec- 

potassium trode saturated 

chloride with hydrogen 

in a dish. gas. 

The potential of such a chain can be determined by the usual physical 
method. Then the relation between the measurement of potential and 
hydrogen ion concentration can be calculated by the following equation :- 

P ~ Q-3377 _ 


-577 + 0.0002 (t 1 8) 
NOTE. (X) = notation of the concentration of ions. 


164 PHYSIOLOGY OF MICROORGANISMS 

where 

PH -the term adopted by S. P. L. Sorensen to express the exponent 

of gm.- equivalent of hydrogen ions per liter. 
P the total E. M. F. of the chain. It can be determined by the 
following equation, having the apparatus arranged as it is 
shown in the diagram : 

P = s : in which RI the bridge reading for the chain against 

an accumulator. 

R - -the bridge reading- for the ac- 
cumulator against the normal ele- 
ment. 

1.0189 the voltage of the normal element at 
1 8 (standard). 

0.3377 the sum of potential of calomel electrode (N/io KC1) and 
hydrogen electrode in a solution where the hydrogen 
concentration is normal (H) = i or PH = o. 

0.0577 thermodynamical factor at 18 which is influenced by tem- 
perature, 0.0002 for each degree centigrade, or it changes 
as follows : 

0.0577 H~ Q-OOO2 (t 1 8), of which t equals temperature 
at the time of determination. 

After PH is determined it is necessary to understand the value of 
H-ion concentration, although the experimental results are generally 
expressed in P H . It will be shown at the end of an example, illustrating 
the application of the formula as well. 


Example. 

f = ig.2C (constant during the experiment). 
RI = 307.0 (constant reading on the bridge at five minute 

interval). 

R = 500.2 (as above). 
E. M. F. of the normal element = 1.0189. 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 165 

Then the total E. M. F. of the chain can be calculated as follows:- 

30^0 x 500.2 N.E. N.E.= normal element. 

1000 Ac.' 1000 Ac. Ac. = accumulator. 

307.0 Ac. , . x = the chain. 

1000 1000 = scale on bridge. 

500.2 N.E. 
1000 Ac. 

1000 N.E. 


Ac. = 

500.2 

1000 X 1.0189 
500.2 

Substituting (2) in (i), 


(2) 


307.0 1000 X 1.0189 
1000 500.2 

= 0.6254 volt, which is expressed p. 

Substituting the value for p in the formula, 

0.525 -0.3377 


PH = 


0.0577 + 0.0002 (19.2 1 8) 

= 4.967 


or in terms of H ion concentration, 


PH = 4-967 = - 4-967(log. H) 
IO - 4 -967 = 1>0 g x io~ 5 


H = 0.000010789 

Besides the apparatus listed below, a H-generator was employed, 
which is a good-sized Kipp's generator used with a series of washing 
bottles and drying tube, consisting of (a) 30 per cent. KOH, (b) alkaline 
pyrogallic acid, (c) cone . H 2 SO 4 and soda lime in U-tube. Since a consid- 
erable amount of CO 2 is produced during the course of metabolism, the 
same precaution is taken as with blood. For this purpose Hasselbach's 
electrode with shaking arrangement is employed. 

In setting up the apparatus special attention should be paid to 
rigidness, insulation and temperature. In order to meet with these 
requisites the apparatus was placed on a big central table in the labora- 
tory. First, one dozen large glass rings of the same height were 


i66 


PHYSIOLOGY OF MICROORGANISMS 


distributed over the top of the table. These supported a thick glass 
plate on which several blocks of paraffine for each piece of apparatus 
were placed. Thus it was possible to obtain a perfect insulation. 

In preparing the different parts of the apparatus extreme care should 
be exercised to obtain an accurate result. The method for the prepara- 
tion of the normal element, calomel electrode, gas cell, and also calibra- 
tion of the bridge wire, etc., is described in detail in Findlay's " Practical 


FIG. 102. Apparatus employed in determination of H-ion concentration. 

DESCRIPTION OF DIAGRAM 

LI Lippmann's capillarimeter. 83 Two-way switch. 

L 2 Tungsten lamp. C Calomel electrode. 

A Accumulator. K Concentrated KC1 cup. 

N Western normal element. G Gas cell. 

Si Switch with quick short circuiting key. B Bridge. 

82 Three-way switch. P Thick glass plate. 

Physical Chemistry." Every contact should be carefully made, so that 
accurate readings can be obtained. It is worthy of mention that the 
diffusion potential between n/io KC1 calomel electrode and the solution 
to be tested is reduced by interposing the saturated solution of KC1 as it 
is indicated by K on the diagram. For the standardization of the elec- 
trode it was first platinized with general precaution; then the hydrogen 
ion concentration of the mixed solution (7 c.c. of m/i5 KH 2 PO 4 , 3 c.c. of 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 167 

m/ 15 Na 2 HPO 4 ) was determined at different intervals. After the read- 
ings became constant there was a difference of 0.0005 volts between the 
theoretical data and the results obtained. 

With the above facts in mind it becomes possible to enter upon a 
more intelligent discussion of the methods involved. It has been stated 
previously that most microbiological experiments, having for their 
purpose the study of reaction upon microbial life, fall under the follow- 
ing procedures : 

(a) Kisch's method. 

(b) Ordinary titration method. 

(c) Colorimetric method. 

It is well known that Kisch's method is a dilution method wherein a 
certain number of gram molecules of an acid or alkali are diluted to a defi- 
nite quantity for the purpose of ascertaining the influence of the reac- 
tion upon the life of bacteria. There are two distinct ways to apply 
Kisch's method, namely: (a) immersing the bacteria in different dilu- 
tions of acids or alkalis in pure water for different periods of time by 
means of silk threads or any other convenient agents, and then testing 
their vitality; or (b) adding a known percentage of acids or alkalis 
directly to the culture medium (usually solution). In either case the 
results obtained by Kisch's method indicate neither the influence of 
"true reaction" upon microbial life nor the influence of molecular 
concentration, because, as Lingelheim has shown, different acids of 
the same molecular concentration have varying influence upon micro- 
organisms, and the degree of influence is parallel to the dissociation 
constant of an acid or alkali. This is especially true in the case of 
the second manner of application, (b) , where adsorption is caused by the 
culture medium. 

The ordinary titration method is generally employed in adjusting 
reaction of culture medium, and also to measure the amount of acid or 
alkali produced in the course of physiological tests. This method is 
inaccurate in the study of physiological liquids containing more or less 
amphoteric substances and a comparatively small quantity of H or OH 
ions. In other words, it is impossible to determine the " true reaction' 
in such a liquid by this method. Fuller's and Schiiltz's methods of 
adjusting the scale of reaction of culture media are scientifically con- 
demned by the recent investigation of Clark, who showed the fallacies 
of the titrimetric method. Again, the adsorption phenomenon caused 


I OS PHYSIOLOGY OF MICROORGANISMS 

by the amphoteric substance in the course of titration is well known, 
and, in the case of albumin, is usually expressed in the following 
manner: 

+ 

In acid solution H. albumin. OH = H albumin + OH 

+ 
In alkali solution H. albumin. OH = Albumin OH + H 

The correctness of the above statement has been experimentally 
demonstrated by Sorensen, Clark and others. 

In many cases the colorimetric method gives fairly accurate results, 
but it has been noted that the presence of neutral salts as well as ampho- 
teric substances interfere with the determination. It may, however, be 
employed successfully if it is standardized for the particular liquid. 
Lately Clark and Lubs employed the principle of the colorimetric method 
for the differentiation of the colon- aerogenes family, using suitable 
indicators. They have based their experiment upon the wide diver- 
gence of the hydrogen ion concentration in a culture of one group and 
of the other, and distinguished this difference by means of paranitro- 
phenol or methyl red. The use of this method for physiologic work 
other than for microbiology has been practiced by many. Sorensen 
and Palitzsch determined the hydrogen ion concentration of sea water. 
Henderson and Palmer used it in determining the acidity of urine to 
diagnose normal and abnormal conditions. In any case, the colori- 
metric method should be standardized previous to its use, by means of 
the hydrogen electrode. 

Examining these methods critically in the light of physical chemistry 
they are not satisfactory for the purpose of ascertaining the influence of 
the so-called 'true-reaction' 1 upon microbial life. The hydrogen 
electrode was devised to determine the hydrogen ion concentration, 
and it has been used successfully in biologic fields. 

SURFACE TENSION 

Due to such forces as cohesion and adhesion the particles of bodies 
have a tendency to come together in the same manner as bodies fall to 
the earth. This property appears to lie within the molecular forces 
of the body and seems to have a circumscribed and limited area of 
action. If a center is assumed in the form of a molecule, this area 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 169 

over which an influence of attraction is exerted would be in the form 
of a sphere and would be recognized as the sphere of molecular action. 
The layer of a liquid representing its surface plane with a depth 
equal to the radius of the sphere of molecular action would be the surf ace 
film. If a particle lies within or inside of this surface film it follows 
that with this particle as a center, the radius of its sphere of activity 
will extend beyond and above the surface film, but if this particle lies 
without and below this surface film the molecular forces on all sides will 
be equal and an equilibrium established. 


B 


FIG. 103. Illustrating surface forces. 

This is illustrated in Fig. 103. AB is the plane surface of a liquid. 
TV is a particle with its circumference indicated in which all forces are 
equalized. A T/ is a particle in which the forces downward are greater 
than the forces upward. The forces lying above the plane surface of 
the liquid AB appear to be less than the forces operating immediately 
below the plane surface AB in the liquid, yielding a considerable 
increase of pressure in the liquid. This increased pressure is known 
as the molecular pressure of the liquid. 

The surface film described above possesses a pull or is under tension 
or is the surface tension of the liquid. If an iron ring has stretched across 
its interior surface a soap film and a silk- thread loop is carefully rested 
upon it and run to the iron ring, the film inside the silk loop may be 
broken readily by any penetrating substance when the sides of the loop 
will spread out in the fullest degree drawn by the soap film without. 
Much like this is the floating of a rubber band on water. If a rod 
dipped into alcohol is touched to the surface of the water within the 
band the water film without pulls the band into its full circular form 
(Fig. 1046) through the reduction of the surface tension of the water 


1 70 PHYSIOLOGY OF MICROORGANISMS 

within by the addition of alcohol. This pull of the water without may 
be broken by the addition of a trace of alcohol. In this case the rubber 
band again resumes its former shape (Fig. 1040). 


a. 

FIG. 104. Illustrating surface pull. 


In the case of an oil drop on water the oil runs to a ball because of 
the cohesive forces within the oil and the lack of sufficient gravitational 
and molecular forces or pulling forces within the water film. Mercury 
for the same reason distributes itself in many small globules when split. 
On the other hand if the forces below or upward attraction has a 
stronger pull than the cohesive forces, then the oil would spread out as 
on a clean glass. 

The definite reactions resulting from experiments as employed in 
demonstrations of the above nature at once establish the possibility 
of accurate quantitative measurements. It has been found that 
substances vary very materially in their surface tensions. Kimball* 
gives the following table: 

SURFACE TENSIONS IN DYNES PER CENTIMETER 

Air Water Mercury 

Water ................ 73.5 412 

Mercury ................... ............ 539 . o 412 

Olive oil ............................... 34.3 20.6 335 

Alcohol ................................ 24 . 5 ..... 

Ether ................................. 17.6 ..... 

* "College Physics." For Method of Measurement, also consult Kimball. 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 1 71 

The possible effect surface tension may have upon the outer layer of 
protoplasm constituting a cell and in the formation of a membrane, 
its relation to nutritional functioning and in cellular movements, its 
suggestiveness in connection with form and its probable importance 
with alterations of various kinds render it a topic of prime import- 
ance although its values are very much dimmed by incomplete 
knowledge. 

ADSORPTION 

Spongy platinum has the power to take up considerable quantities 
of hydrogen gas and also oxygen gas into its mass; charcoal takes color- 
ing material from solutions; it also takes up gases; platinum black takes 
up acetic acid; calcium carbonate takes up sodium nitrate. When 
substances are so taken they are said to be adsorbed. This power seems 
to be resident in the adhesive forces of the extensive surfaces which 
exist through the multiplicity of particles in the substance as in charcoal. 
It has been defined as the local concentration or condensation of dis- 
solved substances at the interface between two phases. For instance, 
the interface existing between the dispersoid phase and dispersion 
means intensifies the surface action to such an extent that there is a 
concentration, a condensation. Reactions are apparently accelerated. 
The contact of hydrogen and oxygen in spongy platinum produces 
water. The action many times is that of catalysis as the oxidation of 
alcohol to acetic acid by platinum black. The adsorbing substance 
does not seem to enter into the chemical reaction which may occur but 
may be recovered intact. 

These reactions are influenced by temperature, pressure, electric 
forces and nature of the substance. 

By this phenomenon of nature soluble salts are held back in soils 
and not washed away by rains. The action of certain disinfectants is 
explained by the deposition or concentration on the surfaces of micro- 
organisms; the reaction of toxin with antitoxin simulates adsorption 
phenomena more closely than mass action; the sensitization of bacteria 
by opsonins and the ingestion by leucocytes also resemble adsorption 
acts; the peculiar reactions of enzymes are regarded as similar to ad- 
sorption; the formation of a membrane upon exposed protoplasm in 
the case of a crushed protozoon also appears to be the result of the 
adsorptive action of certain substances. 


172 


PHYSIOLOGY OF MICROORGANISMS 


BROWNIAN MOTION 

This phenomenon is familiar to students of microbiology. When 
studying some bacteria in a hanging-drop under one-twelfth oil immer- 
sion objective, this movement may be seen. It is not only visible with 
some of these living organisms but extends to many substances existing 
in very fine particles and suspended in certain media. It is a common 
phenomenon among colloidal solutions. 


FIG. 105. Illustrating Brownian movement (After Perrin}. 


The character of the movement is well illustrated by Perrin* (Fig. 
105) who has made a special study of the subject. The path is a 
straight line until opposed when it rebounds in another straight line 
producing a zig-zag route. 

The cause of the motion appears to be inherent in the molecular 
movements of the dispersion means of a colloid, of the liquid in which 
the particles are suspended. The direction of the particles as stated 
above, is that of a straight line until a collision with the invisible mole- 
cules takes place when the rebound sends the particles in a straight line 
in another direction. This process continues indefinitely. The 

* Perrin, M. Jean, Brownian Movement and Molecular Reality. 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 173 

particle subject to these molecular movements and forces responds on 
the whole as a football might be knocked indiscriminately about a field 
by a group of unorganized school-boys. 

Such movements of colloidal particles are supposed to render 
colloidal solutions more stable. This taken together with the density 
of the dispersion means, its viscosity, the size of the particles in which 
surface action becomes more evident, and the electric charge probably 
accounts in large part for the permanency of the dispersoid state. The 
velocity of the movement of particles depends upon many of the factors 
associated with colloidal permanency. An increase of temperature 
quickens the movement not through convection currents but by the 
molecular activity; viscosity acts in a seeming frictional capacity; 
the density acts as if there was a tendency to close in on the particles 
with forces which are made effective through the multiplicity of mole- 
cules; size apparently is much like keeping a small ball in the air as 
compared with a large ball. 

Again, the size of particles which are subject to exact measurement 

^ 

is related to^the rapidity of their movement. Exner has made this 
comparison :- 

Diameter of par- Velocity of particle 

ticle in y. in n per second 

i-3 2 -7 

o.Q 3-3 

0.4 3-8 

It will be seen that the smaller they are the more rapidly they move. 

Brownian motion, because of the forceful drive furnished by the 
molecules, appears to be an important factor in diffusion and osmotic 
bearings. 

DIFFUSION, OSMOSIS, DIALYSIS, PERMEABILITY 

If a twelve per cent, warm gelatin solution is brought in contact 
with water of the same temperature, currents, not convection currents, 
are seen radiating, spreading and extending from the gelatin solution 
into the water until finally they merge with the water and are lost to 
sight, when the entire mass becomes uniform and homogeneous. A 
strong salt solution, when placed in the bottom of a cylinder and water 
carefully poured above it, will little by little work up into the water 
until the whole is one homogeneous concentration. This would also 


174 PHYSIOLOGY OF MICROORGANISMS 

be true if the water in the former case were substituted by a weaker 
solution of gelatin or, in the latter case, by a weaker solution of salt. 
There is a tendency to equalize or become uniform and homogeneous. 

Microbiologists are also familiar with certain special phenomena. 
Litmus agar becomes reduced by the growth of microorganisms. Oxy- 
gen has been consumed. When the culture is allowed to remain ex- 
posed to the air for a time, the microorganisms cease to grow and 
multiply; the litmus, beginning at the top, gradually resumes its color 
as the air works its way down through the culture. There has been a 
gradual diffusion of the air throughout the litmus agar. - Many cul- 
tural phenomena could be recalled in this connection. One will suffice. 
The heating of culture media to drive off the air for anaerobic cultivation 
is of frequent occurrence, for it is well known how the air soon penetrates 
when media are allowed to stand. 

Apparently there are encountered in the first two paragraphs dis- 
tinct phenomena or a single phenomenon modified in the one or the 
other instance. The usual explanation, however, is covered by the 
word "diffusion" 

The recent developments in the understanding of diffusion attribute 
to diffusion the same forces operating in gases. It is the drive possessed 
by the molecules to expand or press out until equalization or equilibrium 
is established. This movement is from the more concentrated solution 
toward the less concentrated or toward the pure solvent. The nature 
of a substance, difference in concentration and temperature materially 
influence this movement. 

This accords with the forces of osmosis as well: The pressure upon 
the obstructing membrane through which the particles, molecules or 
ions of a substance are attempting to make their way is called osmotic 
pressure; the particles are held back or restrained in their movements 
outward. It has been found, however, that " the osmotic pressure of a 
dissolved substance is exactly the same as the gas-pressure which 
would be exerted if the solvent were removed and the dissolved sub- 
stance in gaseous form were left behind to occupy the same volume 
at the same temperature." It is also known that " where two liquids 
which will mix are separated only by a porous membrane there is a 
movement of the liquid in both directions through the, membrane. 
The greater movement is usually from the less dense to the more dense 
so as to cause the line of the more dense liquid to rise above that of the 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 175 

less dense. This action increases with the temperature and is pro- 
portional to the concentration of the solution. In the illustration of 
diffusion above by means of gelatin and salt, diffusion follows its natural 
course; but in the case of oxygen penetrating litmus agar or any other 
medium the action may be regarded as modified diffusion or osmosis in 
which the medium acts as a barrier to the medium-content but allows 
the gas (air) in its drive onward to pass and diffuse throughout. This 
leads to the significance of permeability of membranes. 

Much attention has been given to the study of membranes as 
they relate so closely to the membranes of cells which are concerned 
with living processes. It is more or less simple to demonstrate 
the passage of water and the restraining of a substance like sugar 
by means of parchment paper. This is a common experiment. In a 
thistle tube with its mouth covered with parchment paper place a 
sugar solution to the neck. When plunged into water, the water will 
pass in and appear in the rising line. At the same time no sugar 
passes through and out into the water. The molecules appear too 
large to pass through the pores. This membrane is semi-permeable 
since it permits water to pass but restrains sugar. A membrane or 
anything which does not allow anything to pass, as glass, would be 
called impermeable. 

Whether dialysis (passage through a membrane in the separation 
of colloids and crystalloids) or the permeability of membranes is 
traceable to its sieve-like nature, its chemical reaction, or to its solvent 
action or to more than one of these is a mooted problem of prime 
interest but out of place in this consideration. Some data throwing 
light on the action of membranes may be helpful, however. The 
Bechhold ultra-filters made of collodion, which may be graded to vary- 
ing porosities, have been employed in such a manner as to illustrate the 
permeability of membranes. Some substances will pass while others 
will not until the size of pores are adjusted. The membrane resulting 
by the contact of potassium ferrocyanide with copper sulphate allows 
water and potassium chloride to pass while it withholds potassium sul- 
phate and other salts. In nature membranes may be permeable to 
certain salts at times and impermeable at other times. Osterhout 
has demonstrated many of the possibilities of protoplasmic permea- 
bility. Speaking in very general terms, permeability as manifested in 
living cells and measured by electric conductivity, as has been the case 


iy6 PHYSIOLOGY OF MICROORGANISMS 

with Osterhout's investigations, may be decreased in its reaction to 
sodium chloride by alkaloids, as caffein, nicotine and cevadine, by bile 
salts as sodium taurocholate, and by acids as hydrochloric acid; on the 
other hand, .it is increased by alkalis, by certain isotonic combinations 
of salts or balanced solutions and by acids following the first stimula- 
tion. Protoplasm may vary widely from the normal in its permeability 
and both vegetable and animal cells respond in much the same general 
manner. 

Although these specific facts may be very limited compared with 
the entire field of permeability possibilities to which a living organism 
is exposed, they do, however, indicate that the membrane or protoplas- 
mic protective surfaces have the power to act in a selective manner 
per se or to yield to environing forces or influences which control or 
make life possible by antagonisms, reactions, neutralizations and other 
agencies among themselves.* 

Osmotic pressure, following the laws of gas pressure, represents 
the pressure exerted by the particles of a given volume of a solution. 
The particles, molecules, or ions, of the solution, as in gas are constantly 
on an outward drive, an expansive drive, and they carry with them 
much force which is proportional to the concentration of the solution 
and is subject to the influence of temperature as stated previously. 
Also the osmotic pressure of a given quantity of substance is inversely 
proportional to the volume (p. 174). When, therefore, a solution of a 
great concentration is separated from that of less concentration with a 
semipermeable membrane between, the pressure exerted on each side of 
the membrane will be proportional to the concentration of the solutions. 
The pressure will be influenced by temperature and there will be a 
stirring of the unequal forces to gain an equilibrium. If only the solv- 
ent in the two solutions of different concentration, as just referred to, 
passes the membrane, then there will be movement toward and a grad- 
ual dilution of the more concentrated until it becomes equalized with 
the other; if both solvent and solute pass there will be by the passage of 
both through the not truly semipermeable membrane an effort to 
equalize with more or less exchange from both solutions as in the case 
of obstructed diffusion. 

* The writers call especial attention to Osterhout's work and that of his students as published 
in the Journal of General Physiology, Journal of Biological Chemistry, Science and the Botanical 
Gazette. 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 177 

In the discussions of osmotic pressure there has been constantly in 
mind the action of solutions upon microorganisms. Either a cell-wall 
or membrane exists as a distinct structural part as in the yeast cell or 
the protoplasm comes in contact with its surrounding medium without 
any distinctive cell-wall or membrane as in the amoeba. Whether 
there is a layer of protoplasm on the outer surface of the amoeba which 
has the functioning capacities of a distinctive cell-wall may not be easily 
asserted for there is evidence pointing to the two possibilities. Inas- 
much, however, as the passage of materials into the substance of the 
cell is really that of diffusion or a modification of it, and species and 
varieties respond differently to this diffusion, it is easily seen that 
every species at least must be considered by itself in this respect and 
values likewise determined. 


-p 


FIG. 106. Plasmolysis in cells (After DeVries from Macleod). 

It is well known that water will pass into some cells and cause them 
to swell or fill out when apparently the substance of the cell or its fluid 
content is more concentrated than the surrounding medium. On the 
other hand, when the medium without is more concentrated than the 
cell-contents, water flows from the cell toward the more concentrated 
solution outside of the cell and accordingly the cell shrinks. This is 
many times made evident by the contraction of the protoplasm. This 
process in which the water is abstracted from the cell through osmotic 
pressure is known as plasm oly sis. 

COLLOIDS AND CRYSTALLOIDS 

Since the time of Thomas Graham who established these two 
classes of substances there has been a growing interest in them. At 
present, however, instead of dividing substances into two classes 

placing one substance in one class, as colloids, and another distinct 
12 


1 78 PHYSIOLOGY OF MICROORGANISMS 

substance in the second class, as crystalloid, one and the same substance 
may exist in both classes. Therefore, two conditions or states of the 
same substance may be found, the one the colloidal, the other the 
crystalloidal condition or state. Consequently, substances cannot be 
divided in accordance with the early views of Thomas Graham, but 
the conditions or state under which they exist, may be so divided into 
colloids and crystalloids. The resolution of these classes, as will be 
seen, is fraught with many difficulties. 

The usual ultimate chemical and physical conception of matter is 
molecular and atomic. Associated with this are physical properties 
and qualities. Comparatively recently, matter has taken on new 
interpretations for the molecule and atom have extended to the electron 
and sub-electron possessing definite electric potentialities. In the 
opposite direction there appears to be an aggregating or massing power 
along with the solvent belonging to the molecule in which the atom and 
electrons may be active. This aggregating power does not seemingly 
manifest itself in the same manner with all substances; in other words 
the particle resulting from this aggregation in the case of hydrated 
silicic acid may not be executed in the same manner as in the case of 
ferric hydroxide; in the case of gelatin, as in the case of casein; in the 
case of particulate gold as in the case of particulate carbon. Such 
aggregate particles, apparently, are different from the molecular or 
atomic particles in their structures and reactions and the term aggre- 
gate does not convey the true structural nature. In molecular reactions 
chemistry follows its usual course; in the particulate reactions, physical 
manifestations form the basis of recognition. These differentiations, 
while helping to distinguish between the well known structures met in 
crystalloidal chemistry and the more or less amorphous structures of 
colloidal chemistry cannot be held as a fast cleavage line because they 
merge into each other and too little is understood of the structure of 
colloids. They, however, are suggestive, directive and helpful. 

Crystalloids form, as a rule, true molecular or ionic solutions (see 
Solutions, p. 156) while colloids form solutions of a more or less mechani- 
cal character; the former produce a uniformly dispersed homogeneous 
system not separable mechanically, the latter give rise to a solution 
mechanically separable and not uniformly dispersed a heterogeneous 
system. Also the former give rise to a one-phase system while the 
latter yield a polyphasic system. The solution of colloids is concretely 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 179 

illustrated by reference to casein in milk, or gelatin in aqueous solution, 
which is easily grasped to differ from a solution of salt, a crystalloid, in 
water. In colloidal solutions the particles are referred to as the disperse 
phase, the medium in which they are found, the dispersion means and 
the solution as a whole, a dispersoid. In the event that gold be reduced 
so fine that its suspension gives rise to a colloidal solution, gold would be 
the disperse phase, water the dispersion means and the solution or 
suspension as a whole, the dispersoid. The gold would also represent 
one phase and the water another phase, resulting in a diphasic hetero- 
geneous system. Where the gold particle and the water meet or at the 
point the disperse phase and dispersion means come together or are in 
contact is the so-called interface so important in surface energy. Some- 
times the disperse phase is called the internal phase and the dispersion 
means the external or continuous phase. 

Dispersoids exist as suspension-colloids or suspensoids and emulsion- 
colloids or emulsoids. The former designate the disperse phase to be a 
solid and the dispersion means a liquid (lyophobic colloids)', the latter 
designate the disperse phase to be a liquid and the dispersion means also 
a liquid (lyophilic colloids). As an example of the former, colloidal gold 
as the disperse phase and water as the dispersion means is satisfactorily 
typical; as an example of the latter, gelatin as the disperse phase and 
water as the dispersion means qualifies, although the gelatin is very 
close to a solid at times but probably still in a hydrated condition. 

This attempt to divide the colloidal condition or state into two 
classes is quite general. In the above paragraph Von Weimarn* and 
Ostwald have made the division into suspensoids and emulsoids, 
Perrinf into lyophobic and lyophilic. NoyesJ contributes another 
division: "As types of these I would draw your attention to these 
aqueous solutions of gelatin and of colloidal arsenious sulphide. The 
former class possesses a much greater viscosity than that of water; the 
latter does not appreciably differ from it in this respect. The former 
gelatinizes upon cooling or upon evaporation, and passes again into 
solution upon heating or addition of the solvent; the latter does not 
gelatinize upon cooling, and if gelatinized by other means it does not 
redissolve upon heating. The former is not coagulated by the addition 
of salts (unless in excessive amount), the latter immediately gives an 

* Von Weimarn, Grundzuge der Dispersoid Chemie (Steinkopff, Dresden), 1911. 

t Perrin, J., J. Chim. Phys., 3, 5O, 1905. 

J Noyes, A. A., Jour. Amer. Chem. Soc. 27, 2. p. 85, 1905. 


I So PHYSIOLOGY OF MICROORGANISMS 

abundant precipitate. We have therefore to distinguish the viscous, 
gelatinizing, colloidal mixtures, not coagulated by salts, from the non- 
viscous, non-gelatinizing, but readily coagulable mixtures. The 
former class I shall designate colloidal solutions, the latter colloidal 
suspensions" Other divisions of much the same character have been 
suggested. All lack in fundamental significance. They follow much 
the same cleavage line but it possesses a ragged fringe. Whether of 
great or permanent value or not, it is useful until a more definite, basic- 
ally sound, division can be established. 

Colloidal solutions may exist in which the disperse phase may be 
found in other dispersion means than water. These with water are 
generally known as sols. When the dispersion means is water, the so- 
lution or suspension is specifically called hydrosol; in alcohol, alcosol; 
in glycerol, glycersol; etc. If the disperse phase takes up a certain 
amount of water, it may enter into a jelly-like condition when it is 
generally called a gel. In this instance, it would be called specifically 
a hydrogel. It is possible to have as well alcogels, sulphogels, etc. 
Gelatin may exist in a colloidal solution as a hydrosol and also as 
a hydrogel depending upon the amount of water employed. There 
also always exists the possibility of the disperse phase taking up 
some of the dispersion means and the dispersion means actually in- 
corporating some of the disperse phase. To what extent this may 
be carried is problematical. 

It has already been indicated that colloidal solutions differ from 
crystalloidal. The crystalloidal solutions are true molecular or ionic 
solutions. The molecule may or may not divide into ions. Sodium 
chloride passing into solution breaks into ions carrying with them 
a positive and negative electric charge which in turn create a cur- 
rent of electricity. The cane sugar molecule on the other hand does 
not break up but goes into a molecular solution; there are no positive 
and negative ions, consequently no electric dissociation. Substances 
which ionize as sodium chloride are called electrolytes while substances 
as cane sugar are non-electrolytes because they do not ionize. The 
colloids, too, like sugar, are non-electrolytes and do not ionize, yet they 
respond to a current of electricity passed through a solution. The 
particles of a colloid have a tendency to pass to one pole or the other 
depending upon the nature of the colloid. This reaction is called 
electrophoresis. Further, it may be said that, if colloids pass toward 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES l8l 


the anode, they are negatively charged, if toward the cathode positively 
charged. The significance of this movement of the particles of different 
colloids in response to an electric current passed through a solution does 
not seem to be clearly understood. 

The size of the particles existing in a suspensoid or an emulsoid or 
even in a molecular solution is of considerable importance from the 
standpoint of stability, reaction to light and many other phenomena. 
Ostwald* presents the matter very tersely in the following diagram 
which has been slightly modified by the writers. 

Dispersoids 


s 

True or coarse 

dispersions 

(suspensions, emulsions, 
etc.) 

Size of the particles of 

disperse phase greater 

than o.iu 


\ 


Colloidal solutions 

(Suspensoids, emulsoids, 

etc.) 


\ 

Molecular or 
supermolecular 
dispersoids 


Size of particles of the Size of particles of the 
disperse phase between disperse phase about 

o.ifj. and i MM I MM r less 


Colloidality decreases 


Degree of dispersion increases. 
FIG. 107. An arrangement of dispersoids. (After Ostwald.) 

This graphic presentation can be still better understood by giving also 
the illustration provided on page 30 of the same publication (Fig. 8) 
of this publication (Fig. 108). 

By use of the ultramicroscope developed by Siedentopf and Zsig- 
mondy it has been possible to employ Tyndall's phenomenon which 
makes the visibility of rays of light passing through a medium depend- 
ent upon solid particles as dust in the air of a room. The light must 
enter into a dark room as a ray from one side only to illuminate the 
particles and render the demonstration successful. In the same man- 
ner particles suspended in a transparent medium may also be illumin- 

* Ostwald, Wolfgang. Handbook of Colloid Chemistry, p. 33. 


182 


PHYSIOLOGY OF MICROORGANISMS 


ated. The ultramicroscope makes it feasible to use Tyndall's phe- 
nomenon effectively in revealing particles of some colloidal substances 
and solutions having particles of larger dimensions. Siedentopf and 


Anthrax 
bacillus 

aboum 
6fj long 


Particles or colloid gold 


D Precipitated parhcle 
oF gold, about- 75 ^Jfj 


Starch Chloroform Hydrogen 
molecule molecule molecule 
about0.8uu abou 


tn/argemenf- 1 000 000 to 1 


Particles of a fine mastic suspension 


Enlargement- 3333 to 1 


FIG. 108. Comparison of particles of different sizes. (Ostwald.} 

The large circle corresponds to the diameter of a human red blood corpuscle 
(about 7.5 M); the large pentagon to that of a starch granule of medium size (about 
7.0 n). The particles enclosed in a frame are, in comparison with the rest of the 
figure, enlarged 333 times. 

The figure has been constructed from data and tables given in R. Zsigmondy 
(Zur Erkenntnis der Kolioide, Jena, 1905). The values for the mastic suspension 
are taken from /. Perrin's studies [Kolloidchem. Beihefte I, 221 (1910)]. 

Zsigmondy find that the microscope has its limitation of visibility at 
about o.ifj. and the ultramicroscope at about i.ojuju (submicron) or 
o.ooiyu. There are particles existing beyond the reach of the ultra- 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 183 


microscope which are designated in size by the term amicrons. Accord- 
ing to Zsigmondy the size of the particles covered in colloids ranges 


a O 


1 


FIG. ioga. Arrangement of ultramicroscope. (After Bayliss.} 


FIG. 1096. Rays of light in ultramicroscope. (After Bayliss.} 


from o.i/-t to 
cules : 


Ostwald gives the estimated sizes of certain mole- 


Hydrogen gas ................................... 0.067-0.157^ 

Water vapor .................................... 0.113^ 

Carbon dioxide ........................ .......... o . 285^ 

Sodium chloride .................................. o . 26^ju 

Sugar ........................................... o . 7/j.fj. 

Some conception of the size of molecules and colloidal particles, 
although they may not be absolute and even subject to great range or 
variability, contributes to an understanding of colloidal and molecular 
solutions, osmotic action, life-activities, lower limits of size of micro- 
organisms and other natural phenomena. 

The "disperse phase" of colloidal solutions suggests at once the 
extensive surface made possible by the particles in suspension and must 
likewise suggest the extent of surface energy present in the form of 
surface tension and adsorption. These factors are largely involved in 


184 PHYSIOLOGY OF MICROORGANISMS 

colloidal reactions and life-functions. Their bearing has been already 
indicated (See 168). 

It has already been said that Thomas Graham made the distinction 
between colloids and crystalloids by means of dialysis through a mem- 
brane, the colloids are withheld and crystalloids pass through. This 
movement on the part of these substances follows the laws of diffusion 
which, in turn, conform with the laws of expansion of gases. In the 
case where the membrane obstructs the movement of colloids and 
permits the crystalloids to pass there can be recognized an interference 
with free movement. Whether the colloidal molecule is larger than 
the crystalloidal molecule, which appears to be a fairly satisfactory 
undemonstrated reason, or not, does not materially alter the situation; 
or whether some chemical transition or obstruction accounts for this 
phenomenon of passage and check, in our present position, does not 
contribute much without a real working knowledge of what is involved. 
The facts remain: Colloidal substances do not pass while crystalloids 
do. This significant condition may be actually responsible for the 
cell-entities which incorporate the mechanism of life. 

In colloids, diffusion is slow, slower than in the case of the crystal- 
loids. This enables the crystalloids to penetrate or diffuse through the 
colloidal substances as protoplasm and sustain what must be regarded 
as a more or less fixed substance, protoplasm, through the very nature 
of its powers. 

The microbial cell is generally a unicellular organism which secures 
its nutrition and performs its respiratory functions through the surface 
layer of the cell. This outer layer in most microbial cells takes the 
form of a membrane and where no membrane exists the cell seems to 
respond in much the same manner through its protecting surface layers 
of protoplasm. A yeast cell prepares its food which is not assimilable 
through its cell-wall by secreting suitable enzymes to produce diffusible 
nutrition. Such portions of this solution are assimilated through the 
cell- wall as are needed in cell-construction and are converted by similar 
processes within the cell substance while in transitional route to 
protoplasm itself. In the case of an amoeba the particle of food is 
often taken within the protoplasm by means of its pseudopodia and 
after digestion is assimilated as in the yeast cell. This process in the 
amoeba cannot be regarded as at all different from that of the yeast for 
the digestive-preparatory process and assimilation are much the same. 


PHYSICAL FORCES INVOLVED IN BIOLOGICAL ACTIVITIES 185 

When food is prepared it is probably in the form of a molecular or 
ionic dispersoid which enters the substance of the protoplasm and 
diffuses readily. The ionization of the cell is, according to many 
authorities, dependent upon the ionic or molecular dispersoids which 
are found in the cell substance, whether they are on their way to become 
protoplasm or are the products of cell activity. When these ionic or 
molecular dispersoids of the cell are of a nature and possess the affinity 
to attach themselves to molecules of protoplasmic structure, their 
diffusibility is lost and they become anchored; if, however, there exist 
diffusible substances which are cast off from the protoplasmic molecules 
by metabolic action and no longer possess the affinity for attaching 
themselves, their dissipation by elimination is assured. The change of 
starch, glycogen, protein, as food, to diffusible products by regulation 
digestive processes and the elimination, as waste products, of diffusible 
substances have a tendency to confirm this vital interpretation. 

Literature freely consulted and recommended for extended study. 

BAYLISS, The Principles of General Physiology. 

BURTON, Physical Properties of Colloidal Solutions. 

CLARK, W. M., The Determination of Hydrogen Ions, 1920. 

HATSCHEK, Colloids. 

HOBER, Physikalische Chemie der Zelle und der Gewebe. 

ITANO, The Relation of Hydrogen Ion Concentration of Media to the Proteolytic 

Activity of B. subtilis. 
JONES, Nature of Solutions. 
KIMBALL, College Physics. 

MACLEOD, Physiology and Biochemistry in Modern Medicine. 
McCLENDON, Physical Chemistry of Vital Phenomena. 
MICHAELIS, L., Die Wasserstoffionenkonzentration. 
NICHOLS and FRANKLIN, The Elements of Physics. 
NORTHRUP, Laws of Physical Science. 
OsxwALD-FiscHER, Handbook of Colloidal Chemistry. 
PERRIN, Brownian Movement and Molecular Reality. 
PHILIP, Physical Chemistry. 

SORENSEN, S. P. L., Ergebnisse d. Physiologic, Bd. 12, 1912. 
VON PROWAZEK, Physiologie der Einzelligen. 
THOMSON, The Corpuscular Theory of Matter. 
THOMSON, Rays of Positive Electricity. 
WALKER, Introduction to Physical Chemistry. 
WASHBURN, Principles of Physical Chemistry. 
WELLS, Chemical Pathology. 


CHAPTER III 

CHEMICAL STUDIES OF THE CONTENT OF MICROBIAL 

CELLS* 

Microorganisms have a widely variable chemical .composition. 
They differ so much in their requirements their habits, their food 
needs, their moisture demands, their environmental atmosphere, and 
their capacity for change that their great deviation from a constant 
nature, as manifested by superficial expressions, perhaps, does not 
awaken unexpected mental responses. They also undergo much 
alteration in their compositional nature as well as in their structural 
nature while passing stages in their individual developments. The 
vegetative or growing forms do not seem to have the same composition 
as the spore-forms or resting forms although it may be quite possible 
that fundamentally the exact composition exists in both and only more 
superficial substances are detectable; old cells differ from young cells 
and capsulated forms from uncapsulated forms. Food influences 
greatly the products found in protoplasm both quantitatively and 
qualitatively. While such products which are referable to food may 
not be strictly a part of what is contemplated in the composition of the 
cell, yet it is difficult many times to make the distinction. Doubtless 
most influencing agents whether external or internal have some power 
over the substances now recognized in cellular composition. 

If, however, constancy in species is to be maintained, it is necessary 
to assume that there is to be found in every species a constant group or 
nucleus of chemical atoms or molecules whether existing independently 
or acting in consort in forming congeries of molecular complexes, and 
that substances fluctuating in their presence or in their amount must 
be regarded as more incidental to the basic life-processes. Species, 
therefore, even when undergoing all the recognized variations to which 
it is subjected ageing, developmental stages, reproduction, environ- 
mental factors as food, reaction, oxygen supply, temperature, and others 

* Prepared by Charles E. Marshall and Arao Itano. 

1 86 


CHEMICAL STUDIES OF THE CONTENT OF MICROBIAL CELLS 187 

remains basically constant, apart from its evolutionary possibilities, 
to its line of descent. 

The student, too, should not be led to interpret the products found 
by the chemists as the substances constituting protoplasm or any of its 
differentiated parts but rather as substances entering into the formation 
of the protoplasmic molecule, or as substances resulting from metabolic 
processes, or as substances connected in some way with the food supply 
as reserve material or as substances essentially foreign, having entered 
the cell by means of its mechanical functional acts. Ultimate analyses 
may reveal the percentages of N, C, H, 0, P, S and other elements; 
certain chemical methods may demonstrate the presence of proteins, 
amino acids, carbohydrates, and fats, and the ash may contain definite 
mineral constituents, yet such revelations are only the initial steps 
which will take the wandering industrious scientist or student to the 
museum of nature wherein are found the depicted substances and acts 
involved in living protoplasm. However, besides striving to obtain 
an .insight into the very nature of life and its operating processes, much 
has been accomplished by such studies in ameliorating the conditions of 
man's existence and in helpfulness. By having even this very limited 
knowledge as will be gathered from the study of metabolism, soil, food, 
immunity and infectious diseases, extending to agriculture, medicine 
and the industries, great progress is possible and has been made. 

ANALYSES 

Moisture. The moisture content of microorganisms has a very 
wide range. In the mother-of-vinegar made up largely of acetic 
bacteria, the moisture content reaches 98.3 per cent.; in Bact. pneu- 
monia,* 85.55 P er cent.; in the alga, Chlorella vulgaris,* 63.06 per cent.; 
in the spores of molds, 39 to 44 per cent. From this very brief survey 
it will be seen that all microorganisms vary greatly in their moisture 
content. The amount seems to be largely dependent upon the medium 
in which development takes place, unless it is in the case of spores which 

* Nicolle, M., and Alilaire, E., in Ann. Inst. Pasteur, T. 23, p. 555, furnishes the following 
moisture determinations in per cent.: Bact. mallei, 76.49; Bact. cholera gallinarum, 79-35; 
Msp. comma (Bombay), 73.38; Bact. dysenteries, (Shiga), 78.23; Proteus vulgaris (B. proteus), 
79.99; B. typhosus, 78.93; Bact. anthracis (asporogenic), 81.74; Bact. pseudotuberculosis, 78.83; 
Bact. pneumonia, 85.55; B. colt, 73.35; B. prodigiosus, pathogenic (de Fortineau), 78.00; B. 
psittacosis, 78.05; Bact. diphtherias, 84.50; B. pyocyaneus, 74.99; B. lymphangitis (de Nocard), 
77.90; yeast (Frohberg), 69.25; Chlorella vulgaris (alga), 63.6. 


1 88 PHYSIOLOGY OF MICROORGANISMS 

incorporate an amount which is difficult to remove and which has 
some relation apparently to their high degree of resistance. 

Molds have, as a rule, a greater moisture content than yeast and 
yeast a greater content than bacteria, yet these organisms have no 
constancy or uniformity in their moisture content. The protozoal 
forms are as dissimilar as others and their range of moisture content 
assumes no fixed boundaries. 

Although there is a minimum limit and a maximum limit as indi- 
cated on the one hand by desiccation and on the other hand by an 
inability to absorb more moisture, still retaining life one is forced to 
believe in a very restricted amount of moisture as essential to life- 
processes. Beyond this essential amount, in the case of too little, the 
metabolic activities cannot take place, and, in the case of an excessive 
amount, proper functioning is interfered with or a modification of 
physiological reactions gradually becomes more and more evident. 

Proteins and other nitrogenous substances. Nitrogenous compounds 
are present in varying amounts and are assumed to be the basis of 
protoplasm. The approach in the study of this class of substances 
has been made through the determination of nitrogen, then converting 
the nitrogen into terms of protein by the use of the recognized factor; 
by the recognition of definite nitrogenous compounds which may 
represent certain portions of the protein molecule; and by the use of 
reagents long employed to detect the presence of protein, largely 
qualitatively. All of these can furnish only inadequate means for the 
recognition of the nitrogenous materials which may enter into the 
formation of the active life-substance, protoplasm. However limited 
may be the knowledge available in this particular subject, there is now 
at hand sufficient to point the way for more and for certain directive 
practical purposes. The per cent, of nitrogen* found by Vaughan and 
his associates and by Nicolle and Alilaire ranges from 3.96 (dry weight, 

* Vaughan and Wheeler. "Protein Split Products in Relation to Immunity and Disease," 
by Vaughan, contributes the nitrogen determinations in per cent, for several bacteria: Typhoid, 
11.55; colon, 10.65; tuberculosis, 10.55; anthrax, 10.285; subtilis, 5.964; Proteus vulgaris, 6.791; 
Ruber of Kiel, 10.655; megaterium, 8.349; pyocyanus, 10.843; violaceus, 11.765; Sarcina 
auranliaca, 11.46. 

Nicolle, M., and Alilaire, E., in Ann. Inst. Pasteur, 23, 555, give the following nitrogen re- 
sults in per cent, (based upon dry weight), Bad. mallei, 10.47; Bact. cholerce gallinarum, 10.79; 
Msp. comma (Bombay), 9-795 Bact. dysenteries (Shiga), 8.89; B. proteus (Proteus vulgaris), 
10.73; B. typhosus, 8.28; Bact. anthracis (asporogenic), 9.22; Bact. pseudotuberculosis, 10.36; 
Bact. pneumonias* 8.33; B. coli, 10.32; B. prodigiosus (pathogenic) (de Fortineau), 10.55; B. 
psittacosis, 9-55; B. pyocyaneus, 9-791 B. lymphangitis (de Nocard), 9.17; yeast (Frohberg), 
10.00; Chlorella vulgaris, 3.96. 


CHEMICAL STUDIES OF THE CONTENT OF MICROBIAL CELLS 189 

in Chlorella vulgar is (alga) to 10.73 in B. proteus (Proteus mdgaris). 
In the protozoon, Noctiluca miharis, there was present 7. 74 per cent, of 
nitrogen as determined by Emmerling. * Molds and yeasts appear to 
lie between the alga named and many of the bacteria as indicated by the 
work of Marschall and Nageli.f 

The compounds of nitrogen which have been determined are quite 
numerous although it must be allowed that the analyses have not always 
been satisfactory. RuppelJ claims to have determined nucleic acid, 
nucleoprotamin, nucleoproteid, albuminoids (keratin etc.) in dried 
Bact. tuberculosis. Nishimura|| found nuclein bodies as xanthin, 
guanin, adenin in a water bacillus. Vaughan and his associates have 
been able to demonstrate the presence of various amino acids. The 
work of Emmerling* also contributes much which aids in our under- 
standing of definite substances in the protoplasm of protozoa. 

* Emmerling, O., Biochem. Zeitschr., 1909, gives this analysis of Noctiluca miharis: In 100 
grams of ash free substance there was 7.74 grams of nitrogen (Taken from S. von Prowazek: 
Physiologic der Einzelligen.) 

Lysin 0.212 with o . 040 grams nitrogen 

Arginin i .6492 with o .432 grams nitrogen 

Histidin 3.4762 with 0.938 grams nitrogen 

Tyrosin 0.5271 with 0.041 grams nitrogen 

Glycocoll 15 .9000 with 2 .956 grams nitrogen 

Alanin 2 . 4000 with 0.378 grams nitrogen 

Leucin o . 4200 with o . 044 grams nitrogen 

Prolin 4 .6000 with o .556 grams nitrogen 

Asparagin acid, o . 1700 with o .020 grams nitrogen 


Total 5 405 grams nitrogen. 

t Marschall, Arch. f. Hyg., 28, 19, estimates the protein in Aspergillus at 30.4 per cent., in 
Penicillium at 40.2 per cent., and Mucor at 43.4 per cent, (based upon dry weight). In Arch, 
f. Hyg. 28, 1917, 17, the per cent, of protein in molds is placed at 38.0. 

Nageli and Loew., Jour. Prakt. Chem. N. F., 17, determined 47.0 per cent, of protein in 
yeasts. 

% Ruppel. Zeit. f. Physiol. Chemie, XXVI, 1898, out of 100 grams of dried Bact. tuberculosis 
secured the following substances: 

Nucleic acid (tuberculinic acid) 8.5 grams 

Nucleoprotamin 25.5 grams. 

Nucleoproteid 23.0 grams 

Albuminoids (keratin, etc.) 8.3 grams 

Fatty matter 26.5 grams 

Ash 9-2 grams 

!l Nishimura, Arch. f. Hyg. XVIII, 318, 1893, reports the finding of 0.17 per cent, xanthin, 
0.08 per cent, adenin and 0.14 per cent, of guanin in his water bacillus. 

Vaughan, V. C. and associates, loc. cit., have noted the presence of certain diamino and 
monamino acids. 


I go PHYSIOLOGY OF MICROORGANISMS 

The protein substances vary in amount in different species of micro- 
organisms. Vaughan* compares the compounds of B. coli and Bad. 
tuberculosis indicating that no similarity of ammo acids exists in the 
protoplasm. Duclauxf has found in the analysis of yeast, 15 years old, 
only 2.7 per cent, of nitrogen as compared with the yeast (Frohberg) 
analyzed by Nicolle and Alilaire which contained 10 per cent, nitrogen. 
Age, it seems from this, changed the amounts of nitrogenous material 
present in the cell. Then, again, the medium upon which the micro- 
organisms are cultivated has a decided influence. Cramer J determined 
69.25 per cent, protein in Msp. comma when grown in bouillon and only 
35.75 per cent, when grown in Uschinsky's solution. He also noted 
that the dry matter from this organism was greater when grown at 
body-temperature than when grown at room-temperature. 

Carbohydrates. Substances which correspond to the reactions of 
carbohydrates have been recognized. Some of these substances 
exist as distinctive carbohydrates and some enter into the formation 
of compounds as gly co-proteins. Their relation to the protoplasmic 
molecular structure and to nutritive processes is still more obscure. 

Glycogen has been reported by A. Fischer || in B. subtilis and 
B. coli. Levene has found it in Bact. tuberculosis. Marschall in the 
study of molds records the presence of 3.7 per cent, starch. How- 
ever, glycogen is so much like starch that confusion has arisen. 
Glycogen in molds and yeasts, much like that of animal glycogen, 
is cla'med by several workers. (Glycogen has been commonly 
known as animal starch from the time of Claude Bernard.) In proto- 
zoa glycogen has been determined by Sosnowski^f mParamecium and 
by Biitschli in Gregarina. 

* Vaughan, V. C. and his associates, loc. cit., compare the amino acids of B. coli and Bact. 
tuberculosis. 

B. coli, Bact. tuberculosis, 

Per cent. Per cent. 

Glutanic acid 3 . oo 0.20 

Glycocoll 0.33 o . oo 

Alanin i . oo i . 40 

Valin i . 60 4.60 

Leucin 2 . oo 1.82 

Phenylalanin 0.20 o . 50 

fDuclaux, E.: Kruse, "Allgemeine Mikrobiologie," p. 59. 
tCramer, E., Arch. f. Hyg. 28, i. 

||Fischer, A.: Vorlesungen iiber die Bakterien, Jena, 1903. 

Levene, Jour. Med. Research, 6, 135, 1901. Scheibler, Zeitsch. f. Rubenzuckerindustrie. 
XXIV, 309, 1874. Marschall, Arch. f. Hyg., 28, 19, 1897. 
HSosnowski, Centralblatt f. Physiologic, 13, 1899. 


CHEMICAL STUDIES OF THE CONTENT OF MICROBIAL CELLS IQI 

Cellulose, so bound up with plant life and at one time so 
much used to differentiate plant and animal life, has not been 
positively demonstrated in any microorganism, even in molds and 
yeasts. Substances, giving suggestive reactions, have been studied 
and, at times, have been called cellulose, or some modified form of 
cellulpse, yet recent analysts seem to think there is really no substantial 
ground for this assumption. Vaughan* in his extensive analyses of 
bacterial cells has never been able to identify cellulose. On the other 
hand Vaughan calls attention to two carbohydrate bodies, one of which 
furnishes a reducing sugar when boiled with dilute mineral acid and 
the other does not. 

From time to time there have been detected suggestive traces of 
various carbohydrate substances to which special names have been 
attached but they seem to lack definiteness and individuality in their 
chemical features. Chitin,t a substance quite generally found in 
microbial cell- walls, consists apparently of a carbohydrate- amine or 
glucosamine polymerized. Much emphasis is now placed upon this 
substance as representing the most important constituent not only of 
microbial cell-walls but of wings and coverings of insects and of many 
lower animal forms. 

Fats. Many analyses indicate variable amounts of fat in all classes 
of microorganisms. Whether this fat is the result of degradation proc- 
esses at times, whether it may be ready for assimilation, whether it 
exists as a reserve product, or whether it is the yield of direct absorption 
cannot be asserted off-hand. Probably there are times when it may 
answer to each of these explanations and times when indications are 
such as to furnish a positive understanding. 

Fat globules may be readily revealed by the use of certain stains 
as osmic acid and Sudan III when present in comparatively large 
microbial cells, but in the case of bacterial cells this procedure is un- 
availing, making it necessary to employ recognized chemical methods. 

In the analysis of molds, MarschallJ has obtained the following 

Aspergilhis Penicillium Mucor 

Ether extract 4.7 4.1 4.0 

Alcoholic extract 18.5 1 1 . 8 1 1 . 8 

*Vaughan, V. C. and his associates, loc. cit. 

tChitin when hydrolyzed yields glucosamine and acetic acid. The equation CigHsoX^Ois + 
zO = 2CH 2 OH.CHOH.CHOH.CHOH.CHNH 2 .CHO + sCHsCOOH, has been suggested. 
JMarschall, Arch. f. Hyg., 28, 19, 1897. 


192 PHYSIOLOGY OF MICROORGANISMS 

results from the ether and alcoholic extracts in terms of per cent, of 
dry substance. Nageli and Loew H found 5 per cent, in a bottom- 
fermentation beer yeast. The Bact. tuberculosis has always occupied 
a conspicuous place on account of its fat-content. Klebsf estimated 
20.5 per cent, of a red fat and 1.14 per cent, of a white fat. In amoebae, 
fat globules are frequently detectable in very large numbers. 

Apparently the fatty materials found in different organisms are of 
diverse natures. Hammerschlag | believed most of the fatty substances 
of Bact. tuberculosis consist of tripalmitin and tristearin. De Schweinitz 
and Dorset || obtained palmitic and arachidic acids. Bandraus recog- 
nizes stearin and olein together with the lipoids, cholesterin and lecithin, 
in the same species. It is a matter of determination that stearin, 
palmitin, cholesterin, lecithin have also been recognized in molds, 
yeasts, and protozoa. There is no characteristic uniformity existing 
between species other than certain fatty substances are more commonly 
met with in some than others. In the same species the fat content or 
amount is subject to wide variations. It was noticed by Meyer If that 
in B. tumescens there was an increase of fat till spore production when 
the fat completely disappeared. There was no fat in the spores. 

The Ash Elements. It is exceedingly difficult at the present time to 
determine the number, kinds and limitations of inorganic elements 
included in the compositional structure of protoplasm. Both qualita- 
tive and quantitative studies fail in solving the values and relationships 
of these elements in vital processes. From the nutritional viewpoint 
certain elements may be recognized as very important and others as 
incidental. Uniformity, however, exists only within certain bounda- 
ries, if it exists at all. The elements which stand out most con- 
spicuously are phosphorus, potassium, sodium, calcium, sulphur, 
magnesium, iron, silicon, but manganese, aluminum, copper and others 
have been recognized at times. 

The finding of an element does not establish its relation to proto- 
plasmic synthesis. Attempts have been made to substitute other 
elements for those considered essential but such efforts cannot be 

Nageli and Loew, Sitzgsber. d. Kgl. Academie d. wiss. in Munchen, 1878. 
tKlebs, Cent. f. Bakteriologie, XX, 488, 1896. 
tHainiiu isc hlag, Monats f. Chem., X, 9, 1899; Cent. f. Klin. Med., XII, 9, 1891. 

Srhwcinitz and Dorset, Jour. Amer. Chem. Soc., XVII, 605, 1895; XVIII, 449, 1896 
XIX, 782, 1897; XX, 618, 1898. 

13andraus, Compt. rend. ac. sc., 142, 657, 1906. 
IfMeyer: Flora, 432, 1889. 


CHEMICAL STUDIES OF THE CONTENT OF MICROBIAL CELLS 193 


regarded on the whole as eminently satisfactory. Illustrating, no 
comment is needed to place nitrogen in its many connections and 
phosphorus seems to be very intimately bound up with the complex 
molecule of protein, yet when potassium and iron are considered it may 
be far more difficult to formulate definite conceptions of relationships. 
It is safe to say, however, that ash constituents are required in life- 
processes even if a more detailed analysis is barred or blurred for the 
time being. 

The extent to which ash elements are found is well set forth by 
Kruse*in a comprehensive review in which he considers molds, yeasts 
and bacteria. In the analyses presented, phosphoric acid appears to 
exist in greater proportion than all other elements. Potassium and 

*Kruse's review is here offered in abbreviated form (Allgemeine Mikrobiologie, pp. 86-87). 
Zopf (Pilze, 1 1 8). Higher Molds. 

Phosphoric acid 40 . o per cent. 

Potassium 45 . o per cent. 

Sodium 1.4 per cent. 

Magnesium 2.0 per cent. 

Calcium 1.5 per cent. 

Silicic acid i . o per cent. 

Iron oxide i . o per cent. 

Sulphuric acid 8.0 per cent. 

Chlorine i . o per cent. 

Mayer, Ad. Garungschemie, Aufl., 5, 118, 1902. Yeast. 

Phosphoric acid 51.0 -59 . o per cent. 

Potassium 28.0 -40 . o per cent. 

Sodium ! 0.5 - 1.9 per cent. 

Magnesium 4.0 - 8.1 per cent. 

Silicic acid o.o 1.6 per cent. 

Calcium i.o - 4.5 per cent. 

Iron oxide o.i -- 7.3 per cent. 

Sulphuric acid 0.6 - 6.0 per cent. 

Chlorine o . 03- i . o per cent. 

Kappes, (S. Anm. zu Taf. I, 5, 52), Cramer (Arch. f. Hyg., 28), De Schweinitz and Dorset 
(Cent. f. Bakt., 23, 993)- 

B. xerosis B. prodigiosus, B. tuberculosis, Cholera spirillum, 

per cent. per cent. per cent. per cent. 

: i 

Phosphoric acid 34.0 36.0 55.2 10.0-45.0 

Potassium n.o n.o 6.4 4.0-6.0 

Sodium 24.0 28.0 13.6 27.0-34.0 

Magnesium 6.0 7.0 i r . 6 0.1-0.6 

Calcium 3.0 4.0 12.6 0.3-1.3 

Silicic acid 0.5 0.5 0.6 

Sulphuric acid .... o.o 1.0-8.0 

Chlorine 0.6 5.0 o.o 5. 044 . o 


13 


1 94 PHYSIOLOGY OF MICROORGANISMS 

sodium occupy very prominent places; yet the relations of these two 
elements are sometimes reversed. Calcium and the other constituents 
are subject to considerable fluctuation. If any inference is to be drawn 
from this work, it must mean that phosphorus is a very important 
element, serves an essential role, and is of consequence to protoplasm, 
probably as a basic constituent. Potassium, sodium, magnesium, and 
calcium are uniformly constant ingredients, are concerned in nutritional 
exchanges and may in a limited manner be bound in the structure of the 
protoplasmic molecule. 

The concentration of the culture-medium and brine solutions are 
known to influence the amount of ash-content of microorganisms. 
Cramer,* using a i per cent, sodium carbonate bouillon, a 4 per cent, 
sodium phosphate bouillon and a 3 per cent, sodium chloride bouillon 
obtained in the case of Msp. cholera respectively 9.3 per cent., 22.3 
per cent., and 25. 9 per cent, ash (dry weight). 

Other substances are found present in microbial cells. These should 
be referred to here although more extensive consideration will be given 
some of them later. 

Enzymes are found in all microbial cells. They are agents employed 
in metabolism and in the preparation of food for incorporation in the 
body of the cell and incidentally produce changes which result in 
products of fermentation as alcohol. They act very specifically inas- 
much as a particular enzyme is needed for every substance changed 
as cane sugar, malt sugar, starch, protein, fat, etc. They cause change 
apparently without altering their nature. They are influenced by 
many conditions of temperature, reaction, accumulated products, etc. 
An organism is capable of secreting or containing within its protoplasm 
several enzymes, each being produced only when the cell is specifically 
stimulated. 

Toxins much like enzymes may be found within the cell substance 
or in the medium in which the microorganism may be growing. They 
are associated with disease-production and pathogenesis. Their force 
as a poison (the meaning of the word) is incomparably great. Only a 
small number of microorganisms are able to produce toxins. 

Vitamines are substances, somewhat intangible, which have been 
found in some microorganisms and quite generally in food substances. 
They are seemingly essential to life. Their recognition at the present time 
is largely by solubility and physiological determination upon animals. 

"Cramer, Arch. f. Hyg , 28, i. 


DIVISION II 
NUTRITION AND METABOLISM 

INTRODUCTION * 


The nutrition and metabolism of microorganisms are based on many 
of the same principles which regulate animal and plant metabolism; 
in many ways microorganisms are more closely related to animals than 
to plants, if viewed from the standpoint of their food, their mode of 
digestion, and their general physiological nature. Aside from the 
many specific physiological processes peculiar to microbial life as 
in the case of life without oxygen (anaerobiosis) and in the ability of some 
species to use free nitrogen gas, the functioning of microorganisms 
accords with the cellular metabolism and nutritive principles of 
the more highly developed organisms. Since it will be desirable 
frequently to refer to plant and animal nutrition in the course of 
this discussion, these principles, therefore, are briefly discussed in 
the following paragraphs. 

Green plants feed only on inorganic substances. They assimilate 
carbon dioxide (CO 2 ) from the air which unites with water, nitrates, 
potassium, calcium, and other salts of the soil and form the body sub- 
stances of the plant. The cellulose, starch, sugar, protein and all other 
compounds constituting the plant cells are produced from these simple 
inorganic substances. Animals feed upon animals and plants. Unlike 
plants they utilize the oxygen of the air and give off carbon dioxide 
(CO 2 ). Out of these materials, together with water, life is sustained. 
Although in details animals, plants and microorganisms differ quite 
widely, the general laws of nutrition and metabolism are very similar. 

The methods by which microorganisms secure their food vary. 
Molds take up their food through the mycelium after it has been pre- 
pared by the action of digestive agents, enzymes, secreted by the cells. 
If the food be suitable for the life of the cell without change, of course, 
these digestive agents are not needed. When properly altered, such 

* Prepared by Otto Rahn. Revised by Editor. 

195 


196 NUTRITION AND METABOLISM 

compounds enter as are permitted by the cell-wall and protoplasm by 
means of osmotic pressure. They then diffuse throughout the proto- 
plasm of the cell. Other digestive agents within the cell make the food 
assimilable. In molds the food may apparently pass along the myce- 
lium or hyprue, in other words be transmitted for some distance through 
the organism. In the case of the yeast cell and bacteria the process is 
very similar but the transmission of nutritive material beyond a single 
cell is not known to take place and perhaps there is no need for it. 
Whether food is conveyed from one cell to another in colonies has not 
been" determined so far as the writer knows. 


Food 

Waste 
Secretions 

Water 


FIG. no. Illustrating cell activities. 

Waste products resulting from the metabolism of protoplasm leave 
the cell through the cell- wall, also by means of osmosis, and this process 
appears to be the same for the ingestion of food as for the egestion of 
waste products. 

Some microorganisms live upon dead matter, some upon living 
matter and some may make use of either. The greater portion, by far, 
require or prefer organic substances. When organisms, as protozoa, 
feed upon living organisms they are said to be holozoic in their mode of 
life, in other words they follow closely the methods employed by ani- 
mals. Then there are those protozoal organisms which simulate plants 
in their manner of nourishment. These are called holophytic. This 
latter class is associated with the formation of chlorophyll-bodies within 
their structure. There are those organisms, too, which consume 
organic matter which is rendered suitable by nature or decay, called 
saprozoic or saprophytic, depending upon whether the organism is 
designated as animal or plant. Whenever organisms require living 
tissues to sustain life, in the form of a host, they are called parasitic. 


INTRODUCTION 


197 


Many of these microorganisms absorb their nutrition directly from the 
fluids of the tissues while others, amoebae, are able to devour cells. 

Protozoa are very much like all microorganisms in their manner of 
living but there are details which belong to them as a class and should 
be pointed out specifically. 


....... 

- 

. 


FIG. in. A, Amoeba proteus; Na, a food particle; Cv, contractile vacuole; X, nucleus. 

(After Doflein.} 

'The ingestion of food is accomplished in some protozoa by 
pseudopodia; the protozoon simply flows around and so encloses a food 
particle (Fig. m). In the same way, these protozoa flow away from 
waste particles which are to be eliminated. Other protozoa have defi- 
nite mouth areas for the ingestion of food, and definite anal areas for 
the discharge of residual material. Those protozoa which ingest solid 
food, digest it within gastric vacuoles by the aid of enzymes and of 
acids, just as is the case in many-celled animals. The most important 
of the disease-producing protozoa live within nutrient fluids, for ex- 
ample the blood, and they obtain their nourishment from the fluid in 

* Prepared by J. L. Todd. 


198 NUTRITION AND METABOLISM 

which they live, by osmosis; consequently, they have no definite mouth 
area, nor gastric vacuoles. 

*"Some of the protozoa, for example, some amoeba? and ciliata, pos- 
sess contractile vacuoles. A contractile vacuole is a clear cavity which 
appears in the cytoplasm, grows slowly, empties itself by a rapid con- 
traction of the fluid which has drained into it and forms again. The 
fluid which it ejects contains the soluble waste products resulting from 
the metabolism of the protozoon. One function of the contractile vacu- 
oles is, therefore, excretion; in some protozoa, they are probably also 
concerned with respiration. Contractile vacuoles are usually absent 
in protozoa which are parasitic within other animals. 

1 The process of respiration in the protozoa is in general similar to 
that of higher animals. Most of them require oxygen and eliminate 
carbon dioxide. The contractile vacuole which is found in certain 
forms is believed to have a respiratory function. Respiration may 
consist of the liberation of energy through oxidation or through the 
breaking down of complex molecules. In organisms of an anaerobic 
habit the respiration is probably through internal molecular changes 
affecting material stored in the cytoplasm. 

' In addition to the expulsion of solid undigested material from 
the cytoplasm there is evidence that waste products other than CC>2 
are excreted by contractile vacuoles. Many organisms also secrete 
material either of the nature of chitinous membranes on their surface 
or metabolic products in the form of granules, etc., within their bodies. 

'* Derangement of function may be produced associated with it 
are visible degenerative changes. It has also been found that certain 
protozoa have the ability to recover from injury and to regenerate lost 
parts." 

* Prepared by J . L. Todd. 


CHAPTER I 
ENERGY REQUIREMENTS IN CELLULAR NUTRITION* 

The formation of organic compounds from inorganic compounds 
requires a certain amount of energy. If a certain quantity of sugar is 
burned to carbon dioxide (CO 2 ) and to water (H 2 O), a certain amount of 
energy is liberated in the form of heat. The heat given off in this case 
is also a distinct product of combustion. This heat is always obtained 
in the same amount regardless of the method chosen in burning the 
sugar. It has been definitely determined to be 674 calories for i g. 
molecule (180 g.) of sugar. The complete equation of sugar combus- 
tion is therefore written 

C 6 H 12 O 6 + 1 2 O = 6CO 2 + 6H 2 O + 674 Cal. 

Consequently the same amount of energy will be needed to produce 
sugar from carbon dioxide and water; for the law of the conservation 
of energy requires that, if a certain process liberates a certain quantity 
of energy, the reverse process will require the same quantity of energy. 
Green plants get their energy from the sunlight; exactly the opposite 
proceeds in the equation which should read from right to left; CO 2 
and H 2 O are absorbed by the plant resulting in the formation of sugar. 
But it is evident from the equation that CO 2 and H 2 are not sufficient 
to produce sugar since it takes 674 calories of heat in addition. The 
radiant energy of light is transformed by the chlorophyl granules of the 
plant leaves into chemical energy which causes the formation of organic 
compounds from the simple inorganic or mineral matter. Chlorophyl 
is the green coloring substance of plants, and only green plants can use 
the energy of sunlight for their growth. 

The growth of green plants is a storing of the energy of light in the 
form of organic matter; their metabolism is largely synthetic, i.e., 
building up. Plants without chlorophyl, however, like mushrooms, 
molds, yeasts and bacteria, have to provide for their energy by some 
other means. 

* Prepared by Otto Rahn. 

199 


200 NUTRITION AND METABOLISM 

Animals construct their bodies mainly of organic matter. Their 
body substances as protein, fat, etc., are derived from the protein, 
fat, cellulose, etc., of plants or of animals. Nevertheless, a certain 
amount of energy is required in this assimilation process, since the 
animal protein and fat are somewhat different from the plant protein 
and fat. Consequently, complex chemical changes and rearrangements, 
which require some energy, are necessary for growth. Energy is also 
lost by radiation of heat and by locomotion. Animals, being entirely 
unable to use the sunlight as a source of energy, obtain their energy 
from the digestion of organic food. The larger part of this food is 
oxidized completely; this part provides the energy. The smaller 
part of the food is used for building the tissues of the body; it becomes 
part of the animal itself. Animal metabolism is largely analytic, i.e., 
destructive although a limited amount of energy is required for the 
chemical changes and molecular rearrangements which are essential 
to animal tissue formation a synthetic process. Accordingly more 
organic matter is decomposed than is formed. Often the same sub- 
stance can serve both purposes; the meat eaten by a dog furnishes to 
it energy as well as material for growth. In othe r cases, certain food 
compounds execute only one function and not the other. This dis- 
tinction between food for energy and food for growth must also enter 
into the interpretation of microbial metabolism. 

It might appear from this discussion that energy is needed only by 
growing cells, as the full-grown cells do not increase in size or weight 
or number. They also need energy, for in all living cells, there is 
noticed a continuous breaking down (katabolism) and rebuilding 
(anabolism) of the cell constituents. This process is commonly called 
metabolism. The katabolic processes (the breaking down) in a cell 
will continue even if the cell receives no food. The cell loses in weight 
and the starvation which follows will ultimately result in the death of 
the cell. All living cells require food for the maintenance of life. 

In the first part of this book, microorganisms have been divided 
into plants and animals, but attention has been called in various places 
to the fact that it is often hard to determine whether the plant char- 
acters or the animal characters prevail. This holds true not only 
with the morphology, but also with the physiology of microorganisms. 
Since none of the plants discussed in this text-book possesses chlorophyl, 
none of them can use light as a source of energy, therefore they depend 


ENERGY REQUIREMENTS IN CELLULAR NUTRITION 2OI 

entirely upon chemical energy obtained by the digestion of food. This 
means that they require organic food almost entirely, since inorganic 
food furnishes energy only in exceptional cases. In this respect they 
resemble the animals very much. 

The source of energy in microbial life is always of chemical origin. 
The simplest processes are the oxidations, and simplest among these 
the inorganic oxidations. A number of different types feeding ex- 
clusively on minerals has been discovered during the last twenty years, 
and some of them are of great economic importance. They resemble 
plants in as far as they build their cells exclusively from carbon 
dioxide, nitrates and ash. The food used for building material is 
quite different from the food used for the provision of energy. 

Two typical examples are the nitrifying organisms in soil which 
oxidize ammonia to nitrates. This process, according to Winogradski, 
is divided distinctly into two phases: the Nitrosomonas oxidizes the 
ammonia to nitrous acid, 

NH 3 + 3<3 = HNO 2 + H 2 O + 78.8 Cal. 
and the Nitromonas oxidizes the nitrous acid to nitric acid, 

HN0 2 + O = HNO 3 + 18.3 Cal. 

These oxidation processes yield a certain amount of energy which 
enables the bacteria to build their cells from carbon dioxide, ammonia, 
and certain mineral salts. Without ammonia or without nitrous acid, 
respectively, these bacteria cannot grow for lack of energy; they would 
be like a plant without light. It is evident in this case that the food for 
energy is also used to some extent as food for growth. The nitrogen 
necessary to the bacteria is supplied by the ammonia or the nitrous acid. 
As an example distinguishing strictly between the food for growth 
and the food for energy may be mentioned the hyposulphite bacterium 
studied by Nathanson. This organism oxidizes hyposulphites to sul- 
phates and sulphur, largely following the formula 

Na 2 S 2 O 3 + O = Na 2 SO 4 + S + x Cal. 

Hyposulphite Sulphate Sulphur 

Besides, some more complex compounds, like sodium tetrathionate 
(Na 2 S 4 O6), are formed. The bacterium builds its cells exclusively from 
nitrates, carbon dioxide, and mineral salts; organic food is rejected. 
The hyposulphite can hardly be used for the construction of the cell, 
and must be considered entirely a food for energy. 


2O2 NUTRITION AND METABOLISM 

This distinction is not confined to mineral decomposition only. 
The urea bacteria get their energy from the decomposition of urea into 
ammonium carbonate which is hydrolysis. 

(NH 2 ) 2 CO + 2H 2 = (NH 4 ) 2 Cp 8 + 14-3 Cal. 

Urea Ammonium 

carbonate 

But the urea and mineral salts are not sufficient for the development of 
the urea bacteria. They cannot use urea as a material for building the 
cells, and they cannot use carbon dioxide or carbonates; they cannot 
grow unless a suitable material for cell construction is added. Sohngen 
demonstrated that a few milligrams of malic acid favor a ggod develop- 
ment of the bacteria. The malic acid is used entirely for the forma- 
tion of cell substances. The energy for this formation came from the 
urea fermentation. This example shows clearly the different require- 
ments for cell growth and for the energy supply. 

With the urea fermentation, we have changed not only from inor- 
ganic to organic food, but also from oxidation processes to other 
decompositions. 

Microorganisms differ from the higher animals by their less complete 
metabolism. The food in the animal, if digested at all, is oxidized as a 
rule to the final products of combustion, CO 2 and H 2 O, the only excep- 
tion being the nitrogen which leaves the body still in organic combina- 
tion as urea. With bacteria, yeasts and molds, this is not always the 
case. Though some of these organisms will bring about complete oxida- 
tion of the food we find more commonly incomplete oxidations or 
changes which require no oxygen at all, but still yield energy to the cell. 
The biochemical side of these changes of which the alcoholic fermenta- 
tion is the best known will be discussed in the chapter on oxygen 
requirements. 


CHAPTER II 

MECHANISM OF METABOLISM* 

GENERAL THEORY OF METABOLISM 

ANABOLISM, KATABOLISM, METABOLISM. It has been stated that 
microorganisms need food for at least two different purposes: building 
material and building energy. They may need it for other purposes 
also, e.g., for motion. The sum of all changes which the food undergoes 
in the body, including the deterioration of the cells, is called metabol- 
ism. Metabolism consists of several separate functions: One of 
them is the construction of new cells, or parts of cells, called anabol- 
ism, another the deterioration of cells, called katabolism, and the most 
important quantitatively is the fermentation or respiration. The 
fermentation or respiration processes are fairly well understood; many 
of them can be produced in the chemical laboratory without micro- 
organisms. Katabolism is the sum of many processes some of which 
are well understood while others are still unknown. The synthetic, 
anabolic processes of the cell, however, are almost entirely unknown, 
and we can only speculate regarding the various means by which the 
cell grows. The explanations of the different cell activities began, 
as in most other fields of theoretical microbiology, with a close analogy 
with animal and plant metabolism, but owing to the comparative 
simplicity of the microorganisms, they led to the establishment of new 
facts and theories which proved afterward useful for the understand- 
ing of the metabolism of the more complex organisms where the multi- 
plicity of facts prevented a clearer insight into the separate processes. 

INTRA- AND EXTRA-CELLULAR FERMENTATION 

DECOMPOSITION OF INSOLUBLE FOOD. Many microorganisms feed 
upon cellulose, starch, fat, gelatin, keratin and other insoluble com- 
pounds. Microorganisms, with the exception of some protozoa, 

* Prepared by Otto Rahn. 

203 


204 NUTRITION AND METABOLISM 

depend upon soluble food since they have no means of incorporating 
insoluble compounds into their protoplasm. The protoplasm, however, 
must be considered the center of metabolism, and the digestion of food 
and the formation of energy must take place in the protoplasm if the 
cell is to profit by it. Since the food cannot diffuse into the cell, and 
the protoplasm does not diffuse out, the food must be dissolved. This 
is accomplished by the cell itself by secreting certain agents with 
peculiar qualities. These agents, the so-called enzymes, act upon the 
insoluble foods, changing them into soluble compounds which then can 
diffuse into the cell where they are digested or fermented. The final 
digestion or fermentation of the food must take place within the cell. 
Energy production outside the cell serves the same purpose as a stove 
outside the house. The dissolution of insoluble compounds by cell 
secretions must be considered a preparatory process which has no direct 
relation to intra-cellular food digestion or fermentation. Enzymes are 
not produced by microbial cells exclusively. All living cells produce 
enzymes. They were known before the science of microbiology had 
been established. In fact, microbial activity was considered for a 
long time as an enzymic chemical process. Enzymes in the animal 
and plant body serve largely the purpose of metabolic changes. In 
the animal body, many enzymes help to dissolve the insoluble food 
which cannot pass from the alimentary canal into the body except by 
diffusion through the mucous membrane. There is diastase in the 
saliva which acts upon starch, there is pepsin in the stomach and 
trypsin in the intestine, both dissolving protein bodies; there is ereptase 
for the peptones, lipase for the fat, invertase for the saccharose, and 
many other enzymes. The object of all these enzymes is apparently 
to prepare the food for passing through the membrane into the proto- 
plasm of the cells, where the final changes which liberate energy take 
place. The same processes occur with microorganisms but in a more 
simple manner. Surrounded by a liquid medium, they secrete enzymes; 
these dissolve certain insoluble foods, which then diffuse through the 
cell wall to be decomposed further. 

The food-preparing processes are all supposed to be simple hydrolytic 
processes. For some of these changes the chemical equations are well 
known. The hydrolyzation of starch to maltose by means of diastase is 
represented by the equation 

2(C 6 H 10 5 ) n + nH 2 = nCi 2 H M Oii. 


MECHANISM OF METABOLISM 20 


The splitting up of a fat molecule into glycerin and fatty acid is also a 
well-known process 


= C 3 H 5 (OH) 3 + 

Tristearin Glycerin Stearic acid 

Proteolysis is not so well known and the general supposition that 
the first stages of protein degradation are hydrolytic is largely based 
upon analogies. Some of these enzymes which are secreted by the 
microbial cells act upon soluble compounds. Imertase decomposes 
saccharose into dextrose and levulose: 

Ci2H22On 4~ H 2 O = CeH^Oe -f- CeH^Oe. 

Other disaccharides are hydrolyzed in the same way by other enzymes; 
glucosides are decomposed by emulsin; soluble proteins are changed to 
peptones. It is not necessary that the enzymes act upon the soluble 
compounds outside the cell since these compounds can diffuse into the 
cell; these enzymes are found only occasionally within the cell. It 
may be said, however, that the smaller molecules of the products of 
enzymic action diffuse more readily than the larger molecules of the 
original food compound. 

PROPERTIES OF ENZYMES.- -These secretions of cells are treated in a 
group by themselves because they differ distinctly in many respects 
from any other chemical substance. Probably the most notable differ- 
ence may be discovered in the fact that their action does not follow the 
law of mass action which supposes that all substances reacting upon 
each other diminish in quantity. Rennet will coagulate many hundred 
times its weight of casein, and still the whey will contain rennet. Con- 
sidering that part of the rennet is physically absorbed by the coagulum, 
the amount of rennet is found to be the same as before, though it has 
changed a comparatively enormous quantity of casein. The same is 

A 

true with other enzymes. The enzyme is not destroyed by acting 
upon other substances. This exceptional quality furnishes a reason for 
treating enzymes as a separate group or apart from other chemical 
substances. But there are still other qualities which distinctly separate 
them from the well-known chemical bodies, and show at the same time 
their relation to proteins and toxins (page 248). One of these is 
their sensibility to such outside influences as will destroy life. Enzymes 
are inactivated by exposure to temperatures above 50 to 80, and 


206 NUTRITION AND METABOLISM 

can, like coagulated albumin, by no means be brought back to their 
original state. This temperature is very near the coagulating tempera- 
ture of albumin. It is believed from this resemblance that enzymes 
are of an albuminous nature. Another similarity is the fact that both 
enzymes and albumins are precipitated by concentrated salt solutions. 
Enzymes can further be inactivated by poisons. The same sub- 
stances which kill living cells, like formaldehyde, hydrocyanic acid, 
mercuric chloride, phenol, will also inactivate enzymes, though usually 
stronger solutions are required for the destruction of the enzyme than 
for killing the cell. It is the same with heat; a higher temperature is 
generally required to destroy the enzyme than to kill the cell which 
secreted it. Light will also affect enzymes considerably. The great 
similarity of enzymes and microorganisms in these respects, the simi- 
larity of their reactions and the extreme minuteness of the bacteria 
render it explicable why the chemists of eighty years ago could not 
determine the difference between microorganisms and enzymes, and 
called them both "ferments." 

With the toxins, the enzymes have in common the great sensibility 
to heat, light, and chemicals. Both of these groups are resistant to 
drying to a limited extent. So far as body reactions are concerned these 
two groups seem to belong to one physiological group of compounds. 
When toxins are injected, the body responds by the production of anti- 
toxins which inactivate the toxin. In the same way the body responds 
to enzymes by the production of anti-enzymes which prevent the action 
of the enzymes. It may be mentioned that against protein compounds, 
precipitins are produced by the body which precipitate only that protein 
which was injected. This " specific" action is also true with toxins and 
enzymes. The anti-body will inactivate only the specific kind of toxin 
or enzyme that was injected. 

What an enzyme really is cannot be defined. An enzyme is known 
only by its reactions. Many chemists have tried to prepare pure en- 
zymes by continuously dissolving and precipitating, by dialyzing and 
other means, but there are two great difficulties existing; there is no test 
for the purity of enzymes, and they lose in activity if treated with 
chemicals. The more they are freed from the protein bodies which 
always accompany them, the more sensitive they are to injurious in- 
fluences. Mineral salts seem essential for their action, because con- 


MECHANISM OF METABOLISM 207 

tinued dialyzing weakens the activity which can be restored only by 
adding salts. 

ENZYMES OF FERMENTATION. It has been demonstrated in the 
above paragraph that food is prepared for digestion or fermentation by 
enzymes. The final decomposition, the process which yields the energy 
for cell life, must take place within the cell. 

The difference in importance of food preparation and fermenta- 
tion may be illustrated by the example of Rhizopus oryzce. This 
mold attacks starch, changes it, by means of diastase, to maltose, 
the maltose to dextrose, dextrose to alcohol and carbon dioxide. The 
mold grows well in a starch medium, without sugar; it grows equally 
well in maltose, and equally well, or better, in dextrose; it does not 
grow at all with alcohol and carbon dioxide. The last change, dex- 
trose to alcohol, is absolutely necessary for this organism; it is the 
source of its life; the others are incidental processes, not absolutely 
necessary under all circumstances, in fact greatly suppressed if dextrose 
is given together with starch. The fermentation must take place in 
the cell; the preparation of food may take place in the cell or outside; 
it is not essential where it happens. 

The investigations of recent years have demonstrated that fermenta- 
tions also are caused by enzymes. It has been proved beyond doubt 
that in the alcoholic, lactic, acetic and urea fermentations the fermen- 
tation process may continue after the death of the fermenting cells. 
In the case of alcoholic fermentation, the fermenting agent was 
separated first by Buchner from the lacerated cells and was 
filtered through porcelain filters without losing its ability to act. 
This proves the enzyme-nature of the fermenting agent which, once 
being formed, remains and acts independent of the cell. These en- 
zymes are called zymases. They remain within the cell as long as it 
is alive. They are much more sensitive to injurious influences than 
the above-mentioned food-preparing enzymes. Much skill and pa- 
tience was required to demonstrate their independence of the living 
cell. After these enzymes were found in microorganisms, similar 
enzymes were discovered in the cells of higher plants and animals. 
Many of the biochemical changes taking place in the final dissociation 
of food within the cell are known to be the result of enzymic 
action; heretofore these reactions were believed to be a part of the 
life processes, inseparable from the living cell. Even some of the 


208 NUTRITION AND METABOLISM 

> 

oxidations and many reducing processes have been recognized as caused 
by enzymes, and it is quite probable that the whole process of intra- 
cellular food decomposition in all organisms is accomplished entirely 
by means of enzymes. 

CLASSIFICATION OF ENZYMES 

Since the chemical nature of enzymes and of their action is largely 
unknown, they can be arranged for convenience only according to the 
compounds they act upon. It is possible, however, to distinguish 
between the following four groups: Hydrolyzingj zymatic, oxidizing, 
reducing enzymes. This definition is not quite exact, since the urea 
fermenting enzyme is also a hydrolyzing enzyme, and the acetic fer- 
mentation is caused by an oxidizing enzyme. The distinction between 
endo-enzymes (intra-cellular) and exo-enzymes (secreted) is not exact, 
either, since invertase and lactase are retained in the cells of some 
organisms and secreted by others. 

The following classification is used in the further discussions: 

I. Hydrolytic Enzymes. 

1. of carbohydrates: cellulase (cytase), diastase (ptyalin, amylase), invertase, 
lactase, maltase. 

2. of fats: lipase (steapsin). 

3. of proteins: 

(a) proteolytic (proteases): pepsin (peptase), trypsin (tryptase), erep- 

sin (ereptase). 

(&) coagulating (coagulases) : thrombase, rennet (chymosin). 
II. Zymases. 

1. of carbohydrates: alcoholase, lactacidase. 

2. of other nitrogen-free bodies: vinegar-oxidase. 

3. of proteins: endo-tryptase, autolytic enzymes, amidase, urease. 

III. Oxidizing Enzymes. 
Vinegar-oxidase, tyrosinase. 

IV. Reducing Enzymes. 

Katalase, reductases of nitrates, sulphur, sulphites, telluric salts, methylene 
blue, litmus. 

Several different names have been given to some of the enzymes; 
these are found in parenthesis in the above classification. 

The general action of enzymes being explained in the preceding 
pages, it remains to describe more in detail the different enzymes of 
microbial origin. 


MECHANISM OF METABOLISM 2OQ 

HYDROLYTIC ENZYMES 

ENZYMES or CARBOHYDRATES. Enzymes which decompose carbo- 
hydrates are very commonly found in nature, because carbohydrates 
constitute a very extensive and common group of organic matter. 
By far the largest part of the dry plant consists of cellulose, starch 
and sugar. To decompose them, enzymes are necessary. The chem- 
ical reaction of these enzymes is hydrolytic; in other words, the larger 
molecule is broken into smaller ones by the simple addition of water. 
Thus, the cellulose-destroying enzyme, called cellulose or cytase, de- 
composes the cellulose into soluble sugars after the following formula : 

CeHioOs -f- H 2 O = CeH^Oe 

or, considering that the cellulose molecule is really many times 
CeHioOs, the formula will be more accurately written 

(C 6 H 10 05) n + nH 2 = nC 6 H 12 6 

which indicates at the same time that one cellulose molecule gives 
many sugar molecules. 

Cellulase is an enzyme which is quite difficult to obtain. Though 
it must be produced by all the cellulose destroying molds and bacteria, 
experiments have failed in some instances to prove its presence. It 
is found in some wood destroying fungi and in some of the bacteria 
causing the rot of vegetables. The organisms of certain plant diseases 
force their way into the cell by dissolving the cellulose membrane by 
an enzyme, while certain molds are able to puncture the cell wall 
mechanically. 

j 

Diastase, or amylase, is the starch-dissolving enzyme which is one 
of the most common enzymes in nature. It is found in all green plants, 
and it forms during the sprouting of starchy seeds. Many molds 
and a few bacteria produce this enzyme, while yeasts generally cannot 
decompose starch for lack of diastase. Starch has the same formula 
as cellulose, and it is broken up into soluble sugars in the same way. 
Much attention has been paid to this process by the chemists, and it 
is found that the process is a gradual one, giving first dextrins, and 
finally maltose (Ci 2 H 22 On). The hydrolysis of starch expressed in 
chemical symbols may be presented as follows: 

2(C 6 H 10 06)n + nH 2 = nC 12 H 22 O n . 

Starch Maltose 

14 


210 NUTRITION AND METABOLISM 

The disaccharides or double sugars, having the chemical formula 
CizH-zzOn are broken up into single sugars, monosaccharides, by the 
following process: 


The two molecules of CeH^Oe are different with different sugars. 
If the disaccharide is saccharose, the two monosaccharide molecules 
are dextrose and levulose. Lactose will yield dextrose and galactose, 
and maltose will give two molecules of dextrose. For each of these 
sugars, there is a special enzyme which can hydrolyze only its par- 
ticular sugar and none of the others; like a key, made for one lock, 
it will not open another lock. Maltase will split only maltose mole- 
cules, not lactose, while the lactase cannot attack the maltose. /- 
vertase (or sucrase) will decompose nothing but saccharose. This 
decomposition of the complex sugars into the simple sugars was be- 
lieved to be necessary because only sugars of the type CeH^Oe can 
be fermented directly by the fermenting enzyme in the cell, be it an 
alcoholic or lactic or gassy fermentation. This explains why beer yeast 
cannot ferment lactose; it produces no lactase, and therefore cannot 
attack the lactose molecules; they would be easily attacked, if besides 
the yeast, some lactase were added. Certain lactic bacteria cannot 
ferment saccharose, because they do not form invertase. Recent 
experiments have shown that bacteria exist which ferment lactose 
and saccharose but not dextrose or levulose. An explanation for this 
cannot be given. 

Invertase is, like diastase, a very common enzyme in green plants. 
It is also produced by most molds and yeasts, and bacteria. Maltase 
is not quite so common, and lactase is limited to a few species of 
microorganisms. A few organisms are known which do not secrete 
these enzymes but retain them within the cell. This is especially 
true of lactase, but is also known, in a few instances, of invertase. 
The enzyme can be obtained from the broken cells. Such enzymes 
are called endo-enzymes . 

The decomposition of carbohydrates has been followed from the 
most complex representatives to the simplest ones, the monosacchar- 
ides. If these are decomposed further, the resulting product is no 
longer a carbohydrate. The simplest sugars are decomposed by zy- 
mases, inside the microbial cell, into compounds which are generally 


MECHANISM OF METABOLISM 211 

called fermentation products; these may result from alcoholic, lactic, 
butyric fermentations or some other. 

Emulsin is an enzyme which is able to hydrolyze glucosides. Gluco- 
sides occurring in plants are complex bodies which contain a sugar- 
radical. Emulsin splits glucosides liberating the sugar, usually dex- 
trose. The typical example for emulsin action is the hydrolysis of 
amygdalin to hydrocyanic acid, benzaldehyde and dextrose. 


C 20 H 27 OiiN + 2H 2 O = C 6 H 5 COH + 2C 6 H 12 O 6 + HCN. 

Amygdalin Benzaldehyde Dextrose Hydrocyanic acid 

Emulsin is found in many molds and bacteria, and recently has 
been found in yeasts. Glucoside-splitting enzymes play an important 
role in the fermentations of coffee-beans, cocoa, mustard and indigo. 
In most of these fermentations, however, the emulsin is probably not 
formed by microorganisms, but by the plant, from which the ferment- 
ing material is derived. 

ENZYMES OF FATS. All the enzymes, acting on fat, decompose it 
in the same manner; the fat molecule takes up three molecules of water, 
breaking up into glycerin and three molecules of fatty acid, as indicated 
on page 239. It is possible that there are several fat-splitting enzymes, 
but the result of the cleavage process is always the same. The name 
formerly assigned to enzymes of fat is steapsin, but this term is now 
almost exclusively substituted by the more significant word lipase. 
Occasionally they are called lipolytic enzymes which expression is 
analogous to the proteolytic enzymes; in the same way, the term 
amylolytic enzyme is used for diastase. 

ENZYMES OF PROTEINS. The enzymes composing protein bodies, 
generally called proteolytic enzymes or proteases, have been known 
for nearly a century. Though the difficulty of analyzing protein bodies 
accurately prevents an absolute knowledge of proteolysis, much effort 
has been made to become acquainted with the very important group 
of enzymes which accomplish the digestion of protein food. Naturally 
most experimenting has been conducted with pepsin and trypsin 
of the animal body and accordingly these are better understood than 
others; only little work has been done with microbial enzymes. There 
is so far as can be determined little appreciable difference between 
the proteolytic enzymes obtained from different organisms, whether 
low or high in the plant or animal world, consequently many experi- 


212 NUTRITION AND METABOLISM 

ences with animal pepsin and trypsin can be applied to microbial 
enzymes. 

The specific chemical action of these enzymes is referable to hydro- 
lysis; the large protein molecule is broken up into smaller molecules 
by addition of water. Various proteolytic enzymes differ in the extent 
of decomposition. While some, like pepsin, produce mainly peptones, 
trypsin is able to split protein to amino-acids and even to ammonia. 
Mavrojann is tested for the intensity of gelatin decomposition with 
formaldehyde. The peptones of gelatin will solidify with formalde- 
hyde while amino-acids are not affected. 

Proteolytic enzymes were first divided into two groups: pepsins, 

\ 

which act best in slightly acid solutions, and trypsins, which act best 
in slightly alkaline media. The names are derived from pepsin (peptase) 
the proteolytic enzyme of the animal stomach, and from trypsin (tryp- 
tase) which is found in the small intestine of animals. This classifi- 
cation cannot be used for the enzymes of microorganisms because 
there is no definite line established by the acidity. Some enzymes 
work in either acid or alkaline media equally well, preferring a neutral 
reaction. Enzymes should be classified according to the substances 
they act upon or perhaps according to the nature of the products 
resulting from the fermentation. This would bring pepsin and tryp- 
sin into one class, both acting upon protein bodies as such; they, 
however, differ in the intensity of action as shown by their products, 
the pepsin forming mainly peptones, the trypsin carrying on the 
decomposition as far as amino-acids and traces of ammonia. Another 
class recently recognized is ereptase (erepsin) which cannot decom- 
pose protein, but readily attacks peptones, decomposing them much 
in the same way as trypsin. Pepsin, trypsin and erepsin do not 
break up amino-compounds. 

The presence of proteolytic enzymes in microorganisms is readily 
tested by cultivation on nutrient gelatin. The proteolytic enzyme 
secreted by the cells will liquefy the gelatin. Generally, an organism 
that liquefies the gelatin will also decompose the casein of milk and the 
protein of blood serum. There are some exceptions, however, as is 
shown in the following table, after Frost and McCampbell. A + 
sign means proteolysis, a sign means no action, 


MECHANISM OF METABOLISM 


213 


Milk 


^oriim ^8S PiK*in 


Coag. Digest. 

1 


>erum album Fibrin 


Bact. anthracis 


1 

+ + + 


4- 4- 


Microspira comma 


+ + + 


+ + + 


M. pyogenes var. aureus 


+ + + 


Pseudomonas pyocyanea 


+ + + 


+ + - 


B. violaceus 


B. mycoides 


+ + + 


+ 


B. prodigiosus 


- + + 


+ + + 


Aspergillus niger 


+ + 


Aspergillus oryzcc ... 


+ + 


+ + 


Apparently not all organisms which liquefy gelatin are able to de- 
compose egg albumin; we must conclude that the enzyme liquefy- 
ing gelatin is different from the proteolytic enzyme dissolving egg- 
white. 

COAGULATING ENZYMES. The blood-clotting enzyme (throm- 
base) does not occur in microorganisms. Rennet, however, is found 
in many species. Rennet is extracted from the stomach of calves 
and pigs and used to set the curd in milk for cheese making. The 
enzyme acts upon the casein in milk, decomposing it into paracasein 
and some soluble protein. The time of coagulation depends upon 
the temperature of the milk and the concentration of the rennet. 
This coagulation of milk is quite different from the acid curd, where 
the insoluble casein is precipitated by the acid. If enough acid is 
added, the milk curdles immediately; if there is not enough acid, 
there will be no curd, not even after a long time. An acid curd can 
be brought back to the original state by an addition of alkali, while 
a rennet curd by no means can be changed back to casein. Rennet- 
forming bacteria are found in milk and dairy products, in soil and other 
habitats. They will coagulate milk without causing any appreciable 
increase of acidity. They all seem to digest the curd after it is formed 
(see the above table). The relation between proteolytic and rennet 
enzymes will be discussed in a later chapter. 

Rennet is sometimes called chymosin; the Society of American 
Bacteriologists uses the German word "lab." 


2F4 NUTRITION AND METABOLISM 

ZYMASES 

The zymases are the agents which furnish the energy for cell life 
by causing fermentative decompositions. As has been stated before, 
the processes which provide for energy must take place inside of the 
cell. Consequently, all fermenting enzymes are endo-enzymes. The 
difference between the soluble enzymes and the endo-enzymes is very 
plainly shown in the following table, giving the energy liberated by 
the various enzymes by acting upon i g. of substance. 

ENERGY LIBERATED FROM i G. OF SUBSTANCE 

Soluble Enzymes Endo-enzymes 

Pepsin, trypsin o calories Lactacidase 80 calories 

Lipase 4 calories Alcoholase 120 calories 

Maltase, invertase 10 calories Urease 230 calories 

Lactase 23 calories Vinegar-oxidase 2,500 calories 

The microbial cell does not lose much energy by the activity of 
the soluble enzymes outside of the cell, because their energy yield is 
insignificant. 

The first zymase known was urease, the enzyme which changes 
urea to ammonium carbonate. The actual investigation of the 
zymases did not start until Buchner had demonstrated that yeast can 
be ground with infusorial earth until all cells are lacerated, and then 
can be pressed and the juice filtered without losing the power of alco- 
holic fermentation. Such fermentation cannot be due to anything 
but a soluble compound of the yeast cell. Thus the alcoholase was dis- 
covered. It was found later that yeast may be killed by alcohol, 
ether or acetone without losing its fermenting power. 

This last method was applied later to lactic bacteria, and it was 
proved that the lactic acid is also produced by an enzyme, lactaci- 
dase. It is possible to kill the lactic bacteria so that they do not 
multiply but still continue to form acid. It seems quite probable 
that other fermentations of carbohydrates, like the butyric and the 
gassy fermentations, are really due to enzymes. It is very difficult 
to give the experimental proof, however. These enzymes are so un- 
stable that it requires much experience to separate them from the cell, 
and it is also quite difficult to obtain bacteria in quantities large 
enough for such experiments. 


MECHANISM OF METABOLISM 215 

The vinegar oxidase is an enzyme which remains in the cell of the 
acetic bacterium, oxidizing alcohol to acetic acid. Its independence of 
the living cell has been demonstrated by killing the cells with acetone. 

The PROTEOLYTIC ENDO-ENZYMES of yeasts, only, have been studied 
extensively. That such enzymes exist is recognized by the observa- 
tion that certain microorganisms do not liquefy the gelatin until 
after they are dead and the proteolytic enzymes diffuse out through 
the deteriorating cell membranes. That yeast in the absence of 
sugar loses in weight, and that leucin and other cleavage-products of 
protein are formed, was the first indication of a proteolytic process in 
the yeast cells. By pressing the juice out of the ground yeast cells, 
a liquid is obtained which liquefies gelatin, digests casein, albumin and 
fibrin. The living yeast cell does not attack these compounds, be- 
cause they cannot diffuse into the cell and the enzyme cannot diffuse 
out. The proteolytic endo-enzyme of yeast is called endo-tryptase. 
Its object is apparently the regulation of the protein-content of the cell 
and perhaps it has some bearing on the formation of cell plasma. 
The possible relation between enzymes and growth is discussed in a 
following sub-chapter. 

If yeast is mixed with a weak antiseptic (chloroform, toluol) 
the proteolytic process takes place quite rapidly. This process is 
called autolysis (self-digestion). Similar autolytic enzymes are found 
in other microorganisms. Autolysis is a well-known process in the 
higher animals. To this is due the ripening of meat. 

Proteolytic endo-enzymes must be expected in all microorganisms 
which depend upon protein as food material only. These organisms 
will secrete certain enzymes which decompose the insoluble protein 
into bodies which diffuse easily into the cell. Here, proteolytic endo- 
enzymes further decompose these products. Such an endo-enzyme is 
the amidase discovered by Shibata in the mycelium of Aspergillus 
niger which forms ammonia from urea, acetamid, oxamid, biuret. 
Endo-erepsin and amidase were also found in Penicittium camemberti 
by Dox. 

Similar to these proteolytic enzymes is the urease which is formed 
in large quantities in the so-called urea bacteria, but it is also present 
in the mycelium of some molds. An endo-enzyme, splitting hippuric 
acid into benzoic acid and glycocoll, is found in the mycelium of a few 
molds. 


2l6 NUTRITION AND METABOLISM 

OXIDIZING ENZYMES 

The most typical example of an oxidizing enzyme is the mnegar- 
oxidase, because its chemical action is well known. Most of the oxi- 
dases known act upon complex organic compounds, changing them to 
colored bodies. Such an oxidase is the tyrosinase which forms a 
black, insoluble compound in tyrosin solutions. It is produced by 
several bacteria, especially by chromogens, and its application in test- 
ing for small quantities of tyrosin has been suggested. A number of 
oxidases are known to act upon the leuco-bodies of certain organic dye- 
compounds, as aloin, guaiac, phenolphthalein, and others. Hydro- 
chinon is oxidized by the dead cells of a few molds. Strange seems 
the oxidation of potassium iodide to iodine by the endo-oxidase of 
a mold. Many other oxidations are supposed to be of enzymic nature, 
but their independence of the living cell has not been proved. 

Many higher organisms are known to contain oxidases, the best 
studied are those of certain mushrooms which change the white mush- 
room meat into a bluish or brownish color as soon as it is exposed to 
the air. Oxidases are very common in most of the tissues of higher 
animals. 

REDUCING ENZYMES 

Among the reductases, one enzyme stands apart from all the others, 
that is the katalase or peroxidase which reduces the hydrogen peroxide 
to water by liberation of oxygen. 

H 2 O 2 + katalase = H 2 O + O. 

Katalase is one of the most commonly found enzymes; it is formed 
by practically all plants and all animals and is contained by all but a few 
bacteria. Among these exceptions is the Strept. lacticus. The ab- 
sence of katalase in this species has been recommended as a diagnos- 
tic test. It is possible that this enzyme is necessary for intra-cellular 
oxidations. 

A number of other reductases are known. Nearly all of the re- 
ductions mentioned in the paragraph on the products of mineral 
decomposition are proved to be of enzymic nature; these processes 
will take place after the cell is killed by a disinfectant or is ground to 
pieces. This can be readily demonstrated by lacerating the cells 


MECHANISM OF METABOLISM 2iy 

with quartz sand. They will then reduce nitrates to nitrites, sulphur 
to hydrogen sulphide. The decolorization of litmus, methylene 
blue, indigo, and other organic dyes is due in microbial cultures to 
enzymes which are almost exclusively endo-enzymes. 

ENZYMIC THEORY OF KATABOLISM 

Regarding katabolism as the sum of all destructive processes of 
the living cell substance, i.e., of the protoplasm, and considering the 
cell substance to be decomposed and renewed constantly as long as 
the cell is performing the normal functions of life, there must be a reno- 
vating and a destructive process continuously going on in the proto- 
plasmic molecules. If the food supply ceases, anabolism ceases with 
it, but it has been demonstrated that katabolism may continue just 
the same for some time. By this method, the products of katabolism 
can be obtained separate from the products of food digestion which 
would obscure the results of experiment on katabolism in normally fed 
cells. 

It is difficult to determine to what extent katabolism is controlled 
by endo-enzymes, the so-called autolytic enzymes, which have been men- 
tioned in the above paragraph. Unquestionably, the katabolic processes 
are similar to enzyme processes, since katabolism is checked by heat 
or poison just like enzyme processes. 

ENZYMIC THEORY OF ANABOLISM 

ANABOLISM AND INTRA-CELLULAR ENZYMES. All changes dis- 
cussed in the previous chapters are processes in which organic or 
inorganic compounds are broken up to smaller molecules. These 
processes are exothermic, i.e., liberating heat or energy in other forms. 
The opposite is true of the anabolic processes which build up complex 
molecules from simple compounds. These synthetic processes are 
endothermic, absorbing heat or other energy. Growth is the typical 
manifestation of anabolism. It is the formation of new cells from dead 
organic or inorganic matter, and it means the formation of all the com- 
pounds necessary for cell life. Of all the substances found in the cell, 
practically none are contained in the food, and it is wonderful that 
in such a small unit as a microbial cell, there are contained the powers 
of making protoplasm, enzymes, nuclear bodies, chromatin bodies, 
the substance of the cell wall and probably many other unknown 


2l8 NUTRITION AND METABOLISM 

compounds. All these complex substances are generally made from 
simple food compounds as amino-acids, carbohydrates and others. 
These synthetic processes of the cell will, like most endothermic 
processes, take place only if energy is provided. This condition is 
usually fulfilled in the living cell, due to the fermenting processes 
going on continuously. There is a strange interaction between 
anabolism and mtra-cellular fermentation proceeding in the pro- 
toplasm and this linking together of destructive and constructive 
reaction is the basis of life processes. The life processes decompose 
certain substances, the energy liberated allows the formation of proto- 

i 

plasm, which again liberates energy. Thus a continuous formation of 
protoplasm is secured. 

An explanation of anabolism based upon chemical experiments is 
not possible at the present time. In the study of mtra-cellular destruc- 
tion it is possible to trace most processes back to enzymic action. 
There our knowledge ceases because the nature and mode of action 
of enzymes is unknown. In the study of anabolism our knowledge 
has not even progressed so far. The most promising explanation at 
present is based upon the reversibility of enzymic action. 

REVERSIBILITY OF ENZYMIC ACTION 

Chemical reactions between organic compounds proceed quite 
rapidly at first, then become slower and slower until the reaction 
stops entirely. The reaction is not complete at the time it reaches 
an equilibrium. If the equilibrium is disturbed by adding more of 
the reagents, the process will continue. If, however, the products of 
reaction are added, the reverse process will take place. Reactions 
between organic compounds can proceed either way, depending upon 
the relative concentrations of the reacting substances. The standard 
example is esterification. Acetic acid plus alcohol gives ester plus 
water, 

CH 3 COOH + CH 3 CH 2 OH<=CH 3 COOCH2CH3 + H 2 O. 

Acetic acid Alcohol Ester 

The process goes to a certain equilibrium and stops. If ester is mixed 
with water, it gives acid plus alcohol, until the same equilibrium is 
reached. If acid and alcohol are added to a system in equilibrium, more 
ester will be formed. If ester is added, more alcohol and acetic acid 


MECHANISM OF METABOLISM 2ig 

will be formed. The same is true with enzymes, at least with some 
enzymes. Maltase will decompose maltose into two molecules of 
dextrose. In a concentrated solution of dextrose, however, maltase 
will form maltose, or a similar sugar, isomaltose. Lipase is able to 
produce fat from glycerin and fatty acids. A solution of albumose 
with trypsin or pepsin gives a precipitate of a body which is more com- 
plex than albumose and which gives the protein reactions. It is 
believed by many physiologists that pepsin and rennet are the same 
body. Under certain conditions, it has a dissolving power, under other 
conditions it has the power to coagulate. 

The reversibility of enzymic action has given rise to much specula- 
tion about assimilation and growth. It seems reasonable to suppose 
that the cell forms its protoplasm from amino-acids by the reversed 
action of proteolytic enzymes. In the same way, cellulose may be 
formed from dextrose, fat from glycerin and fatty acids. Nearly all 
phases of growth can be accounted for in this way. This is nothing but 
theoretical speculation, and the only fact to support it is the reversi- 
bility of certain enzymes. The conditions under which chemical reac- 
tions take place inside of the cell are very largely unknown. There 
are so many processes going on at the same time that it is absolutely 
impossible at the present time to obtain a perfect understanding of all 
these reactions. Thus, our knowledge of growth is largely based 
upon analogy and speculation. 

GENERAL ENZYMIC CONSIDERATIONS 

Enzymes are produced only by living cells. After they are once 
formed, they act like chemical compounds, independent of the cell 
which produces them. Even the endo-enzymes follow only the law of 
enzyme-action and are not influenced by the cell which contains them. 
The enzymes are mostly influenced by their own products, and when 
a certain yeast ceases to ferment sugar at the concentration of 8.5 
per cent of alcohol, this means that the alcoholase of this yeast cannot 
tolerate more than 8.5 per cent of alcohol. The inability of the cell 
to regulate enzymic action may account for the fact that often a 
culture produces an amount .of fermentation products sufficient to 
kill all cells. This is observed in the lactic, acetic and alcoholic fer- 
mentations, and, perhaps, occurs in many others. 


220 NUTRITION AND METABOLISM 

Probably all cells produce several enzymes Microorganisms 
feeding upon various foods must form various enzymes. Frequently 
several enzymes are necessary for the decomposition of one com- 
pound. Rhizopus oryzcB uses three enzymes in order to form alcohol 
from starch, first the diastase to change starch to maltose, then 
maltase to change maltose to dextrose and finally alcoholase 
to change dextrose to alcohol and carbon dioxide. The number of 
enzymes formed by certain microorganisms is surprising. Asper- 
gilhis niger has the reputation of forming almost all enzymes which 
have ever been found in microorganisms. Penicillium camemberti 
produces (after Dox) erepsin, nuclease, amidase, lipase, emulsin, 
amylase, inulase, rafrmase, invertase, maltase and lactase. It has 
been believed for a long time that certain enzymes are regular products 
of the cell while others are formed only if the substance upon which 
they act is present. According to Dox's investigations with Peni- 
cillium camemberti, there is no evidence that enzymes not normally 
formed by the organism in demonstrable quantities can be developed 
by special methods of nutrition. The addition of a particular 
food compound does not develop an entirely new enzyme, but stimu- 
lates the production of the corresponding enzyme which is normally 
formed, although in small amounts, under all conditions. 


CHAPTER III 
FOOD OF MICROORGANISMS* 

MOISTURE REQUIREMENT 

Moisture may be called the most important factor of life. Not 
only bacteria, but every microscopic and macroscopic being requires a 
considerable amount of moisture. Living organisms contain on the 
average between 70 per cent and 90 per cent of water, and only 10 per 
cent to 30 per cent of solid matter. Microorganisms which live 
entirely submerged in liquids need water not only within but without 
the cells. Bacteria, yeasts, molds, and some protozoa obtain their food 
by diffusion through the cell-membrane; their food-substances must 
be soluble and dissolved. No other liquid can take the place of water. 

The amount of water required by microorganisms cannot be stated 
briefly. Several factors have to be taken into consideration, as the 
osmotic pressure, the insoluble and the colloidal substances, the species 
of organisms, temperature, and perhaps others. (See pp. 184, 203.) 

AMOUNT OF FOOD REQUIRED 

The amount of food that is ordinarily decomposed by microorgan- 
isms and the amount that is absolutely necessary, differ widely. The 
quantity of organic and inorganic matter just sufficient to support a 
very weak growth is certainly very small, since a few species will 
multiply to some extent in ordinary distilled water. Such water, after 
having stood for some time, is found to contain several thousand 
bacteria per c.c. It may seem to the layman that in such water it 
would be possible to detect easily the organic and inorganic matter of 
the microorganisms so that it could not be considered distilled water. 
An estimate of the weight of bacteria demonstrates, however, that this 
is not the case. If we suppose the average bacterial cell to be a 
cylinder whose base measures i square micron and whose height is 2 
microns (which is a high estimate) the volume of such a cell would be 
1X1X2 cubic microns = o.ooi X o.ooi X 0.002 mm. = o.ooo,- 

* Prepared by Otto Rahn. 

221 


222 NUTRITION AND METABOLISM 

000,002 cu. mm. The specific gravity of bacteria being very nearly i, 
the weight of one bacterium would be 0.000,000,002 mg.; 100,000 cells 
per c.c. means 100,000,000 cells per liter, which would weigh 0.2 mg. 
Of this total weight, at least four-fifths is water and only one-fifth is 
solid matter. The total solid matter in i liter of water containing 
100,000 bacteria per c.c. amounts to the immeasurable quantity of 
0.04 mg. Such water will pass the tests for distilled water. How 
much food the bacteria in distilled water have used is impossible to say, 
since besides the traces of minerals in the water, they obtain some food 
from volatile compounds of the air like carbon monoxide (CO), 
carbon dioxide (02), ammonia (NH 3 ), hydrogen (H), and perhaps 
methane (CH 4 ). Under all circumstances the amount of food used is 
very small. 

On the other extreme, the maximum amount of food cannot be 
stated very definitely. Usually bacteria cease to cause decomposition 
because of the accumulation of noxious metabolic products. The 
ordinary bacterium from sour milk will not form more than about one 
per cent of lactic acid, because this is the highest acid concentration 
that this bacterium can endure. If this acid is neutralized, the in- 
hibiting cause is removed, and the lactic fermentation starts anew 
until the maximum acidity is reached again. The amount of food 
decomposed depends largely upon the power of the organism to resist 
its own products. If the food is too concentrated, however, physical 
influences may interfere with the metabolism of the cell (page 254). 

FOOD FOR GROWTH 

The total weight of a large bacterial cell is estimated in the pre- 
ceding paragraph to be about 0.000,000,002 mg., of which only about 
one-fifth is dry matter. The smallest quantity that can be weighed 
accurately on ordinary analytical balances is o.i mg. This corre- 
sponds to about 250,000,000 bacteria. MacNeal and associates found 
that the dry matter of 550,000,000 cells of B. coli weigh o.i mg. The 
amount of food that is used as the building material for the cell is 
probably larger than the weight of the cell itself, since there will always 
be present waste products, but it is of the same order of magnitude, i.e., 
very small and often hardly measurable. The example of the urea fer- 
mentation (page 202) illustrates this point very well. 

SOURCES OF CARBON.- -The compounds which can serve as building 
stones for the cell vary greatly with the species. The source of carbon 


FOOD OF MICROORGANISMS 223 

for all green plants is carbon dioxide (CO*). Animals cannot use this, 
for they all require complex compounds, such as carbohydrates, fats 
or amino-acids. Bacteria exist between the plants and animals in 
this respect. Some bacteria have already been mentioned (page 201) 
as being able to use carbon dioxide (CO 2), as the only source of carbon; 
they are the mineral-oxidizing species. Such bacteria are called 
autotrophic in their relation to carbon, since they use it in the inorganic 
form. A bacterium feeding on carbon, as such, would be called 
prototrophic; bacteria of this class are said to exist. The vast majority 
of microorganisms are heterotrophic, using carbon in organic form. 
Organic acids and sugars are excellent sources of carbon for micro- 
organisms, although proteins and their decomposition products seem 
to be equally satisfactory as construction material. 

SOURCES OF NITROGEN. The sources of nitrogen are equally varied; 
the green plants use nitrates; animals must have a number of different 
amino-acids; the microorganisms again are found between plants and 
animals. We know autotrophic bacteria, and especially molds and 
yeasts which can grow with nitrates or ammonium salts as the only 
source of nitrogen. There are three groups of prototrophic bacteria 
in their relation to nitrogen the B. amylobacter group, the Ps. radicicola 
group and the Azotobacter group. These bacteria are of the greatest 
importance to agriculture; soil fertility depends, to a large extent, 
upon the last two groups, for they take nitrogen gas from the surround- 
ing air, form their own protoplasm from it, and thus increase the 
amount of chemically combined nitrogen in the soil. Details of their 
relation to soil fertility can be found in Chap. Ill, page 400. The 
majority of bacteria are heterotrophic, requiring organic nitrogen. Urea 
is not well adapted for this purpose; amino-acids or the peptones from 
which amino-acids are derived are the best compounds for most 
organisms. Asparagin is very commonly used if for some reason 
peptones are to be omitted. 

SOURCES OF HYDROGEN AND OXYGEN. The sources of hydrogen are 
hardly ever discussed with bacteria since hydrogen bears such a close 
and peculiar relation in water and organic food supplies. The ulti- 
mate association of hydrogen with oxygen in the molecule of water 
(H 2 O) and with carbon in organic substances (CH 4 ) establishes its 
importance in all life processes. There are many prototrophic bacteria, 
using oxygen as such; others are able to reduce such compounds as 


224 NUTRITION AND METABOLISM 

nitrates or sulphates, which would be autotrophic, thus providing for 
their needs. Heterotrophic bacteria are not unusual. In this connec- 
tion it may be said that it is often difficult to distinguish between oxy- 
gen needed for cell construction and oxygen needed for energy formation. 
SOURCES OF MINERALS.- -The amount of mineral matter necessary 
for the construction of the cell is very small; potassium and phos- 
phorus seem to be among the most essential elements. It is customary 
to consider a tap water with 0.02 per cent of di-potassium hydro- 
gen phosphate (K 2 HPO4), sufficient in mineral matter of all kinds to 
provide for fair growth. Some of the common materials used in the 
preparation of nutrient media, such as meat extract and peptone, also 
contain considerable amounts of mineral matter. 

FOOD FOR ENERGY 

As all food in its decomposition results in products of some form or 
other, it may not seem justifiable to separate a paragraph on food 
from another on products. The essential difference lies in the fact that 
we consider food from the viewpoint of the cell, while products are 
commonly considered apart from the construction processes of the cell 
and only from their application, or, it may be, from the viewpoint of 
usefulness to man. 

Animals provide for their energy by oxidations, and almost exclu- 
sively by complete oxidations. Some bacteria, and most molds, do 
the same. The range of materials which can serve as food for this pur- 
pose is surprising. With animals, the food is practically limited to 
plant and animal tissue. With bacteria, we find the strangest sub- 
stances, such as hydrogen, carbon monoxide, coal, marsh gas, hydrogen 
sulphide, ammonia, nitrites, formic and oxalic acids, alcohol and thio- 
sulphates serving this purpose. The fact that many gases are used 
as food makes us realize that oxygen is not such an extraordinary 
compound as animal physiology seems to indicate, but that it should be 
classed merely as one of the many food compounds. This is especially 
significant since it will be shown later that free oxygen is not necessary 
for microbial life, and that many organisms can exist without it. 

The oxidations are not always complete. The formation of nitrous 
acid from ammonia, the oxidation of alcohol to acetic acid are such 
examples. Some organisms are highly specialized in their food require- 
ments, especially the mineral-attacking bacteria are usually limited 
to one source of energy. The microorganisms oxidizing organic com- 


FOOD OF MICROORGANISMS 225 

pounds have, as a rule, the ability to decompose several compounds, 
and some bacteria are common scavengers, able to feed on organic acids, 
sugars, fats and proteins. 

Oxygen Relations. It is characteristic of many microorganisms to 
provide for their energy without using free oxygen. One such example 
has already been given in urea fermentation. 

(NH 2 ) 2 CO + 2 H 2 = (NH 4 ) 2 C0 3 

Urea Ammonium carbonate 

Very common is the decomposition of sugars without oxygen. 
The two most typical fermentations of this type are the alcoholic and 
the lactic fermentations. 

C 6 H 12 O 6 = 2 C 2 H 5 OH + 2 CO 2 + 22 Cal. 

Sugar Alcohol 

C 6 H 12 O 6 = 2C 3 H 6 O 3 + 15 Cal. 

Sugar Lactic acid 

In fermentations of this type, the changes take place without an 
oxygen gas partaking in the reactions. These fermentations seem to 
be essentially reactions of the oxygen atoms within the sugar molecule. 
One side of the molecule is reduced while the other side is oxidized. 
In the sugar molecule, each carbon atom has one oxygen atom. In 
the products of fermentation, carbon dioxide has two oxygen atoms to 
one carbon atom, and in alcohol there is only one oxygen atom for two 
carbon atoms. In the lactic fermentation, the oxygen, which is dis- 
tributed evenly in the sugar, is shifted to one side of the molecule in 
lactic acid. 

H H H H H O 

O O O O O || 
Dextrose, H C C C C C C 

H H H H H H 

H H O 

o I! 

Alcohol, HC CH C Carbon dioxide, 

H H || 
O 

H H 
H O O 

Lactic acid, HC C C 

H H || 
O 

15 


226 NUTRITION AND METABOLISM 

In some of the more complex fermentations, we find simultaneous 
formation of hydrogen or methane and carbon dioxide; the one is 
the end product of reduction, the other the product of complete oxida- 
tion. This also indicates that the oxidation of one part of the molecule 
takes place at the expense of the other. 

In a similar way, some organic acids, e.g., tartaric and lactic acids, 
can be fermented by certain bacteria without requiring oxygen. Some 
bacteria have the ability to attack proteins and decompose them 
completely in the absence of oxygen. 

Bacteria, having the ability to provide for their energy without 
oxygen gas, may live in the complete absence of oxygen, and may 
multiply indefinitely without it as long as there is sufficient food. But 
some microorganisms, such as yeasts, seem to grow only for a limited 
time in the absence of oxygen. Finally, they cease growing, and 
we may well assume that they need oxygen for cell construction which 
can be used in no other form except as molecular oxygen. The urea 
bacteria also belong in this group. 

A large number of bacteria and yeasts, and also a few molds, can 
provide for their energy by either oxidation or decomposition in the 
absence of oxygen. Very commonly a great variety of compounds can 
be found which may be oxidized while but very few can be intra- 
molecularly fermented without oxygen. This is easily understood: 
all organic compounds will yield heat upon oxidation, while exothermic 
intramolecular changes require a special structure. Carbohydrates 
are the most excellent substances for such intramolecular decomposi- 
tions. S. ceremsice and B. coll can live in sugar-free broth only if ex- 
posed to the air. They provide for all their needs by oxidation of the 
protein. If oxygen is excluded, growth depends upon sugar, or a 
similar fermentable compound. We test for the absence of sugar in a 
given solution by pouring it in a fermentation tube and inoculating 
with B. coll: if the liquid in the closed arm remains clear, i.e., if B. coll 
does not grow without oxygen, it is a good indication that no sugar is 
present. 

It is usually assumed that in fermentations of this nature, the 
oxygen atoms are shifted within the same molecule. In other cases, 
oxygen is taken from one molecule and used for the oxidation of 
another. This results in one of the molecules being reduced. Nitrates 
are reduced in this way to nitrites, or ammonia, or nitrogen gas; sul- 


FOOD OF MICROORGANISMS 227 

phates to hydrogen sulphide, and litmus or methylene blue to the 
colorless leuco-compounds. Such removal of oxygen from a molecule 
requires energy, and is possible only when the bacterium by using the 
oxygen for oxidation of organic matter can obtain a larger amount 
of energy. The following example shows such a possibility: 

2KN0 3 + 36.6 Cal. = 2 KNO 2 + 2 
C 2 H50H + O 2 = CH 3 CO 2 H + H 2 O + 115 Cal. 

This process leaves an energy balance of 115 36.6 = 78.4 Cal. for 
the needs of the bacterium. 

Such decompositions are sometimes referred to as reducing fermen- 
tations" but this term is not correct, as the reduction must always be 
accompanied by a simultaneous oxidation process. 

The amount of energy liberated by a fermentation without oxygen 
is much smaller than that furnished by complete oxidation; the intra- 
molecular change always leaves organic compounds which contain a 
considerable amount of the total energy. Yeast, in presence of very 
much oxygen, oxidizes sugar completely to water and carbon dioxide. 

C 6 H 12 O 6 -f 120 = 6CO 2 + 6H 2 + 674 Cal. 

while in the absence of oxygen it will change the sugar to alcohol and 
carbon dioxide. 

C 6 H 12 6 = 2C 2 H 5 OH + 2CO 2 + 22 Cal. 

The energy gained in the first process is about thirty times as large 
as that gained in the second process. This was demonstrated as early 
as 1 86 1 by Pasteur. He grew yeast in sugar solutions, varying only 
the amount of oxygen in contact with the medium. At the end of 
the experiment, the weight of the dry yeast and the decomposed sugar 
was determined, and the amount of sugar necessary to produce one 
part of yeast was computed. He found: 

In a closed flask, without any air i part yeast required 1 76 parts sugar. 

In a closed flask, with large air space i part yeast required 23 parts sugar. 

In a thin layer, a few mm. thick i part yeast required 8 parts sugar. 

In a very thin layer, in 24 hours i part yeast required 4 parts sugar. 

This experience led Pasteur to the conclusion that fermentation 
corresponded to the respiration process of animals, that fermentation 
was respiration without oxygen. 

It is quite evident that since the utilization of the food in the 


228 NUTRITION AND METABOLISM 

absence of oxygen is very high, the organisms have to decompose 
much more food. This accounts, to a great extent, for the enormous 
destructive power of bacteria, when comparisons of the great quantity 
of food decomposed are made with the very insignificant weights of 
cells. It has been estimated that the lactic bacteria decompose their 
own weight of sugar in one hour. 

Summing up the relation of oxygen to microorganisms, some 
bacteria, and especially the molds, are found depending upon oxygen as 
an indispensable part of their food. Three groups are recognized: 
Those, a large number, organisms in the presence of oxygen producing 
oxidations; those able to sustain life without oxygen; and those de- 
pending entirely upon decompositions which require no oxygen. 
The lactic bacteria and the butyric bacteria belong in the last group. 

In considering the oxygen requirements, it is customary to in- 
clude another influence of oxygen upon bacteria. This has really 
nothing to do with its food value, but deals with the poisonous qualities 
of oxygen. Oxygen in this light may well be called a poison as it will 
kill bacteria in very low concentrations. Ordinarily it is regarded as 
constituting over 20 per cent of our atmosphere. But if a study is 
made of its effect upon bacteria, it is necessary to measure it in the 
same way food is measured, and consider the concentration in which 
it is offered to the cell. Microorganisms obtain their oxygen not as 
gas, but as dissolved oxygen. The solubility of oxygen is very small, 
about 0.0009 P er cent a t 20. Practically all bacteria die readily if the 
oxygen concentration is raised to thirty times the atmospheric pressure. 
This would mean a concentration of 0.027 P er cent. It shows that 
oxygen is about as poisonous as formaldehyde or bichloride of mercury. 

Some bacteria are extremely sensitive to oxygen, and will die if 
exposed to ordinary atmospheric oxygen. They grow only if oxygen 
is almost completely removed. These organisms are called the 
strictly anaerobic or obligate anaerobic bacteria. They are contrasted 
with the facultative anaerobic bacteria which thrive with oxygen as well 
as without, and the strictly aerobic bacteria which have to have oxygen 
for their normal life processes. 

No strict limits can be drawn between aerobic and anaerobic 
bacteria. Even the most sensitive of organisms will be able to tolerate 
traces of oxygen, while the strictly aerobic bacteria can multiply also 
if the oxygen concentration is below that of a saturated solution. The 


FOOD OF MICROORGANISMS 


229 


limits of growth for the anaerobic bacteria are the limits of tolerance of 
the poisoning oxygen; the lower limit of growth for the aerobic bacteria 
is a question of too scanty food supply. The relation between bacteria 
and oxygen is graphically represented in the following diagram, after 
Kruse: 


Oxygen Pressure* 
o.i OH o.k oS 10 


2.0 


3.0 


i 

i 

3 

f 
S 


FIG. 112. Influence of oxygen upon microorganisms. 

The lines indicate the oxygen concentrations where growth is possible. Line 
i is a strict anaerobe; 2 is not quite so strict; 3 is still less sensitive though it 
cannot grow if exposed to direct influence of the atmosphere; 4 is a facultative 
bacterium such as B. coli; 5 is another one which can tolerate still more oxygen; 
6 can grow only with oxygen but can get along with very little : it might be one 
of the urea bacteria; 8 is more dependent upon oxygen and the line would corre- 
spond to average molds; 7 is a peculiar type needing oxygen and yet being very 
sensitive to it. The sulphur bacteria, e.g., the Beggiatoacea, belong to type 7. Type 
9 is said to be representative of B. abortus. 


i.o indicates the normal atmospheric oxygen content (about 21 per cent by volume). 


CHAPTER IV 
PRODUCTS OF MICROBIAL ACTIVITIES* 

GENERAL CONSIDERATIONS 

The great difference in the metabolism of animals and of bacteria, 
even though they feed essentially on the same foods, is t}ie incomplete 
metabolism of most bacteria, contrasting sharply against the very 
complete oxidation of food in the animal body. The food of the animal 
is decomposed by the body cells to carbon dioxide, water and urea. It 
is the most complete decomposition possible, excepting urea which, 
however, is very near the final decomposition product, ammonium 
carbonate. Microorganisms, on the contrary, are characterized by 
incomplete metabolism. They do not commonly oxidize their food to 
the end products but many of them produce organic compounds which 
are not farther decomposed by them. It is this partial decomposition 
of organic matter which makes microorganisms play such an important 
role in life and industries. Our modern microbiology is dated from the 
time when Pasteur showed that the alcohol in the beer fermentation, 
the lactic acid in the souring of milk, the acetic acid in the vinegar 
fermentation are products of microbial activity. The existence of 
microorganisms had been known for nearly 200 years, but they were 
considered largely as a curiosity; as soon as they were recognized as 
the cause of fermentations, and of toxins, they received at once the 
greatest attention. Not all bacteria cause incomplete decompositions; 
some oxidize as completely as animals do. Others, again, form first 
intermediary products, which they later decompose completely; among 
these, are found many molds, the sulphur bacteria, and some species of 
the vinegar bacteria. 

THE CHEMICAL EQUATIONS OF FERMENTATIONS 

The metabolism of all organisms is considered to be a chemical 
process which follows in most respects the laws of chemistry. That 
we are not familiar with all the changes taking place in the cell is not 

* Prepared by Otto Rahn. 

230 


PRODUCTS OF MICROBIAL ACTIVITIES 231 

because we are dealing with unknown forces, but simply because we 
do not know all the factors involved in the process. Some of the 
chemical changes caused by the living cell can be imitated exactly by 
the chemist in a test-tube. This may be illustrated by the oxidation 
of alcohol to acetic acid, the decomposition of urea to ammonium 
carbonate and of ammonia to nitrate. Some other processes are not 
as fully understood and not as easily imitated. The alcoholic and 
acid fermentations of sugars are of such nature. There is no reason 
to suppose, however, that these processes are other than chemical 
changes. Since a chemical process can always be expressed by a 
chemical equation, we should expect the same with the fermentations 
and decompositions caused by microorganisms. 

This formulation is not always simple, because the greater number 
of microorganisms decompose organic substances in more than one way. 
Also, certain compounds may be produced in such small quantities as 
to escape the chemical analysis entirely, since the determination of 
many organic compounds is a very difficult task. Again, part of the 
decomposed material will usually be assimilated in the growth of the 
cells; hence more material disappears than can be accounted for by 
the fermentation products. There are several possibilities for dis- 
crepancies; accurate equations can be given only for the simplest fer- 
mentations, the products of which can be analyzed more or less exactly. 

The best studied microbial process is the alcoholic fermentation. 
The simplest equation for the decomposition of dextrose into alcohol 
and carbon dioxide by yeast is 

C 6 H 12 6 = 2C 2 H 5 OH + 2 C0 2 
1 80 92 88 

According to this formula, TOO parts of dextrose should give 51.11 parts 
of alcohol and 48.89 parts of carbon dioxide. The actual yield comes 
very close to these numbers, but does not reach them; the largest 
amounts found were 46-47.5 per cent of carbon dioxide and 47.5-48.67 
per cent of alcohol. Under the most favorable conditions, the total 
yield of the products of fermentation was only 95 per cent of the 
theoretical yield. 

Other products are formed besides the alcohol and carbon dioxide. 
The amount of glycerin found in fermented liquids varies very much 
with the conditions of fermentation; it reaches from 1.6 to 13.8 per cent 


232 NUTRITION AND METABOLISM 

of the alcohol or from 0.8 to 6.9 per cent of the fermented sugar. A 
small quantity of succinic acid is also formed, usually about 0.6 to 0.7 
per cent of the fermented sugar. Traces of acetic acid and of lactic 
acid seem to be normal products of the process of fermentation, and we 
always find fusel oil. The latest investigations seem to indicate that 
glycerin and succinic acid are produced by yeast cells even in the absence 
of sugar. This discovery makes it probable that the glycerin and suc- 
cinic acid are derived from the reserve substances of the yeast cells, 
such as lecithin, and are not direct products of fermentation. This 
accounts also for the variation of the proportion between alcohol and 
glycerin. Fusel oil is now believed to be a waste product of cell 
construction. 

Similar are the experiences with the lactic fermentation which has 
been studied almost as extensively as alcoholic fermentation. If it is 
supposed that the formation of lactic acid follows the equation 

Ci2H22On -f~ H 2 O = ^CsH-sQs 

342 18 360 

Lactose Lactic acid 

the actual yield of acid is found to be between 90 per cent and 98 per 
cent of the theoretical. The other 2-10 per cent are either used for 
cell-growth or for products which thus far have escaped chemical de- 
termination. Small discrepancies will also be found in fermentation 
of urea and in the nitrifying process, where small amounts of the 
nitrogenous material are used for cell-growth. 

Another difficulty in finding the chemical equation of a microbial 
fermentation is the fact that this process may change with the age of the 
culture. In those fermentations where several gases, as carbon dioxide 
and hydrogen, are produced, the relative proportion of the two is not 
always constant. In the butyric fermentation of dextrose by B. 
amylozyma, Perdrix tries to account for this change by assuming three 
different phases of the process at various ages of the cultures, repre- 
sented by the following equations: 

First stage: 56C 6 H 12 O 6 + 42H 2 O = ii6H 2 + ii4CO 2 + 3oCH 3 COOH + 

Dextrose Acetic acid 

36CH 3 CH 2 CH 2 COOH. 

Butyric acid 

Second stage: 46C 6 H 12 O 6 + i8H 2 O= ii2H 2 + 940)2 + i5CH 3 COOH + 
38CH 3 CH 2 CH 2 COOH. 

Third stage: C 6 H 12 O 6 = 2H 2 + 2 CO 2 + CH 3 CH 2 CH 2 COOH. 


PRODUCTS OF MICROBIAL ACTIVITIES 233 

Kruse has called attention to the fact that these complex equations 
can well be explained as the simultaneous occurrence of the following 
simple fermentations: 

C 6 H 12 O 6 = 2H 2 + 2 CO 2 + CH 3 CH 2 CH 2 CO 2 H 

C 6 H 12 O 6 = 3CH 3 CO 2 H 

C 6 H 12 O 6 + 6H 2 O = 6CO 2 + i2H 2 

The first fermentation continues when the others have already ceased, 
and thus the last stage of Perdrix's equations is very simple. Brede- 
mann also found that the proportion of the various products formed by 
B. amylobacter varies greatly with the conditions, and the same has been 
recently established in the fermentation of B. coll. 

Other complications occur when an organism is able to use its own 
products as food, as is the case with some acetic bacteria. They will 
at first produce considerable amounts of acetic acid and after a while 
they oxidize the acid completely. It becomes impossible to account for 
microbial activity by a chemical equation when several organic com- 
pounds are decomposed at the same time as is found to occur in some 
foods, as butter, cheese, ensilage and in sewage. It is also impossible 
to formulate exactly decompositions which are caused by mixed cultures. 
The complications become so great and the relations between different 
organisms are so little known that it is useless to make the attempt. 

PRODUCTS FROM NITROGEN-FREE COMPOUNDS 

SUGARS. It would be entirely beyond the limits of this book to 
give an account of all the different ways in which sugars and other 
compounds can be decomposed by microorganisms. It is much more 
important for the beginning microbiologist to acquaint himself with 
the main types of sugar fermentations and with the characteristics 
of the organisms which bring about these changes. 

In the action of microorganisms many distinguish somewhat crudely 
six common types: 

Complete oxidation. 

Partial oxidation. 

Alcoholic fermentation. 

Lactic fermentation. 

Acid gas fermentation. 

Butyric fermentation. 
Most of these types have been mentioned previously. 


234 NUTRITION AND METABOLISM 

Complete oxidation of carbohydrates is observed most commonly 
among molds and mycodermas, and also in a few bacteria, e.g., in A zoto- 
bacter. It is possible only where there is a ready oxygen supply, as, 
e.g., in soils of an open texture, in trickling filters, and on the surface 
of decaying fruits. 

The incomplete oxidation is, as a rule, more common in nature. 
Frequently microorganisms produce first an incomplete oxidation, but 
later oxidize the intermediate products completely. The molds are 
typical examples. Aspergillus niger is noted for its formation of oxalic 
acid. If it is grown in a sugar solution, it will bring about at first a 
rapid increase in acidity, but after a while, it decreases again, when the 
acid is oxidizing completely. The following processes may be noted: 

C 6 H 12 6 + 90 = 3(C0 2 H) 2 + 3 H 2 

Oxalic acid 

(C0 2 H) 2 + O = 2 C0 2 + H 2 O 

The intermediate product can be accumulated by precipitating it with 
lime which neutralizes the acidity. This principle is used in the com- 
mercial manufacture of citric acid by Citromyces, a mold closely 
related to the genus Penicillium. This mold oxidizes sugar to citric 
acid according to the following equation: 

C 6 Hi 2 6 + 30 = C 6 H 8 7 + 2H 2 

Citric acid 

This fermentation is much more complicated than this equation indi- 
cates, on account of the entirely different chemical structures of citric 
acid and dextrose. The practical yield in the factory is only about one- 
half of the theoretical, since complete oxidation cannot be avoided 
altogether. 

The oxidation processes, just recited, can take place only in the 
presence of oxygen; the other four types of carbohydrate decomposi- 
tion require no oxygen, and take place as well in the absence of oxygen; 
the butyric fermentation is brought about only in the absence of oxygen. 

Alcoholic fermentation is caused only by yeasts and a few molds; 
no bacterium produces alcohol according to the well-known equation 
mentioned above. Alcohol is formed by several bacteria but only in 
small quantities and always together with several acids; this is a 
distinctly different type of decomposition. 

In the above groups and the following groups of microorganisms, 
there appears to be a close agreement between the morphological 


PRODUCTS OF MICROBIAL ACTIVITIES 235 

characters of the organisms involved and the specific type of fermenta- 
tion. Practically all the alcoholic organisms are yeasts, and the lactic 
acid-producing organisms are streptococci or closely related bacteria. 
The lactic bacteria, as they are briefly named, such as are responsible 
for lactic fermentation, are readily recognized by their scanty growth 
on agar, and their excellent growth in milk, bringing about a solid 
curdling in one to three days. They change sugar to lactic acid only. 

C 6 H ]2 6 = 2C 3 H 6 O 3 

No gas and no volatile acids are formed by these bacteria. The best- 
known representative of this group is the organism which causes the 
normal souring of milk. It was originally called Bacterium lactis acidi, 
but on account of its very close relation to the streptococci, it is more 
commonly now named Streptococcus lacticus. Many streptococci will 
produce the true lactic fermentation. 

The last two groups of organisms, alcoholic and lactic, represent 
complex fermentations. There are several products formed, and as has 
already been pointed out in the paragraph on the equation of fermen- 
tations, the entire fermentation cannot be described accurately by one 
equation, for different fermentations operate independently and simul- 
taneously in the same cell. Under slightly different experimental 
conditions the one or other of these simultaneous fermentations may be 
favored, accordingly a varying proportion of the products is formed. 

The typical representatives of the acid-gas forming group of micro- 
organisms which cause acid-gas fermentation are B. coli } and its near 
relative, Bact. aero genes. Many of the gas-formers in nature belong in 
this group; the bacteria of the fermentations of pickles, sauerkraut, 
salt-rising bread, the gassy fermentation of milk are some of the many 
representatives. They are distinct rods, with good surface growth, 
and do not liquefy gelatin. They are commonly spoken of as the coli- 
aerogenes group. Some of them have peritrichate flagella, while 
others are not motile. 

The fermentation of dextrose brought about by these organisms 
has been described originally by Harden in the equation: 


2C G H 12 6 + H 2 = 2C 3 H 6 03 + CH 3 C0 2 H + C 2 H 5 OH + 2 CO 2 

Dextrose Lactic acid Acetic acid Alcohol 

Harden himself stated later that this equation holds only for one 
strain, and that we have several different strains distinguished by a 


236 NUTRITION AND METABOLISM 

proportion of products quite different from the one suggested by the 
equation. Recently Kamm has shown that a good mineral food 
(probably phosphates are the essential agent) favors a formation of 
gas and of volatile acids, while a scant supply of minerals causes the 
bacteria to produce mainly lactic acid. We must assume, therefore, 
at least two simultaneous independent fermentations: 

C 6 H 12 O 6 = 2C 3 H 6 O 3 
and 

C 6 H 12 6 + H 2 O = CH 3 CO 2 H + CH 3 CH 2 OH + 2 CO 2 + 2 H 2 

i 

The first equation is already known to us; it is the true lactic fermenta- 
tion. The second equation may be divided still further into several 
simpler equations. 

B. typhosus, causing typhoid fever, is closely related to B. coli, but 
does not form gas. It forms, however, formic acid, HCO 2 H, which, if 
decomposed, would give H 2 + CO 2 . 

The last type of sugar fermentations is the butyric fermentation, 
in which butyric acid is the most conspicuous, but not the only fermen- 
tation product. Acetic acid, hydrogen and carbon dioxide, and, with 
some organisms at least, ethyl and butyl alcohols are formed along with 
butyric acid. As already mentioned in the paragraph on the equation of 
fermentation, Kruse believes this fermentation to consist of several 
simultaneous fermentations, of which the most interesting at this stage 
is the one showing the formation of butyric acid. 

C 6 H 12 O 6 = 2H 2 + 2 CO 2 + C 4 H 8 O 2 

The organisms producing butyric acid are mostly strictly anaerobic 
spore formers with a tendency to form spindle-shaped cells; they stain 
bluish-black with iodine and Bredemann gave the clostridium group 
one species name, B. amylobacter, as he found no distinct and char- 
acteristic differences between the many strains which he studied. 
Many members of this group have the ability to fix nitrogen, i.e. 
to build up their protoplasm without using any sources of nitrogen 
other than nitrogen gas. Most of the so-called "Clostridium" species 
belong in this group. Butyric acid is also formed by B. tetani and by 
B. botulinus, the latter of which causes the most dangerous kind of meat 
poisoning. 

Of other sugar fermentations may be mentioned here only by name, 


PRODUCTS OF MICROBIAL ACTIVITIES 237 

the slimy fermentations, as manifested in ropy milk and the mannit 
fermentation. The latter is one of the very few reduction processes 
brought about by bacteria, and one which causes trouble in wine. 

What has been stated broadly for sugars holds to some extent true 
also for the alcohols derived from sugars, including glycerin. Many 
bacteria fermenting dextrose can also ferment mannit and glycerin 
with a slight variation of the products, but some do not do this. 

Among disaccharides there is a great variation of fermentation. 
Some groups ferment lactose readily as the coli organisms and Strept. 
lacticus, while among yeasts, fermentation of lactose is rare. Practi- 
cally all yeasts ferment saccharose, however, and among the lactic 
bacteria and the coli group many strains cannot ferment saccharose. 

STARCH. Quite different is the fermentation of the insoluble carbo- 
hydrates of which we can mention only starch and cellulose. Insoluble 
compounds can be fermented only after being made soluble by an 
enzyme, the amylase (see mechanism of metabolism). Amylase is 
produced by most molds, by none of the fermenting yeasts, by a few 
torulas, and perhaps mycodermas, and by a great many of the bacteria. 
The sugar thus produced from starch is decomposed according to the 
main types mentioned under sugars. The lactic bacteria and the coli 
bacteria do not attack starch, but some acid-gas fermentations of 
starchy foods do take place. Butyric fermentation of starch is com- 
mon. Alcoholic fermentation can be accomplished only by some of 
the Mucors, and Aspergilli. 

CELLULOSE is decomposed only by very few organisms; these must 
be very active and very numerous, to judge from the enormous amounts 
of cellulose produced and destroyed every year on earth. Molds and 
higher fungi play probably the main role in its decomposition; the 
products have not been determined, but we may well assume a complete 
oxidation, since no intermediate products have ever been mentioned. 
No yeast is known to decompose cellulose, and among the bacteria we 
find but very few species. Some species have recently been isolated 
which decompose cellulose in the presence of air; the products have not 
been determined; we can, however, assume a partial oxidation, eventu- 
ally a complete oxidation. Besides the aerobic fermentation, we have 
two types of anaerobic fermentation which are ordinarily described as 
the hydrogen fermentation and the methane fermentation. In these 
fermentations the gases mentioned, together with carbon dioxide, are 


238 NUTRITION AND METABOLISM 

liberated, and butyric and acetic acids are formed at the same time. 
The marsh gas of the marshes originates in this way. 

Summing up all the products formed from carbohydrates, we find 
several acids, among them lactic and acetic acids most commonly, 
and ethyl alcohol, rarely other alcohols, besides carbon dioxide, 
hydrogen and water. The variety is not so great, but with these few 
compounds, a number of different combinations are possible, and the 
complication of the study of such fermentations lies mostly in the 
simultaneous formation of several of the compounds. 

ACIDS AND ALCOHOLS. The organic acids and alcohols can be 
decomposed further by bacteria and molds, also by some yeasts, to 
simpler compounds. Ordinarily, this decomposition consists in the 
complete oxidation. Thus, Oidium lactis will destroy the lactic acid 
of sour milk and of soft cheeses by complete combustion. 

C 3 H 6 3 + 60 = 3 C0 2 + 3 H 2 

By the same process, the acidity of sauerkraut, ensilage, pickles is 
reduced by mycoderma species. Another Mycoderma is known to 
destroy acetic acid and thus spoil vinegar or fruits and vegetables kept 
in vinegar; the yeast grows in a thin, dry white scum over the surface, 
and oxidizes the acetic acid. 

CH 3 CO 2 H + 4 O = 2 CO 2 + 2 H 2 O 

The oxidation of alcohols is not always complete. Especially ethyl 
alcohol is usually oxidized first to acetic acid; this is the common vinegar 
fermentation. Many different kinds of vinegar bacteria are known, 
some forming gelatinous masses of cell membranes called mother-of- 
vinegar, while others remain as separate small cells. They all oxidize 
alcohol first to acetic acid. 

CH 3 CH 2 OH + 2 O = CH 3 CO 2 H + H 2 O 

But most of them will oxidize later the acetic acid completely to carbon 
dioxide, after the alcohol is all exhausted, unless the oxygen supply is 
shut off. This behavior reminds one of the formation and destruction 
of oxalic acid by Aspergillus, mentioned previously. It may be re- 
marked here that the vinegar bacteria cannot attack the sugar directly 
to any appreciable degree, and the manufacture of vinegar from sugar 
requires two agents, the alcohol-forming yeast, and the alcohol-oxidizing 
bacterium. 


PRODUCTS OF MICROBIAL ACTIVITIES 239 

Some of the acids can also undergo an anaerobic fermentation. 
This is possible only with hydroxy-acids. The fermentation of the 
calcium salt of tartaric acid was the first anaerobic fermentation 
observed by Pasteur, and the fermentation of lactic acid to butyric 
acid has a reputation for its chemical peculiarity. A compound with 
four carbon atoms is formed from a compound with only three carbons, 
a very unusual thing in fermentation. 

FATS. The decomposition of fats is comparatively simple. All fats 
are glycerides of organic acids, and if they are attacked at all by micro- 
organisms, they are first split into glycerin and free acid. 

H 2 C - O - CO - Ci 5 H 3 i HOH H 2 COH HO 2 C-C 15 H 31 
HC - O - CO - Ci 5 H 3 i + HOH = HCOH + HO 2 C-Ci 5 H 31 


H 2 C - O - CO - C 15 H 31 HOH H 2 COH HO 2 C-Ci 5 H 31 

Fat Water Glycerin Acid 

This brings about the liberation of three molecules of free acid from 
a neutral fat molecule. It is customary to test for the splitting of fat by 
determining its acidity. The glycerin is readily used up by the micro- 
organisms, while the fatty acids are oxidized but very slowly. 

The number of organisms which can attack fat is quite small. 
Most molds can destroy it; one torula has been found in butter which 
attacks it, and perhaps a dozen species of bacteria will do the same, 
among them B. fluorescens and B. prodigiosus, which cause occasionally 
the rancidity of butter. 

PRODUCTS FROM NITROGENOUS COMPOUNDS 

On account of the complexity of the protein molecule, the products 
of protein decomposition by microorganisms are little known. Some 
products are conspicuous through their odor, others can be told by cer- 
tain color reactions, but as we cannot, at the present, give the structural 
formula of proteins, there is no possibility of stating protein decomposi- 
tions in equations similar to those of carbohydrate fermentations. 
The discussion must be limited, for this reason, to the enumeration of the 
most important products, and to the general types of decomposition. 

As in the carbohydrates, soluble compounds are more easily de- 
composed than the insoluble. The keratin bodies of hair, epidermis 
and horn are slowly attacked by a very few organisms. Gelatin, 


240 NUTRITION AND METABOLISM 

casein and serum albumin are more readily decomposed, though their 
solubility is quite limited. Peptones which are readily soluble are 
used by the vast majority of microorganisms. Of interest in this con- 
nection is the fact that the fresh white of egg is poisonous to most bac- 
teria, and fresh blood and animal tissues as well as freshly drawn milk 
have also germicidal properties which are lost by heating or upon 
standing. 

PROTEIN BODIES are as numerous as plants and animals. Each 
species of organism seems to have its particular protein which differs 
from that of other species. With the more highly developed organisms, 
there are several distinctly different proteins found in the same individ- 
ual in different parts of the body. The constituents, carbon, oxygen, 
hydrogen, nitrogen, and sometimes sulphur and phosphorus can be 
determined in their relative amounts without, however, furnishing any 
knowledge of the structure of the molecule. The molecular weight of 
proteins is estimated to be at least 10,000, while the weight of the very 
large molecule of saccharose is only 342. The protein molecule can be 
broken up into smaller molecules. This cleavage is generally believed 
to be a hydrolytic process similar to the decomposition of starch to 
maltose. The first products of protein decomposition do not differ 
essentially from the original protein, but they can be hydrolyzed again 
and again, until finally products of a crystalline nature are found which 
are well-defined chemical bodies. Among the very first products of 
protein degradation it is usually impossible to determine single com- 
pounds, but several groups of compounds may be separated by certain 
precipitants, as acetic acid, ammonium sulphate, zinc sulphate, copper 
sulphate, tannic acid and others. In order to determine the degree of 
protein degradation, e.g., in the analysis of cheese, it is customary to 
determine the nitrogen of compounds precipitated by these various 
reagents, and state it in percentage of the total nitrogen. Thus the 
terms "water-soluble nitrogen," "acid-soluble nitrogen" and others 
originated, meaning the nitrogen of the compounds soluble in water or 
in acid respectively. Some of these groups of degradation products 
have been named and defined more accurately, of which the albumoses 
and peptones are the most common and best described compounds. 
Their chemical nature and structure is, however, just as little known as 
that of the protein bodies. We speak of peptonisation of proteins, 


PRODUCTS OF MICROS IAL ACTIVITIES 241 

e.g., in the clearing of milk or the gelatin liquefaction, meaning that the 
insoluble protein has been made soluble. 

The amino-acids are the first well known compounds of protein de- 
composition. They are organic acids, in which a hydrogen atom is 
substituted by a NH 2 radical. Some of them are simple compounds, 
as the amino-acetic acid NH 2 CH 2 COOH and also the amino-capronic 
acid usually called leucin (CH 3 ) 2 CH CH 2 CH(NH 2 ) COOH. Others 
are of a more complex nature, such as the tyrosin or hydroxy-phenyl- 
aminopropionic acid, C 6 H 4 (OH) CH 2 CH (NH 2 ) COOH, and the 
tryptophan or indol-amino-propionic acid, CgH 6 N CH 2 CH(NH 2 ) 
COOH. 

Of other nitrogenous products which are not amino-acids, a few 
are of striking significance. The very disagreeable odor of putrefying 
proteins and of excreta is due to indol (CsH 7 N) and methyl-indol or 
skatol (CsH 6 N CH 3 ). Indol gives a rose color with nitrites in acid 
solution, and this convenient reagent is used in the identification of 
bacteria. Another group are the amins. The simplest amins are 
the methyl-amins, of which the tri-methylamin (CH 3 ) 3 N is produced 
by several bacteria. The fishy odor of the brine of salted herring is 
largely due to this compound. In this group belong also a large number 
of the so-called ptomains. 

The ptomains (page 592) are alkaloid-like bodies of basic character 
and of more or less well-known structure. Some of them are notorious 
for being very strong poisons, while others are quite harmless. These 
bodies are called ptomains because they were first discovered in 
putrefying corpses. The best-known compounds of this character 
are the putrescin or tetra-methylen diamin [NH 2 (CH 2 ) 4 NH 2 ] and the 
cadaverin or penta-methylen-diamin [NH 2 (CH 2 )5NH 2 ], which can 
scarcely be considered poisonous. The methyl-guanidin 

NH 2 
HN = C/ 

NHCH 3 

may be mentioned as an example of a very poisonous ptomain. Another 
poisonous ptomain is the neurin CH 2 = CH N(CH 3 ) 3 OH which has 
been found frequently as a product of putrefaction. 

Ammonia is the end product of protein decomposition, as far as 

16 


242 NUTRITION AND- METABOLISM 

the nitrogen-containing fragments of the protein molecule are con- 
cerned. That ammonia is formed by many microorganisms, is well 
known. In some decaying proteins, e.g., in old Camembert cheese, 
ammonia can be very easily detected by the smell. As all proteins 
conta'n many amino-groups as well as ac'd-amid groups, it is easily 
understood how the ammonia originates through the hydrolysis of pro- 
tein. In the complete oxidation of proteins the nitrogen is always left 
as NH 3 or (NH 4 ) 2 CO 3 , respectively, never, so far as known, in any other 
form. No bacterium is known to produce urea, as most of the higher 

animals do. 

In the products of protein degradation mentioned' above only 
those compounds have been considered which contain nitrogen. It is 
quite evident, however, that in the cleavage of the large and complex 
protein molecules, certain parts of the molecule will contain no nitrogen. 
Many organic acids, like acetic, butyric, capronic, benzoic and phenyl- 
acetic acids are quite generally found among the products of putre- 
faction. Alcohols too, especially benzene derivatives like phenol and 
cresol, are not unusual. Gas is often formed in putrefaction, especially 
carbon dioxide and hydrogen; occasionally these gases are mixed with 
traces of nitrogen and methane. 

Many protein compounds contain, besides the organic elements, 
larger or smaller amounts of phosphorus and sulphur. The phos- 
phorus compounds may be changed to phosphine (PH 3 ), which is a gas 
of a strong disagreeable garlic odor. Generally, however, the phos- 
phorus of protein after its degradation is found as phosphoric acid 
(H 3 P0 4 ). Very little is known about the phosphorus of organic 
compounds and the changes it may undergo in the putrefactive process. 

The sulphur of proteins is commonly changed to hydrogen sulphide 
(H2S). Some microorganisms are able to form mercaptan (CH 3 SH), 
a compound of very foul penetrating odor. 

After this enumeration of the products, the main types may be 
considered briefly; since much less work has been done on protein 
decomposition than on carbohydrate decomposition, the groups are 
not so well denned. We might consider the following types: 

Complete Oxidation. --This is brought about by many molds, by 
yeasts if they depend upon proteins only, and by many bacteria, of 
which the large, aerobic spore-forming rods, such as B. mycoides, are 
the main representatives. The products of oxidation are CC>2, H 2 O, 


PRODUCTS OF MICROBIAL ACTIVITIES 243 

NH 3 and H 2 S0 4 . The nitrogen is never changed to any oxidation 
product, but is found as NH 3 , while the sulphur is oxidized. 

Incomplete oxidation is caused by other bacteria, and perhaps molds 
and yeasts. Quite a large number of organisms live on sugar-free 
media if they have oxygen, but they do not oxidize their food com- 
pletely. We can distinguish at least three different groups of micro- 
organisms here. 

B. proteus is the collective name for a number of closely related 
forms which belong to the most common organisms found on decaying 
organic matter, especially when protein is abundant. They produce 
leucin, tyrosin and tryptophane, but no skatol, or phenol. Indol 
and hydrogen sulphide are formed in certain media. Less important, 
but also very common are the pigment-forming rods among which B. 
fluorescenSj B, prodigiosus, Ps. pyocyanea are the best-known repre- 
sentatives. Their metabolism is a little different; amins and ammonia 
are formed, while hydrogen sulphide, phenol and indol are absent. 
As a third group, B. coli may be mentioned which forms indol, but no 
ammonia from peptone, and whose proteolytic powers are very weak 
as it does not even liquefy gelatin. 

Anaerobic decomposition of proteins is limited to very few species; 
there is a great difference in the availability of proteins and of carbo- 
hydrates as a source of energy, protein being available only to a few 
species, most of .these preferring carbohydrates if they are present 
together with protein. B. putrificus is the main representative, but 
other forms exist. B. putrificus is strictly anaerobic, and a spore former, 
very common in nature. Among the products are skatol, hydrogen 
sulphide, ammonia and other very offensive compounds. 

UREA, URIC ACID, HIPPURIC ACID, are the end products of protein 
metabolism of the higher animals. The decomposition of urea to 
ammonium carbonate has been mentioned in several places, mainly 
on page 202. It is a simple hydrolysis 

CO(NH 2 ) 2 + 2 H 2 = (NH 4 ) 2 C0 3 . 

This change can be brought about by only a few bacteria which are 
commonly grouped together as "urea bacteria." These organisms 
have hardly anything else in common, however, and the group is not a 
well-defined one. There are rods and coccus forms, motile and non- 
motile organisms, spore-formers and non-spore formers, and even molds 


244 NUTRITION AND METABOLISM 

have recently been found to hydrolyze urea. All urea bacteria can 
live without urea, feeding on organic matter like other bacteria, but 
most of them require an alkaline medium. 

Hippuric acid is split by certain bacteria to benzoic acid and 
amino-acetic acid which can be oxidized completely. Uric acid can be 
changed in several ways. In some of these changes, urea is found as 
an intermediary product. 

PRODUCTS FROM MINERAL COMPOUNDS 

Minerals are used freely by microorganisms for cell construction, 
consequently, they do not leave the living cell like fermentation 
products. But a few organisms can actually decompose mineral 
matter and when this takes place mineral products are secreted. Two 
main processes can be distinguished, oxidation and reduction. 

OXIDATIONS are the result of the organisms seeking a supply of 
energy. Several oxidations of minerals have been indicated previously, 
as the oxidation of ammonia to nitrites, of nitrites to nitrates, of hypo- 
sulphites to sulphates, of hydrogen sulphide to sulphur and of sul- 
phur to sulphuric acid, of ferrous salts to ferric salts. All these 
microbial changes are simple processes and can be followed by chem- 
ical analysis much more easily than organic fermentations. The 
organisms which cause these changes, do not, as a rule, thrive in 
organic substances and for this reason pure cultures can be obtained 
only with difficulty. Their activity is of great importance in soil 
fertility. 

REDUCTIONS of minerals, too, are of great significance. As a typical 
example, nitrates may be reduced to nitrites, to ammonia, to nitrogen 
gas, and, rarely, to nitrogen oxides. The reduction may be performed 
either by the direct removal of oxygen, or by the formation of free 
oxygen. The reduction of nitrates to nitrites can be written in the 
following three ways: 

KNO 3 - O = KNO 2 
KNO 3 = KNO 2 + O 

KN0 3 + 2H = KN0 2 + H 2 0. 

The result in all three cases is the same. Many bacteria can reduce 
nitrates to nitrites or to ammonia. A few can reduce them to nitrogen. 


PRODUCTS OF M1CROBIAL ACTIVITIES 245 

These "true denitrifiers " are found in soil and in old manure. Their 
reducing process is as follows: 

Ca(NO 3 ) 2 - 5O = CaO + 2N. 

Nitrates are reduced through the efforts of the organism to secure a 
supply of oxygen. The denitrifying bacteria have strong oxidizing 
properties; they take oxygen from all sources possible. If cultures of 
denitrifying bacteria are well aerated, as in soils with a proper mois- 
ture content, they scarcely attack the nitrates, while they will reduce 
them in ordinary liquid cultures so fast that the escaping nitrogen 
gas forms a froth on top of the nitrate solution. Denitrifying bacteria 
need the oxygen to oxidize organic matter. They cannot live without 
organic food. 

Sulphates are reduced in a very similar way to hydrogen sulphide 

H 2 SO 4 - 40 = H 2 S. 

Tap-water, containing calcium sulphates, often forms hydrogen sulphide 
if shut off from the air for some time. 

While only a few bacteria reduce sulphates, many reduce sulphites or 
sulphur to hydrogen sulphide. The potassium and sodium salts of 
selenic and telluric acid (H 2 SeO 4 and H 2 TeO 4 ) are reduced by certain 
organisms and not by others. The reduction results in a colored 
precipitate; this reaction has been suggested as a diagnostic means to 
distinguish different species. The reduction of arsenious oxide to 
arsin (AsHs) is used as a very delicate test for arsenic; it is applied in 
the detection of arsenical poisoning. The material to be tested is 
sterilized and inoculated with Penicillium bremcaule (page 53, the 
"arsenic mold"). This will reduce most arsenious compounds to arsin 
(AsH 3 ) or to diethyl arsin, AsH(C 2 H 5 ) 2 , both of which are easily 
recognized by their very pronounced garlic odor. 

UNKNOWN PRODUCTS OF PHYSIOLOGICAL SIGNIFICANCE 

Among the products of microbial action, there are certain substances 
which must be mentioned because of their importance, though their 
quantity is insignificant compared with the ordinary products of fermen- 
tation. These substances can be divided into four groups: pigments, 
aromatic compounds, enzymes, and toxins. The chemical structure of 
pigments and of many aromatic substances is scarcely known; and as 


246 NUTRITION AND METABOLISM 

far as enzymes and toxins are concerned, it is not even determined 
whether or not they are of protein nature. The last two groups are 
known only by their actions, while the pigments are very conspicuous 
and cannot possibly be overlooked. 

PIGMENTS have naturally attracted the attention of microbiologists 
ever since pure cultures were known, and many investigators have tried 
to explain the nature and the meaning of pigments. All experiments 
concerning the purpose of pigment-formation by microorganisms have 
been without results. It is not known that the pigment is of any 
material advantage to bacteria; for it is possible to cultivate colorless 
strains of pigment bacteria which grow apparently as well as the original 
pigmented culture. Again, pigments cannot take the place of the 
chlorophyl in plants except perhaps the bacteriopurpurin of the purple 
bacteria. It does not even protect the cells against intense light, 
because the pigmented organisms are not more resistant than the corre- 
sponding colorless "sports." The only exception are the colored spores 
of the molds, especially Penicillium and Aspergillus, which are very 
resistant to light, while the spores of Oidium are killed just as easily as 
the mycelium. Pigments cannot be considered as reserve substances, 
since many pigments are excreted and remain outside the colorless 
cells. Pigment production may be incidental. It is possible that the 
waste products of certain organisms happen to be colored. 

After Beyerinck, the chromogenic bacteria may be divided into three 
classes: 

1. Chromophorous bacteria, in which the pigment is placed in the cell 
and has a certain biological significance analogous to the chlorophyl 
of higher plants. In this division belong the green bacteria discovered 
by Van Tieghem and Engelmann and the red sulphur bacteria or purple 
bacteria. 

2 . Chromoparous or true pigment-forming bacteria, which set free the 
pigment as a useless excretion, either as a color-body or as a leuco-body 
which becomes colored through the action of atmospheric oxygen. The 
individuals themselves are colorless and may under certain conditions 
cease to form pigments. To this class belong B. prodigiosus, B. cyano- 
genes, Ps. pyocyanea, and others. 

3. Parachrome bacteria, which form the pigment as an excretory prod- 
uct but retain it within their bodies, as B. janthinus and B. molaceus. 

When the pigment is soluble in water, as those produced by Ps. 


PRODUCTS OF MICROBIAL ACTIVITIEP 


247 


pyocyanea and the fluorescent bacteria, it diffuses through the medium. 
When the pigment is not soluble, it either lies within the cell wall or 
between the individuals. 

This classification furnishes some details concerning the methods of 
pigment production, which depends upon the presence of certain media. 
According to Sullivan, sometimes certain mineral salts, sometimes sugar 
will stimulate chromogenesis. The same is true with molds. Very 
brilliant colors appear with certain species of molds if grown on cellu- 
lose or on fat, while on gelatin the pigment is not produced. The tem- 
perature is an important factor. A large number of chromogens 
produce no pigment when grown in the incubator. It is possible to 
obtain non-pigmentation with many species by propagating them 


FIG. 113. Bacteriopurpin, from a Rhodospirillum, crystallized from a chloroform 

solution. (After Molisch.} 

through many generations at high temperatures. Oxygen also is 
necessary for the chromogenesis of many bacteria. Some need a short 
exposure to daylight in order to produce their pigment, while cultures 
grown in absolute darkness may remain colorless. Strong sunlight, 
however, will check pigment production in the same degree as do 
antiseptics and other harmful influences. 

The chemical nature of microbial pigments is little known. They 
are distinguished according to the solubility in various liquids, as water, 
alcohol, ether, chloroform, benzol, and other solvents, and according 
to the change of color caused by acid and alkali. A group of 
carotin bodies, named because of their similarity to the pigment 
of carrots, the prodigiosin bodies, named after B. prodigiosus, the 


248 NUTRITION AND METABOLISM 

, 

fluorescent pigments and perhaps a few other groups are distinguished, 
but their chemical nature is rather vague as yet. The absorption of 
distinct lines of the spectrum by solutions of these pigments is claimed 
to be a very reliable means of distinguishing the pigments of different 
species. 

AROMATIC SUBSTANCES constitute another group of metabolic prod- 
ucts. The chemical analysis accomplishes more with these com- 
pounds than with pigments, since they are frequently well-known 
compounds. The main difficulty arising in their identification is in 
the very minute quantities of the products available. Some substances 
with strong, mostly very disagreeable odors have already been men- 
tioned: indol, skatol, hydrogen sulphide, mercaptan, the amins and 
ammonia, butyric acid, and some of the higher alcohols. There re- 
main to be mentioned certain oils and esters giving rise largely to 
pleasant aromas. The formation of aromatic oils has been established 
although their nature is entirely unknown. The same is true with the 
esters. The substance causing the fishy flavor in butter is volatile 
with steam and is neither of an alkaline nor acid nature. The strong 
odor of freshly plowed earth is caused by an Actinomyces; the odor 
can be traced to a very volatile oil the nature of which has not been 
determined. The aroma of fermented liquids wines, beers, and 
many others is partly due to compounds constituting the fermenting 
material, and partly to the fermenting agent. Some yeasts are 
known to produce fruit-esters, as succinic-acid-ethylester and the 
corresponding esters of malic and other acids. Besides, some glucosides 
may be split and traces of hydrocyanic acid and benzoic acid may be 
liberated. The change of flavor with the aging of wines is probably 
more a chemical than a biochemical change. 

ENZYMES AND TOXINS. Among the most interesting and least 
understood products of microbial action are the enzymes and the toxins. 
These two groups are related in many respects The enzymes have 
been discussed extensively in a preceding chapter and toxins will be 
treated more extensively on pages 676, 740. Toxins and enzymes are 
formed by the cells in such small quantities that they would never have 
been discovered by ordinary chemical means were it not for the unusual 
effects which they produce, the enzymes acting upon food substances, 
and the toxins acting physiologically upon organisms. Toxins and 
enzymes are chemically unknown. It is assumed that they are chemical 


PRODUCTS OF MICROBIAL ACTIVITIES 249 

bodies, but even this has not been proved. A pure toxin has never 
been obtained and we have no criterion for its purity. The presence 
of a toxin is recognized only by an animal test and in this way the com- 
parative concentration can be determined approximately. Such 
standardization of toxin solutions is only comparative, however, and 
gives no clue as to the actual amount of toxin present. Not all ani- 
mals are sensitive to all toxins. It is quite possible that all bacteria 
produce compounds with chemical qualities similar to toxins, and only 


a few of them happen to react upon men or animals. 

Toxins are not always the product of microbial action. Vegetable 
toxins or phytotoxins are known, among which the ricin of the castor- 
oil bean is perhaps the most studied representative. The best-known 
zootoxin is the rattlesnake poison. These non-microbial compounds 
have the same quality as the microbial toxins they are extremely 
poisonous. Toxins are the cause of disease in diphtheria, tetanus and 
botulism. If a culture of these organisms is filtered through a porcelain 
filter which removes all bacterial cells, the filtrate injected into an 
animal will cause the disease with all its accompanying symptoms 
though there are no microorganisms introduced into the animal body. 
If the filtrate is heated, however, no effect will take place after the in- 
jection, because heat destroys the toxin. The amount of toxin that will 
kill an animal is extremely small. .000005 m S- f the purest tetanus 
toxin will kill a mouse, .0007 mg. of ricin will kill a rabbit, less than 
.23 mg. of tetanus toxin will kill an adult man. The body of an animal 
or man forms an anti-body against the toxin which neutralizes its 
poisonous action. Anti-bodies are also formed against enzymes 
injected into an animal. 

Toxins are very sensitive to heat. A short exposure to temperatures 
between 80 and 100 will inactivate them. They are also very sensi- 
tive to light. While some toxins are secreted, others are retained within 
the cells of microorganisms, and never leave them until the cells die or 
disintegrate. Ptomains, which are also metabolic products of micro- 
organisms and sometimes cause poisoning, differ from the toxins in their 
resistance to heat and light (page 241). Ptomains differ in no way 
essentially from ordinary organic compounds; the animal or human 
body produces no anti-ptomains to counteract their poisonous effects. 
There is no chemical relation whatever between toxins and ptomains, 


250 NUTRITION AND METABOLISM 

and the physiological effects are also quite different, though they both 
cause poisoning. 

Toxins are not essential products of the metabolism of pathogens. 
Strains of pathogenic bacteria can be bred which do not produce toxins 
as chromogens can be bred without pigment, or lactic bacteria which 
do not produce acid. The strains which lose their pathogenicity grow 
better on artificial media but are. less able to produce disease in the 
animal. They may regain the power of producing toxin if passed 
through the body of the animal. The real object of toxin production 
by microorganisms is not known; the microorganisms derive no ap- 
parent benefit. 

PHYSICAL PRODUCTS OF METABOLISM 

PRODUCTION or HEAT. It has long been known that fermentation 
produces heat. The rise of temperature is usually not very great. In 
lactic fermentation it amounts to about i, in alcoholic fermentation to 
2 or 3, but in certain processes the heat liberated is considerable, as 
in the fermentation of manure, of ensilage, of vinegar, and in others. 

The cause of heat formation is quite evident from the discussion on 
page 199. Decomposition of organic matter means a liberation of 
energy which is used for the continuation of life processes; the utiliza- 
tion is, as a rule, incomplete, and a part of the energy appears in the 
form of heat. The amount of heat produced can be measured directly 
with the thermometer if great care is taken that no heat is lost by 
radiation or by evaporation of water. 

Much heat is produced in the vinegar fermentation. In the quick- 
vinegar process (page 644) the temperature rises sometimes as high as 
10 to 15 above the temperature of the room and the vinegar manu- 
facturer uses the heat produced by the bacteria to keep the generators 
at the optimum temperature. If the process is not controlled carefully, 
the vinegar bacteria are likely to produce sufficient heat to kill 
themselves. 

The heat produced in the fermentation of manure, especially horse 
manure, is used in the hot-beds to cultivate and force young plants. 
In the manure pile, great heat production is not desirable because high 
temperatures will volatilize the ammonia; the tight packing of manure 
which keeps out the oxygen will prevent too strong bacterial action. 
The highest temperature in silos which has been recorded is about 70, 


PRODUCTS OF MICROBIAL ACTIVITIES 251 

but the best silage is secured by keeping the temperature below 50. 
Ensilage fermentation is not thoroughly understood, however, and no 
accurate statements can be made as to the cause of the increase in 
temperature. Sometimes the temperature in silos does not exceed 
35. The curing of hay is usually accompanied by a rise of temperature. 
For some time it was believed that the spontaneous combustion of hay 
was mainly due to microorganisms, but it has been shown recently 
that even sterile hay will show a rise of temperature under certain 
conditions. This does not exclude the formation of heat in hay by 
microorganisms under other circumstances. The heating of tobacco, 
of green or moist grain or corn is not of bacterial origin, but due to 
the respiration of the living plant-tissue. 

PRODUCTION or LIGHT. The light-producing or photogenic organ- 
isms are quite numerous and occur more frequently than is generally 
believed. The phosphorescence of decaying tree stumps and leaves in 
the woods and of meat and fish in the cellar are well-known phenomena. 
The phosphorescence of wood and leaves is generally caused by Hypho- 
mycetes; certain mushrooms have this quality in a very high degree. 
The light of meat and fish is usually generated by bacteria, of which at 
least twenty-six species have been described. 

The most obvious evidence of liberation of energy in the physiology 
of protozoa is seen in their movement. Certain protozoa, Noctiluca 
for example, however, emit light and produce the phosphorescence 
often observed in sea water. From analogy with higher animals it is 
to be supposed that heat and electrical changes are also produced." 

Many experiments have been carried on in order to discover the 
nature and origin of the light, but, so far, few results have been obtained. 
The phosphorescence is due to an oxidation process; all photogenic 
organisms cease to generate light when the oxygen is removed. As 
soon as they come into contact with oxygen again, they produce light 
immediately, and this sudden flashing is used occasionally by physiolo- 
gists as a very delicate test for oxygen. The light appears to be pro- 
duced always within the cell; no cell product has ever been found to 
give rise to light outside the cell. It is possible that a chemical com- 
pound is formed in the cell which generates light when in contact 
with oxygen. 

The life processes of the photogenic microorganisms are not neces- 
sarily connected with the formation of light. Photogenic bacteria are 


252 NUTRITION AND METABOLISM 

known to lose the power of light production as the chromogenic bacteria 
may lose the power of pigment production. Phosphorescence has, like 
pigmentation also, no bearing upon the development of the cell, and the 
light-giving compounds may be regarded as incidental waste products. 
Certain chemical bodies stimulate light production, while others favor 
the growth only. One of the most important factors in the production 
of light is sodium chloride. 


CHAPTER V 

PHYSIOLOGICAL VARIATIONS ASSOCIATED WITH METABOLISM AND 

NUTRITION* 

The great variability of microorganisms in morphological respects 
has already been pointed out in Part I of this book. A similar variation 
and adaptation are noticed in their physiology, especially with the food 
substances of bacteria and consequently with their metabolic products. 
Microorganisms change their physiological properties very readily with 
the environment; the new variety may keep its acquired properties for 
some time even if brought back to the original conditions. It is stated 
frequently that microorganisms tend more toward variations than the 
more complex organisms. It should be considered, however, that the 
experiences in the variations of green plants and animals are based on 
individuals, while in the case of microorganisms these experiences are 
gained almost always from millions of cells. A simple illustration is the 
development of bacteria in salt solutions. If a broth culture of B. coli 
is transferred into broth containing 8 per cent of salt, a large number of 
cells will die, often more than 99 per cent. The surviving bacteria begin 
to multiply after a certain length of time and a new variety is created 
which can tolerate the salt. At first, only about one out of one hundred 
cells had the power to tolerate salt, but, since the dying cells are not 
usually counted or considered at all, it is customary to say that bacteria 
easily adapt themselves to an 8 per cent salt solution. If only one 
single plant out of one hundred could be adapted to a certain high 
temperature, it could not be said that it adapts itself easily. This mis- 
take is quite commonly made with microorganisms. 

The best illustration for the variability of cultivated microorganisms 
is the enormous number of varieties of Saccharomyces cerevisice. Nearly 
every large brewery has a yeast type of its own which differs from others 
by the amount of alcohol and aromatic substances produced, by time 
and optimum temperature of spore-production, by the appearance of 
the budding yeast in the hanging drop, and also in other respects. The 

* Prepared by Otto Rahn. 

253 


254 NUTRITION AND METABOLISM 

cultivated organisms are not alone in showing this tendency toward 
variation. The transferring of a soil or water bacterium into the ordi- 
nary laboratory media is a complete change of conditions; the different 
cells of the same species may react differently and give several varie- 
ties. A lactic bacterium on meat medium without sugar does not thrive 
well in the first generations, but it gradually becomes able to grow on 
this medium. By this treatment, it loses gradually the power of pro- 
ducing acid and does not thrive as well in milk. The attenuation of 
pathogenic bacteria by cultivation on media, as potato, very different 
from the blood and muscle upon which they grow most naturally, or 
by growing them at low temperature, or above the maximum, furnishes 
another example. The decrease and finally the entire loss of patho- 
genicity is caused by a change of metabolism, by a loss of the power to 
produce toxin. 

As by certain diet the metabolism can be changed, so certain 
physiological properties of bacteria can, by proper cultivation, be 
increased. By the frequent transferring of an organism on gelatin, its 
liquefying qualities can be increased, provided it had some at the start. 
By continued passing of a bacterium through an animal, its virulence 
can be increased. Strains of bacteria which will produce a very high 
acidity can be bred; this is illustrated by the quick- vinegar process 
and by the strong alcohol-producing yeasts of the distillery process. 
By continued cultivation of an organism upon a certain medium, it 
will become so acclimatized that it degenerates readily when the con- 
ditions become unfavorable Such specifically trained strains of 
microorganisms are used in alcoholic and lactic fermentation, in patho- 
genic bacteriology and in the inoculation of leguminous plants with 
nitrogen-fixing bacteria. 

FACTORS INFLUENCING THE TYPE OF DECOMPOSITION 

In the chapter on products of metabolism, it has been shown 
that the same compound can be decomposed in many different ways, 
and the question may well be asked what decides the type of decomposi- 
tion. Since bacteria are widely distributed, it must be expected that 
there are certain conditions which are most favorable to a given type 
of fermentation, while under changed conditions, other types are more 
likely to dominate. The fact that sugar in cider nearly always under- 
goes alcoholic fermentation, while in milk it undergoes lactic fermen- 


PHYSIOLOGICAL VARIATIONS 255 

tation, has its reason in the physiology of the bacteria, and in their 
reaction upon the environment. 

Cider is acid, and acid is not well suited for the growth of most 
bacteria. The vinegar bacteria can grow in fruit juices, and a few other 
bacteria, especially those causing trouble in wine, are not retarded by 
fruit acids, but the common types attacking proteins and causing 
organic decay are not able to grow on fruits. Yeasts, however, and 
molds thrive well only in acid media. They can exist in neutral 
solutions if in pure culture, but in nature they are easily crowded out 
by bacteria. Acidity of the medium is therefore one of the most 
important factors regulating the type of microbial decomposition. 
This principle is commonly utilized by preserving foods of all kinds in 
vinegar, and by making butter from sour cream rather than sweet 
cream; the keeping qualities of hard cheeses depend upon their acid 
content. 

In acid environment, the two most common types of decomposition 
are oxidation, complete or incomplete, and alcoholic fermentation. 
The oxidation is brought about by molds or organisms closely allied to 
yeasts. The latter are very common on all sour foods, especially on 
foods containing lactic acid, such as cottage cheese or sauerkraut. 
The kind of acid decides the type of mold; wherever there is lactic acid, 
there is Oidium, while malic and tartaric acids favor Penicillium and 
Aspergillus. 

If the decaying materials contain no acid, the type of decomposi- 
tion depends mainly on the presence or absence of carbohydrates, 
especially sugar. It is an old experience, recently verified through 
a large number of experiments by Kendall and Walker, that practically 
all bacteria will decompose sugar in preference to proteins. If a leaf 
contains sugar and protein (cabbage) the sugar decomposition will be 
conspicuous, and the protein is not attacked very readily. Putrefac- 
tion in the presence of sugar or of acid does not take place. Meat will 
not putrefy if mixed with sugar, while milk putrefies readily if the sugar 
is removed by dialysis. The three types of sugar decomposition which 
come into consideration in neutral media, are the lactic, the acid-gas and 
the butyric fermentations. The latter is a strictly anaerobic fermenta- 
tion, and thus limited to special conditions. Of the other two, the acid- 
gas fermentation is the most common, and the souring of vegetables 
of all kinds is due to this type of fermentation (pickles, sauerkraut, 


256 NUTRITION AND METABOLISM 

ensilage, salt-rising bread). Sometimes the acid-gas fermentation 
is followed by a butyric fermentation. The true lactic fermentation 
is not common, and is limited almost entirely to milk. This is ex- 
plained by the circumstance that the organisms causing this decom- 
position are parasitic in their habits, causing disease or living in the 
intestine of animals. In the absence of acid and sugars, putrefaction 
is the most common type of decomposition. 

Many factors aside from the chemical composition of the medium 
are essential. Oxygen has already been mentioned as preventing buty- 
ric fermentation. It will also prevent the acid-gas fermentation if too 
abundant. Ensilage is trampled and pressed down to avoid air spaces 
as much as possible, for molds will outgrow the acid-forming bacteria 
if air has free access. Absence of oxygen will prevent mold growth, 
and for this reason, jelly is paraffined, and butter wrapped tightly into 
impermeable paper. The influence of oxygen upon the type of protein 
and of cellulose decomposition has been pointed out previously. 

The moisture content is of great importance. As will be shown 
later, not all organisms have an equal need of moisture; some molds 
will grow on foods too dry for bacteria and yeasts. Molds are es- 
pecially adapted for growing on dry media, as only part of their cell 
substance is immersed in the medium. Their thread formation enables 
them to search a dry medium, such as flour, for moisture, the extreme 
of adaptation being Rhizopus, and the construction of the fruiting 
bodies shows that they are destined by nature to be spread by air and 
wind. It is no wonder that damp organic matter, if it can be de- 
composed at all, will show molds, and nothing else, regardless of the 
chemical composition, for there is no competition. Flour, moist seeds, 
incompletely dried fruit, damp milk powder will always become 
moldy. The same holds true with very concentrated sugar solutions 
such as syrups, jellies and jams, while in concentrated salt solutions, 
molds cannot thrive, and the torula yeasts are best adapted to such 
conditions. 

A very important part is also the structure of the material. Micro- 
organisms act mainly upon organic matter, and since this comes 
from living organisms, it has usually definite structure, exceptions 
being milk and blood. The structure of all living organisms is such 
as to prevent the intruding of microorganisms. The body of plants 
and animals is surrounded on the outside by tough and dry layers of 


PHYSIOLOGICAL VARIATIONS 257 

epithelial cells, and the cavities of the animal body also have their 
protective membranes. Microorganisms cannot enter the tissues 
if these membranes are perfectly sound, and we know that, as a rule, 
the tissues of healthy plants or animals are free from bacteria. Thus, 
a healthy apple or potato or egg will not be infected and decomposed 
by microorganisms if handled carefully, meat will begin to decom- 
pose on the outside, and the inner parts may be still good when the 
outer layer is already in a state of decay. 

In the plants, each cell is surrounded by its special cell membranes 
which are a barrier to infecting organisms. If we prick the skin of a 
healthy apple with a pin infected with yeast, the infection will not 
spread though we know that yeast will grow most abundantly in cider; 
in the apple, however, it has no means of spreading from one cell to 
the other. Molds possess this means; they can puncture cell walls, 
and forcing their way from one cell to the other, they will soon 
bring about the rotting of the entire fruit after it once becomes 
infected. This protection seems especially necessary in the plant's 
roots which are greatly exposed to injury from insects and other animals 
in the soil and surrounded by billions of microorganisms. They are 
attacked only by fungi which can force their way from cell to cell, 
or by bacteria which can dissolve the membranes by means of enzymes, 
and thus cause a softening of the root tissue. The bacteria causing the 
various rots of vegetables belong to this type. 

There is, then, a great variety of factors deciding the type of 
decomposition of organic matter in nature, and by knowing the chemical 
composition as well as the structure and other physical conditions, 
it is possible to foretell which group of organisms is most likely to 
attack the compounds in question. 

Another quite important factor, the temperature, will be dis- 
cussed in more detail in one of the following chapters. 


17 


CHAPTER VI 

NUTRIT ION OF MICROORGANISMS AND THE ROTATION or ELEMENTS 

IN NATURE* 

All organic matter on earth is undergoing continuous change. Or- 
ganisms grow and decay. The same carbon and nitrogen atoms which 
constitute the organic world of to-day constituted it thousands of 
years ago. The amount of carbon, nitrogen, hydrogen and of all 
other elements of life on earth is limited, and the same atoms will 
be used for the future generations of life that constitute the present. 
There must be continuous destruction to enable new construction. 
Construction is mainly the task of green plants, enabled by the chloro- 
phyl to use the energy of sunlight in building up organic substances 
from minerals, water and carbon dioxide. Destruction is caused 
mainly by animals and other organisms which have to break down 
organic matter in order to exist. These two factors keep the atoms of 
the organic world in perpetual rotation. 

In this circulation of the elements it is necessary that all compounds 
of organic nature be decomposed finally to a form available for plant 
food. If this were not the case, the indestructible compound would 
sooner or later accumulate in such enormous quantities that the 
elements constituting this body would be removed entirely from 
general circulation. Let us suppose, as an illustration, that for some 
unknown reason, all urea bacteria on earth would die. Urea could be 
decomposed no more, and the plants, unable to use urea as a source of 
nitrogen in place of nitrates, would get but little benefit out of stable 
manure. All urea would pass gradually undecomposed into rivers, 
lakes, and finally into the ocean where it would accumulate con- 
tinuously. The enormous quantities of nitrogen taken out of cir- 
culation would cause a decreasing growth of plants, and life would 
soon cease because of lack of nitrogen. For this reason all products of 
living organismsjnust be further broken up by some other organisms, 

* Prepared by Otto Rahn. 

258 


NUTRITION OF MICROORGANISMS 259 

and we find that the destructive work is to a large extent the task of 
microorganisms. Many products of organic life cannot be broken 
down by organisms other than bacteria, and therefore bacteria are 
absolutely necessary for the circulation of the elements and for life on 
earth. Bacteria and green plants are an absolute necessity for the 
maintenance of life, the one breaking down, the other building up, 
one dependent upon the products of the other; animals, however, could 
be excluded from the circle without interfering with a continuation of 
life on earth. 


ranisms 


Carbohydrates 
/'at, frotein 

FIG. 114. Carbon cycle. 

CARBON CYCLE. Carbon is the main element in organic nature, and 
the study of its cycle might be begun with its simplest compound, 
the carbon dioxide of the air. It is absorbed in this condition by the 
green plants, and is changed by the chlorophyl granules of the leaves to 
organic compounds of various types, either to carbohydrates (cellulose, 
starch, sugars) or to fats, or to protein substances, occasionally to 
organic acids or other compounds. The plants will either die and decay, 
or will be eaten by animals. In the first case, the decay will be caused 
exclusively by microorganisms; if the plants are eaten, they will be 
digested; part may be used to build up the animal body or stored as 


260 


NUTRITION AND METABOLISM 


reserve substances, largely fat and protein. If the animal dies, a 
decomposition process will take place, which breaks down the organic 
compounds to simpler products and finally the carbon will be com- 
pletely oxidized to carbon dioxide. Even the marsh gas which might 
be liberated in this process will find organisms that oxidize it to carbon 
dioxide and water. Every product will find an organism to break it 
up further until it is completely disorganized and the carbon atoms can 
start the same circulation anew. Undoubtedly as long as organic 
life has existed on earth, microorganisms have been present, in order 
to render the dead organic matter again available for plant and animal 
life. Fig. 114 gives a schematic illustration of the carb'on cycle; the 
microbial activity is marked by heavy lines. 


fnfes 


Dead 
Organisms 


te 


rates 


Prot 


roiem 


Protein 


FIG. 115. Nitrogen cycle. 

NITROGEN CYCLE. Nitrogen shows the same continuous change 
as carbon. Plants take up nitrogen in mineral form usually as nitrates. 
The plants change this mineral nitrogen to the most complex bodies, 
proteins, where it is combined with the other elements of organic nature. 
The plants may be eaten by animals; part of the protein is then digested 
to urea or hippuric or uric acid, which in turn are readily decomposed 
by microorganisms to ammonia (Fig. 115). Part of the protein will be 
stored in the growing animals, and if the animal dies, the body will 
decay or putrefy, and the nitrogenous compounds of that body will 
pass through the various stages of decomposition to the final product, 


NUTRITION OF MICROORGANISMS 261 

ammonia. Ammonia is then oxidized to nitrites and nitrates, when 
the nitrogen cycle is completed. 

There is, however, one discrepancy in this cycle. It has been 
mentioned already that some organisms are able to reduce nitrates to 
nitrogen gas. This is one of the " leaks" in the rotation of elements 
which would be disastrous to organic life on earth if there were no means 
to compensate for the loss of nitrogen in circulation. Imagine what 
would happen if there were no such compensation. Part of the nitrate 
in the soil is destroyed, the nitrogen gas escapes into the air and is as 
indifferent as the nitrogen of the atmosphere lost to organic life forever. 


nyolroaen -*Julpn/de 

-/ ^^HBrifeK^^ta_ / 


Dead 
Urwan/sms 


FIG. 116. Sulphur cycle. 

More nitrates would be produced from decaying organic matter and 
would eventually be destroyed. After a certain time, this continuous 
loss of nitrogen would become quite noticeable in the growth of plants; 
there would be a scarcity of nitrogen in soil, since part of it is lost continu- 
ously. Finally, the plants would cease to grow because the nitrogen in 
the soil would be exhausted. 

The compensation for this destruction of available nitrogen is found 
in the nitrogen-fixing bacteria, which, either living in symbiosis with 
leguminous plants or growing independently in the soil, have the power 
to use the atmospheric nitrogen for the formation of their own proto- 


262 NUTRITION AND METABOLISM 

plasm. Thus, organic nitrogen is produced from nitrogen gas and the 
continuance of organic life is guaranteed. 

SULPHUR CYCLE. Little more can be said about sulphur, since the 
rotation is quite similar to that of nitrogen. Plants will take sulphur 
usually in the form of sulphates and make protein compounds contain- 
ing a certain amount of sulphur (Fig. 116). These bodies are either 
digested by higher animals or broken down by putrefaction to the 
final product, hydrogen sulphide, which is oxidized by the sulphur 
bacteria first to sulphur, then later to sulphates. 

PHOSPHORUS CYCLE. The cycle of phosphorus has not been worked 
out completely, but from the discussion in the last pages, it is plainly 
seen that a simple cycle very much like the ones above must exist. It 
is probably much simpler because phosphorus does not enter as easily 
into organic compounds as nitrogen. 


DIVISION III 
PHYSICAL INFLUENCES 


CHAPTER I 
WATER AS A PHYSICAL FACTOR* 

It has been indicated already that water has the capacity as a 
solvent way beyond any other substance; it has a function, closely 
associated with its solvent powers as a carrier, in which solution and 
mechanical mixture are equally important; it possesses the property 
of diffusion, which enables its solutions to extend where other solvents 
find no entrance; it possesses much surface energy, having a very high 
rating; and it fosters ionization, the full value of which in life's reactions 
is not known. Living cells have been shown to consist of a high per- 
centage of water. It appears as if water were the body-medium for all 
physiological reactions. [See pp. 187] 

OSMOTIC PRESSURE. In the organic world we find very commonly 
membranes which will allow water to pass through but retain some 
compounds dissolved in the water. Such so-called semi-permeable 
membranes are found surrounding the protoplasm of cells. They are 
not the cell wall, but separate the protoplasm from the cell wall. 
Similar properties are found in parchment paper, pig's bladder, and 
other organic membranes. 

If a salt solution is poured in water, the two liquids will mix in a 
short time and soon every smallest portion of the mixture will have the 
same concentration. If a salt solution and water are separated by a 
membrane which does not allow th* salt to pass, the water will go 
through the membrane toward the salt with a certain amount of 
pressure. This pressure depends upon the nature of the dissolved 
substance as well as upon its concentration. 

The pressure increases in direct ratio with the number of molecules 
in solution. Therefore, a compound with large molecules (cane sugar) 

* Prepared by Otto Rahn. 

263 


264 PHYSICAL INFLUENCES 

will produce a lower osmotic pressure than one with small molecular 
weight (glycerin) if we compare solutions of equal concentration. 
The osmotic pressure of protein, starch and peptone solutions can be 
measured only with the finest instruments, while the pressure of a 30 
per cent dextrose solution is 22 atmospheres.* [See pp. 173-177.] 

PLASMOLYSIS. If a cell is brought into a strong solution of a sub- 
stance which cannot pass the plasma-membrane, this substance will 
cause an osmotic pressure and the concentration in the cell being lower 
than in the medium, the water will pass out from the cell until the pres- 
sure inside and outside is the same. This causes a shrinking of the 
protoplasm, while the rigid cell wall keeps its shape. Such plasmolyzed 
organisms are illustrated in Fig. 69, page 89. 

While plasmolysis is easily demonstrated with the cells of higher 
plants, microorganisms do not show it so readily. In fact, many bac- 
teria, like B. subtilis, Bad. anthracis, cannot be plasmolyzed by any 
concentration of salt in solution. Others, as B. coli, B. fluorescens, 
react promptly. But even though many are killed, the rest recover 
from plasmolysis after a few hours, and appear normal. This indicates 
that the salt passes slowly through the plasma-membrane and thus 
increases the pressure inside the cell until finally the inside and outside 
pressure are the same again. 

The fact that many microorganisms show no plasmolysis whatever 
is explained in the same way. These organisms probably have plasma- 
membranes so constructed that the salts diffuse through nearly as fast as 
the water. An absolute exclusion of all soluble substances by the mem- 
brane is impossible since the food can get into the cell only by diffusion 
through the membrane. 

The resistance of various microorganisms against concentrated 
solutions depends upon the organism as well as upon the dissolved sub- 
stance. The sodium and potassium salts of the common mineral acids 
act upon a culture nearly in proportion to their osmotic pressure, but 
the potassium salts always retard growth a little less than the sodium 
salts. The effect of salts upon microorganisms is therefore not due to 
the osmotic pressure only; the chemical constitution of the salts also 
plays an important r61e. 

The different functions of life are influenced in different degrees by 
concentrated solutions. Some bacteria will multiply but not form 

"One atmosphere equals the pressure of i kg. per square centimeter or about 15 pounds 
per square inch. 


WATER AS A PHYSICAL FACTOR 265 

spores in salt solutions. Molds will sometimes show a good growth in 
concentrated sugar solutions but fail to produce spores. Bad. anthracis 
loses its virulence in sea water. Often, the form of microorganisms is 
affected by concentrated solutions. Some bacteria grow more spherical, 
others become elongated or distorted. The deforming influence is not 
due to the osmotic pressure only, but depends mainly upon the chemical 
character of the salt; magnesium salts especially have a tendency to 
produce such involution forms. 

Salt and Sugar Solutions. Most experiments on the influence of 
concentrated solutions have been carried on with sodium chloride, be- 
cause of its wide application in the preservation of foods. Most micro- 
organisms, especially the rod-shaped bacteria, are suppressed by a salt 
concentration of 8 to 10 per cent. At 15 per cent only few cocci develop 
slowly, while some species of TorulcR grow without a very noticeable re- 
tardation. Above 20 per cent the Torula are practically the only 
organisms which can develop. They are, therefore, found in all food 
products- which are preserved by salt, as salted pork, beef, fish, butter, 
and pickles, often in nearly a pure culture. It seems that they are 
easily overpowered by other organisms in the absence of salt, but in 
salted food, this competition is eliminated. 

The selective influence of salt is used in some fermented products to 
prevent undesirable fermentations. This is true in sauerkraut and 
brine pickles, where the desirable bacteria can grow in the presence 
of salt while the undesirable ones are kept away. Possibly the salting 
of butter has the same effects. 

Another compound of great practical importance is cane sugar, 
which is the standard preservative for fruits and condensed milk. Its 
action has been studied mainly upon molds. Theoretically, dextrose 
should be expected to have twice as strong a preserving action as saccha- 
rose because it has only half the molecular weight and consequently 
produces twice as strong an osmotic pressure in the same percentage of 
concentration. Its preserving effect is indeed a little higher than 
that of saccharose, but the proportion is not nearly 1:2. The common 
molds are extremely resistant to strong sugar solutions, about 60 to 70 
per cent of cane sugar seems to be the limit of growth for PenicilUum 
and Aspergillus species. Yeasts can also grow and ferment in very con- 
centrated solutions while bacteria in general do not tolerate solutions 
higher than 15 to 40 per cent, though many exceptions are known. 


266 PHYSICAL INFLUENCES 

Colloidal Solutions. In order to determine the amount of water 
which is absolutely necessary for microbial proliferation, only such 
media can be used which do not cause osmotic pressure. If B. prodigio- 
sus does not develop in a 10 per cent salt solution, this is not due to lack 
of moisture, because the same bacillus will grow in a 30 per cent sugar 
solution which contains 20 per cent less moisture. Another factor be- 
sides the water content enters, which can be avoided only in solutions 
without osmotic pressure. 

A few substances are known to give such solutions, namely, colloidal 
bodies which have a very large molecular weight. Their osmotic pres- 
sure even in very concentrated solutions would not be high enough 
to interfere with microbial growth. Among these colloidal bodies 
are found egg albumin, gelatin, peptones, all protein substances; 
also starch, dextrin and gum arabic among the carbohydrates. None 
of these substances has a retarding influence upon bacteria; some of 
them can be mixed with water in all proportions; consequently, they 
are the ideal medium to test the water requirements of microorganisms. 
Experiments carried on with gelatin, powdered meat, crackers, 
bread and potato, vary but little in results. A few bacteria cannot 
grow in a medium with only 60 per cent water, but most organisms 
develop slowly even with 50 per cent water and some may be able to 
develop with only 40 per cent. Molds can grow very scantily in even 
more concentrated media. Protozoa probably have to have a more 
diluted medium for their development though no experiments bearing 
upon their water requirements are known to the author. 

The fact that in a colloidal solution growth will cease if the moisture 
is below 30 to 40 per cent does not necessarily indicate the conclusion 
that any substance with less than 30 per cent water cannot be decom- 
posed. The above statement refers only to solutions, while in natural 
media as dried foods or soil, a combination of solid and dissolved 
substances is involved. Butter is an excellent medium for many bac- 
teria, yeasts, and molds, though it contains only 12 to 15 per cent of 
moisture. If butter fat were soluble in water, the concentration of 85 
parts of solid in 15 parts of liquid would certainly prevent any growth 
whatever, but fat is insoluble, and the fat particles do not interfere 
at all with the growth of microorganisms in the droplets of buttermilk 
distributed all through the butter. The concentration in these small 
droplets is the deciding factor. If the growth of microorganisms in 


WATER AS A PHYSICAL FACTOR 267 

butter is to be prevented by salt, it is unnecessary to give any attention 
to the fat; the bacteria live only in the water and not in the fat globules. 
In adding 3 per cent of salt to a butter with 15 per cent of moisture, a 
brine of 3 parts of salt in 15 parts of water is produced; in other words, 
a 20 per cent brine, because salt does not dissolve in the fat. Similar 
considerations will come up in the preservation of fruit, vegetables, meat, 
milk, and other food substances by drying or condensation. 

DESICCATION. Microorganisms do not die immediately after the 
removal of the water, and they do not die all at once after a given time. 
Death through drying is a slow and regular process. Paul and his 
associates found that the number of bacteria dying in the unit of 
time is, under constant conditions, proportional to the number sur- 
viving. If we had 1,000,000 cells per gram in the beginning, and the 
death rate were 90 per cent per day, there would be, at the end of each 
day, 10 per cent of the original number surviving. This would give the 
following numbers for one week: 

Beginning 1,000,000 cells per gram. 

After i day 100,000 cells per gram. 

After 2 days 10,000 cells per gram. 

After 3 days 1,000 cells per gram. 

After 4 days 100 cells per gram. 

After 5 days 10 cells per gram. 

After 6 days i cell per gram. 

After 7 days o.i cell per gram. 

This table shows graphically the mode of death of dried bacteria. The 
number of cells approaches zero without ever (at least theoretically) 
reaching it. From one cell per gram after six days we do not come to 
o on the seventh, but to one cell in 10 g. and on the eighth day one 
cell in 100 g. The total number dying in the first day is much larger 
than that dying on the sixth day, but the rate is constant, 90 per cent 
of the number surviving. This regularity has been found with bacteria 
dying from various causes, and it is commonly compared with the 
simplest chemical processes, the monomolecular reactions. 

Paul and his associates found further, that the death through drying 
is caused by an oxidation process; in pure oxygen bacteria died much 
faster. The poisonous effect of oxygen upon moist bacteria has already 
been pointed out on page 228. 

Most resistant to drying are the spores of bacteria; mold spores, 


268 PHYSICAL INFLUENCES 

too, show considerable resistance, while some bacteria, e.g.,B.carotarum 
and Ps. radicicola, are readily killed. 

The resistance of microorganisms is influenced greatly by the me- 
dium on which they are placed for drying. Hansen found that yeast 
cells dried on cotton were still alive after two to three years, while if 
dried on platinum wire some died in five days and others lived as long as 
100 days. Compressed beer-yeast mixed and dried with powdered char- 
coal kept as long as ten years; Ps. radicicola dried on a cover-glass 
or filter paper died within twenty-four hours ; on seeds, this same organism 
was still alive after fourteen days and in the dried nodules of legumes a 
few cells were able to reproduce after more than two years. Soil con- 
taining an average number of 17,000,000 bacteria per gram was dried for 
two years; the total number of organisms averaged then 3,250,000, 20 
per cent of the bacteria, therefore, could resist desiccation. Dried cul- 
tures of microorganisms are commonly sold for several purposes, as 
dairy-starters and the so-called "magic yeast" and "yeast foam" used for 
bread-making. Such cultures are dried on milk, sugar, starch, flour or 
similar porous and absorbing material. Starters are usually guaranteed 
only for a certain 'length of time, from one to twelve months. The 
advantage of the dry culture is its better keeping qualities. Liquid 
cultures produce substances harmful to themselves, and die rapidly 
after a short time, while the dry cultures show little change. 

The resistance of pathogenic bacteria to desiccation is of consider- 
able importance in the spreading of contagious diseases. Many patho- 
genic bacteria die after desiccation of a few hours to a few days, and 
spreading of such diseases by dust is highly improbable. Protozoa of 
soil decrease in number by drying, but all are not killed. 


CHAPTER II 

INFLUENCE OF TEMPERATURE* 

Temperature, as well as moisture, is one of the most important fac- 
tors of life. It is so important that the most highly developed animals 
protect themselves by a very complicated mechanism of regulation 
against changes of temperature; the life processes of such animals will 
take place at a temperature nearly constant from birth to death. This 
causes the metabolism of warm-blooded animals to be different from 
that of all other organisms. The metabolism of the warm-blooded 
animals takes place at a constant temperature. The required amount 
of food is constant except for the part that is used for heating the body; 
at lower temperatures, more heat-producing material is used and the 
result is that warm-blooded animals require more food at lower tempera- 
ture. All other organisms, reptiles as well as bacteria, have the tem- 
perature of their environment and the decrease of temperature will 
decrease the intensity of metabolism as it retards any other chemical 
process. The lower the temperature, the less food is required by all 
lower organisms. 

There are, of course, limits to the favorable influence of high tempera- 
tures. Growth and metabolism of microorganisms will increase with 
rising temperature to a certain point, called the optimum temperature, 
and beyond this point the rate of growth will fall off rapidly and soon 
cease entirely. The highest temperature at which growth can take 
place is called the maximum temperature. Correspondingly, the mini- 
mum temperature of an organism is the lowest point at which growth can 
take place. 

THE OPTIMUM TEMPERATURE which allows the fastest growth will be 
quite different for different species. Groups of bacteria are known 
which develop only at very high temperatures and others for which room 
temperature is too high. The temperature requirement is largely de- 
pendent upon the natural habitat of the organisms. The bacteria of 

* Prepared by Otto Rahn. 

269 


2JO 


PHYSICAL INFLUENCES 


the polar sea and of a lagoon near the equator will very probably 
have different optimum temperatures because of the acclimatization 
and selection which has been taking place for centuries. 

The great majority of bacteria and related organisms, in fact of all 
living organisms, except in a few instances, has its optimum tem- 
perature between 20 and 40. The optimum temperature of an 
organism is generally somewhat higher than the average temperature 
of its natural habitat. 

The following table shows the data obtained for a few microor- 
ganisms. 


Temperatures 


Species 


Minimum 


Optimum 


Maximum 


Penicillium glaucum 


i-5 


25-27 


O fO 

31-30 


Aspergillus niger 


7-io 


33-37 


40-43 


Saccharomyces cercvisics I 


i-3 


28-30 


40 


Saccharomyccs pasteurianus I 


o-5 


2S-30 


34 


Bacterium phosphor eum 


below o 


i6-i8 


28 


Bacillus subtilis 


6 


30 


50 


Bacterium anthracis 


10 


30-37 


43 


Bacterium ludwigii 


50 


5S-57 


80 


THE MINIMUM TEMPERATURE or the lowest limit of growth is usually 
farther from the optimum than the maximum temperature. It will 
vary with the organisms just as do the other cardinal points. But 
there is a natural limit drawn by the freezing-point of the nutrient 
liquid. Not all organisms can grow at such low temperatures, in fact 
the greater number does not develop below 6 to 10. Those that can 
grow at the freezing-point will be inhibited by the solidification of the 
water in the nutrient medium, for if the water is frozen, food cannot 
diffuse into the cells and therefore, all life processes are checked. If 
freezing is prevented by adding salts or other soluble substances which 
lower the freezing-point, growth may continue even below o. Milk 
freezes at about - - 0.5. Bacteria are found to multiply in it as long as 
it is not entirely solid. A certain yeast multiplied slowly in salted but- 
ter kept at about 6. 


INFLUENCE OF TEMPERATURE 271 

The number of microorganisms that developed at the freezing- 
point was found to be: 

In i c.c. of market milk, up to 1,000 germs. 
In i c.c. of sewage, up to 2,000 germs. 

In i g. of garden soil, up to 14,000 germs. 

THE MAXIMUM TEMPERATURE is usually about 10 to 15 higher 
than the optimum. The development of microorganisms above the 
optimum temperature is not quite normal; there is a great tendency 
toward involution forms. The mycelium of molds grown near the 
maximum temperature appears unhealthy and pathogenic bacteria 
lose part of their virulence. This loss of virulence is made use of in 
the preparation of attenuated cultures for vaccines. 

The maximum temperature varies with different species of bac- 
teria. Most bacteria do not grow above 45, but with some of them 
the maximum temperature is considerably lower. Bact. phosphoreum 
dies if exposed for a few hours at 30; others may require still lower 
temperatures. The average organisms found in water, soil, milk, and 
the body, which have their optimum near 30 to 38, do not grow higher 
than about 45. There are very noticeable exceptions to these, such 
as the physiological group known as thermophilic bacteria. 

These extraordinary organisms have their maximum between 70 
and 80, a temperature which coagulates albumin. Corresponding to 
the high maximum the thermophiles have a very high optimum, and 
the minimum lies with most of these species above 30. These or- 
ganisms are found in soil, sewage, ensilage and occasionally in milk. 
They find the temperature suitable for their life only under extra- 
ordinary circumstances, as in fermenting manure piles, in silos, in 
self-heating hay and similar organic material that develops a high 
temperature by fermentation. Some hot springs have a very remark- 
able flora of thermophilic bacteria. 

The range of temperature within which growth is possible, is very 
uniformly 35 to 45; the starting points and end-points of this range 
vary greatly, while the total range is quite constant, except for some 
bacteria adapted to special conditions, such as some pathogenic bac- 
teria. The temperature relations of bacteria can be shown graphically 
by using as ordinate the rate of growth, as abscissa the temperature 


272 PHYSICAL INFLUENCES 

BIOLOGICAL SIGNIFICANCE or THE CARDINAL POINTS OF TEMPERA- 
TURE. The importance of the temperature requirements of certain 
organisms to the role they play in nature can be illustrated by a few 
examples. Most molds cannot cause disease in man and warm- 
blooded animals because their maximum temperature is below the 
body temperature. Exceptions are some Aspergilli and Mucorinece. 
Pathogenic microorganisms must have their optimum temperature 
coincide with that of their host. 

Organic substances may undergo a different change at different 
temperatures. The biochemical changes in soil may not be the same 
in northern Canada and near the Gulf of Mexico. Even the warm and 
cold season of the same climate is apt to change not only the rate of 
decomposition but possibly the products. Perhaps the most striking 
example in this respect is the decomposition of ordinary market milk 
kept at different temperatures. Such milk contains a great variety 
of microorganisms; at various temperatures different types will pre- 
dominate, while the remainder are retarded or inhibited by unfavor- 
able temperature conditions and by the products of the dominant type 
of bacteria. If milk is kept at about the freezing-point, only a few 
organisms will develop slowly, but after a certain time their number 
will increase to many million cells per c.c. There is, however, no appar- 
ent change; no acid or deterioration can be discovered by the taste 
though chemical analysis proves the presence of hydrogen sulphide 
and ammonia. Between 15 and 25, milk will sour in about thirty- 
six to forty-eight hours, giving a firm curd of an agreeable flavor 
without whey or gas; later Oidium lactis destroying the acid develops 
on the surface. Near body temperature the milk will lopper in twenty- 
four hours, the curd is usually contracted, a large quantity of whey 
is extruded, and much gas is produced by Bact. aerogenes and B. coll. 
The odor is disagreeable and later butyric acid is produced; eventu- 
ally the lactic acid increases further by the action of Bact. bulgaricum. 
If kept above 50 the milk either keeps permanently, or a decomposi- 
tion by thermophilic bacteria begins which is either an acid fermenta- 
tion followed by digestion or a complete putrefaction, depending upon 
the species of thermophilic organism that happens to be in the milk 
sample. Thus there can be induced in the same substance, contain- 
ing the same organisms at the start, four entirely different types 
of decomposition merely by the difference of temperature. 


INFLUENCE OF TEMPERATURE 273 

This indicates the importance of temperature regulation in the fer- 
mentation industries. Even pure cultures may give different products 
if working at different temperatures. Cream ripened with a pure 
culture starter at too high a temperature will have a sharp acid flavor. 
The cold curing of cheese has become a very common practice because 
of the much improved flavor. Bioletti claims that the value of the dry 
California wines would be doubled if the fermentation were carried 
on generally at a lower temperature. 

END-POINT OF FERMENTATION. Another question is the relation 
between the end-point of fermentation and the temperature. Of the 
few data existing, many indicate that at a lower temperature the final 
fermentation goes farther than at a higher temperature. Miiller- 
Thurgau found that under exactly the same conditions with the tem- 
perature as the only varying factor the following final amounts of 
alcohol were produced by a pure culture of yeast: 

At 36 3.8 per cent alcohol. 

At 27 7.5 per cent alcohol. 

At 1 8 8 . 8 per cent alcohol. 

At 9 9.5 per cent alcohol. 

Concerning the lactic fermentation some investigators find no differ- 
ence in the end-point, while others obtained results similar to the re- 
sults with alcohol. With three strains of Bad. lactis acidi were ob- 
tained after thirty-four days, by C. W. Brown: 

A B C 

At 37 0.89 per cent 0.87 per cent 0.60 per cent of lactic acid. 

At 30 i. oo per cent 0.96 per cent 0.81 per cent of lactic acid. 

At 1 8 i. 08 per cent i. 06 per cent 0.88 per cent of lactic acid. 

At 6 0.70 per cent 0.73 per cent 0.62 per cent of lactic acid. 

These results are quite logical and perhaps can be explained by 
the recognized experience that all products of fermentation tend to 
check the process of fermentation, and that any chemical product 
or substance acts the more vigorously upon any life process the higher 
the temperature. The same amount of alcohol that will still allow a 
slow fermentation at 10 may check the fermentation entirely at 20. 
Naturally the rate of fermentation in the beginning will be higher at 
the higher temperature but the end-point is lower. The end-point of 
the lactic cultures A, B, and C at 6 is probably not final, because 

18 


274 PHYSICAL INFLUENCES 

thirty-four days is a short time of growth at so low a temperature. 
Above the optimum, the rate of decomposition will decrease rapidly 
with the rising temperature and the end-point will also be lower. 

FREEZING. The discussion of the relation of temperature to 
microorganisms has so far considered only the temperatures within 
the limits of growth. However, the temperatures below the minimum 
and above the maximum are also of greatest importance. If bacteria 
are cooled below their minimum temperature they do not die immedi- 
ately. They remain alive in a dormant condition ready to multiply 
as soon as the temperature rises. Even the freezing of a liquid will 
not kill them immediately. Of course, they cannot multiply in ice, 
because they have no water, consequently no food, and they cannot 
thaw the ice to get their water and food for lack of body temperature 
of their own. As long as liquids are frozen solid the bacteria in them 
will remain dormant much like dried organisms, and like them their 
number will decrease very slowly. An example is given in the follow- 
ing table relevant to the number of bacteria in frozen milk (after 
Bischoff). The decrease in numbers is not very uniform, since there are 
many different bacteria in milk, but the general tendency is the same 
as in the dried bacteria. 

Milk kept at 3 to - 7 

Freshly frozen 200,000 bacteria per c.c. 

After i day 105,500 bacteria per c.c. 

After 2 days 72,300 bacteria per c.c. 

After 3 days 62,000 bacteria per c.c. 

After 4 days 46,400 bacteria per c.c. 

After 7 days 44,000 bacteria per c.c. 

After 14 days 40,500 bacteria per c.c. 

After 21 days 30,300 bacteria per c.c. 

After 35 days 22,500 bacteria per c.c. 

After 49 days 14,200 bacteria per c.c. 

The table shows plainly that it is impossible to sterilize milk by 
freezing, but as long as it is frozen it will keep; there is no possibility 
of any microorganisms decomposing a frozen liquid, for the organisms 
need water above all. If food substances change in cold storage 
(and some food products do deteriorate), this must either be due to 
changes other than microbial or the material was not completely 
frozen as is probably the case with salted butter. 


INFLUENCE OF TEMPERATURE 275 

After bacteria are once frozen, they do not seem to be affected by 
any lower temperature. Macfadyen and Rowland found that they 
tolerate very low temperatures remarkably well. Many bacteria 
were not killed by a twenty hours' exposure to the temperature of 
liquid hydrogen ( 252). Yeasts are not quite so resistant and the 
mycelium of most molds is easily destroyed by freezing, while the spores 
are hardier. 

THERMAL DEATH-POINT. Heating above the maximum tempera- 
ture is quite harmful to bacteria, and the amount of injury increases 
with the temperature. Recent experiments have shown that heat does 
not kill bacteria instantaneously, but that we have an orderly process 
as in the case of death by drying. This can be observed only in a 
very narrow range of temperature, however, since the death rate rises 
very rapidly with the increase of temperature. 10 increase may make 
the death rate ten to one hundred times as great, and death is almost 
instantaneous. For most practical purposes, it is sufficient to state 
the time and temperature necessary to bring about complete sterili- 
zation. It has become customary to define, as the thermal death- 
point, the lowest temperature at which a culture will be killed in ten 
minutes. As most bacteriologists will use very nearly the sametech- 
nic, they will have fairly uniform numbers of cells to start with, 
and therefore obtain fairly uniform results. 

The thermal death-point does not depend upon the species and 
the temperature only. It varies with the age of the culture since 
older cells are less resistant than younger ones especially if heated in 
their own products. The medium in which the organisms are heated 
is also of great significance. The fact that acid liquids, as fruit juices, 
are more easily sterilized than neutral meat or vegetables is largely 
due to a chemical (poisonous) action of the acids upon the bacteria. 
But the greater resistance of tubercle bacteria in the sputum compared 
with those suspended in salt solution cannot be so readily 
accounted for. 

A necessary factor for the prompt destruction of organisms by 
heat is the presence of moisture. The resistance of dry organisms 
is remarkably higher than that of the same organisms in a liquid cul- 
ture. The following table shows the death-point of yeast cells and 
spores in a dry and moist state. 


276 


PHYSICAL INFLUENCES 


THERMAL DEATH-POINT OF DRY AND MOIST YEAST 


{. 


;eiis 


sp<. 


>res 


. 


Moist 


Dry 


Moist 


Dry 


Pale ale yeast 


6c 


o^-io^ 


6s-7o 


Iir- I2 c- 


Hofbrau yeast 


v o 

S<5 


y j ^j 

8s- 00 


w o / w 
6cr 


* A x * O 
Iiq-I20 


Saccharomyces pasteurianus 


O J 

5o-55 


ioo-io5 


"O 

60 


H5 


RESISTANCE OF SPORES. The organisms most resistant to heat are 
the spores of certain bacteria. In the chapter on moisture require- 
ments attention has been called to the great resistance of spores to 
drying. We find the same exceptional resistance to high temperatures. 
Boiling heat will not kill spores readily. Some bacterial spores can 
stand the temperature of 100 for several hours. In order to kill spores 
in one heating the temperature must rise to about 110 for fifteen to 
thirty minutes; this can be accomplished only by heating under pres- 
sure. This is not always advisable for sterilizing food substances. 
While vegetables are usually sterilized under pressure without losing 
much of their palatability, other foods like milk are changed materially 
in taste and appearance. To prevent these changes, discontinuous 
sterilization is sometimes used. This is based upon the following 
principle. 

If milk or any other medium is heated to 100 for about fifteen min- 
utes, all living cells of bacteria, yeasts and molds will be killed except a 
few spores of bacteria. After cooling, these spores will germinate under 
suitable conditions and the vegetative cells thus appearing instead of the 
resistant spores are easily killed in a second heating. A third heating 
is necessary in order to kill any vegetative cells which may have devel- 
oped from spores not yet germinated before the second heating. It is 
essential to have the time between two heatings long enough to allow the 
germination of spores, and not too long to permit formation of new 
spores. It is customary to heat on three successive days for fifteen 
minutes each time. In this case, sterilization is usually complete, 
while a forty-five minutes' heating at once is not sufficient to guarantee 
sterilization. Among the substances that are very easily sterilized are 
cider and other fruit juices, while milk and soil are the most difficult 
materials to sterilize. 


INFLUENCE OF TEMPERATURE 277 

Dry spores will resist still higher temperatures than moist spores. 
Some dry spores survive an exposure to 140 or 150 for ten minutes. 
It requires a very high temperature to sterilize glass, cotton, gauze, and 
instruments with dry heat. A discontinuous sterilization of dry mate- 
rial is useless, since the spores will not germinate without moisture, 
therefore their resistance remains unaltered. 

The spores of molds are more resistant than the mycelium, but if 
moist, they all die at 100. The dry mold spores can tolerate a some- 
what higher temperature, but not as high as the spores of many bacteria. 
Yeast spores and yeast cells are very much alike in their resistance to 
heat. The table on page 276 shows hardly any difference between their 
resistance. 


CHAPTER III 

INFLUENCE OF LIGHT AND OTHER RAYS* 

Microorganisms in their natural environment are temporarily but 
not usually exposed to light. The organisms of decay, living in soil, in 
foods, in the intestines of animals, will only occasionally come in con- 
tact with the direct rays of the sun. Water bacteria and the organisms 
on the surface of plants and animals are more commonly exposed to the 
sun. 


FIG. 117. These plates were heavily inoculated with B. coli and B. prodigiosus 
respectively and then were exposed, bottom side up, to the direct rays of the sun, 
for four hours. On the instant of exposure, a figure O cut from black paper was 
pasted to the plate shading the bacteria underneath. After one, two and three hours 
the corresponding figures were pasted to the plates. The above picture was taken ZA 
hours after exposure, proving that three or four hours of direct sunlight weaken and 
and may even kill bacteria. B. prodigiosus proved more sensitive than B. coli. 
(Original.} 

The influence of light varies with its intensity. Direct sunlight 
has a very harmful effect upon microorganisms. Most bacteria are 
killed by direct sunlight in a few hours; the time depends upon the 
organism as well as upon the intensity of light; this again varies with 

* Prepared by Otto Rahn. 

278 


INFLUENCE OF LIGHT AND OTHER RAYS 


279 


the amount of moisture and dust in the atmosphere, with the time of 
the day and with the season; an absolute measure for the action of light 
cannot be fixed, therefore, as easily as with the action of heat in the ther- 
mal death-point. The different colors of the spectrum do not act 
alike; the part of the spectrum from red to green is practically without 
influence upon microorganisms, while the blue light acts strongest 
and the intensity decreases in the violet and ultra-violet. In carrying 
on experiments with the influence of light, it must be remembered that 
glass absorbs ultra-violet rays, and further that the heating of the 
medium by direct radiation must be avoided (Fig. 117). 


FIG. 1 1 8. Phototropism of Rhizopus nigricans. The mold is grown on gelatin with 
diffused light coming from right side. (Original.} 

Yeasts, molds, and bacteria and probably Protozoa are equally sensi- 
tive to light. Even the spores of most bacteria do not show a greater 
resistance to light, while the mold spores are an exception. The col- 
ored spores of the Penicillmm, Aspergillus and Mucor species can be 
exposed to light for a long time without being killed, but the colorless 
spores of Oidium and Chalara show no increased resistance. It is sup- 
posed that the pigment in mold spores is a protection against light. This 
is not true with the pigment of bacteria. The colored and colorless 
strains of pigmented bacteria show no difference in their resistance to 
light. The only exceptions are the so-called purple bacteria. These 
peculiar organisms, many of which feed on hydrogen sulphide, seem to 


280 


PHYSICAL INFLUENCES 


thrive better in light than without it. Direct sunlight does not kill 
them, it rather attracts them and they move toward the light. This is 
called phototaxis or heliotaxis. The pigment, bacteriopurpurin, does 
not take the place of chlorophyl, however, since the bacteria do not pro- 
duce oxygen in light and always need organic food. 

The effect of light upon microorganisms is mainly brought about by 
a chemical change in the protoplasm, and also, to some extent, by a 
chemical change in the medium, namely the formation of a peroxide or a 
similar oxidizing agent. 

The germicidal action of light is of importance in the purification of 
rivers. It is applied also in curing diseases of the skin, as lupus and 


FIG. 119. Two cultures of an Aspergillus^on.? grown in the dark the other in 

diffused light, showing rings. (Original.} 

leprosy, by exposing the diseased parts to a very concentrated light of 
the electric arc. This light contains plenty of blue and violet rays and 
is preferable to sunlight because it is always ready for use and its com- 
position and intensity can be controlled easily. Ultra-violet light is 
used in the sterilization of water and of milk. 

Diffuse light is not nearly as harmful to microorganisms as direct 
sunlight. Long exposures to diffuse light will kill most bacteria, while 
molds are not at all sensitive. They rather like a very dim light, and 
many molds grown in a dark room with light only from one side will 
grow toward the light. This property, which is characteristic for all 
green plants, is called heliotropism or phototropism (Fig. 118). It has 


INFLUENCE OF LIGHT AND OTHER RAYS 281 

been found that molds produce mycelium mostly in the dark, while in 
daylight sporangia are produced mainly. This difference in the devel- 
opment during the day and during the night accounts for the concentric 
rings which are quite commonly found in older mold colonies, and 
which indicate the age of the culture (Fig. 119). Similar rings are 
occasionally found with yeast and bacterial colonies, and are possibly 
due to the same influence of light. 

X-RAYS. Of other rays, the invisible X-rays and the radium rays 
have attracted the attention of bacteriologists and physiologists. It 
is known that the X-rays will destroy living tissue by long exposures; 
microorganisms cannot be considered less resistant. X-rays are used 
in the treatment of microbial diseases of the scalp and skin. 

RADIUM RAYS are not so well known, and their bactericidal action is 
doubtful. The treatment of certain bacterial diseases has been 
attempted, but it has not been applied as generally as yet as the X-ray 
method. The sterilization of milk and possibly other foods by this 
method has been suggested, but the practical application is at present 
quite improbable because of the cost and the uncertainty of the results. 


CHAPTER IV 

INFLUENCE OF ELECTRICITY* 

The influence of electricity upon microorganisms is much less than 
one might perhaps expect, if the electricity as such is considered. A 
direct electric current passing through a nutrient medium will, of course, 
cause electrolysis which is usually manifested by the formation of acid 
on the positive pole and of alkali on the negative pole. The acid and 
alkali will kill microorganisms, as is discussed in the chapter on chemical 
influences. In this case, it is not the electricity itself that destroys the 
bacteria. It is also possible to kill bacterial cultures by passing an 
alternating current through the medium for some time. No electrolysis 
takes place in this case, still it is not the current that acts directly upon 
the organisms, but rather the heat produced by the current passing 
through a medium of high resistance. If the culture is cooled properly 
the influence of the current is insignificant if at all noticeable. When- 
ever electricity is applied against microorganisms the effect is con- 
sidered electrochemical. 

The electrical current is used in a very small way in the purification 
of sewage. The sewage passes between two iron plates which represent 
the two poles of a strong current. The electrical sterilization of milk 
has been patented. Wines are improved by electricity. The steriliza- 
tion of drinking water by ozone is also an application of electricity, 
though of course the ozone once formed by the current acts as a chem- 
ical compound independently of its source, and the same effect would 
be produced if the ozone were manufactured chemically. 

* Prepared by Otto Rahn. 


282 


CHAPTER V 

INFLUENCE OF MECHANICAL AGENCIES* 

PRESSURE. The resistance of microorganisms to mechanical pres- 
sures is very great. Pressures of 3,000 atmospheresf will not kill the 
majority of bacteria in four hours. They are, however, weakened and 
some species will die. A specific difference between the molds, yeasts, 
and bacteria in this particular does not seem to exist. Of the organisms 
exposed to 2,000 atmospheres for ninety-six hours, Bact. anthracis, Bact. 
psendodiphtherice, M. pyogenes var. aureus, Oidium lactis and Saccharo- 
myces ceremsia survived, while seven other organisms lost the power of 
multiplication. Some of these were not dead, however, since they 
retained their motility for several days. It is noteworthy that high 
pressure will destroy one quality (multiplication) and not affect another 
(motility). Pigment-production and virulence of pathogenic bacteria 
were either diminished or lost completely. The resistance against 
high pressure is necessary for the organisms which cause the decay 
of organic matter at the bottom of the oceans. Vertebrates breathe 
oxygen in the form of gas or have at least an organ filled with gas (fish 
bladder) ; the volume of gas is changed considerably by slight changes 
of pressure; this will affect organisms depending on gas. Microorgan- 
isms do not require gas as such. They can absorb gases only in 
solution. A change of pressure therefore will not cause a change of 
volume, since liquids have a very small coefficient of compression. 

The situation is entirely different if the liquid is not exposed to the 
pressure directly, but to compressed air. In this case, the chemical 
effect of the gas is the deciding agent. The higher the pressure, the 
more gas will be dissolved in the culture medium. The fatal pressure 
under these conditions will vary as much as the fatal dose of an antisep- 
tic; it depends upon the chemical qualities of the gas, upon the pressure 
(concentration), upon the temperature, and upon the organism. 

* Prepared by Otto Rahn. 

fOne atmosphere is i kg. pressure per square centimeter (or about 15 pounds per square 
inch). 

28 3 


284 PHYSICAL INFLUENCES 

Some data have been given already in the chapter on oxygen require- 
ments. It was mentioned in that connection that Bad. butyricum can- 
not tolerate more than 0.65 per cent of the total oxygen content in air 
(0.2 atmosphere) ; in other words, an oxygen pressure higher than 0.0013 
atmosphere will kill the organism. The maximum pressure for B. 
prodigiosus was found to be about 5.4 to 6.3 atmospheres. Very few 
experiments have been made with other gases. Carbon dioxide at a 
pressure of 50 atmospheres retards the growth of bacteria in water and 
will sterilize it in twenty-four hours. Suspensions of pure cultures of 
B. typhosus and M sp. comma are killed by 50 atmospheres carbon dioxide 
pressure in three hours. Milk cannot be sterilized by this pressure but 
bacteria do not multiply. Carbonated milk has been recommended as 
a refreshing drink by several investigators. The ordinary market milk 
will keep about two days longer under the pressure of 10 atmospheres 
(150 pounds) than without pressure. If pasteurized it is said to keep 
for a week. 

GRAVITY. Gravity would have a great influence upon the growth of 
microorganisms in liquids if their specific gravity were much greater 
than that of water. This does not seem to be the case however. It has 
been estimated by accurate weighing to vary between 1.038 and 1.065. 
Very much higher results (1.3 to 1.5) have been obtained by centrifuging 
bacteria in salt solutions of varying specific gravity, but these data are 
not exact since the salt solution will diffuse into the cells and thus in- 
crease their weight. The specific gravity being very nearly that of the 
culture medium, it is plainly seen that gravity has but little influence. 
The microorganisms will live suspended in the liquid and sediment out 
very slowly. The slightest current in the liquid will carry them 
around and distribute them through the medium. The motility is of 
minor importance; the actual distance covered by motile bacteria has 
been measured, and under the most careful exclusion of currents in the 
liquid has been found to be about a millimeter in a minute for B. subtilis. 
This is very slow compared with the speed of the circulating water 
moved by changes of temperature or other incidental agents. 

Yeast cells and other gas producers use the carbon dioxide as a ve- 
hicle. The gas bubbling up in the fermenting liquid keeps it constantly 
in motion and moves the yeast cells against gravity toward the surface 
where the gas escapes and lets the cells fall back to the bottom. 

The production of scums and pellicles on the surface by organisms 


INFLUENCE OF MECHANICAL AGENCIES 285 

which are heavier than the liquid they float on, is often accomplished by 
small gas bubbles between the cells (Mycoderma). In other instances, 
it may be just the floating of cells having oily surfaces. 

The growth is influenced by gravity very little. The sporangia of 
molds are the only exceptions, growing decidedly away from the center 
of gravity (negative geotropism). 

AGITATION. For the majority of microorganisms, the quiet, undis- 
turbed growth of the laboratory culture is the normal or the ideal one. 
Such cultures, if shaken for a considerable time, show a decrease of liv- 
ing organisms, and it is possible to sterilize cultures by continued shak- 
ing. The effect is not a simple mechanical breaking or tearing of the 
cells. The bacteria break 'up into the finest particles. This is also the 
case if cultures are exposed for several days to the trembling motion 
caused by the working of very heavy machines. There is no grinding or 
tearing effect but the cells break to pieces just the same. 

A slight and slow agitation seems to be advantageous for many cul- 
tures, only continuous heavy motion proves harmful. Different organ- 
isms show wide variations in their resistance to agitation. 


DIVISION IV 

CHEMICAL INFLUENCES 


CHAPTER I 
STIMULATION OF GROWTH* - 

i 

The influence of chemical substances upon microorganisms may 
be helpful or harmful, or not noticeable. As helpful must be con- 
sidered above all the food compounds. Unless given in such large 
doses as to cause a physical or osmotic effect they will stimulate 
the development. Other substances, not food, can also act as 

stimulants. It is a recognized fact of long stand- 
ing that many poisons in very small doses will 
stimulate. This applies to the most highly 
developed animals and plants as well as to micro- 
organisms. Raulin noticed in 1869 that Asper- 
gillus niger grew very much better in a nutrient 
solution if a small amount of zinc salt was added. 
He considered the zinc, therefore, as a necessary 

' ' 4 

constituent of the mold cells. Alcoholic fermenta- 
. 

Vv tion can be stimulated by metallic salts. It is be- 
lieved by some physiologists that, as a law of nature, 
FIG. 120. Chem- every substance that is injurious in a certain con- 
s'* a /^V (After centra tion is a stimulant in a lower concentration. 

A similar action of certain chemical compounds 
upon enzymes has been noticed, retarding in high concentrations, 
stimulating in weaker solution. 

CHEMOTROPISM AND CHEMOTAXIS. Microorganisms manifest their 
preference for certain foods not by a stimulated growth alone. They 
also make efforts to obtain better food by growing or moving toward it, 
which is not a manifestation of a rudimentary intellect. Such reactions 
of microorganisms may be accounted for largely by chemical or osmotic 

* Prepared by Otto Rahn. 

286 


STIMULATION OF GROWTH 287 

forces. In a solid medium the hyphae of molds will grow toward the 
best source of food supply. This growth on account of chemical 
stimulation is called chemotropism, analogous to the phototropism 
or growth toward light. If some injurious compound is offered, 
the hyphae will grow away from it. Thus we have to distinguish 
between positive and negative chemotropism. The motile organisms, 
bacteria as well as protozoa, demonstrate their preference for certain 
food compounds by swimming toward them. This is called chemotaxis 
(Fig. 120). Here also a positive and negative chemotaxis must be 
distinguished, the latter taking place if injurious substances are present. 


CHAPTER II 

INHIBITION OF GROWTH* 

POISONS, GERMICIDES, DISINFECTANTS, ANTISEPTICS, PRESERVATIVES 

A great number of inorganic and organic bodies will destroy 
life in comparatively weak solutions. These substances are called 
poisons if they are considered in their effect upon man arid animals. In 
their application to microorganisms they are generally called germicides 
(germ-killers), or disinfectants if the emphasis is laid upon the prevention 
of infection rather than upon the actual killing of the microorganisms. 
Analogous to the general term germicides, the terms bactericide and 
fungicide are used occasionally. The term antiseptic means a prevention 
of sepsis which may be accomplished by checking the growth without 
necessarily killing all microorganisms. The meaning of the word pre- 
servative is practically the same, only the latter is used more commonly 
in relation to foods, feeding stuffs and preparations of similar origin 
while the word antiseptic is largely used in relation to microbial diseases. 
A strict line cannot be drawn between any of these definitions. A dis- 
infectant, if diluted, becomes an antiseptic. A strong salt solution is an 
antiseptic for some organisms and a disinfectant for others. Of the 
above expressions, germicide is the most definite, but is not so commonly 
used as the others. 

MODE OF ACTION. The action of a poison upon the cell is generally 
considered an action upon the protoplasm. The poison is supposed to 
combine chemically with the cell plasma producing compounds which 
interfere with the continuation of the life processes and thus cause 
death. If the cell has been subjected to the action of the poison only a 
short time, it can be saved by removing the poison. Bacteria can be 
treated with mercuric chloride (HgCl 2 ) so that they will no longer de- 
velop if transferred to a fresh medium. If the mercuric chloride is re- 
moved from the cell by means of hydrogen sulphide, some of the organ- 
isms may be revived. 

The mode of death through poison is the same as that through 

* Prepared by Otto Rahn. 

2S8 


INHIBITION OF GROWTH 


289 


heat or drying. The number of cells dying in a given time interval is 
proportional to the number of cells surviving. In the last five years, 
this has been tested and found true with practically all disinfectants. 
Fig. 1 2 1 shows the curves plotted from data obtained with Bad. anthracis, 
the full-drawn line representing the number of live spores in .21 per 


4-000 


3500 


o 


3000 


o 
O, 

in 

** 

" 
O 


2500 


9.000 


IOOO 


500 


o A 


10 


FIG. 121 Curve of disinfection. Spores of Bact. anthracis in mercuric chloride 

solution. (After Chick.} 

cent of mercuric bichloride, the dotted line the same in .11 per cent 
solution. 

The (apparent) resistance of the few remaining cells is of great im- 
portance in those applications of disinfection where a thorough kill- 
ing of all bacteria is intended, e.g., in the treatment of drinking water. 
Our ideas of the efficiency of a disinfectant would depend, therefore, 

19 


2QO CHEMICAL INFLUENCES 

upon the accuracy with which we can prove the presence of a certain 
bacterium. 

FACTORS INFLUENCING DISINFECTION. The efficiency of a dis- 
infectant depends upon several factors. Moisture is necessary 
a dry poison has only a very slow action upon microorganisms. For 
this reason, absolute alcohol has not nearly the same germicidal power 
upon dry bacteria as diluted alcohol; the strongest poisonous effect 
is obtained by a 50 to 70 per cent solution. The necessity of moisture 
is further demonstrated in the sterilization with gases, as with formal- 
dehyde. The effect of formaldehyde gas without the provision of a 
very moist atmosphere is surprisingly weak. 

The temperature is also quite an important factor in the study 
of disinfectants. Since poisoning is supposed to be a chemical effect, 
it must be expected that the poisoning process like other chemical 
processes will take place faster at a higher temperature. As a matter 
of fact, the death rate through poisoning is usually doubled or trebled 
by a temperature increase of 10. Above the optimum temperature, 
where the growth is not very vigorous, and when the disinfecting 
power of the poison is increased considerably by the higher temperature, 
a very small amount of poison will have a very strong germicidal effect. 
The combination of high temperatures with a disinfectant has been 
suggested as a means of sterilizing foods. This has been tried in 
the case of milk with hydrogen peroxide at 50 to 60. 

It makes a considerable difference whether the organisms which 
are tested with a certain disinfectant are in a culture with their food 
material, or suspended in water or salt solution without any food. It 
is very probable that part of the disinfectant is acted upon by the food 
products which are partly protein substances and are in many ways 
similar to the protoplasm of the bacterial cells. It is especially diffi- 
cult to poison bacteria in blood, pus, or similar material. The sensi- 
bility of the microorganisms in pure water is remarkable. Very small 
doses which would not be considered efficient under any other condition, 
will destroy microorganisms in pure water. The concentration of 
chloride of lime which is sufficient to sterilize drinking water, does 
not at all suppress the development of bacteria in sewage. 

The influence of the number of cells is evident from the above ex- 
planations of the mode of action, and from the curves of disinfection. 
The concentration of the poison is of course of greatest importance. 


INHIBITION OF GROWTH 2QI 

Recent investigations have shown the rather unexpected fact that the 
efficiency of a poison is not proportional to its concentration. If 
a certain poisonous solution is diluted with an equal volume of water, 
we might expect it to be half as poisonous as before, but depending 
upon the chemical nature of the poison, it may be more poisonous 
than expected, or considerably less. It follows from this that two dif- 
ferent poisons of the same intensity, if diluted in the same proportion, 
may not have the same intensity any more. 

Microorganisms will gradually become accustomed to certain poi- 
sons, and become more resistant. This principle has been utilized 
in the manufacture of distilled alcohol; yeasts have been cultivated 
which can tolerate a high concentration of acid; the acid serves 
to suppress bacteria producing undesirable fermentations. 

The age of the culture and the stage of development will naturally 
change the resistance of a species materially. The old cultures 
which are past the culmination of growth will be much more sensitive 
to any poison unless a spore-producing organism is under test. In 
this case, we find a greatly increased resistance, similar to the 
increased resistance of spores against drying and heat. 

THE CLASSIFICATION OF DISINFECTANTS is very difficult as long 
as we cannot explain completely the process of poisoning. It is im- 
possible to arrange them according to the intensity of action, because 
the intensity of influence depends not only upon the disinfectant, 
but also upon the species of organisms. Some yeasts can resist ten 
times as much alcohol as certain bacteria. Formaldehyde is not 
nearly as strong an agent with molds as it is with bacteria. The dis- 
infectant concentration of a poisonous substance is not absolute. The 
simplest method of grouping is by chemical structure and qualities. 
Of the following natural groups can be distinguished acids (inorganic 
and organic), metallic salts, hydrocarbons (aliphatic and cyclic), 
alcohols (aliphatic and cyclic), aldehydes, anaesthetics, essential oils, 
oxidizing agents and reducing agents. 

The first three groups, acids, alkalies and salts, are distinguished 
from the rest as electrolytes; the strength of acids and alkalies (chemic- 
ally speaking) is measured by the degree of electrolytic dissociation. 
The disinfectant value follows largely the same law. The strongest 
acids in the chemical sense are also the strongest disinfectants. There 
are exceptions, however, where, besides the poisonous effect due to 


CHEMICAL INFLUENCES 

the degree of dissociation, there is a specific effect due to the chemical 
structure, as is the case of nitrous, salicylic and hydrocyanic acids. 
The same is true of alkalies. With metallic salts, the action will depend 
mainly upon the metal in solution, but the electrolytic dissociation 
is also of importance. NaCl will decrease the dissociation of mer- 
curic chloride (HgCU) and decrease also its disinfectant power. Mer- 
curic chloride dissolved in absolute alcohol is not dissociated. In 
this case, it has almost no action upon bacteria. 

Acids are not commonly used as disinfectants, except in the house- 
hold, but they play a certain role in nature. The common fruits con- 
tain so much acid that bacteria cannot easily attack them; the decay- 
ing of fruit is almost exclusively due to molds which have a preference 
for acid media. The acid in the stomach of man and animals plays an 
important role as a sterilizing agent for the food. Many microorgan- 
isms are killed in the stomach. In the household, the natural 
acidity of fruit helps in keeping canned fruit, preserves and jellies. 
Especially in heating, the acid together with the high temperature 
has a very strong germicidal effect. Vinegar is often used to pre- 
serve fruit and vegetables; in some parts of the country, meat is kept 
in buttermilk. Benzoic and salicylic acids are often used in the pres- 
ervation of fruit and vegetables. Their poisonous influence is not 
so much due to the acid reaction but to the specific chemical character 
of these compounds. 

Of the alkalies, only one is used extensively, namely, lime; quick- 
lime (CaO) is considered a valuable disinfectant for excreta in privy 
vaults; it is universally applied as a whitewash in stables, barns, 
poultry houses and similar buildings. Quite commonly, it is used as 
"milk of lime" (one part of slaked lime with four parts of water). 
It should be kept in mind that the calcium oxide unites with the carbon 
dioxide of the air and thus gradually loses its disinfecting power. 

Of the metallic salts, many are well-known germicides. The most 
powerful disinfectant is mercuric chloride (HgCl 2 ) which is one of the 
standard disinfectants. It is generally used in a dilution i : 1000 
which is sufficient to kill all vegetative cells as well as spores in a few 
minutes. Quite commonly, hydrochloric acid or salt is added, to 
prevent coagulation or precipitation of slimy or albuminous matter 
which would protect the enclosed bacteria from immediate contact 
with the poison. The addition of hydrochloric acid or any chloride 


INHIBITION OF GROWTH 293 

decreases somewhat the disinfectant value for bacteria suspended in 
distilled water because it decreases the electrolytic dissociation. 

Another disinfectant of remarkable strength is silver nitrate; it 
is not used commonly because of its high price. It also decomposes 
easily and leaves dark spots on the skin and clothes. Of the other 
metallic salts, copper and iron sulphate are not used extensively, 
though recommended for the disinfection of feces. Zinc sulphate may 
be applied to mucous membrane the same as silver nitrate. Many 
other salts may be used occasionally for disinfecting purposes, though 
the expense or undesirable qualities prevent their common application. 

The alcohols are well known for their poisonous effects, but the 
value of ethyl alcohol as a disinfectant is usually overestimated. It 
takes quite strong alcoholic solutions, more than 20 per cent, to kill 
certain yeasts and the spores of some bacteria in less than a day, 
and a complete sterilization by alcohol in a few minutes cannot al- 
ways be guaranteed even with 50 to 60 per cent solution. It has 
already been mentioned that desiccated organisms are very resistant 
to concentrated alcohol, more so than to a 50 per cent mixture. 
Methyl alcohol is weaker, the higher alcohols, especially amyl alcohol, 
are stronger disinfectants than ethyl alcohol. They all give good 
results in the presence of water while the absolute alcohols have 
scarcely any effect upon desiccated bacteria. None of these alcohols 
in whatever concentration they may be used, can be relied upon to 
kill bacterial spores. 

Stronger germicidal effects can be obtained by the alcohols of the 
benzol group, of which phenol or so-called carbolic acid (CeH 5 OH) 
is the simplest representative. Phenol, like ethyl alcohol, is not as 
effective as is commonly believed. It is applied in solutions from .5 per 
cent to 5 per cent ordinarily, but it usually takes a long time even for 
the 5 per cent solution to kill vegetative cells as Bact. tuberculosis or 
B. coli; it is inefficient against anthrax spores. More powerful are 
the higher cyclic alcohols, of which the cresols are examples. They are 
used extensively as disinfectants and antiseptics. They are, together 
with phenol, coal-tar constituents and are sold commercially under many 
different names, either pure or mixed with soap or other disinfectants 
which make them emulsify readily in water. The cresols are almost 
insoluble in water, and not as effective in solutions as they are in 


294 CHEMICAL INFLUENCES 

emulsions. The disinfecting properties of tar come from the cresol 
contained in it. 

Hydrocarbons are used only for laboratory experiments as very 
weak antiseptics. The aliphatic bodies, as methane, etc., which con- 
stitute a large part of coal gas, have very little if any effect upon bac- 
teria; gas is used occasionally in place of hydrogen for growing anae- 
robic bacteria. Benzol, xylol, and toluol are antiseptics, if shaken 
frequently with the liquid to be protected, but they are not reliable 
as disinfectants. The same is true with the common anaesthetics, ether 
and chloroform. The high prices of these agents forbid their general 
use, but they are sometimes used for laboratory work. ' 

The essential oils have a little more practical importance. Some of 
these are the main constituents of mouth washes, especially the oil of 
peppermint (menthol), of thyme (thymol), and of eucalyptus (eucalyp- 
tol). Their action is very weak, however. The volatile oils of spices 
have to be considered in the preserving of fruit, pickles, catsups, and 
other food products. Though the antiseptic value in general is insigni- 
ficant, certain microorganisms are sensitive to certain spices. The 
bacteria of the mesentericus group are said to be suppressed entirely 
by quite small quantities of garlic, while others, like the lactic bacteria, 
are not affected at all. Cloves, cinnamon and allspice are the most 
efficient spices, while the disinfectant power of black and white pepper 
and mustard is very small. 

The most important disinfectant has not been mentioned, because 
it does not belong to any of the above groups. This is formaldehyde. 
Formaldehyde (HCOH) is a gas, soluble in water to the amount of 40 
per cent at room temperature; it does not attack metal, clothing, wood- 
work, and is, therefore, preferable to many other disinfectants for steril- 
izing rooms. It kills spores of bacteria in a short time in a i : 1000 di- 
lution. Its greatest importance lies, however, in its gaseous nature, 
because it can be applied to rooms and buildings by simply evaporating 
it. The saturated 40 per cent solution can be evaporated directly or by 
generating steam which passes through the formaldehyde solution; this 
latter method has the advantage of saturating the air with moisture, 
which increases the power of the formaldehyde gas. Formaldehyde 
can also be obtained in a dry form; it polymerizes to a white crystalline 
substance, paraformaldehyde ((HCOH) 3 ) which can be changed back to 
formaldehyde gas by gentle heating. This paraformaldehyde is com- 


INHIBITION OF GROWTH 295 

monly used instead of the liquid, because it is more easily handled and is 
quite inoffensive in its solid form, while the formaldehyde solution has a 
very penetrating odor and is exceedingly harmful to the mucous mem- 
brane of the respiratory organs. 

Of the oxidizing agents, oxygen itself has already been mentioned. 
Though it is able to destroy certain anaerobic bacteria, it cannot be 
called a disinfectant. For this purpose, oxygen must be activated ; such 
oxygen can be obtained in the form of ozone (O 3 ). It is formed in air 
under the influence of electric discharges and can be produced at a price 
low enough to allow its application for use in the sterilization of water. 
It has also been recommended for preservation of milk. 

Hydrogen peroxide (H 2 O 2 ) resembles ozone in its chemical reactions ; 
it changes readily to H 2 O + O, and this oxygen atom in the nascent 
state is quite effective as an oxidizing agent. For an antiseptic, it must 
be used in at least a i per cent solution, and for an absolutely reliable dis- 
infectant a still higher concentration is required. It loses its disinfect- 
ing property easily because it is decomposed readily by the peroxidases 
of tissues and organic liquids as blood, milk, and pus. It is used in the 
preservation of milk. Hydrogen peroxide is slowly decomposed by the 
katalase of milk thus disappearing completely. 

Chlorine in its gaseous form is not used as a disinfectant, though its 
germicidal power is quite strong. The so-called " chloride of lime," 
manufactured by absorbing chlorine in slaked lime, gives in water 
hypochlorite and free chlorine; these substances are good germicides 
and chloride of lime is used in the disinfection of privy vaults, and other 
places in which it may be employed without injury. Hypochlorite is 
now used with great success for rendering safe drinking water and 
sewage; it has also become the basis of some commercial dis- 
infectants. 

Potassium permanganate is only incidentally used as a disinfectant. 
Its chemical qualities prevent an ordinary use. 

Sulphurous acid, or sulphur dioxide (SO 2 ) was for a long time a 
standard disinfectant and is still used occasionally for fumigating rooms, 
stables, barns and out-buildings though it is substituted more and more 
by formaldehyde which can be applied almost as easily. The burning 
of sulphur is an extremely simple process, but it requires a moist air to 
disinfect properly, and under these circumstances it will attack metal, 
dyes of clothing and even the fiber itself. 


296 CHEMICAL INFLUENCES 

In addition to these disinfectants which are used outside of the 
human body, or applied to its surface only, there have come into use 
during recent years, several disinfectants which are injected into the 
body to kill the microorganisms in the blood. Among these might 
be mentioned the colloidal metals, mainly colloidal silver which is sold 
under various trade names, e.g., collargol. It is given especially in 
pneumonia, but its action upon the bacteria directly is very insignificant, 
though it greatly stimulates phagocytosis. Further, there is to be 
mentioned ethoxyl, given against the protozoon of sleeping sickness, 
and the latest and most discussed of all, salvarsan, an organic arsene 
compound, against syphilis. 


DIVISION V 
MUTUAL INFLUENCES : 


INTRODUCTION 

The biological relations of microorganisms are of the greatest im- 
portance in nature. Pure cultures in nature are very rare and of excep- 
tional occurrence; they are hardly ever found except in certain diseases 
of man, animals and plants. Generally, nature works with mixed cul- 
tures. All natural fermentations, decompositions and putrefractions 
are accomplished by a number of different species among which perhaps 
one dominates, but is influenced by the rest. The study of the mutual 
relations of microorganisms is in the very first stage as yet; practically 
all laboratory work is done with pure cultures. The experiences obtained 
with pure cultures are not sufficient to explain all microbial activity in 
nature. 

There are many possibilities of mutual influence between different 
organisms. Generally three main cases are distinguished: symbiosis, 
where two organisms profit by the combination; meiabiosis, where one 
profits by the other's action without benefiting the other in return, and 
antibiosis, where one organism injures the other. These cases cannot be 
separated strictly. The relations are not always constant through the 
entire development of the cultures; an originally beneficial influence 
may change to an injurious one in a few days. Many terms have been 
coined to designate all these various possibilities, but in order to avoid 
this multiplicity of more or less indefinite names for the various relations, 
the general term " association " has come into use, especially when the 
relationship is not well understood. 

SYMBIOSIS 

Symbiosis is not very common among microorganisms, and it is 
difficult to find examples where true symbiosis exists through the entire 

* Prepared by Otto Rahn. 

2Q7 


298 MUTUAL INFLUENCES 

development of both organisms. The association of lactic bacteria and 
Oidium lactis in milk is, for a certain period at least, a symbiosis. The 
bacterium will produce only a certain amount of acid, and then it can 
grow no more because the acid is too strong; the mold will destroy the 
acid and thus gives the bacterium a chance for continued activity. The 
bacterium produces the acid which the mold likes; the mold in turn 
removes the excess acid which otherwise would check the bacterial 
activity. 

True symbiosis is more common in the relation of microorganisms 
with higher plants and animals. The standard example in the plant 
kingdom is Ps. radicicola in the nodules of legumes, feeding on carbo- 
hydrates provided by the plant and furnishing the plant nitrogen from 
the air which the plant cannot assimilate directly. The typical exam- 
ple in the animal kingdom is B. coli in the intestine of animals, being 
nourished by the food of the animal and rendering the food more easily 
digestible. 

METABIOSIS 

Metabiosis may be considered a one-sided symbiosis; two organisms 
live together, but only one is benefited, the other remains uninfluenced 
or later may be injured by the association; the latter case is the most 
common. In this relation, one usually prepares the food for the other. 
It has previously been mentioned that the metabolic products of one 
species serve as food for another species, thus breaking up the various 
organic compounds step by step to smaller and simpler molecules. 
Quite commonly, each step is accomplished by a different species of 
microorganism. Consequently, metabiosis is a very common occurrence 
among microorganisms. 

The classical example is the two nitrifying bacteria: the nitrate bac- 
terium is unable to oxidize ammonia, and depends entirely upon the ni- 
trite bacterium to oxidize the ammonia to nitrite; then, and only then, 
can the nitrite bacterium grow. 

The relation between yeasts and acetic bacteria is also very well 
known. The yeast ferments the sugar to alcohol, and then the acetic 
organisms oxidize the alcohol to acetic acid. The yeast is in no way 
helped by the acetic bacteria, while these could not form acetic acid 
from sugar readily. These bacteria depend upon the action of the 
alcohol-forming yeast. Other cases of metabiosis are found in the 


ANTIBIOSIS 299 

association of lactic bacteria with certain protein destroying organisms. 
The lactic bacteria often develop much better if the protein bacteria 
grow together with them or have grown previously in milk. Meta- 
biosis does not require the growth of the two associated organisms at 
the same time. The effect will be the same if first the one and later the 
other develops, and even after the first organism is killed or removed, 
its effect upon the pure culture of the second will still be noticed. This 
does not occur in the case of symbiosis. 

One species can favor the development of another by other means 
than food provision or preparation. Certain bacteria cannot live in 
acid media, and molds or mycodermas destroying the acid will render 
possible the growth of these bacteria though they do not provide them 
with food. This is the case in the ripening of certain soft cheeses. 
Another example is the production of heat by fermenting organisms in 
manure, hay, ensilage, enabling the development of thermophile organ- 
isms. A very interesting and important problem is the growth of strictly 
anaerobic bacteria near the surface of liquids in association with 
some aerobic bacteria. How this is really possible cannot be satisfac- 
torily explained. Though the aerobic bacteria continuously remove the 
oxygen from the water a certain amount will remain, sufficient to pre- 
vent the growth of the anaerobic bacteria under ordinary conditions. 
There seerns to be a certain protective influence derived from the aerobic 
bacteria, the nature of which is unknown. 

ANTIBIOSIS 

The standard examples of antibiosis are the alcohol production by 
yeast in sugar solutions and the acid production by lactic bacteria in 
milk. Fresh cider contains a large number of bacteria, yeasts and 
molds; some of these organisms cannot develop in the acid medium, 
but many will begin to grow. Some of the bacteria will produce or 
destroy acid, others may begin to work on the nitrogenous material of 
the cider, and the yeasts produce alcohol and carbon dioxide. The 
carbon dioxide will soon saturate the cider and begin to bubble up, thus 
removing the other gases. The molds will stop growing if the oxygen 
is taken away, but some of the bacteria may continue growing until 
the alcohol concentration checks their further development. They 
first cease to grow, then cease to produce acid and finally die, while the 
yeast is still continuing in the fermentation. 


300 MUTUAL INFLUENCES 

In the lactic fermentation of milk, Bad. lactis acidi combats all 
other organisms by a rapid production of lactic acid. Though it is pres- 
ent in fresh milk only in very small numbers, its rapid growth and the 
formation of acid which will check and even kill most other bacteria 
soon makes it the dominant organism in the flora of milk, and at the 
time of curdling, it is often difficult to find any other organisms 
besides the lactic bacteria. In the preceding chapter was mentioned 
the metabiosis of certain protein-digesting bacteria with Bad. lactis 
acidi. This metabiosis can be considered as such only from the stand- 
point of the lactic organism. The protein bacteria are killed by the 
acid formed by the rapidly growing lactic bacteria, ^rom the view- 
point of the protein bacteria, the relation is antibiosis. Another illus- 
tration of antibiosis is the acetic fermentation. The formation of 
acetic acid prevents the development of all bacteria and of most yeasts 
and molds. 

In all these cases, the deciding agent is a well-known chemical com- 
pound. In other combinations, the principle is unknown. Bact. lactis 
acidi will check the growth of B. subtilis not only in milk where it forms 
acid, but also in sugar-free broth where acid production is impossible. 
Acetic bacteria act upon the yeast cells not only by means of the acetic 
acid produced, but also by some other, unknown agent, since vinegar 
is more injurious than the corresponding amount of pure acetic acid in 
water. A very remarkable organism is Ps. pyocyanea; it secretes a 
substance, pyocyanase, which will kill and dissolve the cells of other 
bacteria rapidly. 

Parasitism, which would be classified under antibiosis, has not been 
found to exist among bacteria or yeasts; but we know of cases where one 
mold grows on the other; this is especially true with the largest represen- 
tatives of the mucor family, which are often attacked and sometimes 
killed by smaller fungi. 

RELATIONS BETWEEN CELLS OF THE SAME SPECIES 

That cells of the same species will also influence each other, may well 
be assumed. The simplest relation will be the competition for food. 
This will be the case in nature more commonly than in laboratory media 
which are, as a rule, so rich in nutrients that development ceases before 
all food is used up. 


RELATION BETWEEN CELLS 301 

The cause for cessation of growth in a culture is of great theoretical 
and practical interest. Apparently there are various factors concerned 
in this. Lack of food, or of one single essential food compound, may be 
the cause. This is found sometimes in media where it would be least 
expected. Some strains of Strept. lacticus are supposedly limited in 
milk by the lack of available nitrogen; they cannot attack casein readily 
and albumin; besides these proteins, nitrogen compounds are not plenti- 
ful. Addition of peptone increases the maximum number of cells from 
0.7 billion to 2.5 billions per c.c. More commonly, however, growth 
is checked by the accumulation of metabolic products. Yeasts are 
checked by the alcohol, and acid-formers by the acid, urea bacteria 
by the alkali. In many of these cases, the removal, or neutralization, 
of the inhibiting product will bring about new development. 

The harmful products accumulating are not always of such simple 
nature. Some very interesting observations have been made during the 
last ten years. Eijkmann, as the first, found that B. coll reached its 
maximum growth in gelatin at 37 in a few days, and that this gelatin, 
after hardening at 20, would not support growth after streaking with 
a young culture of the same organism; but after this gelatin had been 
heated at 60 for half an hour, B. coll grew on it as well as on fresh 
gelatin. Broth in which B. coll had grown became fit again for growth 
of the same bacillus after filtration through porcelain. The inhibition 
of growth is, in this case, due to a compound which resembles a toxin 
in many respects. The importance of such investigations to general 
physiology is evident. 


PART III 

APPLIED MICROBIOLOGY 


DIVISION I :i 
MICROBIOLOGY OF AIR 


CHAPTER I 

THE MICROORGANISMS OF THE AIR AND THEIR DISTRI- 
BUTION 

The atmosphere is not the normal habitat of bacteria, for growth and 
multiplication cannot take place in it under ordinary conditions. The 
phrase "microorganisms of the air" is therefore somewhat ambiguous. 
The small size of microorganisms enables them to remain suspended for 
considerable periods when physical forces have separated them from the 
substrata on which they have developed. 

MICROORGANISMS PRESENT IN THE AIR. Molds, bacteria, and yeasts 
are all found in the air under certain conditions. The first two are usu- 
ally relatively abundant, the latter are less common. 

The common molds have adapted themselves for the most part to 
wind distribution. They bear spores that are small in size and possess a 
surface that is not readily moistened. These spores are resistant to 
desiccation and light and remain viable for a considerable time even 
under unfavorable conditions. Furthermore, the fruiting bodies of 
many, though not all molds, show a distinct negative hydrotropism,i.e., 
the mycelium remains in contact with the moist substratum while the , 
threads which bear the spores rise at right angles to it. These latter are 
so sensitive that they can detect slight differences in the moisture con- 
tent of the air and grow in the direction which will bring the spores into 

* Prepared by R. E. Buchanan. 

303 


304 MICROBIOLOGY OP AIR 

the driest situations. A slight current of air will detach the spores from 
these structures and carry them long distances. 

Bacteria and yeasts lack the specific adaptations for wind distribu- 
tion found in molds. The material upon which they have been growing 
must be dried and pulverized before they can be blown about. Many 
species produce spores or other resistant cells, and physiologically are as 
well adapted for air distribution as are the molds. 

OCCURRENCE IN THE AIR. Microorganisms are found free in the 
air, attached to particles of dust, or enclosed in minute drops of water. 
Mold spores are commonly free or in unattached clusters. , Bacteria and 
yeasts are usually associated with dust particles, frequently the pulver- 
ized substratum on which they have been growing. Not all dust par- 
ticles have living organisms attached. It has been computed that in 
the air of London during a fog there is only one living organism for over 
thirty-eight millions of dust particles. Microorganisms are some- 
times sprayed into the air with water. Droplets containing bacteria 
are thrown off in the saliva in coughing or in speaking, and from the 
surface of fermenting liquids on which bubbles are bursting. When 
the drop is small enough, the air currents keep it in suspension and the 
water soon evaporates and frees the organism. This brings about the 
condition first discussed, free bacteria in the air. The decrease in 
weight and size incident to this loss of water probably accounts for the 
fact that the so-called " infectious droplets" are sometimes carried for 
considerable distances. 

How MICROORGANISMS ENTER THE AIR. In comparatively few in- 
stances do microorganisms possess mechanical devices for projecting 
the spores or other cells into the air for wind distribution. Usually the 
organism is passive and is freed only by air currents or by mechanical 
agitation. Some molds, as has been stated, release their spores even in 
the presence of moisture, so that complete desiccation is unnecessary for 
their dispersal. Bacteria and yeasts, on the other hand, are not usually 
given off from moist surfaces. Only when dry and pulverized can the 
bacterial medium be readily blown about. Hansen found that in the 
immediate vicinity of a heap of decaying malt, the air was comparatively 
free from bacteria. Winslow has shown that sewer air is frequently 
practically free from bacteria although the surface with which it comes 
in contact teems with bacterial life. Mechanical agitation often throws 
large numbers of organisms into the air. Moving hay and straw, 


THE MICROORGANISMS OF THE AIR 305 

grooming animals, sweeping a floor or carpet will multiply the dust and 
bacterial content of the air many times. In a similar manner, tiny, 
germ-holding droplets may be scattered by the splashing of sewage or of 
fermenting or putrefying liquids, and in speaking, sneezing or coughing. 

CONDITIONS FOR SUBSIDENCE OF BACTERIA. The length of time 
during which an organism may remain suspended in the air is dependent 
upon several factors. Small particles settle out more slowly than large 
for the reason that as the size of an object is decreased, the surface area 
decreases less rapidly, proportionately, than the volume. The lifting 
effect of air currents depends upon the ratio of surface area to volume 
and specific gravity. The smaller the object, therefore, the greater is 
the resistance to subsidence Consequently, bacteria usually settle 
out of air very slowly if free in a quiet atmosphere. The time of sus- 
pension is determined also by the velocity of the air currents. While 
considerable velocity may be necessary to dislodge microorganisms and 
bring them into suspension, a very slight air current will sustain 
them. Winslow has found that a current of 17 inches per minute is 
sufficient to sustain B. prodigiosus. The relative humidity of the air is 
also an important factor. In a supersaturated air solid particles, such 
as bacteria, become foci of condensation for water and quickly settle 
out. When dust is present in considerable quantities, and certain elec- 
trical or moisture conditions exist, flocculation occurs and the larger 
bodies so formed subside rapidly. The character and abundance of 
surfaces with which the suspended particles may come in contact also 
play an important part. Moist surfaces are much more effective in 
retaining particles than those which are dry. 

DETERMINATION OF THE NUMBER OF BACTERIA IN THE AIR. The 
number of bacteria in the air is frequently determined by exposing open 
petri dishes of gelatin or agar in different places for definite periods. 
This is a comparative quantitative method only. The number of colo- 
nies developing upon these plates will give the number of dust particles 
having living spores or cells upon them that fall in the given area under 
the conditions of the experiment. Evidently this is of value only for 
rough comparative work as constantly shifting currents of air usually 
introduce great errors. A somewhat more accurate method is to draw 
measured volumes of air into a flask, the bottom of which is covered 
with a layer of gelatin or agar. The colonies which develop represent 

the number of organisms which settle out from the given volume. More 
20 


306 MICROBIOLOGY OF AIR 

accurate results still may be obtained by drawing measured vol- 
umes of air in small bubbles through liquid gelatin. Practically all of 
the particles will be retained and the number of colonies which develop 
may be counted. This method is sometimes modified by drawing the 
air through a definite volume of water, care being taken to insure suffi- 
cient contact of air and water to remove all dust particles. A propor- 
tionate part of the water is then plated and the number of organisms 
estimated. Air is sometimes drawn through a filter made of sugar, 
sodium sulphate, or sodium chloride, and this material then dissolved 
in water and plated. Sand, asbestos, glass, etc., are sometimes used 
as air filters, then thoroughly washed, and the wash water plated. 

Relative quantitative examination of the air is of more historical 
than practical importance. It has been useful in the development of 
the germ theories of fermentation and of disease and in overthrowing 
the theory of spontaneous generation. There is so little ordinarily to be 
learned by a study of the air flora that a comparison of plates exposed 
directly will usually suffice. Where more accurate results are desired, 
one must resort to one of the filtration methods discussed above. 

Qualitative determinations of the species of air organisms are not 
often made. When necessary it may be done by simple examination of 
the colonies developed on the plates or by animal inoculations made 
from the water used in the air filter. It is sometimes necessary to vary 
the composition of the medium used in order to favor the development 
of certain types of organisms desired, for example, a higher precentage 
of molds will be found and a more luxuriant development will take place 
if wort agar or acid gelatin is used. 

NUMBER OF BACTERIA IN THE AIR. The number of bacteria in the 
air is determined by a variety of conditions. The velocity of air cur- 
rents and the nature of the surface with which these currents will come 
into contact, are probably most important. Bacteria are usually more 
abundant on quiet days in the air of buildings than out of doors, but on 
windy days the reverse is true. They are often more abundant in cities 
than in the country. Fewer are found at high altitudes and over large 
bodies of water. Frankland found that there are fewer in winter than 
in summer. They are washed from the air during rains. Bright sun- 
light destroys many. The nature of the soil and the vegetation cover- 
ing it has a marked influence. The following figures from various 


THE MICROORGANISMS OF THF AIR 


307 


authors are appended to serve as an index to what may be expected in 
the air content of bacteria. 


Locality 


Number of organisms 
per cubic meter 


Observer 


Outdoor air, Boston 100-150 bacteria. 

50- 75 molds. 

Open air 

Open field 

Seacoast 

Mountain altitude, 200 meters 

Mont Blanc 

Spitzbergen (Arctic Regions).. 

Middle of Paris 

Paris Street 

Tailor's Room in Whitechapel 17,000 

Boot Workshop 25,000 


100-150 bacteria. 
250 
100 
o 

4- ii 

o 

4,000 


Sedgwick and Tucker. 

Fischer. 

Uffelman. 

Uffelman. 

Pasteur. 

EUis. 

Levin. 

EUis. 

Fischer. 

Ellis. 

Ellis. 


SPECIES OF ORGANISMS IN THE AIR. Penicillium is the most com- 
mon mold isolated from the air. Next in importance are Mucor, 
Rhizopus, and Aspergillus in the order given. In addition to these a 
considerable number of species of hyphomycetous molds are occasion- 
ally found. Torula, but not true yeasts, are usually common. Bac- 
teria are either spore-bearing soil bacilli or cocci. Of the former, B. sub- 
tills, B. mycoides, and related forms are ubiquitous. Sarcina lutea and 
Sarcina aurantiaca and certain other chromogenic cocci are to be found 
in almost every plate exposed. Since the air does not have a true flora, 
the species as well as the number of bacteria present must depend en- 
tirely upon the character of the environment. 


CHAPTER II 

MICROBIAL AIR INFLUENCE IN FERMENTATION, 

DISEASES, ETC. 

AIR AS A CARRIER OF CONTAGION. There are many popular mis- 
conceptions of the influence of air upon health. Experience early 
taught that exposure to the night air in certain localities or to swamp 
air during certain seasons was generally followed by disease. Natur- 
ally, the air itself was held responsible. We know now that certain 
fevers, malaria, etc., are caused in every instance by infection with 
specific microorganisms and that these organisms are not usually car- 
ried by the air but by insects, such as the mosquito, in water and food. 
Nor can the emanations from decaying organic matter or sewer gas itself 
be held to produce disease directly. Before the establishment of the 
germ theory of disease, leading sanitarians held that sickness was 
induced by the gases from the decaying organic matter, by the effluvia 
from cesspools and by sewer gas. However important the places named 
may be in harboring disease microorganisms, we have learned that the 
air itself rarely acts as a carrier. Sewer gas has been shown to be un- 
usually free from bacteria. Hazen says, " After many years of exper- 
ience and long-continued investigation, there is not the slightest reason 
to believe that infectious diseases are carried by the air of sewers." 

Undoubtedly the air does play some part in the carrying of disease 
germs. In certain diseases, as the exanthemata (smallpox, measles, 
etc.), the infecting agent may be present on the dry skin and may be 
blown about and inhaled. This means, however, is not established. 
In certain nasal, tracheal, and pulmonary infections, the organisms 
may be spread through speaking, sneezing, and coughing, for the infec- 
tious droplets, as has been seen, remain suspended for a time in the 
air. Pyogenic cocci are present in the mouth and care must be used in 
surgical operations that the mouth is so protected that none of these 
organisms gain entrance to wounds. Rarely, if ever, are intestinal 
infections, as typhoid or cholera, spread through the air. We may there- 
fore conclude that air is of secondary importance as a carrier of infection. 

308 


MICROBIAL AIR INFLUENCE 309 

/ 

It may be of importance in a crowded workroom, but even under these 
conditions it is probable that transmission of infection comes about 
more frequently through actual contact or through food and drink. 

ORGANISMS or THE AIR AND FERMENTATIONS. A uniform inocula- 
tion with soil bacteria such as produce the nodules on the roots of leg- 
umes is obtained over considerable areas through the action of the wind 
in blowing dust particles. The bacterial flora of milk is to some extent 
dependent upon air currents as is also the development of the molds 
necessary to the proper ripening of cheese, such as the Camembert. 
Acetic, butyric, and other organisms are likewise distributed in this 
manner. The organisms responsible for putrefaction and decay, the 
molding and spoiling of foods are wind-borne. 

FREEING AIR FROM BACTERIA. Air is most commonly freed from 
bacteria by sedimentation, for this is the ultimate fate of most dust par' 
tides. We have seen that they gradually subside in a quiet atmos- 
phere. When large quantities of pure air are required, dust and bac- 
teria may be removed by passage through a spray of water or through 
various types of niters, such as cotton, glass, wool, etc. A familiar 
example of this type of nitration is the laboratory use of cotton plugs in 
test-tubes. It is sometimes necessary to resort to fumigation to destroy 
the organisms of the air when an undesirable species is present. 


DIVISION II 

MICROBIOLOGY OF WATER AND SEWAGE 


CHAPTER I* 

MICROORGANISMS IN WATERf 

Water is necessary in the life of man. Besides its use as a beverage, 
for cooking, and all domestic purposes, it is largely used in many manu- 
facturing industries; therefore, the study of its chemical and biological 
content is one of the most important features of modern hygiene. All 
natural waters contain microorganisms, which gain entrance from many 
sources. 

Under the influence of the sun, sea water evaporates and forms a 
water vapor, which we call clouds; and these, driven by the wind over 
the land, are precipitated as rain and in the form of snow or hail. 

Most of this water collects from vast areas into brooks, creeks, 
rivers, lakes, or in subterranean streams, and finally reaches the sea 
whence it came. 

The water vapor arising from the sea or land contains no organisms; 
but as soon as the vapor is precipitated microorganisms find their way 
into it. These come from the air and from the soil. Some of them find 
in water sufficient nutriment for their life and growth; and, because of 
their constant presence and evident ability to thrive in water, they are 
sometimes spoken of as belonging to the "water flora" Others, such as 

* Prepared by F. C. Harrison. 

f For specific details regarding methods of analysis and a fuller presentation of the subject, 
readers may consult any of the following excellent books: 

1. Savage, W. G.: The Bacteriological Examination of Water Supplies, London, H. K. 
Lewis, 1906. 

2. Horrocks, W. H.: An Introduction to the Bacteriological Examination of Water, London, 
J. and H. Churchill, 1901. 

3. Prescott and Winslow: Elements of Water Bacteriology, 2d Ed., New York, Wiley & 
Sons, 1913. j 

310 


MICROORGANISMS IN WATER 311 

the soil bacteria, are found only at certain seasons, as after rain or dur- 
ing flood- time, and flourish only for a time; while some few, such as 
intestinal organisms that find their way into water, survive for only 
a short period. 

CLASSES OF BACTERIA FOUND IN WATER 

The bacteria found in water are here roughly divided into : (a) natu- 
ral water bacteria; (b) soil bacteria from surface washings; (c) intes- 
tinal bacteria, usually of sewage origin. But there is no strict divid- 
ing line between these three groups; for some organisms belonging to 
the water flora are found in the soil, and vice versa. Water draining 
from manured land frequently contains intestinal organisms. The 
division, however, is sufficient for all practical purposes. 

NATURAL WATER BACTERIA. The natural water bacteria are gen- 
erally regarded as harmless to man. These organisms are frequently 
numerous in river, lake, and all surface waters; certain species predomi- 
nate at one season, and disappear at another. Some of the best known 
are mentioned below. Several investigators have grouped the bacteria 
found in water into classes according to their biochemical properties. 
Where groups are subsequently referred to, the classification is that 
used by Jordan and followed by many other workers. 

B. fluorescens liquefaciens, Group V, together with some closely allied 
varieties, is probably more frequently found in water than any other 
form, and is easily recognized by the green fluorescence and liquefaction 
it produces in gelatin. 

B. fluorescens non-liquefaciens, Group VI, as the name implies, does 
not liquefy gelatin, but produces characteristic colonies with a fluores- 
cent shimmer, is often very abundant in river waters, and is representa- 
tive of a group comprising B. f. longus, B. f. tennis. B. f. aureus, and 
B. f. crassus. 

Certain organisms which liquefy gelatin and acidify milk classed by 
Jordan in his Group VIII are quite common at certain seasons. 
Some of these are soil organisms and are closely related to the proteus 
group; and some of them are B. liquefaciens , B. punctatus, B. circulans. 

Chromogenic bacilli and cocci (Groups XIII , and XIV) are often 
present in water. Of those producing red coloring matter, the well- 
known B. prodigiosus is the type of the group; others are B. ruber, B. 


312 MICROBIOLOGY OF WATER AND SEWAGE 

indicus, B. rubescens and B. rubefaciens. Several yellow and orange 
organisms are commonly found, such as B. aqualilis, B. ochraceus, B. 
aurantiacus, B. fulvus, etc. 

At certain times, particularly in river and brook waters, organisms 
producing violet pigment are quite common. B. molacem or B. janthi- 
nus, as it is sometimes called, is the prevailing type; others are B. lividus, 
B. amethystinus, and B. coeruleus. 

The chromogenic cocci produce either orange or yellow pigment, and 
as a rule are not numerous in water. Sarcina lutea is the most common 
species. 

Non-chromogenic cocci (Group XV) are more frequent. M . candi- 
cans, M. nivalis, M. aquatilis, are non-liquefying forms, and M . corona- 
tus is the type of those which liquefy gelatin. 

SOIL BACTERIA FROM SURFACE WASHINGS. During times of flood, 
high water, and after rains, numerous soil organisms are found in 
natural waters; and occasionally certain species persist for a consider- 
able time. Among the commonest species is B. mycoides, with its 
characteristic rhizoid colony; also B. subtilis, B. megatherium, and B. 
mesentericus vulgatus, with its allied varieties; likewise B. m.fuscus and 
B. m. ruber- all belonging to Jordan's Group VII, and having many 
characters in common, such as characteristic colonies, followed by 
liquefaction when growing in gelatin, production of spores, etc. 

Cladothrix dichotoma, one of the thread bacteria, easily recognized 
on gelatin plates by the brown halo that surrounds the colony, is often 
found in fresh and stagnant water, and in most soils. It seems to 
flourish wherever there is much organic matter. 

These are the soil organisms most often found when beef peptone 
gelatin is used for isolating purposes; but if other media are used, a 
different flora appears, and we find nitrifying organisms, yellow 
chromogens, etc. 

INTESTINAL BACTERIA, USUALLY OF SEWAGE ORIGIN. Proteus 
Group. There are several groups of sewage organisms found in impure 
water; some of these are very abundant in crude sewage, but are not 
found in such relatively large numbers in contaminated water. Jor- 
dan's Group III contains the organisms belonging to the large proteus 
group, the principal species being B. vulgaris, B. zenkeri, B. mirabilis, 
B. zopfii, the sewage proteus of Houston, and B. cloaca. All these are 
frequently found in impure water, and in sewage. In the latter Hous- 


MICROORGANISMS IN WATER 313 

ton has found as many as 100,000 per c.c. All these organisms are mo- 
tile, liquefy gelatin, and produce gas in dextrose and saccharose broth, 
and little or none in lactose; reduce nitrates, curdle milk, produce indol, 
and give a fecal, disagreeable odor in broth or other media. 

Sewage streptococci. The streptococci found in sewage are probably 
similar to those found elsewhere; but their appearance in contaminated 
water may be regarded as indicative of recent sewage contamination, 
because the bulk of the evidence available seems to show that they are 
delicate organisms, which rapidly die outside of the body. While it is 
easy to ascertain their presence in polluted water, it is almost impossible 
to enumerate them; and they do not furnish such good evidence of sew- 
age pollution as the colon bacillus. They may be said to furnish valu- 
able confirmatory evidence of sewage contamination. 

B. enteritidis sporo genes. This resistant, spore-bearing organism is 
usually present in the intestinal tract of man; is found in sewage, milk, 
and dust; and occurs in foodstuffs, such as wheat, oatmeal, rice, etc. 
On account of its ubiquity and the resistance of its spores, it cannot be 
considered a good indicator of excretal pollution. 

B. coli. The presence of this organism in potable water is gener- 
ally accepted as the best bacterial indicator of sewage pollution. It 
must be remembered, however, that there are many varieties of this 
organism, to which certain investigators have given specific names, even 
when the differences from the type organism have been very slight. It 
may be well to mention some of these, to avoid confusion in the mind of 
the reader. The true colon bacillus, B. coli, or B. coli communis, or B. 
coli communis verus, is a short bacillus with rounded ends, motile, forms 
no spores and is Gram negative, does not liquefy gelatin, produces 
acidity and coagulation in litmus milk, gives rise to acid and gas in 
glucose and lactose media, causes canary-yellow fluorescence in neutral 
red media, and produces indol when grown in peptone water. The term 
" Excretal B. coli" has been suggested as a convenient designation of an 
organism which possesses the above characteristics. 

A saccharose fermenting variety of B. coli has been named B. com- 
munior; and we have a whole series of organisms which differ more or less 
in various biochemical reactions, or lack some of their positive reactions. 
To some of these the name " para-colon" has been given; and the name 
: 'para typhoid" has been applied to those which more closely approxi- 
mate to the cultural peculiarities of the typhoid bacillus. 


MICROBIOLOGY OF WATER AND SEWAGE 


For practical purposes in the analysis of water, these distinctions are 
unnecessary. 

Bad. lactis aerogenes, a short, thick, capsulated, non-motile 
bacterium related to B. coli, is also an intestinal organism, and must be 
regarded as an indicator of sewage pollution. 

B. typhosus. Very few instances are recorded in bacteriological 
literature of the direct isolation of the typhoid bacillus from infected 
water. The organism is not long-lived, even in pure water (eight 
or ten days); and when exposed to the action of sewage bacteria, its 
longevity is greatly diminished (not more than five to six days). A 
few resistant specimens may remain alive for longer periods of time. 

Although the typhoid bacillus has been found so infrequently in 
water, it is well understood at the present time that the purification of 
the water supply of a town or city produces a marked decrease in the 
number of cases and in the mortality from typhoid fever, as the following 
table shows: (See also Fig. 122.) 

DEATHS FROM TYPHOID FEVER PER 100,000 PER YEAR 


Place 


Purification 
by 


Date of 
change 


Five years 
before 
change 


Five years 
after change 


Percentage 
of 
reduction 


Hamburg 


Filtration 


18023 


47 


7 


85 


Zurich 


Filtration 


188* 


76 


IO 


87 


Lawrence, Mass 


Filtration 


1803 


121 


26 


70 


Albany, N. Y 


Filtration 


"yo 

l8oO 


IO4 


28 


73 


Not only has such a marked improvement followed the purification 
of public water supplies in the case of typhoid fever, but it has been 
shown by statistics that "where one death from typhoid fever has been 
avoided by the use of better water, a certain number of deaths, probably 
two or three, from other causes have been avoided." 

In the routine examination of water, no particular effort is made to 
isolate this organism, owing to the difficulty of the task. The tests that 
the present-day investigator has to satisfy are extremely thorough; and 
unless the suspected organism conforms to the whole of these necessary 
tests it cannot be accepted as true B. typhosus. 

Msp. comma. The spirillum, or vibrio, of Asiatic cholera is 
an intestinal organism; and the disease it produces is spread largely 
by water. Epidemics of cholera are more easily traced to their 


MICROORGANISMS IN WATER 


315 


AVERAGE ANNUALDEATH RATEFROM TYPHOID FEVER PER 100.000 OFTHE POPULATION. 
1912 I 10 20 30 40 50 60 

MUNICH 


VIENNA 


BERLIN 
ZURICH 


HAMBURG 


PARIS 


LONDON 


CLEVELAND,*). 


PATERSON.NJ. 


WATERTOWN,N.Y. 


CINCINNATI,0. 


SEATTLE .WASH. 


CHICAGO, ILL. 


ST.LOUIS,MO. 


HINNEAPOLIS,MINN. 
PHILADELPHIA^. 
PITTSBURGH. PA. 
NEW ORLEANS.LA. 
NEWYORK.N.Y 
SPRINGFIELD,MASS. 
BINGHAMPTON,N.Y. 
ALBANY, NY 
LAWRENCE, MASS. 
RICHMOND.VA. 
BALTIMORE.HD. 
HILWAUKEE,WIS. 

TOLEDO.O. 

ATLANTA.GA. 
BIRMINGHAM, ALA. 
WHEELING.W.VA. 
MEMPHIS/TENN. 
ATLANTA,GA. 

An instructive contrast between Altona and Hamburg before the latter filtered 
its water, having learnt its lesson from a sharp outbreak of cholera. 

SCATTERED CASES OF CHOLERA. 

ALTQMA^ 

'HAMBURG. 

POPULATION: 600.000 
CHOLERA CASES: 17.000 
DEATHS: 8.60O 


ALTONA: 

"WATER FILTERED 

HAMBURG: 

"WATER UMFILTERED 


FIG. 122. (After G. E. Armstrong.) 


3i6 


MICROBIOLOGY OF WATER AND SEWAGE 


source than those of typhoid fever, owing to the "explosive" character 
of the disease. At the time of the outbreak of cholera in Hamburg, in 
1892, the cholera vibrios were frequently isolated from the water of the 
river Elbe, which was used to furnish the regular supply of the city. 
The adjoining city of Altona also obtained its water from the same 
river, after it had received some of the Hamburg sewage; yet it remained 
practically free from the scourge, owing to the efficiency of sand filters 
which were used to purify the water (Fig. 122). In times of epidemic, 
the organism has been isolated from rivers, wells, and reservoirs in 
India, a country in which the disease is endemic. 


THE NUMBER OF BACTERIA IN RAIN, SNOW, HAIL, ETC., AND IN WATER 
FROM WELLS, UPLAND SURFACE WATERS, RIVERS, AND LAKES 

RAIN. The number of bacteria found in rain depends upon the 
month of the year and the dryness of the air. When considerable dust 
is present in the air, the first rain beats it back to the soil; and at 
such time rain water contains more organisms than usual. Rain falling 
in densely inhabited cities always contains more microbes than rain 
falling on open farm land or upland pastures. A few figures will be 
sufficient to illustrate. 

NUMBER OF BACTERIA PER LITER OF RAIN WATER 
Figures for Montsouris Park, Paris, France, and the average for two years 


Month 


Number of organisms 
per liter 


Month 


Number of organisms 
per liter 


January 


8,OOO 


Tulv.. 


^,600 


February 


I. 32O 


August. 


jj v ' 

8.300 


March 


2.O2O 


September 


c,77o 


April 


2.IAO 


October 


3,220 


May ... 


*-"'*fr % * 

2.4AO 


November 


O) 

3 2ZO 


June 


*J*TT' W 

^,6OO 


December 


O>* J^ 
A.. 33O 


O)^ 


T-JOO^ 


Yearly average 5,300 per liter per month. 


The average for the interior of Paris corresponds with the larger 
amount of dust in the air, and reaches a total of 19,000 organisms per L. 
With a yearly rainfall of 609.6 mm. (24 inches), the rain washes 
down during the year some 5,000,000 organisms to the square yard. 


MICROORGANISMS IN WATER 317 

SNOW. The results obtained from snow are similar to those ob- 
tained from rain; but as a rule the numbers are larger, a result doubtless 
due to the larger particles of the snow flakes. One investigator has 
found from 334 to 463 bacteria per c.c. of snow water. On the sum- 
mit of high mountains snow is practically sterile, Binot not finding 
a single organism in 8 c.c. of water from mountain-top snow. 

Water issuing from glaciers is of remarkable purity, containing 
only from three to eight organisms per c.c.; but the numbers are larger 
as the distance from the glacier increases. 

HAIL. Hail stones usually contain large numbers of bacteria, 
varying from 628 to 21,000 per c.c. of water obtained from the melt- 
ing hail. Fluorescing bacteria have been found in some samples; 
and the presence of these microorganisms suggests that surface water 
is sometimes carried up by storms and congealed. The presence of 
many molds in hail is due to contamination from the air. 

DEEP WELLS. Deep well water and spring water contain as a 
rule but few organisms, usually less than 50 per c.c. on gelatin at 20, 
and less than 5 per c.c. on agar plates at blood heat. In a series of 
tests of water taken direct from forty- three artesian wells, 152.4 M. 
(500 feet) deep or more, the writer has found an average of 27 per 
c.c. for the gelatin and 1.5 per c.c. for the agar counts. These tests 
have extended over a period of several years; and water from deep 
springs has given similar results. 

SHALLOW WELLS. The bacterial content of shallow wells depends 
greatly on their location and construction. Even in those well lo- 
cated and constructed, the number varies with the amount of rainfall, 
and is often large. In polluted wells, very high numbers of organisms 
are found. 

Sedgwick and Prescott found from 190 to 8,640 bacteria per c.c. 
in unpolluted wells. 

In the same class of wells, Savage found from 10 to 100 per c.c. by 
the blood-heat count, and 100 to 20,000 or more by the gelatin count. 

Sixty polluted wells examined by the writer gave an average 
gelatin count of 740 bacteria per c.c.; and thirty-eight wells which were 
free of contamination gave an average count of 400 per c.c. 

Polluted wells often give counts approximating the higher numbers 
mentioned above; but, of course, the character of the bacterial flora 
is quite different. 


MICROBIOLOGY OF WATER AND SEWAGE 


UPLAND SURFACE WATERS. There are few bacteria in upland sur- 
face waters draining barren uplands. Cultivation, grazing of animals, 
and human habitation produce other conditions. In pure waters, 
50 to 300 per c.c. by the gelatin and i to 10 by the agar count are found. 

RIVERS. The greatest variation in the number of bacteria exists 
in river waters. Many factors, such as sewage contamination, tempera- 
ture, rain fall, vegetable debris, etc., influence the microbial popu- 
lation. A few figures may be given for illustration. 

BACTERIOLOGICAL EXAMINATION OF RIVERS AT AND BELOW LARGE SOURCES OF 

POLLUTION (BOYCE AND CO-WORKERS) 


Distance 


Direction 


Munich. 
River Isar 


Cologne. 
River Rhine 


About 0.6 mile 


Above 
Below 


305 
0,387 


4,786 


About 2.7 miles 


Below 


17. CQ^ 


About 6.0 miles 


Below 


8,764 


30,432 


About 12.0 miles 


Below 


4,706 


12,460 


About 15.0 miles 


Below 


3,602 


oxo? 


About 26.0 miles 


Below 


7,860 


In the Chicago drainage canal, Jordan found 1,245,000 bacteria 
per c.c. at Bridgeport; 650,000 at Lockport, twenty-nine miles below; 
and 3,660 at Averyville, 159 miles below. Below where the sewage of 
Peoria enters, the number rises to 758,000 at Wesley City, and decreases 
to 4,800 at Kampsville, 123 miles from Peoria. 

The River Rhone contains an average of 75 bacteria per c.c. above 
Lyons and 800 below. The Dee, 88 above Braemar and 2,829 per c.c. 
below. Many more similar results are found in the literature. 

LAKES. The water of lakes is generally much purer than river 
water. Near the shore, the bacterial content is higher than farther 
out, showing the contaminating influence of habitation. Thus Lake 
Geneva contains as many as 150,000 bacteria per c.c. near the shore, 
and further out only 38 per c.c. Other figures are as follows: Loch 
Katrine, 74 per c.c., Lake Lucerne, 8 to 51 per c.c., Lake Champlain, 
82 per c.c. 

SEA WATER. There are few bacteria in sea water remote from 
the coast; but near the shore and in the neighborhood of seaports 
there may be large numbers. 


MICROORGANISMS IN WATER 319 

Examples: 350 M. from Naples, sea water contained 26,000 bac- 
teria per c.c. At a distance of 3 KM., only 10. Samples taken from 
depths of 75 to 800 M. at distances from 4 to 15 KM. from shore were 
found to contain from 6 to 78 bacteria per c.c. in surface water, and 
from 3 to 260 at various depths below. 

CAUSES AFFECTING THE INCREASE AND DECREASE OF THE 
NUMBER OF BACTERIA IN WATER 

There is a number of causes which influence the multiplication 
or diminution of microorganisms in natural waters; and while it is 
necessary to discuss each of these causes in detail, it must be remem- 
bered that a number of them may be simultaneously influencing the 
increase or decrease. 

TEMPERATURE. In natural waters, a low temperature probably 
acts injuriously on parasitic bacteria, reducing their numbers; but 
the bacterial content of water during the hot summer months is gener- 
ally not so large as during the cooler seasons. Water collected for 
examination should be analyzed at once; otherwise, contradictory 
results as to numbers will be found. Usually, in most waters, there is 
a reduction in numbers for a few hours, followed by a large increase. 
Very much polluted waters, however, show a marked decrease of 
intestinal organisms, if the samples are kept cool. 

LIGHT. Although the germicidal effect of sunlight is well known, 
yet it has not such powerful effects on the bacteria in water. 
Much depends, no doubt, on the turbidity and speed of the cur- 
rent, the maximum killing effect being produced in shallow, clear 
and slow-moving water. It has been found by experiment that the 
germ-killing power of light extends to a depth of 3 M (about 9.84 feet). 
As a means of purifying water, direct light produces very little effect. 

FOOD SUPPLY. The amount of organic matter in water directly 
influences the growth of bacteria. Where a large amount of this is 
present, the number of microorganisms is also large. Rivers containing 
considerable organic matter derived from vegetable debris, etc., contain, 
as a rule, more organisms than rivers in which there is but little of 
such material. Thus the Ottawa River, which drains a large area of 
forest lands and is characterized as an upland peaty water carrying a 
rather high percentage of organic and volatile matter, contains through- 


320 MICROBIOLOGY OF WATER AND SEWAGE 

out the year a larger number of organisms to the cubic centimeter 
than the water of the river St. Lawrence, which is much clearer and 
contains much less organic matter. Sewage water is rich in organic 
matter, and proportionately rich in bacterial life; and bacterial purifica- 
tion is synchronous with a diminution of organic matter. 

Jordan remarks in this connection that "in the causes connected 
with the insufficiency or unsuitability of the food supply is to be found 
the main reason for the bacterial self-purification of streams." 

OXIDATION. On the surface of waters, in rapids, falls, and tidal 
rivers, much oxygen is absorbed, and much impure matter is oxidized. 
Such oxidation is one of the minor agencies in the purification of water. 

VEGETATION AND PROTOZOA. Low forms of plant and animal life, 
like certain species of algae, river plants, and the numerous protozoan 
forms, bring about a reduction of organic matter in water, and thus 
reduce the amount of food available for bacteria. There is also the 
antagonism between these forms and bacteria. The chemical products 
of the higher forms are considered by some authorities to be injurious 
to bacterial life; and many bacteria are ingested by predatory protozoa. 

DILUTION. Sewage flowing into a river or lake is at once diluted 
with quantities of pure water, and the amount of available food mate- 
rial is thus diminished; the space occupied by a definite number of bac- 
teria is increased; and it is easy to see that the greater the dilution, 
the fewer sewage bacteria will be found. An example will suffice to 
illustrate. The sewage of the city of Ottawa amounts to about 
454 L. (100 gallons) per second; and the gelatin count from it gives 
an average in round numbers of 3,000,000 bacteria per c.c. The 
yearly mean discharge of the river is about 1,364,511 L. (300,000 
gallons) a second; and thus the sewage becomes diluted 3,000 times. 

SEDIMENTATION. Impurities, suspended matter, and bacteria 
having weight, naturally gravitate to the bottom; and the subsidence 
of these matters is spoken of as sedimentation. 

Lake water being still, sedimentation in it is more marked than in 
moving water; and such water contains but few bacteria. In slow- 
moving rivers the influence of this factor is also quite pronounced; 
and, according to Jordan, "The influences summed up by the term 
sedimentation are sufficiently powerful to obviate the necessity for 
summoning another cause to explain the diminution in numbers 
of bacteria" in sewage polluted rivers. The example already given 


MICROORGANISMS IN WATER 321 

of the self-purification of the Chicago drainage canal illustrates Jordan's 
contention. 

OTHER CAUSES. There is a number of other causes, not well 
known nor of sufficient practical importance for more detailed com- 
ment, which may increase or decrease the number of bacteria in water, 
such as the inhibiting action of microorganisms and their products 
on one another, the effects of pressure, etc. 

A peculiar fact, which has never been satisfactorily explained, is the 
quick death (in three to five hours) of the cholera vibrio in the waters 
of the Ganges and Jumna. When one remembers that these rivers 
are grossly contaminated by sewage, by numerous corpses of natives 
(often dead of cholera), and by the bathing of thousands of natives, it 
seems remarkable that the belief of the Hindoos, that the water of these 
rivers is pure and cannot be defiled, and they can safely drink it and 
bathe in it, should be confirmed by means of modern bacteriological 
research. It is also a curious fact that the bactericidal power of 
Jumna water is lost when it is boiled; and that the cholera vibrio 
propagates at once, if placed in water taken from wells in the vicinity 
of the rivers. 

INTERPRETATION OF THE BACTERIOLOGICAL ANALYSIS OF WATER 

In making any analysis of water, all data, such as the kind of 
water and the particulars regarding collection, transmission, sampling, 
rainfall, etc., should be given, as these are a great help in interpreting 
the results. One analysis is rarely sufficient; examinations should 
be regularly and systematically made. 

QUANTITATIVE STANDARDS. No absolute guide can be given to 
determine the potable quality of water from the number of micro- 
organisms in it. It may, however, be safely assumed that high bacte- 
rial counts indicate a large amount of organic matter. The number of 
organisms growing in beef peptone gelatin at 20 to 22, and termed 
the "gelatin count," should be given. For deep wells and springs, 
this should not exceed 50 per c.c.; and for shallow wells and rivers, 
not over 500 per c.c. After rains or floods, these figures might be 
exceeded, and would not necessarily indicate dangerous pollution. 

The number of organisms which develop on beef peptone agar 

incubated at blood heat, commonly termed the "agar" or "blood- 
21 


322 MICROBIOLOGY OF WATER AND SEWAGE 

heat" count, is perhaps more important than the gelatin count, as 
many water bacteria do not grow at blood heat, whereas sewage and 
soil organisms grow readily at this temperature. The agar count 
eliminates the water flora, but obscures the sanitary results by reason 
of the presence of soil bacteria. For deep waters, the agar count 
should generally not exceed 10 per c.c.; and for surface waters, not 
over 100 per c.c. 

QUALITATIVE STANDARDS. The isolation and identification of 
specific disease organisms, such as typhoid and cholera microbes 
from water, is sufficient to condemn such a sample as unfit for use; 
but, on account of many technical difficulties, it is practically impossible 
to make such an examination. Apart from a few special cases, when 
it may be necessary to attempt the isolation of these pathogenic 
bacteria, the presence of the colon bacillus (B. coli) in small amounts 
of water, is generally looked upon as significant and indicative of sew- 
age pollution. The technical methods used in this isolation and 
enumeration are many, and may be found in the works cited; but there 
is considerable difference of opinion as to the number of B. coli which 
should condemn a sample of water. Prescott and Winslow state 
that if the colon bacillus is in "such abundance as to be isolated in a 
large proportion of cases from i c.c. of water, it is reasonable proof 
of the presence of serious pollution." Savage suggests that B. coli 
should be absent from 100 c.c. in the case of water from deep wells 
and springs, and should be absent from 10 c.c. in surface waters, such 
as rivers used for drinking purposes, shallow wells, and upland surface 
waters. 

The streptococcus examination is next in importance as an indi- 
cator of sewage. Streptococci should be absent from the amounts 
of water mentioned above for B. coli; and B. enteritidis sporogenes 
should not be present in 1,000 c.c. of water from deep wells, nor in 
100 c.c. from surface waters. 

SEDIMENTATION, FILTRATION, AND PURIFICATION OF WATER 

As areas become more and more thickly settled and towns and 
cities increase in population, the problem of obtaining sanitary con- 
trol over the water supply increases in importance. Very few towns 
and cities are fortunate to obtain their water supply from an unpol- 


MICROORGANISMS IN WATER 


323 


luted area. Consequently expensive installation must be made, in order 
to purify a suspiciously contaminated water by freeing it from organ- 
isms injurious to health. There are several methods of accomplish- 
ing such purification; and these will be briefly mentioned. 

SEDIMENTATION AND FILTRATION. This method of purifying water 
has been used for nearly a hundred years; but the great impetus given to 
this hygienic measure was due to Koch, who showed in 1893 that the 


- 
. 


FIG. 123. Section of a sand filter. 

proper filtration of Elbe water saved the town of Altona from an epi- 
demic of cholera which devastated Hamburg as a result of drinking un- 
filtered water. In this system of purification, the water is first stored in 
large reservoirs, where the effect of sedimentation and storage reduces 
considerably the number of bacteria. From the reservoir, the water is 
filtered through sand, gravel, and pebbles, etc., arranged as shown 
in Fig. 123. This filtration removes from 97 to 99.5 per cent of the 
microorganisms. 

The action of the filter bed is due to the mechanical obstruction of 
impurities, to oxidation of the organic matter, and to nitrification due 


324 MICROBIOLOGY OF WATER AND SEWAGE 

MEAN OF MONTHLY EXAMINATIONS FOR THE YEAR 


Microorganisms per c.c. 


At source 


After storage 


After filtration 


London, Lambeth Works 


16,138 
16,138 
1,400 

79,000 
186,986 


7,820 
1,067 


75 
34 

60 
630 
400 


London, Chelsea Works 


Berlin, Lake Miiggel 


Paris, Marne 


Paris, Seine 


to the living bacteria in the scum which forms on the top of the layer of 
sand. Of these, the last is the most important; for until this gelatinous 
layer forms, the filter does not act properly in fact, it has little filter- 
ing action, as the following figures show: 

BACTERIAL CONTENT OF WATER BEFORE AND AFTER CLEANING THE SAND FILTER 

Before cleaning, i.e., before removing the scum layer. . . 42 per c.c. 

One day after cleaning 1880 

Two days after cleaning 752 

Three days after cleaning 208 

Four days after cleaning 156 

Five days after cleaning 102 

Six days after cleaning 84 

Thus provision must be made to permit the scum or film to form be- 
fore the filtered water is used for domestic purposes. 

The rate of filtration must be regulated; for if the water is allowed to 
exceed a certain rate (101.6 mm. or 4 inches per hour), inefficiency 
follows. 

COAGULATING BASINS AND FILTRATION.- -This method of purifica- 
tion consists in adding a coagulant, such as basic sulphate of aluminum, 
by means of a mechanical device which regulates the quantity, as the 
water is pumped into the coagulating basins or reservoirs, where it re- 
mains for six to twenty-four hours. The aluminum sulphate is decom- 
d by the lime in the water and forms insoluble aluminum hydrate; 
the sulphuric acid combines with the lime. The hydrate of alumi- 
num is precipitated in large flocculent masses, entangling all particles 
of soil or organic matter; and these, being deposited on the surface of the 


MICROORGANISMS IN WATER 


325 


sand, form the filtering layer. Such filters are very efficient; they re- 
move from 97 to 99.8 per cent of the bacteria from the water. 

POROUS FILTERS. (Fig. 124.) These filters are made either from 
unglazed porcelain or baked diatomaceous earth; the former are known 
as Chamberland, and the latter as Berkefeld filters. These filters 
are usually candle-shaped, require considerable pressure to force water 
through them, and can be used only when a small supply of water 
is needed. Water which is forced through these filters is at first sterile; 
but with repeated use they allow bacteria to pass through the pores and 


FIG. 1 24.- -Unglazed porcelain niters. Chamberland system; A, without pressure; 
B, fitted to main water supply; C, section of a porous porcelain filter. 

thus the filtering efficiency is impaired and will remain so, until the fil- 
ters are cleaned and baked to red heat in a mufHe-furnace. Unless this 
is done regularly, no dependence should be placed on these filters, as 
they only put those who use them off their guard against the danger to 
which they are exposed. 

PURIFICATION BY OZONE. The antiseptic properties of ozone are 
well known. It is used in the purification of the water supply of some 
towns Nice, Chartres, etc. Ozone used for this purpose is usually 
obtained by means of the electric current; and a flowing film of water is 


326 MICROBIOLOGY OF WATER AND SEWAGE 

brought into contact with an upward current of air charged with ozone, 
which current makes the water almost completely sterile. This method 
of purification is efficient, but rather expensive. 

PURIFICATION BY HEAT. By bringing water to the boiling point, all 
harmful bacteria are destroyed; a few spores may resist this treatment, 
but they are harmless. Boiled water is of a flat, insipid taste, due to the 
driving out of the contained gases. The taste may be improved by 
cooling and shaking. The boiling of water is often resorted to as a hy- 
gienic measure in times of epidemic, and for the supply of armies in the 
field. 

PURIFICATION BY CHEMICALS. The addition of a small amount of 
calcium hypochlorite, or potassium iodide, etc., purifies water; but these 
methods are seldom used, except for the use of soldiers on campaign. 
Hypochlorite, however, is now used more commonly in municipal water 
supplies where they can not be otherwise controlled. 

LOCATION AND CONSTRUCTION OF WELLS 

Farms in many sections of this country are practically all supplied 
with surface water collected in shallow wells. Hence farmers should 
understand the principles involved in the location and construction 
of wells. 

Many farm wells are badly located too near such sources of con- 
tamination as outhouses, cesspools, stables, or barnyards; and those 
who locate them give too little attention to the slope of the ground, and 
the nature and slope of the subsoil. There should be at least 22 to 
30 M. (75 to 100 feet) between the well and all probable sources of 
contamination; and this distance is too small, if the soil is very porous, 
or if the surface and subsoil drainage is toward the well, or if the well 
is sunk in fissured rock as it is obvious that there are serious chances 
of contamination in each of the above circumstances. 

In all cases, the surface drainage should be away from the well; and, 
as far as possible, the subsoil drainage also should be from the well. 

Sketches 125, 126, and 127 illustrate these points, the upper part of 
each drawing showing the plan and the lower portion a section through 
the dotted line marked on the plan. Fig. 125, shows that the surface 
drainage is from the house, privy, stables, and barnyard toward the well. 
The section through the line " A" shows the relation of the impervious 


MICROORGANISMS IN WATER 


327 


D 


m en / 


/ 


e 


i 


.5 


i 
^ 


/ 


... .. 


if: 


FIG. 12 v 


g^.,* 


----A 


FIG. 126. 


-- A 


FIG. 127. 


FIGS. 125, 126, and 127. In each figure plan above section through A B below. 
S = soil; B = impervious subsoil or strata, i, House; 2, well; 3, outhouse; 4, 
piggery; 5, stables; 6, stable yard; 7, hen house; 8, sheep stable. Arrow heads 
indicate direction^)! water flow. (Original.) 


328 


MICROBIOLOGY OF WATER AND SEWAGE 


subsoil "B " to the drainage. Water falling on the surface of the ground 
would penetrate through the soil to the upper portion of the subsoil, and 
then move along it in the direction of the greatest slope. In this sketch, 
the subsoil drainage is away from the well; and in this respect the well is 
located properly; but, in respect to the surface drainage, improperly 
located. A better place for the well would be at the letter "X". 

In Fig. 126 the surface drainage including that from the adja- 
cent outhouse at 3, which is too close to the well is toward the barn, 
and away from the well; but the subsoil drainage from all the buildings, 


ff'aslfu'aier drain 

4- 


FIG. 128. Construction of a model well. On the right is brick construction, on 
the left stone construction, as illustrated. (Original.) 

except the house, is in the direction of the well; and thus contamination 
of the water supply is liable to occur. 

Fig. 127 shows a well properly located as regards both surface and 
subsoil drainage. Such a well will supply pure water, if it is properly 
constructed. 

Fig. 128 shows the proper construction of a well with brick or stone. 
Large vitrified drain pipes with cemented joints will answer equally well 
when there is an abundant supply of water; but in case the supply of 


MICROORGANISMS IN WATER 329 

water is limited, a large area is needed, and a stone or brick well is 
necessary. 

Reference to the illustrations will show that every endeavor is made 
to prevent surface water from entering directly into the well. The walls 
are impervious; and the earth or clay is well rammed against the outer 
side of the wall. The curb is carried well above the surface of the 
ground. The waste water is conducted by means of a sloping platform, 
trap, and drain, away from the well; and the well opening is properly 
covered. All water entering such a well must percolate through a con- 
siderable depth of soil, and undergo purification by means of the aggre- 
gations of living bacteria in the soil spaces. Thus the soil around a well 
fulfils the same function in purifying the surface water as the scum 
layer that forms on the surface of gravel filters. 


CHAPTER II* 

MICROBIOLOGY OF SEWAGE 

THE BACTERIAL FLORA OF SEWAGE 

COMPLEXITY or FLORA. Sewage is made up of the miscellaneous 
and varied wastes of human life and activity, and the bacteria which are 
found therein are the result of a haphazard and chance admixture of 
substances of diverse origin and character. The resulting flora is not 
only of great diversity and variability, but it is with few exceptions non- 
characteristic. In brief, the medium with which we have to deal has 
had an origin too indefinite and a history too short to have permitted 
the establishment of anything approaching a constant or characteristic 
bacterial flora. 

TYPICAL FORMS. Our interest in this sewage flora is a very practical 
one, being confined to those organisms which carry on the work of bio- 
logical purification and to certain pathogens which for obvious reasons 
require special treatment. We are interested chiefly in what these bac- 
teria do rather than in what they are, and our classification is influenced 
accordingly. It is based, not upon the species or the genus nor even 
upon the group or type, that proves so convenient in general bacterial 
classification, but upon a sort of physiological or functional type, having 
to do solely with the activities of the organisms in sewage and in its puri- 
fication. Bacteria performing a common function or producing a com- 
mon result are members of one type. Individuals may belong to several 
of our types and there are doubtless a great many that belong to none. 
These latter simply have no place assigned them as yet in the role of 
sewage purification, because they possess none of the recognized typical 
functions. 

Apparent exception may be taken to these general principles in 
the case of such organisms as the B. coli, sewage streptococci and 
B. enteritidis. These are, to a certain extent, characteristic sewage 
bacteria. But interest in them as individuals is confined to water 

Prepared by Earle B. Phelps. 

330 


MICROBIOLOGY OF SEWAGE 331 

bacteriology. If they have any functions in the bacterial changes of 
sewage, they receive attention as members of a corresponding type, not 
as individuals. A study of these sewage types, therefore, is a study of 
the chemical changes induced in the medium by the activities of one or 
the other group of bacteria. 

TYPES OF SEWAGE BACTERIA 

According to the general character of the changes which they bring 
about, sewage bacteria are divided into two large groups, the anaerobic 
or putrefactive bacteria, and the oxidizing bacteria. In regard to the 
former, no attention is paid to the fine distinctions that have been made 
in recent years in connection with the definition of putrefaction. In 
sewage chemistry putrefaction is that change which takes place natur- 
ally in sewage after anaerobic conditions have become established. It 
involves the reduction of urea, the hydrolysis of protein and of cellulose, 
the emulsification of fats, the reduction of nitrates and sulphates and 
possibly of phosphates, and those other changes which are characterized 
by the withdrawal of oxygen and the hydrolysis of complex molecules. 
These changes are always noted in sewage under anaerobic conditions 
and the terms putrefactive and anaerobic change are for the present pur- 
poses practically synonymous. 

The oxidizing reactions on the other hand might be classed under 
the general heading of aerobic reactions, except that they constitute 
only a small portion of the group of reactions which take place normally 
under aerobic conditions. They are distinguished by the fact that oxy- 
gen is added to the molecule, the product always containing more oxy- 
gen than the initial substance. Carbon dioxide, water and nitrates are 
produced, in distinction from methane, hydrogen and ammonia, which 
characterize the anaerobic reactions. A third type, possessing objec- 
tive rather than subjective functions, in sewage, is made up of patho- 
genic and other harmful bacteria. These play no part in our theories 
of purification and the proof of their presence is generally lacking. 
For the protection of the public health, it is assumed that they are 
always present in sewage, and our procedure in sewage disposal is modi- 
fied throughout in accordance with this assumption. 

With these definitions in mind we may proceed to a more detailed 
study of the bacterial types themselves. 


332 MICROBIOLOGY OF WATER AND SEWAGE 

PUTREFACTIVE AND ANAEROBIC BACTERIA. Putrefaction or anae- 
robic fermentation involves the withdrawal of oxygen from one molecule 
or part of a molecule and the subsequent oxidation of another molecule 
or part of the same molecule. The energy released in this process is 
utilized in the vital functions of the organism. This action is neither 
oxidation nor reduction, or more strictly, they are both taking place 
simultaneously. 

A good example of such a process is the fermentation of urea. The 
reaction takes place as follows: 

CO(NH 2 ) 2 + 2 H 2 = (NH 4 ) 2 C0 3 . - 

Carbon is oxidized at the expense of hydrogen, a process which, by itself, 
is endothermic, that is, requires heat or energy for its maintenance. 
But the heat of formation of the final product is greater than that of the 
initial substances and the energy thus liberated becomes available for 
use by the bacteria. It is in this way that hydrolytic changes of 
this character play the same role in anaerobic reactions that is played 
by direct oxidation under aerobic conditions. 

The Liquefaction of Protein. One of the most clearly defined and 
useful types of bacterial activity to be seen in the various sewage 
disposal processes is that which we term liquefaction. This term is 
used to denote broadly all those changes by which solid and insoluble 
organic matter is converted into a soluble condition. The particular 
process known as protein liquefaction is in the main analogous to gas- 
tric digestion. Its one characteristic is the increased solubility of the 
product. The practical importance of protein liquefaction in sewage 
disposal is very great and the value of the liquefying bacteria corre- 
spondingly high. Nevertheless, aside from our knowledge of analogous 
processes in digestion and in bacterial putrefaction of albuminous sub- 
stances, we know almost nothing of the chemistry or the bacteriology 
of this process. An enormous variety of bacteria are included in this 
group. The whole process is doubtless the result of a very complicated 
symbiosis in which various sub-groups of bacteria carry out the initial re- 
action, from which point other groups carry it through successive stages. 
Absence of one or another of these groups or of some important species of 
any group doubtless accounts for the diverse results that are recorded. 
It is well known that the activities within a septic tank, for example, 


MICROBIOLOGY OF SEWAGE 333 

are seldom twice the same. Gross differences readily apparent to the 
senses of one versed in such matters certainly exist, and in actual results 
it is rare to find two tanks doing exactly the same kind of work. Much 
depends of course upon the chemical character of the sewage itself, but 
much, that is still unexplained, must eventually be traced to the great 
diversity of the sewage flora and the complex symbiosis as well as bac- 
terial antagonisms that are involved in the reactions with which we 
are dealing. 

During these reactions proteins and albumins are hydrolyzed by suc- 
cessive stages to albumoses, peptones, amino-acids, amines, and finally 
to ammonia, carbon dioxide, methane, hydrogen, etc. Simultaneously 
ammonia, amines, and carbon dioxide are eliminated at each stage as 
products. The tendency then is toward simple, soluble and gas- 
eous side products, and hence of value in the preliminary resolution 
of the sewage. 

The Fermentation of Cellulose. The fermentation of cellulose is, 
next to protein hydrolysis, the most important work of the anaerobic 
bacteria in sewage treatment. So far as is definitely known this action 
is usually confined to anaerobic conditions. The fact that fence posts 
decay first at the surf ace of the ground, or that wood in general decays 
more rapidly when it is exposed to only a slight degree of moisture, than 
when it is immersed in water is only an apparent contradiction. The 
conditions are aerobic in both cases and aerobic bacteria would not be 
favored by total immersion but the effect in both instances seems to be 
due to fungus growths which are more active in the moist wood. 

The anaerobic fermentation of cellulose is that which is found typ- 
ically in marshes and of which the chief products are carbon dioxide and 
methane or " marsh gas." Nitrogenous food material is also requisite, 
which accounts for the preserving property of reasonably pure water 
upon wood. 

In the septic tank the solution of cellulose is extremely rapid, and 
large pieces of cotton cloth or rolls of paper are completely dissolved 
within a few months. Wood itself is more resistant and withstands the 
action of the tank for years. This is largely due to the fact that the 
wood molecule is much more complicated than a simple cellulose 
molecule, and, among the conifers at least, to the further fact that 
antiseptic intercellular substances are present. 

Chemically considered the action is hydrolytic and can be imitated 


334 MICROBIOLOGY OF WATER AND SEWAGE 

by prolonged boiling in dilute acids. Pectin substances, starches and 
finally sugars are produced while butyric and other organic acids, carbon 
dioxide and methane appear as by-products. Bacteriologically, al- 
though it has variously been ascribed to one or another organism, it is 
probably the result of the activities of many and is possibly not the 
principal activity of any one of these. In other words, cellulose fermen- 
tation is probably a series of side reactions produced during the fermen- 
tation of the nitrogenous material rather than a definite reaction upon 
which the metabolism of any single species depends. This view is 
strengthened by the general observations that this fermentation is in 
most cases due directly to enzymes. Viewed in this light it is easy to 
understand the difficulty that has surrounded the isolation of definite 
cellulose fermenting organisms. Many have been described, chief of 
which are B. butyricus or B. amylobacter, B. omelianski, Sp. rugula. 

The Saponification of Fats. A third great group of type reactions 
occurring under anaerobic conditions is the saponification or split- 
ting of fat. Our knowledge of this process is even less definite 
than of the cellulose fermentations. It is a fact that there does take 
place in sewage a gradual saponification and emulsification by which 
the fat loses its identity and mingles with the liquid. This effect is 
most noticeable in the case of long sewers in which considerable veloci- 
ties are maintained. In quiescent tanks there is a tendency for the fats 
to rise to the surface and thus become removed from the influence of 
this action. Thus in small installations enormously heavy scums form 
upon the tanks and analysis shows a considerable percentage of fat 
in this material. In larger systems on the other hand there is less and 
less evidence of fatty material as such. It is true that there is a deposit 
upon the walls and tops of such sewers and that small floating objects, 
like matches, rolling along such a wall will accumulate layers of grease 
and become eventually the familiar " grease-balls " found in the dis- 
charge, but in the main the fatty material has become well disintegrated 
before the outlet is reached. 

In this case also as in that previously discussed it is not believed that 
the action is a direct result of the activity of any particular organism. 
The proteolytic changes are accompanied by the freeing of alkaline 
products, ammonia and amines, which leads to some saponification. 
and which, in turn, leads to a further emulsification. It has also been 
demonstrated that bacterial activity is commonly associated with fat 


MICROBIOLOGY OF SEWAGE 335 

saponification and decomposition. Whether specific enzymes are pres- 
ent which assist in this final process or not has never been determined. 
It is significant to note, however, that where sewages are slightly acid, 
unaltered fats are much more abundant, even though the acidity is 
insufficient to prevent vigorous putrefactive changes in the sewage 
itself. 

The Fermentation oj Urea. The fermentation of urea has already 
been referred to as a typical and simple case of anaerobic decomposition. 
This reaction has great significance in sewage chemistry since a consider- 
able proportion of the nitrogen of sewage is present initially as urea. 
Owing to the ease and rapidity with which the reaction takes place, 
however, no special effort is necessary to bring it about in sewage 
treatment and it therefore receives brief attention in discussions of the 
chemistry of sewage. The change to ammonia takes place in the small 
sewers of the system and it is difficult and generally impossible to detect 
the presence of urea in sewage. It has even been suggested that certain 
enzymes present in fecal matter are instrumental in bringing about this 
change and that the bacteria are "only indirectly concerned. It is 
known, however, that a large number of bacteria of general occurrence 
have the power to produce this fermentation. Of these the Bact. urea 
(Miquel) may be cited as an example. 

The Reduction of Sulphates and Nitrates. The production of sul- 
phuretted hydrogen during the anaerobic decomposition of sewage 
is commonly noted. This substance may arise in at least two ways. 
Sulphur, being a constituent of most protein substances, is split off 
from the molecule in this form during certain types of fermentation. 
Its formation in these cases is analogous to that of ammonia from 
protein. The amount so produced is small and is usually neutral- 
ized and precipitated by the small amounts of iron and other metals 
always present in sewage. There is therefore no liberation of the 
gas itself and it is often said that sulphuretted hydrogen is not formed 
normally in a septic tank. This conclusion is readily disproved by 
a simple test of the black residue found at the bottom of such tanks. 

A second and more important source of this substance is the sul- 
phate normally present in many sewages. Throughout many parts 
of the country the water supply contains material quantities of mag- 
nesium or calcium sulphate, and upon the sea coast the sewage gener- 
ally receives more or less salt water. 


336 MICROBIOLOGY OF WATER AND SEWAGE 

In these cases the reduction of sulphates to sulphuretted hydro- 
gen is not only of interest bacteriologically but probably exerts an 
influence upon all the reactions that are going on simultaneously. 
In fact this example serves excellently to illustrate the great complex- 
ity of these anaerobic reactions and the mutual interdependence of 
each upon all the others. Sulphates, under anaerobic conditions, 
are a source of oxygen and it is upon oxygen that the course of all these 
reactions depends. Therefore the presence of sulphates and the 
possibility of their yielding oxygen may alter the course of the other 
reactions involved. The products of the protein hydrolysis for ex- 
ample may be profoundly modified by the presence of 'this additional 
source of oxygen. 

The effect upon the bacteria themselves is also to be considered 
as a factor quite distinct from the purely chemical effect just de- 
scribed. It has frequently been observed, and in fact would be ex- 
pected, that the products of anaerobic putrefaction are themselves 
detrimental to the activity of the organism producing the. changes 
in question. The nature of sulphuretted hydrogen makes it appear 
quite probable that we are dealing here with a toxic substance that 
would at least inhibit the activities of certain bacteria and in this way 
further modify the final result. 

The same might be said of almost all the reactions with which we 
have to deal but this example is cited as a typical one. 

It is known in practice that the presence of sulphates in a sewage 
does lead to a distinct type of anaerobic change which is characterized 
by the marked blackening of the sewage, the "formation of secondary 
reaction products which precipitate after the removal of the suspended 
matter of the sewage, the evolution of hydrogen sulphide, an excessive 
amount of mineral or non-volatile residue in the sludge and the forma- 
tion of free sulphur upon subsequent aeration of the sewage. 

Here again, as in the other types of reaction, it is useless for the pres- 
ent to attempt to ascribe this reaction to any particular species. Sp. 
desulphuricans and B. sulphur eus have been isolated. A non-liquefy- 
ing anaerobic bacillus, which reduced sulphates strongly, was isolated 
from Boston sewage in the writer's laboratory by G. R. Spaulding. 
Others have been described and there is undoubtedly a large group of 
organisms capable of bringing about the reaction. 

Just as the reduction of nitrates is a function performed by many, 


MICROBIOLOGY OF SEWAGE 337 

perhaps most, anaerobes, so the reduction of sulphates, although a 
less common function, is still common to many forms. In fact ni- 
trates, sulphates, and phosphates form a series in regard to their 
reducibility and the effect of their presence upon the reaction as a 
whole. The phosphates so far as has been recorded are not ordinarily 
reduced. 

OXIDIZING BACTERIA. The Production of Nitrate and Nitrite. A 
long series of investigations upon the organisms which oxidize 
nitrogen began with the Franklands and Winogradski, and has 
continued to the present day. These have given us much in- 
formation concerning the habits and functions of the nitrifying 
organisms. Winogradski's original types were Nitrosomonas and 
Nitrobacter, the former oxidizing ammonia to nitrite, the latter 
completing the oxidation to nitrate. Work upon these organisms 
constitutes such an important factor in soil bacteriology to-day 
that more detailed discussion of this nitrifying function is left for 
another place. 

In the earlier days of sewage purification great stress was laid upon 
the work of these organisms, which was believed to be fundamental. 
The degree of nitrification was accepted as a measure of the work of 
the filters and little thought was given to the possibility of oxidizing 
reactions by other forms. With the development of modern sewage 
disposal methods, the work of this latter type of bacteria has assumed 
a more important role and the actual work of the nitrifying organism 
has been found to be of only minor and incidental importance. 

Other Oxidizing Reactions. The great groups of aerobic and facul- 
tative bacteria are in general concerned in the oxidation of organic 
matter. There is nothing specific in this reaction and very little that 
is characteristic of any special or smaller groups. Under certain special 
and restricted conditions, typical products are formed by particular 
species, as in the manufacture of vinegar, and it is possible that a care- 
ful study of the complex reactions involved in the oxidation of sewage 
would show a certain sequence in the order of events and certain definite 
work being accomplished by definite groups. In other words, symbio- 
sis and specialization doubtless take place to a limited extent. But 
the fundamental fact remains that the metabolism of the organism 
demands that organic matter be oxidized for the production of energy. 

Even though certain food substances may be preferred and certain 
22 


338 MICROBIOLOGY OF WATER AND SEWAGE 

decompositions be normally produced there is necessarily a great 
latitude and great adaptability. 

For this very reason a study of the individual organism and its 
action upon specific materials throws no light upon the major 
problem, which is, given fifty different types of organisms and fifty 
different fermentable substances, in a mixture, what will be the course 
of the reaction? Here the preferences, the adaptability and the antag- 
onisms all come into play and while it is impossible to say what has 
happened or how, it is readily conceived and, in fact, almost apparent, 
that out of this heterogeneous mixture there will come a homogeneous 
symbiotic family and an orderly sequence of chemical events, in 
which metabolic needs and food supply are all delicately adjusted. 

PATHOGENIC BACTERIA. Prevalence and Longevity. Owing to its 
origin and nature, sewage may at any time contain infectious material 
and for the purposes of the sanitarian it is assumed that at all times the 
germs of disease are present. Such an assumption is possibly in excess 
of the actual facts and is only justified because it supplies the only pos- 
sible hypothesis having an adequate margin of safety. The actual 
prevalence of pathogenic bacteria obviously depends in the first instance 
upon the amount of sickness in the contributing community. Further- 
more, if, as we are coming to believe, a definite proportion of the popu- 
lation are perpetual carriers of typhoid infection then to just as definite 
an extent is the bacterial population of the sewage made up of typhoid 
bacteria from apparently well persons. In addition to these, about 
five one-hundredths of i per cent of the population of American cities 
are suffering from the disease in acute form. Making due allowance 
for the extra precautions that are, or should be taken in the care of 
the dejecta, these persons constitute a definite and fairly constant 
source of infection. 

In the case of the other infectious diseases of the alimentary tract, 
and, possibly to a less extent in the case of tuberculosis, diphtheria, and 
many others, these general statements are equally applicable, so that 
the possibility of the occurrence of infectious material in sewage is 
not a remote one, but definite and almost quantitatively determinable. 

As to the persistence of active pathogenic bacteria in the sewage for 
any length of time the data are less exact. In the case of typhoid fever, 
which has been more carefully studied than any other disease, the germs 
are more persistent in pure water than in impure, but whether this 


MICROBIOLOGY OF SEWAGE 339 

generality can be extended to sewage is debatable. Our best informa- 
tion leads to the belief that any reduction in numbers of typhoid 
bacteria which may take place within the sewer before discharge is of 
minor importance and of slight sanitary significance. 

Discussion of other pathogens must be in even more general terms. 
Information is almost wholly lacking and it can only be assumed for 
purposes of safety that, in so far as organisms of these various types are 
discharged into the sewer, they will persist to a certain extent in the 
sewage until it is finally disposed of. If such disposal be by discharge 
into a stream without purification, then the waters of that stream 
become polluted with infectious material. Studies recently made by 
Sedgwick and McNutt have indicated the possibility that many dis- 
eases, other than the oft-quoted typhoid fever, may be transmitted 
in this way. 

Life in Septic Tanks and Filters. With the introduction of the 
septic tank at Exeter, England, in 1893, the question of the fate of 
pathogenic bacteria in such a tank was raised. It was even suggested 
that bacteria, such as the typhoid organism, might multiply in the 
tank. The question was investigated by Professor Sims Woodhead, 
who concluded that no organisms capable of setting up morbid changes 
in animals were discharged from the tank. This negative evidence, 
however, has little weight in the light of more recent experiments. 
Pickard introduced an emulsion of typhoid bacteria into this same tank 
and noted only a gradual decrease. After fourteen days he was able 
to detect i per cent of the initial number. He also reported a removal 
of 90 per cent of the typhoid organisms introduced into a contact 
filter. These data must be interpreted in the light of two established 
facts. The typhoid organism tends to die at a rapid but diminishing 
rate under any but the most favorable conditions. This results in a 
rapid decrease at first, with a prolonged survival of a few individuals. 
This process takes place in sewers, in streams, and, in fact, under most 
artificial conditions. The second fact of importance is the difficulty 
of recovering the typhoid organism under experimental conditions like 
those described. 

A thorough study of the bacteriology of sewage and of filter effluents 
led Houston to conclude that the biological processes at work in a filter 
or tank were not strongly inimical, if hostile at all, to the vitality of 
pathogenic germs, 


340 MICROBIOLOGY OF WATER AND SEWAGE 

A conservative study of all the evidence bearing upon this impor- 
tant question including the vitality and fate of certain non-pathogenic 
species, such as B. coli, leads to the conclusion that the removal of 
pathogenic bacteria in purification methods is due to two allied causes, 
the efficiency of which can be approximately determined. There is 
first the time element and the known rapid decrease in the numbers of 
certain bacteria such as B. typhosus when placed under conditions that 
preclude multiplication. The rate of decrease varies but is roughly 
about 50 per cent in twenty-four hours. 

The second factor, acting in reality in conjunction with the first, 
is the mechanical hindrance that is offered to the free passage of sus- 
pended materials through the body of a filter. Even fine sand offers 
little straining action as such, since the open channels are thousands 
of times as big as the bacterial cell, but surface tension phenomena 
tend to make all solid material adhere to the medium and thus its 
passage is delayed. This action is prominent although of less impor- 
tance in coarse-grained filters. Actual experiments by the writer have 
indicated that while the liquid may pass through a trickling filter 
in half an hour, small suspended particles such as ultramarine and B. 
prodigiosus cells require an average of over twenty-four hours. In 
this way the actual time of passage is greatly delayed even when coarse 
broken stone is the filter medium, and the times that are now known 
to be necessary for the passage are ample in themselves to account for 
the reductions that have been noted. 

It may therefore be stated as a conservative view of the efficiency 
of purification processes in the removal of pathogenic bacteria, that 
there are no strongly inimical processes at work in the tanks or filters, 
and that the rate of decrease is not materially greater than would be 
observed in the same period of time under the conditions of a running 
stream. 

THE CULTIVATION or SEWAGE BACTERIA 

There are two general methods employed for the cultivation of 
those bacteria which are of assistance in sewage purification. They 
may be cultivated in so-called filters of sand or coarser material, or 
in specially constructed tanks such as the septic or the hydrolytic tank. 
In the former case the bacterial growth occurs upon the special medium 
provided, the sand or stone; in the latter, it takes place in the liquid 


MICROBIOLOGY OF SEWAGE 


341 


itself and a continuous life history within such a tank is possible only 
when the rate of flow is sufficiently slow to permit of the inoculation of 
the incoming stream by the contents of the tank. 

FILTERS. The filtering media most commonly employed are sand 
or crushed stone or other coarse material. In natural sand beds a 


FIG. 129. Sewage Experiment Station, Mass. Inst. Technology. Trickling 
filter in front, sand filter just behind filter, dosing tank just behind sand filter, and 
septic tank just behind dosing tank. 

brief period of treatment with sewage suffices to produce an active 
state of nitrification." By this term is indicated all the complex 
processes of oxidation one index of which is the formation of nitrates. 
After such a filter has once become active in this way it will continue, 
with proper care, to oxidize sewage almost indefinitely. Improper care, 
such as an overdose of sewage or continued flooding of the surface due 
to poor drainage, will soon destroy the activity of the filter. The addi- 
tion of germicidal substances has a similar effect and cold weather some- 


342 


or WATER AND SFWAGE 


what reduces the efficiency. From all this it is apparent that a filter 
is a biological culture medium upon which the various types of bacteria 
are growing and carrying out their functions and that such a medium 
requires careful control and is sensitive to unfavorable changes in 
environment. 

The other filters are similar to this and illustrate the true function of 
nitration. In the case of the sand filter it might be maintained that 
nitration or straining was an essential element in the process, but in the 
case of these coarse-grained media straining action is eliminated. Here 
there is nothing but a pile of stones, varying from i to 3 inches or 
more in diameter, upon the surface of which the bacteria grow. The 


Siphon 
Chamber 


Grit 

Chamber 


FIG. 130. Sketch of septic tank. (Original.) 

sewage trickles slowly over the surfaces, or is held in contact with them 
temporarily, according as we are dealing with trickling or contact filters. 
Solids adhere to the stones or settle upon them, and soluble material is 
" absorbed" by the surface growth and removed from solution. Within 
these gelatinous growths to which the air also has free access, the proc- 
esses of oxidation take place and the products, the semi-oxidized 
organic material, are later "shed" from the stones appearing again in 
the effluent as humus or stable organic matter. 

ANAEROBIC TANKS.- -The cultivation of bacteria in anaerobic tanks 
is not quite as simple a matter as that which has just been described. 


MICROBIOLOGY OF SEWAGE 343 

The sewage is allowed to flow slowly through the tank and after some 
time, from a few days to a month or more, a normal and constant 
flora will have become resident there. This flora will soon have be- 
come so well established that the incoming sewage laden with a flora 
of its own mingles with a liquid in which the established flora is so 
greatly in excess that the former in large measure gives way to the 
latter. In this way, while the sewage itself moves onward and is 
gone within a few hours, the flora is constant and persistent. A further 
aid in preserving this constant flora is the sludge at the bottom, in 
which the bacteria lodge and multiply and from which they are carried 
upward by the ever moving eddies and constantly re-inoculate the 
liquid above (Fig. 130). 

THE DESTRUCTION OF SEWAGE BACTERIA 

BY BIOLOGICAL PROCESSES. Reference has already been made 
to ..the effect of biological processes of purification upon pathogenic 
bacteria. What was stated in regard to the pathogens is equally true 
of the sewage bacteria as a whole. Their destruction is due to time and 
an environment unfavorable to growth, rather than to any specific 
cause. Further evidence of these facts may now be given. Bacteria 
as a whole do pass even the fine-grained filters in large numbers. 
Careful analyses of their types show them to be a haphazard mixture 
from the original sewage flora with little or no observable selection. 
Houston pointed out the relative abundance of the streptococci, sup- 
posedly delicate organisms, and found on the whole that the relative 
abundance of the different kinds of bacteria seemed to be much the 
same in the effluent as in the crude sewage. 

On the whole we may conclude that the biological processes remove 
bacteria not by any specific antagonistic action but by delaying their 
passage and permitting the natural decrease that occurs when multi- 
plication is prevented. The more efficient the mechanism of the 
filter in producing this delay the more complete will be the removal. 

BY CHEMICAL PROCESSES. A much more reliable and economical 
method for bacterial destruction is now available in chemical disin- 
fection of sewage effluents. The writer's studies at Boston, Baltimore 
and elsewhere have shown that the application of hypochlorite of 
calcium in amounts depending upon the character of the effluent, and 


344 MICROBIOLOGY OF WATER AND SEWAGE 

ranging from one to five parts per million of available chlorine (25 to 
125 pounds of bleaching powder per million gallons), will produce a 
bacterial removal amounting to 98 or 99 per cent. This disinfectant 
is the most efficient of the known germicides, cost being considered. 
By this means it is possible to practically eliminate the bacteria, good 
and bad, from an effluent and it is no longer necessary nor desirable 
to seek high bacterial removals in the purification process proper. 
By thus dividing the work of purification into its component parts 
each part can be carried out at a maximum of efficiency and economy. 


DIVISION III" 
MICROBIOLOGY OF SOIL 


CHAPTER I 
MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 

INTRODUCTION 

Rational views on soil fertility were first presented, in a systematic 
way, by Justus von Liebig in 1840. In his " Organic Chemistry in its 
Applications to Agriculture and Physiology ' :i he developed important 
theories on the circulation of carbon and nitrogen in nature, and on 
the function of the so-called mineral constituents of plants. 

When Liebig's book appeared, many of the leaders and students of 
agriculture still believed that humus, the partly decomposed residues of 
plants and animals in the soil, was the direct food of crops. They 
believed that soils could yield poor or rich harvests in proportion to the 
amount of humus present in them; they believed, in other words, that 
plants, like animals, used organic substances as food. 

Liebig rendered a great service to agriculture in emphasizing the 
significance of decay processes. He made it evident that humus as 
such is of no use to plants, and that it becomes valuable only in so far 
as it is resolved into the simple compounds carbon dioxide, ammonia, 
nitric acid and various mineral salts. To be sure, he regarded the 
decomposition of organic matter as a phenomenon purely chemical, 
nevertheless he succeeded in showing that decay, putrefaction and 
fermentation are fundamental facts, connecting links between the 
world of the living and the world of the dead. 

The research of the following decades brought to light the intimate 
relation existing between microorganisms and the decomposition of 

* Prepared by Jacob G. Lipman with exception of sub-chapter on "Soil Inoculation" which 
has been prepared by S. F. Edwards. The author is indebted to Dr. S. A. Waksman for help 
in the revision of a portion of the manuscript. 

345 


346 MICROBIOLOGY OF SOIL 

organic matter. In the realm of soil fertility the new discoveries re- 
vealed the vastness of the task assigned to soil microorganisms in 
providing available food for crops. It was shown that under the attack 
of bacteria and of other microorganisms the various organic debris in 
the soil is split into relatively small chemical fragments; that the 
carbon is restored to the air as carbon dioxide; that the nitrogen is 
changed into ammonia, nitrites and nitrates. It was shown, further, 
that in this breaking down of organic matter the various cleavage 
products, and, particularly, carbon dioxide, hasten, to an amazing 
extent, the weathering of the rock particles and make available thereby 
the mineral portion of plant food. It was shown, likewise, that apart 
from accomplishing the transformation of unavailable into available 
plant food, microorganisms are concerned also in the addition of 
nitrogen compounds to the soil. The evidence gathered slowly by 
many investigators made it plain, therefore, that microbes are an 
important factor in the growing of cultivated and uncultivated plants. 
Hence, the important place assigned to microorganisms in the study of 
soil fertility problems. 

THE SOIL AS A CULTURE MEDIUM 

Arable soils present so wide a range of conditions as to modify, 
materially, the development and predominance of different species. 
Variations as to moisture, temperature, aeration, reaction, food supply 
and biological relations are important, in each case, in determining 
the survival or disappearance of any particular species. For this 
reason, the study of soil microorganisms must reckon with the mechan- 
ical composition of soils, their ability to retain water and their content 
of inert and soluble plant food. 

MOISTURE RELATIONS IN THE SOIL 

AMOUNT AND DISTRIBUTION OP RAINFALL. Precipitation in 
different regions of the earth's surface varies from practically nothing 
to more than 1,524 cm. (600 inches) per annum. A portion of this 
water runs off the surface into the nearest stream, another portion is 
rapidly changed into vapor and is returned to the atmosphere, and the 
remainder passes downward, into the soil and becomes the medium 
in which plant food is dissolved. It is estimated that only about half 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 347 

the total rainfall percolates through the soil. Where the soils are 
open and nearly level the proportion of percolating water is relatively 
greater; where the soils are fine-grained and more or less impervious, 
or the topography broken, the proportion is relatively smaller. 

Bacteria and other microorganisms, as well as the higher plants, are 
directly influenced by the amount of moisture available for their various 
needs. Hence soil microbial activities are affected not alone by the 
amount of rainfall, but also by its distribution. It is obvious, for 
instance, that an annual rainfall of 762 mm. (30 inches) distributed 
rather uniformly throughout the year would produce different soil- 
moisture relations than the same amount of precipitation confined to 
only two or three months. As is pointed out by Abbe, a daily pre- 
cipitation of 2 mm. (.079 inch) distributed throughout the three 
summer months would be quickly changed into vapor, and would 
hardly wet the soil; whereas the total quantity of 180 mm. (7 inches) 
evenly divided into ten or twelve rains would penetrate the soil to a 
considerable depth, and would furnish very favorable conditions for 
microbial development. In a similar manner it is pointed out by Hil- 
gard that Central Montana, and the region in the vicinity of the bay 
of San Francisco, have each a total precipitation of 610 mm. (24 inches). 
But while in Montana the rainfall is distributed over the entire year 
and irrigation becomes necessary, the precipitation near San Francisco 
is limited to the portion of the year that nearly coincides with the 
growing season, and crops are enabled to mature without irrigation. 

RANGE OF SOIL MOISTURE. Any given volume of dry soil consists 
of solid particles separated by empty spaces. The sum of these spaces 
is known as the "pore-space." It varies from about one- third of the 
entire volume in coarse sands to more than two-thirds in pipe clay. In 
peat and muck it may amount to as much as 80 or 90 per cent of the 
entire volume. Under air-dry conditions each soil grain is surrounded 
by a very thin film of moisture designated as hygroscopic water. When 
air-dry soil is moistened the films around the soil particles become 
thicker and finally cease to be isolated. A continuous liquid membrane, 
as it were, is stretched from particle to particle, and the surface tension 
that thus comes into play is capable of lifting large amounts of water 
to the surface. The continuous film of soil water that can hold its 
own against the pull of gravity is known as capillary water. Finally, 
when the liquid films around the soil grains increase in thickness be- 


348 MICROBIOLOGY OF SOIL 

yond a certain point, the attraction between the molecules in the soil 
grains and the more distant molecules of water is no longer great 
enough to overcome the force of gravitation, and the excess of water 
percolates downward. The water more or less readily moved by 
gravitation is called hydrostatic water. 

For any given conditions of the soils the amount of hydrostatic, 
capillary and hygroscopic water is directly dependent on the mechanical 
structure. It is evident that the aggregate surface of the particles in 
a fine-grained soil is much greater than that in a coarse-grained soil. 
Actual determinations have shown that the aggregate inner surface 
of .02832 c.m. (i cu. ft.) of coarse sand may be but a fraction of an 
acre; whereas the same quantity of the finest clay may have an 
inner surface equivalent to 1.2141-1.6188 hectares (3 or 4 acres). 
These differences are to be expected, since, as is shown by Lyon and 
Fippin, i g. of fine gravel may contain 252 particles; i g. of medium 
sand, 13,500 particles; i g. of very fine sand, 1,687,000 particles; i g. 
of silt, 65,100,000 particles, and i g. of clay, 45,500,000,000 particles. 

Since the soil water is spread as a film over the solid particles and 
varies in amount with the fineness or coarseness of the soil, and since 
the quantity of plant food going into solution is determined largely 
by the amount of water in contact with the soil particles, it follows that 
clay soils will, under the same conditions, contain more plant food in 
solution than loam soils and still more than sandy soils. From the 
standpoint of soil microbiology this is important, for the microorganisms 
live and multiply in the film water surrounding the soil particles. The 
concentration of salts in this film water as well as their composition 
must of necessity affect bacterial activities. In the same way, methods 
of tillage and cropping affecting the concentration and composition 
of the film water will modify the chemical changes caused by bacteria 
and other microorganisms. 

EFFECT OF DROUGHT AND OF EXCESSIVE MOISTURE. Optimum 
conditions for plant growth and the development of many important 
soil bacteria are furnished when about half of the entire pore space is 
filled with water. In light sandy soils the optimum moisture content 
may be reached when the wet material contains scarcely more than 8 
to 10 per cent of water by weight; while in silt and clay soils the 
optimum may reach 1 6 to 20 per cent or even more. 

Continued depletion of soil moisture by plant roots and evaporation 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 349 

at the surface causes the film of capillary water to stretch more and 
more. Finally it becomes very thin, breaks, and ceases to be con- 
tinuous. The soil then becomes air-dry and contains only hygro- 
scopic water. It is estimated by Lyon and Fippin that, under average 
conditions of humidity, light sand will contain 0.5 to i per cent of 
hygroscopic moisture; silt loam, 2 to 4 per cent; and clay, 8 to 12 per 
cent. The amount of water present in air-dry muck or peat may range 
up to 40 per cent, or even more. According to Hall the film of hygro- 
scopic moisture is about 0.75^ (0.00003 inch) thick. As the soil 
dries out bacterial activity is suspended and many vegetative cells 
undoubtedly perish. Nevertheless, it will be seen that the moisture 
film even in air-dry material is deep enough to allow the bacteria a 
reasonable degree of protection. This will account for the survival 
of non-spore-bearing bacteria in dry soil for a long time. Indeed, in- 
stances are on record of the isolation of Azotobacter and Nitrosomonas 
from soils that had been kept in a dry state in the laboratory for 
several years. It may be noted, in this connection, that in. the process 
of drying the soluble salts in the soil the moisture may be sufficiently 
concentrated in the films to cause plasmolysis and the destruction 
of individual cells. 

On the other hand, excessive moisture in the soil is not only directly 
unfavorable to aerobic species in that it limits their supply of oxygen, 
but is objectionable because it encourages the formation of reduction 
products that are toxic to these species. It is apparent, therefore, that 
favorable conditions for the formation of available plant food by 
bacteria are created when a certain relation is established between the 
volumes of moisture and air in the soil. The shifting of this relation in 
one direction or another is bound to react on species relationships and 
numbers. 

COLLOIDAL NATURE OF THE SOIL .--The colloidal condition of 
the soil imparts to it the ability to absorb substances from their 
solutions as well as the ability to change them from a flocculated 
to a deflocculated state. Another important colloidal property of 
soil is the formation of a colloidal solution in pure water and coagu- 
lation by the addition of small quantities of electrolytes. Soluble 
fertilizers when added to the soil are adsorbed by the latter: other- 
wise, they could easily be washed out by drainage. The adsorbed 
substances displace others which may be washed out of the soil. 


350 MICROBIOLOGY OF SOIL 

The addition of ammonium and potassium salts, for example, re- 
sults in the displacement of the corresponding calcium salts, which can 
be washed out, and the formation of insoluble nitrogen or potassium 
compounds which remain in the soil. On adding sodium and magnes- 
ium salts to the soil, displacement of some of the insoluble potassium 
salts may take place and these may become available for plant growth. 
The interchanges taking place between the salts existing in the soil 
and those added in the form of fertilizers have an important effect upon 
soil biological phenomena and plant nutrition. On heating or drying 
soils, an increase in the amount of soluble food is produced which is 
probably a result of the change produced in the colloids. It is in this 
colloidal complex of organic and inorganic compounds, saturated with 
water and surrounded by the mineral particles that most of the soil 
biological phenomena take place. 

AERATION 

MECHANICAL COMPOSITION OF SOILS. Soil ventilation is an impor- 
tant factor in crop production. It provides for the proper supply of 
elementary oxygen so essential to decomposition processes in normal 
soils; for the supply of elementary nitrogen required by nitrogen-fixing 
species; for the removal of excessive amounts of carbon dioxide; and 
for the destruction of various toxic substances. The intimate relation 
existing between soil ventilation and the mechanical composition of the 
soil material is bound to react on the microbial factors involved. It is 
well known that the rate of flow of air through soils is inversely propor- 
tional to the fineness of the material; in other words, the fine-grained 
soils, notwithstanding their greater pore space, will not allow air to 
pass through them as rapidly as coarse-grained soils. King shows, for 
instance, that 5,000 c.c. of air passed through a column of fine gravel 
in thirty-seven seconds, whereas in similar columns of medium sand, 
fine sand, loam and fine clay soil the same amount of air required for its 
passage 1,178, 44,310, 282,200, and 2,057,000 seconds respectively. 

AEROBIC AND ANAEROBIC ACTIVITIES. The more rapid diffusion 
of gases from open soils naturally leads to a more frequent renewal of 
their oxygen supply. In its turn, the latter affects the ratio of aerobes 
to anaerobes; it follows, therefore, that in clay soils and clay loam soils 
the activities of aerobic species are retarded to a greater extent than 
they are in sandy loams or sandy soils. It follows, also, that in fine- 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 351 

grained soils the activities of the aerobes are confined to a shallower 
soil layer than in coarser grained soils. The reverse is true of anaerobic 
species. Methods of soil treatment tending to improve soil ventilation 
react both on the amount of chemical change produced by definite 
species, as well as the numerical ratio of different species to one another. 
Among such methods may be included drainage, liming, manuring and 
tillage. 

RATE or OXIDATION OF CARBON, HYDROGEN AND NITROGEN.- 
Experiments carried out by Wollny proved conclusively that the pro- 
duction of carbon dioxide in soils is directly affected by the amount of 
oxygen supplied; that is, by the more or less thorough aeration of the 
soil. In one of these experiments air containing varying proportions 
of oxygen and nitrogen was passed through columns of soil. When 
this air contained 2 1 per cent of oxygen there were produced for every 
1,000 volumes of air 12.51 volumes of carbon dioxide; while with 2 per 
cent of oxygen in the entering air there were produced only 3.62 
volumes of carbon dioxide. Similar observations were made by 
Schloesing in connection with the formation of carbon dioxide and of 
nitric acid. Deherain and many others have recorded the favorable 
influence of aeration on the rate of nitrate formation, while Lipman 
and Koch have observed an increased fixation of nitrogen by Azotobacter, 
consequent upon a better supply of oxygen. 

THE MINERALIZATION OF ORGANIC MATTER. Conditions that favor 
the intense activities of decay bacteria lead to a relatively rapid restora- 
tion of the phosphorus, sulphur, calcium, magnesium and potassium 
that had been made fast in plant tissues, to the stock of available plant 
food in the soil; indeed, in extremely well-aerated soils the decomposition 
of organic matter and its ultimate mineralization proceed too fast. It 
often happens that the farmer is unable to maintain a proper supply 
of humus in these soils because of their openness and is forced to adopt 
measures that will retard soil aeration. He resorts therefore, to rolling, 
marling, manuring and green manuring. 

On the other hand, heavy, fine-grained soils are not sufficiently well 
aerated to allow a rapid mineralization of the organic matter. Under 
extreme conditions the decomposition processes do not keep pace with 
the process making toward the accumulation of organic matter, and a 
more or less considerable increase in the amount of the latter takes 
place. This occurs in low lying meadows, and, more particularly, in 


352 


MICROBIOLOGY OF SOIL 


bogs and swamps. Hence the farmer attempts to intensify aeration 
and the resulting mineralization of the humus by more thorough 
tillage, drainage, liming and manuring. 

TEMPERATURE 

INFLUENCE OF CLIMATE AND SEASON. An illustration of the differ- 
ences that may exist in the soil temperatures of different regions is given 
by a comparison of the mean temperatures of 1901 recorded at Moscow, 
Idaho, and New Brunswick, New Jersey. The soil temperatures were 
taken to a depth of 152 mm. (6 inches). 

SOIL TEMPERATURE,* 1901 


Jan. 


Feb. 


Mch. 


Apr. 


May 


June 


July 


Aug. 


Sept. 


Oct. 


Nov. 


Dec. 


Moscow, Idaho. . . 


32.0 


30.0 


35-0 


40.0 


52.0 


58.0 


68.0 


72.0 


57-0 


50.0 


40.0 


34-0 


New Brunswick, 


3i.S 


28.6 


35-3 


47-9 


S7-9 


72.1 


76.4 


73-4 


68.5 


56.0 


41.1 


33.4 


N. J. 


AIR TEMPERATURE,* 1901 


Moscow, Idaho. . . 


30.0 


30.5 


38.3 


44-0 


56.9 


55-0 


65.5 


69.6 


50.3 


50.5 


39-5 


39.0 


New Brunswick, 


30.8 


24.8 


39.1 


48.3 


59-2 


70.9 


77-4 


74-6 


67.6 


54-6 


38.6 


32.6 


N.J. 


It will be observed that in the months of November to March the 
soil temperatures in the two places were nearly the same. On the 
other hand, in April to October the average temperatures at New 
Brunswick were for soil 14.5 (58F.) and for air 22.5 (72F.), re- 
spectively; and in July they were 20.0 (68F.) and 24.5 (76.4^.) 
respectively. It will also be observed that there is an unmistakable 
relation between the corresponding air and soil temperatures. 

As a further illustration of the relation of climate to temperature a 
comparison may be made of the average daily mean temperatures at 
Bismarck, North Dakota, for the period 1873-1895, and at Key West, 
Florida, for the period 1872-1895. 

DAILY MEAN TEMPERATURES* (AIR) 


Jan. 


Feb. 


Mch. 


Apr. 


May 


June 


July 


Aug. 


Sept. 


Oct. 


Nov. 


Dec. 


Bismarck, N. D.. . 


4-5 


9-5 


22 .6 


42.1 


54-2 


63.8 


69.5 


67-5 


57-0 


43-8 


2S.9 


14.7 


Key West, Fla.. .. 69.7 71.4 72.7 


76.1 79-4 


82.5 


83-9 


83.9 


82.5 


78.5 


74-2 


70.0 


* Recorded in Fahrenheit scale. 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 


353 


It is obvious from the figures given here that, because of the im- 
portant temperature variations of different soil regions, the micro- 
biological activities must be profoundly modified. But apart from the 
climatic variations already indicated there are seasonal variations in 
any particular locality that are of great moment for soil microbiological 
activities. Such differences are demonstrated by the temperatures 
of 1898 and 1902, taken to a depth of 152 mm. (6 inches), at New 
Brunswick, N. J. 

SOIL TEMPERATURES* 


Jan. 


Feb. 


Mch. 


Apr. 


May 


June 


July 


Aug. 


Sept. 


Oct. 


Nov. 


Dec. 


New Brunswick, 


N. J. (1898) ... 


33.2 


33.1 


45-1 


48.9 


59.1 


76.0 


79.3 


77.8 


72.0 


60. 1 


44-6 


33-6 


New Brunswick, 


N. J. (1902) 30.7 


28.9 


41-3 


49.5 


60.4 


68.0 


72.6 


70.5 


65.9 


56.4 


48.6 


34-1 


In this instance, the season of 1898 was not only earlier, but the 
temperatures of June to September were sufficiently higher to favor 
more intense bacterial grow.th and activity. 

EARLY AND LATE SOILS. Under any given climatic conditions the 
warming, up of soils in the spring will depend on their chemical and 
mechanical composition, color, tillage and topography. Because of the 
high specific heat of water, fine-grained soils containing a relatively 
large amount of moisture will warm up more slowly than coarse-grained 
soils containing a relatively small amount of moisture. The differences 
in the specific heat of humus, sand, clay and chalk are less important, 
yet they introduce appreciable variations in the soil temperature 
according to the proportion of each present. The topography of the 
soil introduces a factor of some importance for it affects the inclina- 
tion toward the sun's rays as well as the drainage conditions. Tillage 
operations are of considerable moment, since they influence the rate 
of evaporation, that is, the rate at which heat is lost from the soil by 
the transformation of liquid water into vapor. Finally the color of 
soils exerts an influence on their temperature in that it affects the 
absorption and reflection of heat. 

Taking all of the factors together, it is found that sandy soils and 
sandy loams are early soils, because they part readily with their excess 

* Recorded in Fahrenheit scale. 
23 


354 


MICROBIOLOGY OF SOIL 


of water. Clay soils and clay loams are, on the other hand, late soils; 
it means, therefore, that in the more open soils microbial activities be- 
come intense earlier in the spring. Market gardeners usually attempt 
to improve matters still further by the use of large quantities of readily 
fermentable manure that develops enough heat to raise slightly the 
soil temperature. 

PRODUCTION AND ASSIMILATION OF PLANT FOOD. It was observed 
by Moller that slight amounts of carbon dioxide may be evolved 
from frozen soil. Kostychev could detect a considerable production 
of carbon dioxide at o to 5. In a series of experiments carried out 
by Wollny the amounts of carbon dioxide produced were as follows: 

CO 2 IN 1,000 VOLS. OF AIR 


Water in soil 


10 


20 


30 


40 


50 


6. 79 per cent 


2 .03 


3 . 22 


6.86 


14. 60 


25 . 17 


26 79 per cent 


18.18 


54 22 


63 . ^o 


80 06 


8l. S2 


46 . 79 per cent 


1< .07 


6 1 . 40 


82.12 


91.86 


07 .48 


ty 


y / *r" 


The increased production of carbon dioxide at the higher temperatures, 
as shown in the above table, corresponded with the observations that had 
already been made by Ebermayer, Schloesing and others, that carbon 
dioxide production in the soil is greater in summer than it is in winter. 
These facts, taken together with the early observations of Forster on 
the multiplication of photo-bacteria at o, and the more recent ob- 
servations of numerous investigators on the multiplication of in- 
dividual species, or of mixtures of species in milk, water, soil, butter, 
etc., at o, or even below that, make it evident that bacterial activities 
are not entirely suspended at relatively low temperatures. As the 
latter rises these activities become more intense as gauged by the 
formation of carbon dioxide. 

Coming down to specific groups of soil bacteria, it may be noted that 
at 12 nitrification is already quite perceptible; that urea bacteria grow 
slowly at 5; Ps. radicicola at 4; members of the B. subtilis group at 
6 to 10, etc. At 15 the breaking down of organic matter is fairly 
rapid, and at 25 the optimum is reached for many species. It follows, 
thus, that the production of plant food namely, ammonia, nitrates, 
sulphates, phosphates, etc. gains rapid headway as the optimum tem- 
peratures are approached. The organic matter itself, apart from serv- 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 355 

ing as a source of plant food, furnishes carbon dioxide and various 
organic acids that help to attack the rock fragments and to render 
available compounds of phosphorus, potassium, calcium and mag- 
nesium. It is likewise evident that in warm countries bacterial 
activities are not only more intense at any one time, but they continue 
through a longer period. For this reason, the soils of the South can 
furnish both relatively and absolutely a greater amount of available 
plant food than the soils of the North. 

The production of plant food is necessarily followed by more 
vigorous growth of bacteria and of higher plants. More food is, there- 
fore, assimilated and more moisture used up until the very rank growth 
of the crops hastens the depletion of the soil moisture. In this manner 
the soil may be dried out sufficiently to retard seriously the growth of 
soil bacteria and to retard thereby the decompositon of organic matter; 
under such conditions, moisture, rather than temperature, becomes 
the controlling factor of growth. 

REACTION 

RANGE OF SOIL ACIDITY. Acid soils are very common in humid 
regions. The older soils of Europe include extensive areas whose lime 
content has been restored repeatedly by the application of wood ashes, 
marl, oyster and clam shells, and various grades of burned or crushed 
limestone. In the United States acidity is becoming prevalent in many 
of the cultivated soils, as is shown by the investigations of the Rhode 
Island, Ohio, Illinois, Oregon and Florida experiment stations. These 
investigations, confirmed by experiments in other states, show that 
there is a marked removal of lime and of other basic materials from the 
soil as cultivation and the use of commercial fertilizers become more 
thorough. Knisley shows, for instance, that 38.75 per cent of the 
Oregon soils examined were acid, and that 16.25 per cent were strongly 
acid. Similarly, Blair found that of 189 samples of different Florida 
soils and subsoils examined, 68.22 per cent of the former and 51.35 
per cent of the latter were acid. He also found that virgin soils were 
less acid than cultivated soils. 

CAUSES OF SOIL ACIDITY. Soil acidity may be due to acids or acid 
salts, both inorganic and organic. Under ordinary conditions the 
latter are of much greater importance than the former as a cause of 
soil acidity. This is demonstrated by the extremely acid conditions 


356 MICROBIOLOGY OF SOIL 

ot peat and muck soils that are particularly rich in organic acids. In 
soils left to themselves the formation of basic substances in the break- 
ing down of silicates and other compounds keeps pace with their 
neutralization by acid and their removal in the drainage water. When 
soils are placed under cultivation, lime and other bases are removed 
more rapidly and the inert humic acids are left behind. The loss of 
bases is intensified by application of acid phosphate, potash salts and 
ammonium sulphate, commonly used as fertilizers. This accounts 
for the less extensive acidity in and among virgin soils as compared 
with cultivated soils. Arid soils lose scarcely any of their basic sub- 
stances by leaching and are seldom acid. Residual limestone soils 
may be alkaline, neutral or acid, according to the loss of bases they 
have suffered by leaching. Low-lying soils, including meadows 
and swamps may accumulate large amounts of organic acids because 
of their imperfect aeration. 

The more recent investigations of the nature of soil acidity have 
suggested a physical explanation, namely, that the acidity of the 
soil is due not to the existence of definite humic and other complex 
organic acids, but rather to selective adsorption. According to some 
investigators there is a direct adsorption of the base when a soil is 
treated with a salt solution. Hence, the behavior of the soil towards 
neutral salts is not due to the presence of organic matter, but to inor- 
ganic compounds, probably hydrated silicates. According to others the 
development of acidity in the salt solution is due rather to an exchange 
of bases : aluminum is given up from the soil in amounts approximately 
equivalent to the base adsorbed. 

Through the action of microorganisms in the soil, the organic matter 
is decomposed with the liberation of weak organic acids (oxalic, citric, 
CO 2 , etc.). By the interaction of these acids in the soil solution and 
the basic material held adsorbed by the soil, soluble salts are formed 
which are subsequently removed by leaching: the soil can then adsorb 
more basic material, giving rise to soil acidity. 

SOIL REACTION AND HYDROGEN-ION CONCENTRATION .--The dif- 
ferent methods for measuring the lime requirements of soils are merely 
attempts to measure the total soil acidity, but not the intensity of the 
acidity or the active acidity. The latter can only be determined by 
measuring the hydrogen-ion concentration of the soil. Pure water 
dissociates, producing equal concentrations of H ions and OH ions 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 357 

indicating neutrality. The product of the concentrations of these ions 
(water) is constant, about i X io~ 14 . ' If the concentration of H ions is 
greater than that of the OH ions, the solution is then acid. When the 
concentration of OH ions is greater than of H ions, the solution is alkaline. 
Total acidity (or potential acidity, to use the expression of Sharp and 
Hoagland) or alkalinity may be due to undissolved substances or to 
soluble compounds only partly hydrolyzed or dissociated. 

The hydrogen-ion concentration of the soil can be measured both 
electrometrically and colorimetrically. The work of Gillespie, Sharp 
and Hoagland and others has brought out the fact that soils vary greatly 
in the hydrogen-ion concentration, from a high acidity to a high alkalin- 
ity. There is a definite correlation between the hydrogen- ion concen- 
tration of soils and the occurrence and activities of microorganisms. 
Gillespie has shown that potato scab (Actinomyces scabies) rarely occurs 
in soils having a hydrogen-ion exponent lower than 4.8 to 5.2. 

Gainey called attention to the fact that Azotobacter occurs in soils 
having a hydrogen-ion exponent greater than 6.0, while the more acid 
soils are practically free from this important group of nitrogen-fixing 
organisms. Waksman demonstrated the occurrence of Azotobacter 
in cranberry soils that received an application of lime and gave a de- 
cided increase in crop, while the unlimed soil was too acid for the organ- 
isms to act in; this limiting reaction for Azotobacter corresponded to a 
hydrogen-ion exponent of about 6.0. 

CHANGE OF REACTION PRODUCED BY MICROORGANISMS IN THE 
MEDIUM. Microorganisms modify the reaction of the medium both 
by their ability to produce organic and inorganic acids (in the case 
of sulphur oxidizing bacteria) and also by their utilization of the 
organic acids as sources of energy. 

EFFECT OF REACTION ON NUMBERS AND SPECIES. Some of the 
important groups of soil bacteria including nitro, azoto and ammonify- 
ing species will develop slowly or not at ah 1 , when the amount of acid in 
the medium is increased beyond a certain point. Hence it is realized 
by progressive farmers that a proper supply of lime is essential for the 
satisfactory decomposition of organic matter in the soil, and the abund- 
ant supply of available nitrogen compounds, as well as of other con- 
stituents of plant food to growing crops. The influence of lime on the 
multiplication of soil bacteria is well illustrated, for instance, by the 
experiments of Fabricius and von Feilitzen. These investigators found 


358 MICROBIOLOGY OF SOIL 

only 138,500 bacteria per g. in newly broken and unlimed peat soils; 
whereas in similar soils that had been limed and cultivated for several 
years the numbers averaged about 7,000,000 per g. and reached a 
maximum of 22,132,000 per g. 

FOOD SUPPLY 

ORGANIC MATTER. It may be said truly that a soil devoid of 
organic matter is practically devoid of bacteria. To the fresh and the 
partially decomposed organic matter (humus) the soil organisms must 
look for most of their food and energy. Being largely of plant origin 
this organic matter contains starches, fats, organic acids, higher al- 
cohols, proteins and amino-compounds. Because of the different 
relations that these vegetable substances bear to the several species of 
soil bacteria, a high or low proportion of starch, of cellulose, or protein 
must necessarily modify both numbers and species relationships. For 
instance, observations have been made by Coleman and others that 
small amounts of dextrose favor nitrification, whereas larger quantities 
retard it; similarly, it has been noted that in the spontaneous de- 
composition of protein bodies bacteria are prominent and molds absent 
or relatively few in numbers. But where dextrose is added to the 
decomposing proteins molds soon appear in large numbers. There 
may also be cited, in this connection, the observation of Hilgard that 
humus should contain at least 4 per cent of nitrogen if it is to furnish 
a sufficient quantity of available nitrogen compounds; otherwise, the 
soil bacteria seem to be unable to decompose it, so as to meet the 
needs of the growing plants. Many similar facts could be cited to 
show that as a culture medium the soil is influenced by green manures, 
barnyard manure, commercial fertilizers, lime, tillage and any other 
treatment that will modify the quantity as well as the quality of its 
organic matter. 

THE MINERAL PORTION or THE SOIL. The moisture films sur- 
rounding the soil grains contain in solution substances derived from 
these soil grains. A particle of calcium carbonate will be surrounded 
by a moisture film containing some calcium bicarbonate. In the 
same way particles of feldspar may give rise to a solution of potassium 
bicarbonate; particles of apatite to a solution of calcium phosphate; 
particles of selenite to a solution of calcium sulphate; particles of 
protein to a solution of ammonia, etc. In view of the fact that these 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 359 

reactions are more or less localized and diffusion slow, there are, un- 
doubtedly, in the soil minute zones where individual species are more 
prominent than they are in others. For example, Heinze has found it 
convenient to isolate Azotobacter by inoculating suitable culture solu- 
tions with particles of calcium carbonate picked out from the soil. 
Evidently these organisms were present in much greater abundance 
on these particles than on others of non-calcareous origin. Indeed, 
he occasionally obtained in this manner Azotobacter membranes that 
constituted almost pure cultures. The more general significance of 
this relation is apparent when it is remembered that nitro-bacteria 
are particularly favored by magnesium carbonate; tubercle bacteria 
by gypsum and calcium carbonate; Azotobacter by calcium phosphate 
and calcium carbonate; photo-bacteria by sodium chloride, etc. 

Considerable as must be the local differences in any one soil, they 
are undoubtedly even more pronounced when different soils are com- 
pared. Extreme conditions are met with in certain irrigated soils 
in which a marked concentration of salts occurs. In so far as crop 
production is concerned, it is stated by Hilgard that the upper limit is 
practically reached when the concentration of soluble salts in the irriga- 
tion water is about 4.55 g. (70 gr.) per gallon. Nevertheless, in Egypt 
and the Sahara region irrigation water is occasionally used that con- 
tains more than 13 g. (200 gr.) of soluble salts per gallon. Further 
differences are introduced by the quality of these salts, e.g., the pro- 
portion of sodium sulphate, magnesium sulphate, sodium chloride, 
sodium carbonate, etc. Again, instances are on record, as in the investi- 
gations of Headden in Colorado and California, where the concentration 
of nitrates in the soil water is so great as to kill even relatively resistant 
plants like alfalfa. It is to be shown by future investigations what the 
effect of the concentration and composition of such salts may be on the 
soil bacteria. 

In humid soils conditions are less extreme, yet even here the variable 
concentration and composition of the soil solution are of direct moment 
for the different microorganisms. Granite soils, for instance, are fairly 
well supplied with phosphoric acid and abundantly with potash, but 
when hornblende is lacking they are apt to be deficient in lime. Ill- 
ventilated clay soils may contain reduction products of iron salts, while 
green sand, chalk, slate, shale, sandstone and other soils may have their 
individual peculiarities from the standpoint of a culture medium. 


360 .MICROBIOLOGY OF SOIL 

BIOLOGICAL FACTORS 

MOLDS. Distribution. While the study of the lower bacteria in the 
soil has attracted the attention of many investigators, that of fungi and 
actinomyces received, until recently, but scant consideration. Fungi oc- 
cur in all soils, cultivated as well as uncultivated, rich or poor in organic 
matter, heavy or light in texture. Most of them are obligate sapro- 
phytes, although facultative parasites are found in large numbers in 
the soil, especially where single-cropping or short rotations favor the 
survival of the particular organisms. The isolation of soil fungi has 
been accomplished either by the dilution method, where- a sample of 
soil was shaken with water, and only a certain dilution was used for 
inoculation; and by the direct method, where a clump of soil was inocu- 
lated into a sterile medium, and the fungi developing on it were isolated. 
About 150 different species of fungi have been isolated from different 
soils, and the data accumulated by investigators in this country and 
in Europe seem to point to the fact that many of these fungi are 
universal in their habitat, since the same species are recorded to have 
been isolated from different soil types and in different localities. Most 
of the work done refers to the classification of the organisms isolated. 
The largest group of soil fungi belong to the following genera: Mucor, 
Zygorrhynchus, Rhizopus, Aspergillus, Penicillium, Fusarium, Tri- 
choderma, Cephalosporium, Monilia, Cladosporium, Alternaria, and 
Acrostagmus. Many other genera have been isolated, but to a more 
limited extent. As to the individual species occurring in the soil, 
Hagem, having isolated about 30 mucors from the soil, states that 
certain Aspergitti occur in larger numbers than all the mucors taken 
together. As to quantitative relations, no exact data are available. 
Some investigators report only several hundred fungi per g. of soil, 
while others record as many as 1,000,000 per g. of soil; that is the 
total number of spores and pieces of mycelium that develop on suitable 
media. As to the numbers and types in relation to depth, Goddard 
concluded that there does not seem to be an appreciable variation in 
numbers at the different soil depths. There are very few fungi in the 
soil below 8 inches and one of the most common forms at these depths 
is Zygorrhynchus vuilleminii. It was formerly thought that soil fungi 
are abundant only in acid soils, but recent investigations make it 
appear that also limed and well-cultivated soils have an abundant 
fungus flora. 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 361 

The plate count is not an index of the activity of the molds in the 
soil, but merely indicates the number of spores present. An organism 
that produces a large number of spores, particularly when these resist 
drying, will be found by the plate method in much larger numbers than 
another organism which, though causing a greater degree of chemical 
change in the soil, produces fewer and less resistant spores. A method 
was therefore suggested by Waksman which would permit the separa- 
tion of molds w r hich produce mycelia abundantly and readily from 
those that develop in soils only under special conditions of moisture and 
temperature. The method consists of planting a lump of soil into the 
agar in a Petri dish and observing the development of the mycelia in 
the soil. It is obvious that the mycelia develop more readily than 
the spores and grow out into the agar. In this manner we may separate 
the organisms which actually live in the soil. Moreover, the fact that 
the same species of molds have been isolated from soils in different parts 
of the world would tend to indicate that, when conditions become favor- 
able, molds vegetate in the soil, although at other times they may exist 
there only in the form of spores. 

Ammonification. Miintz and Coudon, and after them Marchal, 
working with pure cultures, proved conclusively that fungi decompose 
organic matter and cause an accumulation of ammonia in the soil. 
Wilson and McLean found that the forms of Monilia&re the most active 
ammonifiers among the several groups of organisms studied, while the 
Aspergilli showed the least ammonifying power. More recent work 
has confirmed the earlier findings and has proved that fungi may 
play an active part in the decomposition of organic matter, and the 
accumulation of ammonia. 

The molds have been shown to be more rapid ammonifiers than the 
bacteria and actinomycetes. Species of Trichoderma have been found 
by Waksman and Cook to transform more than 60 per cent of the 
nitrogen of dried blood and cottonseed meal into ammonia in a period 
of seven to twelve days. This comparatively rapid ammonia produc- 
tion is readily explained in view of the recent information on the energy 
requirements of microorganisms. The molds decompose organic 
matter more readily than do the actinomycetes and many bacteria. 
They consume a great deal more energy and therefore liberate more 
nitrogen as a waste product in the form of ammonia. 


362 MICROBIOLOGY OF SOIL 

Nitrogen- fixation. Experiments on nitrogen-fixation by fungi were 
carried on by Jodin as early as 1862. He observed a rich fungus growth 
on nitrogen-free media, supplied with sugar, tartaric acid, or glycerin. 
Berthelot, Saida, Ternetz, and others also reported- fixation of atmos- 
pheric nitrogen through the activities of fungi, such as Aspergillus 
niger, Alter naria tennis and several species of Monilia, Penicillium, 
Mucorini and others. But other investigators, among them Wino- 
gradsky, Czapek and Heinze, were unable to confirm these observa- 
tions. The careful work of Goddard has also given negative results. 
Duggar and Davis, eliminating all possible errors involved in this 
study, could not demonstrate any nitrogen fixation for Aspergillus 
niger, Penicillium digitatum, Penicillium expansum, and other fungi, 
some of which commonly occur in the soil. Hence, nitrogen fixation 
by soil fungi is at best of very little importance, since even in the case 
of positive fixation the amounts are very slight. 

Nitrogen Utilization --The molds assimilate readily available 
nitrogen compounds in the presence of available carbohydrates. In 
this respect they may readily compete with higher plants in using up 
the ammonia and nitrates formed in the soil by bacteria. 

Cellulose Decomposition. The destruction of cellulose in the soil is 
due to a large extent, to the activities of soil fungi, as has been demon- 
strated by several investigators. Cellulose decomposition by fungi was 
first observed in the study of plant diseases. Van Iterson used filter 
paper for the isolation of fungi, by exposing this medium to the air for 
twelve hours. Thirty-five species of fungi were isolated thus proving 
that a large number of cellulose-destroying fungi may be present in 
the air. Appel found that certain species of Fusarium destroyed in 
fourteen days 80 per cent of the filter paper used. Marshall Ward 
and others recorded that a number of fungi are economically impor- 
tant as wood-destroyers. Spores of a pure culture of Penicillium sown 
on sterile blocks of spruce wood, germinated and grew normally. 
Sections of the wood showed that the hyphse had entered the starch- 
bearing cells of the medullary rays of the sapwood and consumed the 
whole of the starch. MacBeth and Scales found that when the medium 
is slightly alkaline, certain aerobic bacteria will play the principal 
role in the destruction of cellulose. When the medium is acid, molds 
and higher fungi become the active agents of destruction. They also 
found that the cellulose-destroying forms multiply with great rapidity 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 363 

in alkaline soils when cellulose in the form of filter paper is added. The 
power to destroy cellulose is reported for a number of species of Peni- 
cillia, A spergilli, Trichoderma and other organisms which belong to the 
common soil forms. Though the fungi may play an important part 
as cellulose destroyers also in alkaline soils, in acid soils where the 
activity of bacteria is greatly inhibited, fungi probably play a pre- 
dominant role. This fact led Marshall to conclude in 1893 that 
fungi take an active part in the mineralization of the organic matter 
in acid humus soils. 

Mycorrhiza. Apart from the so-called soil fungi, there exists another 
group known as mycorrhizal fungi. These live symbiotically on the 
roots of the higher plants. Many roots of forest trees, when examined 
carefully, show that there is a union between the mycelium of certain 
fungi, usually belonging to the fleshy fungi, and the root of the plant. 
This union is called a " mycorrhiza." The fine filaments of the fungus 
enter the cells of the root. These organisms were thought at first 
to supply the roots with water and soluble plant food from the soil. 
The power to fix atmospheric nitrogen has been ascribed to these organ- 
isms by several investigators. But aside from these useful so-called 
endotrophic Mycorrhizce, there are also the ectotrophic Mycorrhiza 
which probably live only parasitically upon the roots of plants. 

Actinomyces. The study of soil Actinomyces is nearly all of very 
recent origin. Several years ago but two soil Actinomyces had been 
definitely described, viz., Act. albus and Act. chromogenus. The work 
of Krainsky, of Conn and of Waksman and Curtis has demonstrated 
that Actinomyces are widely scattered in cultivated soils. The last- 
named investigators have shown that while the absolute numbers of 
Actinomyces decrease with depth of soil, their relative numbers are 
materially increased so that if at a depth of 25 mm. (i inch) there 
are only 6 to 10 per cent of Actinomyces and 82 to 93 per cent of 
bacteria, at a depth of 750 mm. (30 inches) the Actinomyces form 
40 to 80 per cent of the total microorganic flora of the soil. The 
numbers of Actinomyces in the surface soil vary greatly with the types 
of soil and abundance of plant food. In one instance 1,300,000 Acti- 
nomyces were found in a total of 15,000,000 bacteria per g. of rich 
meadow soil. The actinomycetes are present in the soil both in the 
form of spores and vegetative mycelium. The same species have been 
isolated from North America, Canada, Hawaiian Islands and newly 


364 MICROBIOLOGY OF SOIL 

forming soils of Tortugas Island, indicating the universal occurrence 
of these organisms in the soil. Many species of actinomycetes have 
been demonstrated to occur in the soil to the extent of millions of cells 
per gram. As to the activities of Actinomyces in the soil, Beyer- 
inck has shown that the Act. chromogenus produces an oxidizing sub- 
stance, quinon (C 6 H 4 O 2 ) which may play an important part in the 
oxidation of organic matter in the soil. Munter, Krainsky and Scales 
have demonstrated that many Actinomyces are able to decompose cellu- 
lose in the soil, and that in some instances this ability is very marked. 
Krainsky records that soil Actinomyces need very little .nitrogen for 
their life activities, and that they can get it from any available source. 
If nitrates are present, these are reduced first to nitrites, and then 
utilized. Waksman and Curtis, working with soil sterilized by steam, 
did not find any great accumulation of ammonia through the activities 
of Actinomyces, although different species seemed to show marked varia- 
tion in their power to accumulate ammonia. 

ALG.&. At times the influence of algas in changing the character of 
the soil as a culture medium for bacteria is quite considerable. As 
chlorophyll-bearing organisms they are enabled to manufacture sugar 
and starch with the aid of sunlight, and to favor thus the development 
of Azotobacter and of other microorganisms dependent for their energy 
on the organic matter in the soil. Investigators both in France and 
in Germany have found that the fixation of nitrogen in sand used for 
pot culture experiments occurs in the surface layer possessing a growth 
of alga?. The advocates of bare fallows attribute the greater pro- 
ductivity of fallowed land to the growth of algae, the accumulation of 
nitrogen through their influence and to other changes affecting the soil 
bacteria. 

PROTOZOA. It has been known for a long time that certain species 
of protozoa are common in soils and that their food consists of bacteria. 
To what extent protozoa play a part in soil fertility has not yet 
been fully explained, even though Russell and Hutchinson of the 
Rothamsted Experiment 'Station have maintained that these minute 
animals are extremely important in that they maintain a certain bac- 
terial equilibrium in the soil. Their claim is mainly based on the fact 
that partially sterilized soils (either by means of heat or antiseptics) 
soon come to contain enormous numbers of bacteria, 

It is, therefore, assumed by them that this abnormal increase is 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 365 

made possible by the destruction of the protozoa (which have a lower 
power of resistance to heat and antiseptics than bacteria) that normally 
check the increase beyond a certain point. Under the conditions re- 
corded a causal relationship obtains between an increase in numbers of 
bacteria and the rate of ammonia production, which is considered to be 
an index of fertility. 

This theory has been the basis of considerable investigation, much 
of which has failed to corroborate the above conclusions. The fact 
that there is an increase in bacterial numbers and in consequence, 
enhanced fertility of the soil may not be due to the elimination of 
protozoa but may rather be ascribed to such effects of the partial 
sterilization process as (i) increase in available food for bacteria; 
(2) rendering soil toxins insoluble; (3) destroying bacterio- toxins; 
(4) acceleration of the biological processes. 

It has even been noted in some instances that partial sterilization 
has been responsible for a decrease rather than increase in the produc- 
tion of ammonia. Such considerations, among others, have been in- 
strumental in stimulating investigation in another branch of soil fertility, 
namely, soil protozoology. There has been difficulty in establishing 
suitable methods and technic, as for example the development of 
media favorable for the isolation and culture of soil protozoa, although 
blood meal solution, hay infusion and soil extract have been used to 
advantage. The organisms have been counted in the same manner as 
bacteria, namely, by the dilution method, or by means of a standard 
platinum loop. An adaptation of the apparatus used in the counting 
of blood corpuscles has been successfully employed by Kopeloff, 
Lint and Coleman. 

A study of the morphology and life history of soil protozoa reveals 
the fact that encystment occurs under most conditions which are not 
immediately favorable, as for example slight variations in moisture 
content, or food. In point of fact this period of the protozoan life cycle 
which is analogous to the spore-forming stage of bacteria forms the 
basis for the question which arises as to the existence of protozoa, in 
their trophic form, in field soils. Of the well-defined groups of pro- 
tozoa (page 14), namely, flagellates, ciliates and amoebae, many types 
have been described. Among those occurring frequently are: Colpoda 
cucullus, Boda ovatus, Colpidium colpoda, Amceba terricola, Monas, etc. 
The requirements for maximum development in the soil for these organ- 


366 MICROBIOLOGY OF SOIL 

isms are : ( i ) A high degree of moisture, closely approximating saturation ; 
(2) an abundant supply of organic matter; (3) moderate temperature. 
The thermal death point of active forms has been found by Goodey 
to be 40 to 50, and for the cyst forms of the same organisms about 
72. The optimum temperature for most forms is about 22. 
Encystment of protozoa occurs within wide limits in an alkaline medium 
containing up to .18 per cent NaOH, and in the presence of an acidity 
represented by .09 per cent HC1. 

Protozoa are found in many greenhouse soils, due no doubt to the 
fact that they contain a high degree .of moisture and organic matter. 
However, in dealing with field soils some investigators have failed to 
isolate active forms of protozoa, whereas others record the presence of 
large numbers of these organisms. Their distribution appears to 
parallel that of bacteria, namely, the greatest number of protozoa occurs 
within the upper 100 mm. (4 inches) of soil, with a decrease down to 
300 mm. (12 inches), which represents the lower limit of their activity. 

As regards the occurrence of the various groups of soil protozoa, 
flagellates are found to be dominant over ciliates and amoebae. G. P. 
Koch has found that the development of soil protozoa in artificial 
culture solutions varies (i) with the kind of media employed; (2) the 
quantity of soil used for inoculation; (3) drying of the soil; (4) different 
kinds of soil and different soils of the same kind; (5) the temperature 
of incubation. 

While it is generally accepted that protozoa feed upon bacteria, 
until the relation that obtains between the various types of protozoa 
and the different species of soil bacteria has been more fully investigated 
the direct effect of protozoa upon bacteria must remain, to a degree, 
indeterminate. 

Soil sterilization has had a practical application in eliminating 
various diseases in greenhouses and infested fields. Partial steriliza- 
tion as employed by Russell and Hutchinson while not so drastic, 
involves serious changes in the soil, which might be considered in much 
the same light as the phenomena attending complete sterilization 
by means of heat and antiseptics. It is an established fact that 
sterilization is responsible for increased plant growth, and to explain 
this phenomenon the following theories have been advanced: 

i. R. Koch's theory of direct stimulation to plant growth a 
physiological effect of the sterilizing agency. 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 367 

2. Hiltner and Stormer's theory of indirect stimulation an altera- 
tion of the bacteriological equilibrium resulting in a marked develop- 
ment of numbers after decimation. 

3. Liebscher's view that soil sterilization may be regarded in the 
same light as a nitrogenous fertilizer. 

4. Russell and Hutchinson's protozoan theory of soil fertility. 

5. Pickering and Schreiner's contention that the alteration in 
chemical composition is largely responsible for increased plant growth. 

6. Greig-Smith and others adhering to the bacterio-toxin hypothesis. 
HIGHER PLANTS. Higher plants modify the soil as a culture 

medium for bacteria in at least three ways. The root-hairs come 
into contact with the moisture films surrounding the soil grains and 
not only modify the composition of the film water, by withdrawing a 
portion of the dissolved matter, but also change its character by secre- 
tions from the roots. The changes thus effected must, necessarily, 
modify the character of the soil and the soil solution as a culture 
medium. Again, the rapid removal of water from the soil by growing 
crops causes the film water to become more concentrated in so far, at 
least, as some salts are concerned. Modifications are, also, introduced 
thereby in the proportions of oxygen, nitrogen and carbon dioxide in 
the soil air. Finally, higher plants modify the soil environment for 
bacteria by their root and stubble residues. For example, residues of 
leguminous plants, being richer in nitrogen and possessing a narrower 
carbon-nitrogen ratio than the corresponding residues of non-legumes, 
will affect the soil somewhat differently than the latter. 

BACTERIA. Occupying, as they do, the leading role, bacteria 
demand a more detailed consideration; in fact, most of the biological 
discussions of soil are based upon a knowledge of these organisms. 

Numbers and Distribution (Bacteria in Productive and Unproductive 
Soils). The numbers of bacteria in soils well supplied with organic 
matter usually range from 3,000,000 to 200,000,000 per g., as shown 
by the agar plate method; the microscopic count will show as high as 
900,000,000 per g. of soil. These numbers vary from soil to soil, and 
from season to season for any particular soil. The numbers of fungi 
are also variable and may reach a total of 1,000,000 per g., although 
it still remains to be demonstrated whether the large numbers thus 
found represent organisms which lead an active life in the soil or only 
spores of fungi brought in by external agencies. The numbers of 


368 MICROBIOLOGY OF SOIL 

Actinomyces may reach 1,000,000 or more per g. of soil. The fungi 
almost disappear below 20 to 30 cm., while the actinomyces do not 
decrease rapidly at depths lower than 30 cm. 

Distribution at Different Depths. Most of the soil bacteria are found 
in the stratum in which the organic residues are concentrated, that is, 
in the surface soil. Immediately at the surface the rapid evaporation 
and the germicidal effect of direct sunshine act as disturbing factors, 
hence the numbers in the uppermost 25 to 50 mm. (i to 2 inches) are 
smaller than in the layer of soil immediately below. Beyond the 
depth of 20 cm. or 22 cm. (8 or 9 inches) the numbers diminish rapidly. 
Material from a depth of .6 m. to .9 m. (2 to 3 feet) is nearly sterile in 
humid regions. Differences occur, however, in keeping with the 
mechanical composition of the soil. In light, open soils the bacteria 
are not only carried down to greater depths by the percolating water, 
but can also multiply there, thanks to better aeration. On the con- 
trary, fine-grained compact soils are more effective in holding back 
suspended material and do not allow the bacteria to pass downward as 
readily. Moreover, the less thorough aeration of these soils and the 
accumulation of toxic reduction products in the subsoil serve as an 
effective check in the increase of bacteria in the deeper layers. 

In irrigated soils of the arid and semi-arid regions bacteria are dis- 
tributed at much greater depths. Their occurrence 2 m. to 3. m. 
(8 or 10 feet) below the surface is made possible not only by the better 
aeration of these soils, but by the penetration of roots to great depths 
and the accumulation there of considerable amounts of organic matter. 
The practical significance of distribution appears, among other things, 
in the use of soil for inoculation purposes; for instance, it is reported by 
Salstrom that in making peat soils arable the addition of small amounts 
of fertile loam increases to a very marked extent their crop-producing 
power. The efficiency of the inoculating material decreases as it is 
taken from the deeper soil layers. Similarly, in the use of alfalfa soil 
for the inoculation of new fields the most efficient material is secured at 
a depth between 7.62 cm. and 17.78 cm. (3 and 7 inches). 

Seasonal Variations of Bacterial Numbers and Activities. Conn has 
reported an apparent increase of bacteria in frozen soil. This increase 
seems to be due to an actual multiplication of the organisms rather than 
to a mere lifting of the bacteria from lower depths by capillary action. 
The greatest increase was found to occur during the winter in the slow- 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 369 

growing bacteria and not in those that liquefy gelatin rapidly or in the 
Actinomyces. Conn tries to account for the phenomenon by assuming 
the existence of two groups of bacteria, winter and summer bacteria. 
The latter, he thinks, prevents the former from multiplying rapidly 
in warm weather. Hence, the increase in frozen soils is not to be 
ascribed directly to the low temperature, but to the depressing effect 
of the cold upon the summer bacteria. Brown found that the soil 
bacteria diminish during the fall season with the lowering of the 
temperature, but, when the soil is frozen, an increase in numbers 
occurs. He also found frozen soils to possess a much greater ammoni- 
fying, denitrifying and nitrogen- fixing power than non-frozen soils. 
According to him, the lowering of the freezing-point of the capillary 
water, due in part to the concentration of salts at the time of freezing, 
may account for the abnormal bacterial activities. 

Vass recently pointed out that the apparent increase of bacteria in 
frozen soils is due to the breaking up of the clumps of cells rather than 
to growth and multiplication. The bacterial activities are influenced 
by freezing only in so far as it affects the physical properties of the soil. 

Morphological and Physiological Groups (Morphological Groups) - 
Rod-shaped organisms are numerically the most prominent among soil 
bacteria. They occur at times to the extent of 80 or 90 per cent of 
the total number. Spherical organisms usually constitute less than 
25 per cent of the bacterial flora. Spirilla and sarcinae are present in 
slight numbers. Conditions may occur, however, when the proportion 
of spherical organisms is markedly increased. This happens, par- 
ticularly, when large quantities of composted manure (rich in spherical 
organisms) is added to the soil. 

Conn has shown that non-spore-forming bacteria (mostly immotile 
rods) are the most abundant of all soil microorganisms. Next to them 
in abundance are the various types of Actinomyces, referred to elsewhere 
in this book. Spore-forming bacteria are also quite common, but are 
apparently of no great importance in normal soil. Among the most 
prominent soil bacteria are non-spore-forming, slowly liquefying or 
non-liquefying, short rods; rapidly liquefying, non-spore-forming, short 
rods with polar flagella represented by Ps. fluorescens; spore formers, 
which seem to come from spores instead of from active organisms. A 
few micrococci and members of the B. radicicola group have been 
demonstrated. 

24 


370 MICROBIOLOGY OF SOIL 

Among the rod-shaped species B. mycoides, B. subtilis, B. mesen- 
terictts, B. tumescens and other members of the subtilis group are quite 
prominent. Members of the amylobacter group are seldom absent. 
Members of the proteus group and various fluorescens are always 
present, while Bact. cerogenes and allied species are common inhabitants 
of the soil. 

(Physiological Groups). In the decomposition of organic matter in 
the soil certain important changes in both nitrogenous and non-nitro- 
genous material are accomplished by definite groups of bacteria. The 
breaking down of protein substances is accomplished by the forma- 
tion of ammonia, nitrites and nitrates. These in turn may be trans- 
formed back into more complex amino-compounds, peptones, and pro- 
teins, or they may be destroyed with the evolution of free nitrogen. 
Moreover, there are groups of bacteria capable of joining non-nitro- 
genous organic matter to elementary nitrogen and of producing thus 
nitrogen compounds. Again, there are groups of bacteria bearing 
distinct and important relations to the decomposition of cellulose, or 
the transformation of its cleavage products, methane and hydrogen. 
There are, likewise, definite groups of bacteria concerned in the 
transformation of sulphur and its compounds, and of ferrous compounds. 
( 

METHODS OF STUDY 

METHODS FOR COUNTING BACTERIA- -There are two methods for 
the quantitative determination of bacteria in the soil: the plate method 
and the direct count method. By the use of the plate method we can 
obtain only relative results, since not all soil bacteria are able to grow 
and develop into colonies even on the most suitable media. The plate 
method shows cells of bacteria that are able to develop under laboratory 
conditions but furnishes no direct evidence as to their exact number. 
Conn therefore suggested the direct count method, already employed suc- 
cessfully in the bacteriological examination of milk. A smear is prepared 
by spreading o.i c.c. of the soil infusion over an area of i sq. cm., then 
stained with Rose Bengal in carbolic acid. The bacteria are colored 
deep pink or red, while the mineral particles remain uncolored and most 
of the organic matter is unstained or stained yellow or light pink. 
The bacteria are then counted by means of an oil-immersion objective 
and a high power eye-piece. The actual numbers of bacteria detected 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 371 

by the microscope is probably, according to Conn, 5, 10 or over 20 times 
greater than that indicated by the plate method. The discrepancy is 
due to the failure of many cells to produce colonies rather than to the 
occurrence of large clumps that o not break up in the process of 
plating. 

QUANTITATIVE RELATIONS. Since the early work of Koch in 1881 
many investigators have determined the number of bacteria in soil 
samples, by means of the plate method. It is well known, however, 
that on ordinary gelatin or agar plates kept under aerobic conditions 
but a fraction of the soil organisms produce visible colonies. -The 
anaerobic species do not appear, nor do aerobic Azotobacter, and nitro- 
bacteria, while other common soil organisms form colonies sparingly 
or not at all. By employing synthetic agar media instead of beef broth 
gelatin or agar, Lipman and Brown have succeeded in securing the 
growth of a much larger number of colonies from any given quantity 
of soil, yet even these larger numbers were incomplete for reasons 
mentioned above. 

H. Fischer recommends a simple medium of agar to which nothing 
has been added but soil extract (prepared by extracting with a .1 
per cent solution of Na 2 CO 3 ) and potassium phosphate. Following 
the path of Lipman and Brown in reducing the content of organic 
matter, Temple employed i g. of peptone per 1. as a culture medium 
and obtained satisfactory results. Brown has further modified the 
formula of Lipman and Brown by replacing the .05 g. of peptone with 
.1 g. of albumin, and obtained results which were somewhat superior. 
In a comparison of culture media, Conn considers the former media 
undesirable for quantitative purposes because they contain substances 
of indefinite chemical composition, and offers an agar medium con- 
taining no organic matter except agar, dextrose and sodium asparag- 
inate, and also a soil-extract gelatin which is valuable for qualitative 
purposes. Other media that have been suggested, after a comparison 
of all of the above-mentioned media, are the urea-ammonium nitrate 
agar of R. C. Cook and the tap water gelatine and asparaginate agar of 
Conn. It is evident, therefore, that the results secured in the counting 
of soil bacteria have only a relative value. With the same media and 
methods some information may be secured concerning the influence 
of fertilization, tillage, liming, etc., on certain of the soil bacteria. But 
even this information must be properly discounted, since equal numbers 


372 MICROBIOLOGY OF SOIL 

do not necessarily mean equal amounts of chemical work accomplished; 
for example, there is no certainty that 1,000,000 of decay bacteria 
derived from one soil will accomplish exactly as much decomposition 
as the same number of similar organisms from another soil. Otherwise 
stated, individual cells differ in their physiological efficiency from other 
cells of the same species. 

QUALITATIVE REACTIONS. By modifying the composition of the 
culture media different physiological groups may be favored in their 
development. In this manner the silica jelly medium proposed by 
Winogradski, or the gypsum plates proposed by Omelianski may be em- 
ployed for making numerical comparisons of nitro-bacteria in different 
soils. In like manner Beijerinck's mannit agar may be used for the 
numerical comparison of Azotobacter, and other media can be adapted 
for the quantitative-qualitative determination of urea, denitrifying, 
methane, and still other physiological groups of microorganisms, 
modified Czapek's agar and Krainsky's agar can be used for actino- 
mycetes and raisin agar for molds. 

There is no doubt that the quantitative-qualitative method just out- 
lined may be made to yield valuable information. Yet it, too, possesses 
defects already noted in connection with the more strictly quantitative 
method. Apart from the vast amount of work involved in the prepa- 
ration of a large number of media and in the counting of colonies on 
many plates, this method fails to indicate differences in physiological 
efficiency. Furthermore, the colonies of the specific organisms sought 
are almost invariably accompanied by those of foreign species not 
always easily distinguished. With these limitations properly recognized 
and with further improvement in the constitution of special media the 
method may be made useful in supplementing data secured by other 
methods. 

TRANSFORMATION REACTIONS. Instead of counting soil bacteria in 
accordance with colonies produced in general or special media, soil 
investigators have attempted to measure the bacteriological functions of 
soils by comparing more or less definite quantities of the latter under 
known conditions. This method was employed by Wollny and others 
in studying the factors that affect the formation of carbon dioxide in 
soils. It was also used by Schloesing and Mii-ntz and their followers in 
similar studies on nitrate formation. A method somewhat similar in 
principle but different in its application was proposed by Remy in 


MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 373 

1902. He suggested the use of special media for the quantitative 
estimation of different physiological reactions; thus, making a i per 
cent solution of peptone and inoculating with equivalent quantities of 
soil, he caused the decomposition of the peptone and the formation of 
ammonia, and secured comparisons of the ammonifying power of 
different soils. In a similar manner he used special solutions for com- 
paring quantitatively the transformation accomplished by nitrifying, 
denitrifying or nitrogen-fixing bacteria. 

Remy's method has been extensively tested by Lohnis, Ehrenberg, 
Lipman and others. It has been shown to possess a serious defect in 
that it deals with conditions unlike those occurring in the soil itself. 
For this reason more recent investigations have been carried on in 
weighed portions of soil rather than in culture solutions inoculated with 
10 per cent of soil as is done in Remy's method. 

RATE OF OXIDATION OF CARBON. The rate of decomposition of 
humus or of other organic matter in the soil may be measured, as was 
done by Wollny, by determining the amount of carbon dioxide evolved 
in weighed quantities of material kept under definite conditions. The 
influence of temperature, moisture, aeration, organic matter, anti- 
septics, etc., has been determined in this manner. The same method 
may be used in studying decay, and factors influencing decay, in soils 
in the field. 

More recently Russell and his associates have modified the method 
in that they have determined the rate of oxidation of carbon not by 
measuring the carbon dioxide evolved, but by estimating the amount of 
oxygen absorbed. In either case decay is measured from the carbon 
standpoint. The method based on this principle should find wide 
application in future soil fertility investigations. 

Potter and Synder measured the amount of carbon dioxide evolved 
from sterilized soil when inoculated with soil emulsion or with cultures 
of molds. The latter produced in nearly all cases as much carbon diox- 
ide as the soil suspension and in some cases more. This fact led them 
to suggest that molds are probably active in normal soils. Gainey 
pointed out that there is a similarity and agreement between the curves 
representing carbon dioxide and ammonia formation in soils. The 
relative content and availability of the carbon and nitrogen sources in 
the soil influence greatly the relative amounts of carbon dioxide and 
ammonia produced. 


374 MICROBIOLOGY OF SOIL 

RATE OF OXIDATION OF NITROGEN. Another method or series of 
methods for studying decomposition processes in the soil may be based 
on the determination of nitrogen compounds formed in the breaking 
down of proteins. Two of the derivatives of protein, namely, ammonia 
and nitrate, have been used successfully to gauge the decomposition of 
organic matter in the soil. The recent results secured by Lipman and 
his associates demonstrate that ammonia formation from dried blood in 
weighed quantities of soil may serve as a very accurate measure of 
decay from the nitrogen standpoint. Corresponding determination of 
nitrates may similarly be employed in tracing protein cleavage and 
transformation as influenced by the various factors of season, soil 


and cultivation. 

ADDITION OF NITROGEN. At least one other bacteriological factor 
in soils should be mentioned here as deserving attention in a systematic 
study of soil fertility from the nitrogen standpoint. It is known that 
Azo-bacteria are widely distributed in arable soils, and that they are 
more prominent in some regions than they are in others. The student 
of soil fertility finds it desirable, therefore, to study azotofication in 
different soils, and employs (for this purpose) mannit solutions like 
those proposed by Beyerinck, sand cultures supplied with sugar solu- 
tions like those proposed by Fischer, or weighed quantities of soil mixed 
with sugar as suggested by Koch. 

The methods referred to above make possible thus the study of 
ammonification, nitrification and azotofication under controlled con- 
ditions and permit, thereby, the measure of bacteriological factors in 
soil fertility from the nitrogen standpoint. 

REACTIONS CONCERNING CALCIUM, MAGNESIUM, SULPHUR AND 
PHOSPHORUS. In addition to the purely chemical methods available for 
the study of these constituents, microbiological methods have also been 
suggested. In some of his still unpublished experiments with A zoto- 
bacter Lipman employed solutions of mannit in distilled water, provided 
with small quantities of sterile soils which were to supply the organisms 
with the essential mineral constituents. In this manner interesting 
data were secured on the availability of phosphorus compounds in 
different soils; similarly, Christensen has suggested the use of Azoto- 
bader for determining the lime requirements of soils, and Butkevich 
has experimented with cultures of Aspergillus niger in determining the 
availability of the mineral constituents. 


CHAPTER II 

THE DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 

CARBOHYDRATES 

Origin. The sugars, starches, vegetable gums and allied pectine 
substances, as well as the cellulose, contained in roots and other crop 
residues add large quantities of carbohydrates to the soil. The crop 
residues are augmented still further by green manures and animal 
manures whenever these are used. A good growth of timothy, for 
example, may increase the content of organic matter in the surface 
soil by 250-500 kg. (500 or 1,000 pounds) per acre, and three-quarters 
of this consists of carbohydrates. In the same manner, a green ma- 
nure crop, or an application of barnyard manure may add to the land 
as much as 1,500 pounds, or even more, of carbohydrates per acre. 
These carbohydrates contain a large proportion of cellulose. 

The Decomposition of Cellulose. Pure cellulose (page 237), 
(C 6 HioO5) x is a rather inert substance, as exemplified by the resistance 
of cotton and flax fiber to decomposition processes. It is well known, 
nevertheless, that even cellulose is in the end decomposed and resolved 
into simple compounds. Plant roots, leaves and stems, as well as the 
trunks of fallen trees, gradually disintegrate and vanish. Under favor- 
able conditions this may proceed rapidly, as is indicated by the process 
in septic tanks, or in manure heaps on the one hand, and in open 
sandy soils on the other. The disappearance of cellulose may be ac- 
complished by (a) anaerobic organisms, (b) by aerobic organisms, (c) 
by denitrifying bacteria, (d) by molds and (e) by actinomycetes usually 
classed as higher bacteria. 

The Production of Methane and Hydrogen. The decomposition 
of pure cellulose and the formation of methane and hydrogen mixed 
with other gases was first noted by Popov in 1875. Some years 
later Tappeiner and also Hoppe-Seyler confirmed Popov's observa- 
tions that nearly pure cellulose in the form of Swedish filter-paper, or 
cotton fiber may be fermented by bacteria with the evolution of 
methane, carbon dioxide and occasionally also of hydrogen. These 

375 


376 MICROBIOLOGY OF SOIL 

investigators ascribed the decomposition of cellulose to an organism 
found by Trecul in decomposing vegetable materials, and named by 
him Amylobacter in 1865, because of the blue color assumed by it when 
stained with iodine. 

Subsequent investigations by Omelianski begun in 1894 and con- 
tinued through a period of years demonstrated the presence of specific 
anaerobic organisms in decomposing cellulose. He described two dis- 
tinct species of long, slender bacilli, assuming the clostridium form when 
in the spore stage. Morphologically the organisms can hardly be dis- 
tinguished, but physiologically they show important differences in that 
one causes the fermentation of cellulose with the production of gases 
consisting of carbon dioxide and methane, while the gases produced by 
the other consist of carbon dioxide and hydrogen; hence the one is desig- 
nated by Omelianski as the methane bacillus and the other the hydro- 
gen bacillus. These organisms do not stain blue with iodine, and do not 
belong, therefore, to the butyric bacilli designated as Amylobacter by 
earlier investigators. Omelianski's investigations make it appear that 
the butyric organisms are not capable of fermenting cellulose proper. 

In culture solutions containing mineral salts and nitrogen in the form 
of ammonium compounds the decomposition of filter-paper and other 
forms of cellulose proceeds with considerable rapidity. Calcium car- 
bonate must be added to neutralize the acids formed, otherwise the 
fermentation soon comes to a standstill. In one of Omelianski's experi- 
ments begun in October, 1895, and ended in November, 1896, 3.3471 
g. of cellulose was decomposed by a nearly pure culture of hydrogen 
bacilli. The products consisted of 2.2402 g. fatty acids, .9722 g. carbon 
dioxide and .0138 g. of hydrogen, a total of 3.2262 g. which nearly 
accounts for all of the cellulose destroyed. The fatty acids were made 
up of butyric and acetic acids with a slight proportion of some higher 
homologue, probably valerianic acid. 

In a similar experiment with an apparently pure culture of the 
methane bacillus, begun in December, 1900, and ended in April, 1901, 
fermentation began after an incubation period of about one month, and 
the entire volume of gas gradually evolved was 552.2 c.c. This mix- 
ture consisted of 190.8 c.c. methane and 361.4 c.c. carbon dioxide. The 
products formed from the 2.0065 cellulose consumed included 
1.0223 g. fatty acids, .8678 g. carbon dioxide and .1372 g. of methane, 
or a total of 2.0273 g- The slight difference in weight in favor of the 


DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 377 

fermentation products falls within the limit of error. These experi- 
ments show that about one-half of the fermentation products is 
gaseous and that the other half consists of acetic and butyric acids. 

McBeth has shown that the cellulose-dissolving bacteria are unable 
to produce gaseous products in cellulose or sugar solutions in which 
they make a luxuriant growth. The compounds formed, under natural 
conditions, by the cellulose dissolving bacteria are used by other micro- 
organisms and split into simpler products. The carbon dioxide formed 
is presumably due in all cases to secondary fermentations. The 
organic acids noted by early investigators were, for the most part at 
least, presumably due to secondary fermentations and not to the action 
of the cellulose-dissolving forms. 

The Oxidation of Methane, Hydrogen and Carbon Monoxids. Aside 
from cellulose, methane may also be produced from various other carbo- 
hydrates, organic acids and proteins. Large amounts of methane are 
thus contributed to the atmosphere by swamps, manure heaps and low- 
lying meadows. In a purely chemical way methane may also be set 
free from volcanoes and mineral springs. The constant additions of 
methane, ethane, hydrogen and carbon monoxide represent a consid- 
erable amount of potential energy. It is important to know, therefore, 
whether these materials are at all utilized. 

That methane may be utilized by bacteria as a source of energy was 
first shown by Sohngen in 1905. He isolated an organism named by 
him B. methanicus that showed itself capable of growing in inorganic 
solutions confined over an atmosphere of methane, oxygen and nitrogen. 
The methane gradually disappeared and there were formed considerable 
quantities of organic matter. The ability to oxidize methane has been 
claimed for a number of other organisms by Sohngen and others. 

Early observations on the ability of moist soil to cause the oxidation 
of hydrogen are credited to de Saussure (1838). Many years later 
(1892) Immendorff called attention to the same fact. It was not, 
however, until 1905 that the oxidation of hydrogen was shown to be a 
specific biological process. In that year papers by Sohngen and Kaserer 
reported experiments wherein inorganic solutions confined under an 
atmosphere of hydrogen, oxygen and carbon dioxide and inoculated with 
very small quantities of soil developed a bacterial membrane at the 
surface. The hydrogen was oxidized and organic matter produced at 
the expense of the energy set free. The observations just noted have 


378 MICROBIOLOGY OF SOIL 

been confirmed by other investigators, by means of mixtures and single 
species of soil bacteria. Finally it should be added here that B. 
oligocarbophilus previously isolated by Beijerinck and Van Delden is 
able, according to Kaserer, to oxidize also carbon monoxide. 

The Cleavage and Fermentation of Sugars, Starches and Gums- 
Sugars (page 233) are a very acceptable source of food and energy 
for soil bacteria. A culture solution containing suitable mineral 
salts and sugar ferments readily when inoculated with a small amount 
of fresh soil. When no combined nitrogen is added, Azotobacter, or B. 
(Clostridium) pasteurianus (or both), may come to the fore. The cleav- 
age products then include alcohols, organic acids and carbon dioxide. 
With B. (Clostridium} pasteurianus butyric acid is one of the prominent 
cleavage products. When combined nitrogen is also added to the 
culture solution other organisms will develop prominently, notably 
members of the subtilis group, butyric bacteria, aerogenes, etc. In the 
soil itself the addition of sugar leads to a very marked increase in 
number and, if acid production is favored, molds may subsequently 
become prominent. In general it may be said that butyric, propionic, 
acetic, formic and lactic acid, and ethyl, propyl, butyl and iso-butyl 
alcohol are common cleavage products. 

In the case of starch, pectins and pentosans, similar conditions hold 
good. Diastatic enzymes seem to be produced by various bacteria 
as well as by molds and actinomycetes. Members of the subtilis group 
and B. Huorescens seem to be able to transform starch into sugar with- 
out difficulty. It needs hardly be added here that the vast quantities 
of organic acids and of carbon dioxide thus formed must play an im- 
portant role in the breaking down of the mineral constituents in the 
soil. 

FATS AND WAXES 

Origin and Decomposition. Plant substances contain varying 
proportions of fats and waxy materials. In the dry matter of grasses 
and cereal straw crude fat is usually present to the extent of 1.5 to 
2.0 per cent. In hay made from clover and other legumes the propor- 
tion of crude fat is rather more than 2 per cent. In cereal grains it 
may range up to 4 or 5 per cent while in soy beans the content of 
crude fat is 19 per cent, in germ oil meal 22 per cent and flax seed 
meal 34 per cent. 


DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 379 

Under the influence of enzymes produced by molds, yeasts and 
bacteria the fatty acids occurring as glycerides are decomposed into 
glycerin and fatty acids. The extent of fat decomposition, brought 
about largely by molds in the opinion of some, is shown by numerous 
experiments with peanut cake, olive press cake, cottonseed meal, 
almond oil, corn meal, etc. In a number of these experiments Asper- 
gillus niger seemed to be particularly efficient in decomposing fats. 
Analogous decomposition processes may occur in the soil as proved by 
the experiments of Rubner. 

ORGANIC ACIDS 

Soiirce.--The cleavage products of proteins include large quantities 
of amino-acids. The latter are still further transformed and yield a 
variety of fatty acids. The carbohydrates being present in larger 
quantities than the proteins are still more important as a source of 
organic acids. Finally, the fats, gums, and higher alcohols contribute 
additional quantities of the latter. Among the more simple acids, 
acetic, propionic, butyric, succinic and lactic are common. The extent 
of acid production was already indicated in connection with cellulose 
decomposition by the methane and hydrogen bacilli. Apart from these 
organisms, organic acids are formed by nearly every important species 
of soil bacteria; moreover, the tissues of dead plants and animals are 
not the sole source of organic acids in the soil. According to Stoklasa 
conditions may occasionally occur in the latter, especially when 
atmospheric oxygen is excluded, that favor the excretion by plant roots 
of appreciable quantities of acetic acid. 

Aside from the organic acids produced by bacteria, we must also 
consider the acids produced by molds; among these oxalic and citric 
acids are most important. Certain members of the Aspergillus niger 
group are able to convert as much as 40 per cent of the sugar in solution 
into citric acid; the latter is then further oxidized into oxalic acid. In 
addition to the Aspergilli, several Penicillia, Mucors, Absidia and other 
molds, which have been isolated from the soil, are able to produce citric 
or oxalic acids, or both. The acid produced in the culture medium is 
either allowed to accumulate or is further oxidized. Aspergillus niger 
oxidizes sugar first into citric acid, the latter is then oxidized to oxalic 
acid and finally to carbon dioxide. 


380 MICROBIOLOGY OF SOIL 

Transformation and Accumulation. Salts of organic acids are 
suitable as food for a wide range of soil bacteria. Azotobacter will 
readily make use of acetates, propionates and butyrates. A number of 
denitrifying bacteria will grow vigorously with citrates as the only 
source of organic nutrients. The fermentation of lactates by butyric 
bacteria has been known for a long time. The decomposition of 
malates, succinates, tartrates and valerates may be accomplished by 
various species, and even simple compounds like formates may yield 
food and energy to certain soil bacteria, among them B. methylicus 
studied by Loew and his associates. It is evident, therefore, that 
organic acids are not liable to accumulate in well- ventilated soils. 
Molds, as well as bacteria, destroy them rapidly, and carbonates, 
carbon dioxide and water are the final products of the decomposition 
of non-nitrogenous organic matter. 

Notwithstanding the ready decomposition of the more simple 
organic acids in the soil, it is well known that arable soils are frequently 
acid. This acidity is largely due to the so-called "humic acids," 
organic compounds whose composition is not well understood. They 
are composed, to some extent, of rather complex organic acids or of their 
acid salts. Continued cultivation seems to favor the accumulation of 
these acid compounds, partly on account of the diminished supply of 
lime and of other basic materials in older soils. When these soils are 
limed the humic acids and acid humates are changed into neutral com- 
pounds and are then subject to more rapid decomposition by micro- 
organisms. According to the investigations of Blair the average acid 
soil in Florida requires 1,500 pounds of lime (CaO) per acre to neutralize 
the acidity to a depth of 84 mm. (9 inches), This means an acidity 
equivalent to more than one ton of hydrochloric acid per acre. In 
peat and muck soils the acidity is equivalent to many times this 
amount of hydrochloric acid. 

PROTEIN BODIES 

Amount and Quality. The protein content of farm crops that 
leave residues in the soil is variable, but in all cases quite considerable. 
Dried corn stalks contain 5 per cent of protein, timothy hay 6 per cent, 
red clover hay 12 per cent or more, alfalfa hay 15 or 1 6 per cent. Even 
wheat and rye straw may contain as much as 3 per cent of protein. 
Cotton-seed meal and other oil cakes, tankage, ground fish, hair and 


DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 


wool waste and dried blood (all used more or less extensively as sources 
of nitrogen to crops) are made up in a large measure of protein 
compounds. 

Being derived from plant residues, from microorganic, insect and 
animal remains, and from fertilizers and manures applied, the nitrogen 
in the soil humus exists, for the most part, in the form of protein com- 
pounds. Hilgard reports the following humus and nitrogen content, 
as based on the analyses of a large number of samples of humid, 
semi-arid and arid soils. 


Drnt 
per cent 


( Nitr ogen in (Nitrogen 

humus). soil), 

per cent per cent 


in 


Arid uplands 


O. QI 


I 1 ?. 21 


o. 1^5 


Sub-irrigated arid soils 


i .06 


8.38 


o.ooo 


Humid soils from humid and arid regions 
(California) 


2 .4$ 


"? 2Q 


O. I*<) 


Humid soils from other states 


7 .01 


3.78 


o. 205 


Taking the weight of an acre-foot of dry soil at 2,000,000 kg. 
(4,000,000 pounds) and multiplying the nitrogen by 6.25 (the factor 
usually employed for converting nitrogen into protein) we find the 
protein content of these soils to range from about 11,339 kg. (25,000 
pounds) per acre to nearly three times as much. Similarly, the 
Illinois Experiment Station reports quantities of nitrogen equivalent 
to 3> I 75 to 4,989 kg. (7,000 to 11,000 pounds) per acre to a depth of 
1 01. 6 cm. (40 inches) in gray silt loams, of the lower Illinoisan glacia- 
tion. In the brown silt loams the amount of nitrogen to the same depth 
is usually more than 4,535 kg. (io;ooo pounds) per acre; occasionally 
it is more than 9,071 kg. (20,000 pounds) per acre. In one instance a 
black clay loam of the late Wisconsin glaciation is reported to have 
about 13, 154 kg. (29,000 pounds) of nitrogen per acre, to a depth of 
101.6 cm. (40 inches). This would be equivalent to more than 81,646 
kg. (180,000 pounds) of protein; of course, not all of the nitrogen in the 
soil exists in the form of protein, some of it occurring as amino-com- 
pounds, and a small portion as ammonia and nitrates. Nevertheless, 
by far the greatest part of it occurs as protein compounds. 

The protein compounds of the soil humus must be considered from 
the standpoint of quality as well as from the standpoint of quantity. 
It is well known that fresh plant residues are attacked more readily by 


382 MICROBIOLOGY OF SOIL 

microorganisms than older plant substances. For this reason soils 
frequently supplied with fresh organic material supply greater amounts 
of available food -to crops than similar soils whose organic matter con- 
sists largely of older residues. 

Carbon-nitrogen Ratio -Tine decomposition of organic matter is 
readily influenced by the relative content of nitrogenous and non-ni- 
trogenous compounds. Substances of animal origin yield relatively and 
absolutely more available nitrogen in a given length of time than sub- 
stances of plant origin. The difference noted is due largely to the 
greater proportion of protein in the animal materials; in other words, 
to the narrower carbon-nitrogen ratio. On this basis Hilgard attempts 
to explain the adequacy of the small proportion of humus in arid 
and semi-arid soils. Because of the narrower carbon-nitrogen ratio 
the humus compounds in these soils are decomposed with greater 
rapidity and yield a sufficient amount of ammonia and nitrate to supply 
the needs of the crop. 

But when plant substances alone are considered the statement just 
made requires qualification. It is true that cotton-seed meal or linseed 
meal, having a narrower carbon-nitrogen ratio, will decay more readily 
than corn-meal or wheat flour. It is also true that any given plant sub- 
stance, as it undergoes decay, will lose in proportion more carbon than 
nitrogen. Older humus has, therefore, a narrower carbon-nitrogen 
ratio than humus of recent origin. The former is more resistant to 
decay, however, than new humus. In a concrete way, on the other 
hand, it may be stated that fresh vegetable material of a narrow car- 
bon-nitrogen ratio will decay more rapidly than fresh vegetable material 
of a wide carbon-nitrogen ratio. The reverse, nevertheless, is true of 
vegetable materials in advanced stages of decay. Under any given 
climatic conditions and in any given soil type, the carbon-nitrogen 
ratio may give important indications only as to the availability of the 
humus nitrogen. Lawes and Gilbert, as quoted by Hall, found the 
following carbon-nitrogen ratio in the organic matter of different soils: 

Cereal roots and stubble 43 -o 

Leguminous stubble. 23.0 

Dung 18.0 

Very old grass land 13.7 

Manitoba prairie soils 13 .o 

Pasture recently laid down 11.7 

Arable soil 10 . i 

Clay subsoil 6.0 


DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 383 

Hall concludes, therefore, that humus with a wide carbon-nitrogen 
ratio is more valuable than humus with a narrow carbon-nitrogen ratio, 
since the latter will be attacked more easily by the soil bacteria. Brown 
and Allison indicate that there might be a possibility of applying ma- 
terials of a wide carbon-nitrogen ratio to supply the deficiencies of 
organic matter on the basis that the former may have the same or 
better effect on bacterial activities such as azofication, or non-symbiotic 
nitrogen fixation. 

THE TRANSFORMATION OF NITROGEN COMPOUNDS 

AMMONIFICATION. Experimental Study. By ammonification is 
meant the production of ammonia by bacteria out of protein substances 
or their cleavage products. That ammonia production in the soil is 
a biological process was first demonstrated by Miintz and Coudon in 
1893. These investigators showed that no ammonia is formed in sterile 
soils. They also showed that ammonia may be produced out of nitro- 
genous organic matter by molds as well as by bacteria. Marchal not 
only confirmed these observations, but proved that various micro- 
organisms differ markedly in their ability to produce ammonia. Of 
the several species of bacteria tested by him, B. mycoides (one of the 
common soil bacteria) proved itself particularly efficient in the breaking 
down of nitrogenous materials and the production of ammonia. 

Since the publication of these experiments a large number of investi- 
gators, both in Europe and America, have studied ammonia production 
in culture solutions as well as in the soil itself. It has been shown that 
under favorable conditions the breaking down of protein compounds and 
the formation of ammonia may be very rapid; for instance, in some ex- 
periments carried out by Lipman and his associates the following pro- 
portions of nitrogen were transformed into ammonia in the course of 
six days: 

Dried blood 16 . 74 per cent 

Concentrated tankage 56.66 per cent 

Ground fish 47. 16 per cent 

Cotton-seed meal ; 4-95 per cent 

Bone meal 16 . 65 per cent 

Cow manure, solid and liquid excreta 32 .60 per cent 

Cow manure, solid excreta 5-39 per cent 

The experiments were carried out in equal quantities of soil and with 
equivalent quantities of nitrogen in the different substances. It will 


384 MICROBIOLOGY OF SOIL 

be observed that more than 56 per cent of the nitrogen in the con- 
centrated tankage was transformed into ammonia, whereas under the 
same conditions cotton-seed meal yielded less than 5 per cent. 

Mechanism of Ammonia Production.- -The relatively large protein 
molecules are readily broken into larger or smaller fragments. This 
may be accomplished by purely chemical means, as, for instance, by 
boiling with acids or alkalies, or by biological activities. Among the 
first cleavage products albumoses and peptones are quite prominent. 
These in turn undergo further cleavage and the various amino-acids 
and their derivatives, as well as ammonia, make their appearance. In 
so far as the different species of bacteria are concerned, the "hydrolysis of 
proteins seems to depend, to a marked extent, on the ability to secrete 
proteolytic enzymes. With the aid of such enzymes the proteins are 
more readily hydrolyzed and further changed into amino- and hydroxy- 
acids, ammonia and carbon dioxide. 

Influence of Soil and Climatic Conditions. Ammonia production in 
the soil is affected by (a) its mechanical and chemical composition; by 
(b) the amount and distribution of rainfall; by (c) the prevailing tem- 
peratures; by (d) fertilizer treatment; and by (e) methods of tillage and 
cropping. The mechanical composition of the soil influences the pro- 
portion of aerobic and anaerobic species, while the chemical composi- 
tion, particularly that of the humus, influences the rate of multiplica- 
tion and the character of the chemical transformation accomplished. 
It is well known, for example, that additions of fresh organic matter 
intensify the rate of decomposition of the soil humus, and, likewise, 
ammonia production as has been already demonstrated by Breal. In a 
more general way it was proved by Lipman and his associates that, 
with a constant bacterial factor, ammonia production in soils varies with 
the chemical and mechanical composition of the latter. In some of 
these experiments 100 g. portions of different soils were each mixed with 
5 g. of dried blood, sterilized in the autoclave, cooled and inoculated 
with equal quantities of infusion from fresh soil. The following 
amounts of ammonia nitrogen were produced in six days: 

Soil Ammonia nitrogen found 

A 31.62 mg. 

B 68 . 29 mg. 

C 1 1 7 . 06 mg. 

D 107.16 mg. 

E 156.47 


DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 385 

With all other factors constant, chemical and mechanical differences 
in the soil used were responsible for striking variations in ammonia 
production, as indicated by the figures given above. 

The influence of temperature and moisture conditions is fully as 
important as that of the chemical and mechanical composition of the 
soil. The following data secured by Lipman may be cited in this 
connection as showing the effect of moisture: 

One-hundred-gram quantities of air-dried soil were each mixed with 
3 g. of dried blood and varying amounts of water added. The ammonia 
formed was distilled off and determined at the end of eight days. 
The amounts of ammonia nitrogen found were as follows: 

Water added Ammonia nitrogen found 


c.c ...................................... 4-13 

1 c.c ...................................... 4-13 mg. 

3 c.c ...................................... 5 . 40 mg. 

5 c.c ...................................... 10 . 64 mg. 

7 c.c ...................................... 26.37 mg. 

10 c.c ...................................... 49-57 mg. 

12 c.c ...................................... 70.71 mg. 

15 c.c ...................................... 93.90 mg. 

It appears, therefore, that ammonia production in soils rises or falls 
as the rainfall or irrigation is increased or decreased, or as the soil water 
is more or less thoroughly conserved by proper methods of tillage. In 
the same way, seasons of high temperature favor ammonification while 
seasons of low temperatures discourage it. This point is well illustrated 
by the observations of Marchal that at o to 5 only traces of ammonia 
were formed in his culture solutions; that at 20 ammonia production 
was quite marked, and that at 30 the maximum was reached. More- 
over, apart from the seasonal variations in any one locality, there is a 
wide range in ammonia production, as we pass from the torrid to the 
temperate and from the latter to the frigid zones. 

Species and Numbers. Ammonia production is a function common 
to most soil bacteria. In the earlier experiments of Marchal, seventeen 
out of the thirty-one species tested were found capable of producing 
ammonia. Prominent among these ammonifiers were B. mycoides, 
B. (Proteus) vulgaris, B. mesentericus vulgatus, B. janthinus, and 
B. subtilis. Of a considerable number of soil bacteria tested by 
Chester all but one were observed to produce ammonia. In Gage's 
experiments with sewage bacteria, seventeen out of twenty species 

25 


386 MICROBIOLOGY OF SOIL 

tested proved to be ammonifiers. Similarly, a number of species tested 
by the writer, among them B. coli, B. choleras suis, B. (Proteus) vulgaris, 
B. subtilis, B. megatherium, etc., all produced ammonia in meat infusions. 
A mass of additional data, accumulated by different investigators, 
furnishes further proof that ammonia production is a common function 
of soil bacteria. 

The more prominent ammonifiers, including members of the B. 
subtilis group and certain strep tothrices, are numerically important in 
all arable soils. Their numbers are affected, however, by the amount 
and composition of the soil humus. It has been found, for instance, 
that additions of straw and of strawy manure increase markedly the 
numbers of B. subtilis and of other members of the group. An increase 
in the numbers of certain ammonifiers is caused also by additions of 
lime or of green manure. For example, in experiments carried out by 
Lipman and his associates portions of fertile soil inoculated with B. 
mycoides were found to contain, a month later, 2,000,000 of bacteria per 
g. of soil. In similar soil portions that had also received additions 
of grass the number was twice as great. 

More recent investigations (Temple, Waksman) have shown that 
ammonification tests are of little value in determining the nature of the 
microbial soil flora, since the rate of ammonia production is largely 
controlled by the soil medium. If the soil is suitable, there will usually 
be found enough microorganisms capable of changing the protein nitro- 
gen into ammonia. Temple has suggested the use of ammonification 
as a test for soil fitness. 

Ammonification should be studied not only in the light of decompo- 
sition proteins and protein derivatives in the soil, but also from the 
point of view of energy sources in the soil. Microorganisms can use 
both carbohydrates and proteins as sources of energy. There is a great 
deal more of the carbon compounds oxidized to supply the required 
energy than there is nitrogen consumed in the normal metabolism of 
the microbe. The addition to any soil of definite amounts of protein 
with varying amounts of available carbohydrates will lead to the 
following results: ammonia will be accumulated in the soil to which the 
protein alone has been added, the amounts of ammonia increasing with 
the period of incubation up to a certain point; where only small quanti- 
ties of carbohydrates have been added there will be at first no ammonia 
produced, but soon the ammonia will begin to accumulate, so that the 
actual quantities of ammonia may become in a few days even greater 


DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 387 

than in the soil where no carbohydrates have been added; in soils to 
which, aside from the protein, large amounts of available carbohydrates 
have been added, no ammonia or only traces of it will be found. 

Ammonia is produced by microorganisms chiefly in the deanniza- 
tion of the amino acids; when the carbon part of the molecule is used to 
supply the energy required and the nitrogen part is not consumed in the 
process of metabolism, it is left as a waste product in the form of 
ammonia. When there is in the soil, in addition to the proteins and 
protein decomposition products, a sufficient amount of available carbo- 
hydrates, the microorganisms will use the latter as a source of energy 
and will attack the proteins only in so far as they need nitrogen for their 
metabolism. In that case no ammonia will accumulate in the soil; 
such as is produced will probably be assimilated by the microbes. 
But, when there is an insufficient amount of available carbohydrates, 
the microorganisms are compelled to use the proteins not only as sources 
of nitrogen, but also as sources of energy. More carbon will then be 
oxidized to supply the necessary energy than there will be nitrogen 
consumed; the excess of nitrogen will be left in the medium as a waste 
product in the form of ammonia. The presence of only small amounts 
of available carbohydrates will check for a short period the accumula- 
tion of ammonia, but will also result in more active microbial flora 
The latter, after all the carbohydrate is used up, will attack the proteins 
present and may produce larger quantities of ammonia than if no 
carbohydrate had been added. 

Rate of Ammonia Production. Miyake, using the results obtained 
by Lipman, and Waksman, in his work on the ammonia production 
byAspergillusniger, have shown that the rate of ammonia accumulation, 
whether by a pure culture or by a mixed culture, is an autocatalytic 
reaction. The rate of ammonia accumulation is at first slow, then it 
begins to fall off and finally comes to a standstill. 

Relative Efficiency of Different Species. In Marchal's experiments 
already referred to, the species employed showed marked differences in 
their ability to produce ammonia out of egg albumin. The following 
proportions of the protein nitrogen were converted into ammonia in 
twenty days: 

B. mycoides 46 per cent B. subtilis 23 per cent 

B. (Proteus) milgaris 36 per cent B.janthinus 23 per cent 

B. mesentericus vulgatus.. 29 per cent B. fluorescens putidus 22 per cent 

Sarcina lutea 27 per cent B. fluorescens liquefaciens . 16 per cent 


388 MICROBIOLOGY OF SOIL 

Furthermore, apart from the variations from species to species, differ- 
ences have been observed by Marchal and many other investigators 
between one strain and another of any single species isolated from the 
same or different soils. It must be remembered, therefore, that in the 
study of ammonification in soils and culture solutions, due considera- 
tion should be given to differences in physiological efficiency as they are 
manifested by strains and species of microorganisms. 

Apart from the ammonifying bacteria already mentioned there is a 
group of organisms studied by Muller, Pasteur, van Tieghem, Leube, 
Miquel, Beyerinck and others. These are the so-called urea bacteria, 
capable of intensive transformation of urea and allied compounds into 
ammonium carbonate, by means of the enzyme urease. 

NH 2 

CO + 2 H 2 = (NH 4 ) 2 CO 3 

NH 2 

Morphologicall ythese organisms include spherical and rod forms, 
spore-bearing and non-spore bearing species. Most of the urea bacteria 
are particularly prominent in the transformation of animal manures. 

Ammonifying Efficiency. Lipman and Burgess have found marked 
differences in the ammonifying efficiency of fifteen organisms in pure 
cultures using peptone, bat guano, sheep and goat manure, dried 
blood, tankage, cottonseed meal and fish guano. The nature of the 
soil as well as the nature of the nitrogenous material markedly modify 
an organism's ammonifying power. B. tumescens on the whole appears to 
have been the most efficient organism tested. Comparing these findings 
with those of Marchal the former have obtained results in soils, while 
the latter 's work was with solution cultures, the application of which 
to soil conditions is not always permissible. In point of fact the am- 
monifying efficiency of organisms is greater in sandy soil and possi- 
bly in others than in solutions, as Lipman and Burgess have obtained 
a transformation of 41.98 per cent of peptone in nitrogen and 36.06 
per cent of bat guano nitrogen into ammonia by Sarcina lutea and 
B. mycoides, respectively, in twelve days at temperatures between 
27 and 30, while Marchal obtained similar transformations in 
thirty days at 30 in albumen solutions. 


DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 389 

It is also of interest to note that investigations with soil fungi have 
revealed the fact that certain species are even more efficient am- 
monifiers than B. mycoides. McLean and Wilson, Waksman, Cole- 
man and Kopeloff have worked with organisms like Trichoderma 
koeningi which is capable of transforming more than 50 per cent of 
the nitrogenous material added in such experimentation. 

NITRIFICATION. Experimental Study. --The term nitrification refers 
to the oxidation either of ammonia or of nitrites to nitrates. In a 
broader sense nitrification may be defined as the production of nitrates 
from decomposing organic matter. Saltpeter or niter, the terms 
formerly applied to potassium nitrate, possessed, for a long time, a 
peculiar interest because of its relation to gunpowder. Whether it be 
true or not that gunpowder was known to the Chinese before the be- 
ginning of the present era, there is no doubt that for several centuries 
it played an important part in the political and economic history of 
Europe. The large quantities of gunpowder consumed in the almost 
incessant wars created a steady demand for saltpeter that was not 
readily met by the saltpeter refiners of India, Hungary and Poland. 
European nations, particularly France, were therefore thrown on their 
own resources and were forced to develop the domestic production of 
saltpeter. The industry came under government control and experts 
were appointed to study the so-called saltpeter plantations and the 
conditions affecting the appearance and increase of nitrates in com- 
post heaps and in the soil. Much knowledge was thus gained about 
nitrification even though it was not suspected that living organisms 
were concerned in the process. 

With the rapid development of chemistry in the latter half of the 
eighteenth century a nearer approach was made to the understanding 
of the true character of nitrification. The observations of Cavendish 
in 1784 that potassium nitrate is formed when electric sparks are passed 
through air confined over a solution of potassium hydrate formed the 
starting point for various theories that attempted to account for nitrate 
formation on the basis of purely chemical reactions. The formation of 
nitric acid and of its salts was thus assumed to be due to electric dis- 
charges in the atmosphere, to combustion processes in nature, or to the 
oxidation of organic matter and of calcium, magnesium, iron and man- 
ganese compounds in the soil. Much credence was given to the latter 
explanation because of the almost universal occurrence of nitrates in 
arable soils. 


390 MICROBIOLOGY OF SOIL 

The first indication that nitrate production in the soil and in de- 
caying organic matter is due to biological activities was given by 
Pasteur in 1862. A few years later Miiller expressed his belief in the 
biological origin of nitrates and nitrites in sewage and drinking water. 
It was not, however, until 1877 that the true character of nitrification 
was made clear. In that year Schloesing and Miintz demonstrated 
that dilute solutions of ammonia could be changed into nitrate by being 
passed slowly through long tubes filled with soil. The amounts of 
nitrate nitrogen found in the leachings corresponded almost exactly 
to the amount of ammonia nitrogen used up. When the soil in the 
tubes was first sterilized by heating or by means of chloroform and other 
germicides, the ammonia passed through unchanged. But when soils 
sterilized by heat or chloroform were reinfected with small quantities 
of fresh soils nitrification again proceeded in a normal manner. 

The biological nature of nitrification having been thus established 
numerous investigators tried to isolate the specific organisms in pure 
culture. A large amount of work in this direction was done by 
Schloesing and Miintz, Celli and Marino-Zuco, Munro, Warington, the 
Franklands and many others. A large number of bacteria, yeasts and 
molds were tested with negative results. Warington, who gathered 
a great mass of valuable information about nitrification, almost 
succeeded in securing pure cultures of nitrifying bacteria. Finally, 
Winogradski showed in 1890 not only that nitrification is caused by 
specific bacteria, but explained also why the others failed in securing 
pure cultures. He proved that these organisms do not develop colonies 
on the ordinary gelatin and other organic media, a fact whose recog- 
nition was largely responsible for his successful solution of the problem. 
The medium subsequently employed by him consisted of silica jelly 
properly supplied with inorganic nutrient salts. After him other in- 
vestigators proved that agar, deprived of its soluble organic matter, 
gypsum and sandstone disks, filter-paper pads, etc., could be used 
effectively as solid media. 

Nitrous and Nitric Bacteria. Winogradski' s investigations led to 
the conclusion, foreshadowed by the earlier work of the Franklands and 
Warington, that the oxidation of ammonia proceeds in two stages, viz., 


(1) 2 NH 3 + 3O 2 = 2HNO 2 + 2 H 2 O 

(2) 2 HNO 2 + O 2 = 2 HNO 3 


DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 391 

The organisms oxidizing ammonia to nitrites, and designated as 
nitrous or nitrite bacteria, were called by Winogradski Nitrosomonas 
and Nitrosococcus. The former include species or varieties isolated 
from soils in Europe, Asia and Africa, and the latter those isolated from 
soils in America and Australia. The organisms oxidizing nitrites to 
nitrates and known as nitric or nitrate bacteria, were included by 
Winogradski in the genus Nitrobacter. 

Apart from these bacteria there is an organism, according to Kaserer, 
that can oxidize ammonia directly to nitrate. He named it B. nitrator. 
The reaction is illustrated by the following equation : 

NH 3 + H 2 CO 3 + O 2 = HNO 3 + H 2 O + CH 2 O - 41 Cal. 
CH 2 O + O 2 = H 2 CO 3 + 132 Cal. 

Enough energy for the completion of the reaction is obtained by the 
oxidation of the formaldehyde (CH 2 O). Beyond the preliminary 
announcement of Kaserer's there are no experimental data to prove 
the existence of this organism, even though other evidence of an 
indirect nature may be construed to lend support to his theory. 
But whether it be proved or not that ammonia may be oxidized 
to nitrate by a single species, it is evident that the number of species 
concerned in nitrate production is relatively small. 

Relation to Environment. The conditions that affect nitrate forma- 
tion in soils may be classified under the following heads: (a) supply of 
oxygen; (6) range of prevailing temperatures; (c) amount and dis- 
tribution of moisture; (d) quantity of lime and of other basic materials; 
(e) quantity of soluble mineral salts; (/) character and amount of 
organic matter; (g) presence of toxic substances; (ti) association with 
other organisms; (i) physiological efficiency of the nitrifying bacteria. 

The rapid disappearance of organic matter from sandy soils is due in 
large measure to their better aeration. On the other hand, the decom- 
position of vegetable and animal substances in heavy, ill- ventilated soils 
is materially retarded by the limited supply and very gradual renewal of 
oxygen. An intimate relation exists here between air and water in that 
the latter replaces the former to a more marked extent in heavy than in 
light soils. The influence of both aeration and the range of moisture is 
illustrated by an experiment of Lipman's in which equal quantities of 
soil were kept in large boxes under different moisture conditions. At 


392 MICROBIOLOGY OF SOIL 

the end of a year the following quantities of nitrate nitrogen were 
found: 

Moisture / 

I 0.52 per cent 14.75 per cent 18.62 per cent 22.05 per cent 22.12 per cent 

Nitrate f 

nitrogen { 697 mg. 823 mg. 720 mg. Trace Trace 

found [ 

In examining the figures recorded above, we find that moisture was the 
controlling factor in the development of the nitrifying bacteria, when 
the proportion of water in the soil was 6.52 per cent. As the amount of 
water increased to 14.75 P er cent there was a marked increase in the 
amount of nitrate produced. Beyond that, however, the further in- 
crease in the amount of water began to limit the supply of oxygen, and 
the production of nitrate nitrogen with 18.62 per cent of water in the 
soil was somewhat decreased. A still further addition of water up to 
22.05 P er cen t led, practically, to saturation, and the encouragement of 
reduction rather than oxidation processes. Hence, no nitrate was al- 
lowed to accumulate in the soil. The data in question thus help to 
explain why care was taken, on saltpeter plantations, to keep the 
compost heaps moist, yet not too wet. 

The influence of temperature on nitrate formation has been observed 
by many investigators. Schloesing and Miintz recorded that at 5 
nitrification is quite feeble, at 12 marked and at 37 at its best. 
Other investigators have obtained substantially the same results, except 
that the optimum has been found to be considerably lower, often be- 
tween 25 and 30. Under field conditions nitrification seems to take 
place at relatively low temperatures, as is indicated by the rapid 
oxidation of ammonium salts in the Rothamsted experiments in Eng- 
land; and the rapid decay and nitrification of clover and of other 
legume residues in the experiments at the New Jersey Experiment 
Station. These facts have, therefore, an important bearing on the 
nitrogen feeding of crops in tropical, subtropical and temperate zones. 

The influence of lime and of other basic substances including the 
carbonates of magnesium, potassium and sodium, and of the oxides of 
iron is of far-reaching importance in all nitrification processes. It is 
well known that applications of magnesian and non-magnesian lime, 
marl or wood ashes promote nitrification in the soil and in compost 
heaps, a fact that was well recognized by the ancient niter refiners. The 


DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 393 

favorable action of lime is readily explained by its ability to neutralize 
organic and mineral acids and to render, thereby, the soil reaction 
favorable for the rapid growth of ammonifying, as well as of nitrifying 
bacteria. Furthermore, the reserve of basic material serves to neutral- 
ize the acid formed by the bacteria and prevents thus the accumulation 
of an undue amount of acidity. 

The role of certain mineral salts in promoting nitrification is quite 
significant. Small amounts of sodium chloride have been found to favor 
nitrification in the experiments of Pichard and also those of Lipman. 
The former showed also that sulphates not only promote nitrification, 
but that different sulphates display marked variations in this respect. 
In the same manner nitrate formation was shown to be favorably 
affected by phosphates in bone meal, Thomas slag, and acid phos- 
phates. Generally speaking, therefore, nitrifying bacteria are stimu- 
lated in their development by a proper supply of available mineral 
nutrients. 

The exact relation of organic matter in the soil to the activities of 
nitrifying bacteria is but beginning to be properly understood. Earlier 
observations made it manifest that heavy applications of animal 
manures, or of green manure may not only retard nitrification, but may 
actually cause the disappearance of a part or of all of the nitrate in the 
soil. Subsequent experiments by Winogradski and by Winogradski 
and Omelianski showed that in pure cultures the presence of even slight 
amounts of soluble organic matter may depress or even suppress the 
development of the nitrifying bacteria. It was, therefore, concluded 
by these authors that relatively small amounts of soluble organic 
matter may inhibit nitrification. These conclusions, based on the 
study of liquid cultures only, were given a very broad application by 
many writers on agricultural topics. More recent experiments make 
it certain, however, that in the soil itself small amounts of soluble 
organic matter, e.g., dextrose, are not only harmless, but may really 
stimulate nitrification. It was shown, likewise, that humus and 
extracts of humus may, under suitable conditions, stimulate nitrifica- 
tion to a very striking extent. 

Certain substances in the soil may exert a toxic effect on nitrifying 
bacteria. Ferrous sulphate, sulphites and sulphides may thus act in- 
juriously, as may also calcium chloride and excessive concentrations of 
sodium carbonate, sodium bicarbonate, sodium chloride, magnesium 


394 MICROBIOLOGY OF SOIL 

sulphate, etc. Injury by ferrous compounds, as well as by organic 
acids, is not uncommon in low-lying fields and bogs; while injury from 
excessive concentration of soluble salts may occur in the so-called 
alkali lands. 

Finally nitrification in the soil should be considered from the stand- 
point of the organisms themselves. There is no doubt that continued 
growth under extremely favorable conditions leads to the develop- 
ment in the soil of nitrifying bacteria, possessing a very marked phy- 
siological efficiency. On the other hand, in ill-aerated, sour soils the 
environment would depress the physiological efficiency of the nitrify- 
ing bacteria. Differences are thus undoubtedly established under 
actual field conditions, as is made probable by the variable behavior 
of soils from different sources when used as inoculating material in 
recently reclaimed or peat swamp lands. 

Accumulation and Disappearance of Nitrates. As shown above, the 
rate of formation of nitrates in the soil is dependent upon moisture, 
temperature and aeration, as well as on the presence of organic matter 
and basic substances. On the other hand, the accumulation of nitrates 
depends, under any given conditions, largely on the character of the 
growing crop. Observations on the rain gauges at Rothamsted showed 
an average annual loss of 14 kg. (31.4 pounds) of nitric nitrogen per acre 
in the drainage water from uncropped soil. In one of King's experi- 
ments, land that had been fallowed contained 137 kg. (303.24 pounds) 
of nitric nitrogen per acre, to a depth of 4 feet. Adjoining cropped 
land contained only 26 kg. (57.56 pounds) of nitric nitrogen per acre 
to the same depth. Stewart and Greaves found in limestone soil in 
Utah 64 kg. (142 pounds) of nitric nitrogen per acre, under corn; 
98 pounds under potatoes, and only 12 kg. (27 pounds) under alfalfa. 
Under the same conditions fallow land contained 74 kg. (165 pounds) 
of nitric nitrogen per acre. The smaller amount of nitric nitrogen found 
under alfalfa bears out the observations already made by a number of 
other investigators that the accumulation of nitrates under legumes is 
smaller than it is under non-legumes. While several explanations have 
been offered to account for this fact, it is generally agreed that legumes 
assimilate nitrate nitrogen more rapidly than non-legumes. Unusual 
circumstances may favor, at times, the accumulation of quantities of 
nitrate large enough to destroy all vegetation. It is reported, for 


DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 395 

instance, by Headden that he has found in limited areas in Colorado as 
much as 90,718.5 kg. (100 tons) of nitrate per acre foot of soil. 

The amount of nitrate nitrogen in the soil is influenced by the grow- 
ing crop not alone because of the nitrogen absorbed by the latter, but 
because of the moisture relations as affected by growing plants. It is 
quite apparent that a large crop dries out the soil more rapidly than a 
small crop. When the soil moisture is sufficiently depleted, nitrifica- 
tion stops and the further accumulation of nitrates becomes impossible, 
while their disappearance is hastened by the constant demands of the 
crop. The disappearance of soil nitrates is, likewise, hastened by the 
leaching action of rain and by certain species of bacteria that transform 
them into other nitrogen compounds. 

DENITRLFICATION. Experimental Study. Denitrification may be 
defined as the reduction of nitrates by bacteria, involving the evolu- 
tion of nitrogen gas or of nitrogen oxides. In a more general way, 
denitrification has been defined as the partial or complete reduction of 
nitrates by bacteria. The term direct denitrification has been sug- 
gested for complete reduction, and indirect for the partial reduction 
to nitrites or ammonia. The term denitrification should not be em- 
ployed to designate losses of nitrogen gas due to the oxidation of 
ammonia, or to the disappearance of nitrates following their conversion 
into proteins by microorganisms. 

The reduction of nitrates in the presence of fermenting organic 
matter was noted by Kuhlmann as early as 1846. The same fact was 
recorded many years later by Froehde and by Angus Smith. In 1868 
Schoenbein expressed the belief that nitrates may be reduced to nitrites 
by fungi. For more than a decade after that, data were rapidly accu- 
mulating in support of Schoenbein's contention, until in 1882 Gayon 
and Dupetit made it certain that nitrate reduction with the evolution 
of nitrogen gas may be caused by a "ferment." Finally, in 1886, the 
same investigators described two organisms, B. denitrificans a, and B. 
denitrificans /3, capable of completely reducing nitrates. Subsequently 
the studies of Giltay and Aberson, Burri and Stutzer, Severin, van 
Iterson, Jensen, Beyerinck and of many others not only greatly in- 
creased the number of known denitrifying bacteria, but added much to 
our knowledge concerning the development and activities of these 
organisms. It has been shown that a very large number of species 
can reduce nitrates to nitrites and ammonia; moreover, a considerable 


396 MICROBIOLOGY OF SOIL 

number of organisms are already known that can cause the complete 
destruction of nitrates with the evolution of nitrogen gas or nitrogen 
oxides. The following reactions illustrate diagrammatically the com- 
plete or partial reduction of nitrates. 

2HNO 3 = 2HNO 2 + O 2 
HNO 3 + H 2 O = NH 3 + 2O 2 

4HNO 2 = 2H 2 O + 2N 2 + 3O 2 

In the soil, manure or other culture media the denitrifying bacteria 
which are, for the most part, aerobic develop also under anaerobic 
conditions and transfer the oxygen of nitrates and nitrites to carbon 
compounds. This is illustrated by the equations suggested by van 
Iterson: 

5 C + 4 KN0 3 + 2H 2 = 4 KH C0 3 + 2 N 2 + CO 2 
3 C + 4KNO 2 + H 2 O = 2KH CO 3 + K 2 CO 3 + 2 N 2 

When nitrates are reduced to nitrites in the presence of amino- 
compounds, or even of ammonium compounds, elementary nitrogen 
may escape as shown by the following reactions: 

C 2 H 5 NH 2 + HNO 2 = C 2 H 5 OH + N 2 + H 2 O 
NH 4 C1 + KNO 2 = KC1 + 2 H 2 O + N 2 

An organism has been described by van Iterson that can decompose 
nitrates in the presence of cellulose: 

5C 6 H 10 O 5 + 2 4 KNO 3 = 2 4 KHCO 3 + 6CO 2 + i2N 2 + i 3 H 2 O 

Still more interesting is Thiobacillus denitrificans described by 
Beyerinck as capable of reducing nitrates in inorganic media. The 
nitrate oxygen is used to oxidize elementary sulphur: 

6KNO 3 + 58 + 2CaCO 3 = 3K 2 SO 4 + 2CaSO 4 + 2CO 2 + 3 N 2 

The Actinomyces reduce nitrates to nitrites, but they do not cause 
any loss of free nitrogen, for the nitrites are utilized by the organisms, 
and complete denitrification does not take place. Thus these organ- 
isms may prevent the leaching out of nitrates and nitrites in the soil, 
or the active denitrification by other organisms. 

Relation to Environment. Nitrate reduction is favored by insuffi- 
cient aeration, as well as by an abundance of readily decomposable 
organic matter. In fine-grained, compact soils nitrate formation and 
nitrate reduction may alternate, depending upon the more or less 


DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 397 

complete replacement of soil air by water. Similarly, in soils receiving 
excessive amounts of animal manure denitrifying bacteria may cause 
the reduction of nitrates. In greenhouse soils excessive moisture, as 
well as excessive amounts of organic matter, may be present and may 
prevent the accumulation of nitrates. It has also been shown by 
Niklevski that, contrary to opinions previously held, denitrification 
may occur in manure heaps. In the better aerated surface portion of 
manure heaps conditions are favorable for the oxidation of ammonia 
to nitrites and nitrates. The nitrous acid may combine with ammonia 
to form ammonium nitrite, the latter decomposing, spontaneously, into 
water and nitrogen gas. It is very likely, also, that the nitrites and 
nitrates are reduced by the denitrifying bacteria in manure. On the 
other hand, in manure kept moist under the feet of cattle nitrite and 
nitrate formation is prevented and losses by denitrification are not 
likely to occur. 

The economic significance of denitrification was overestimated at 
one time, on account, largely, of the assertion of Wagner in 1895 that 
in all soils receiving applications of horse manure, the nitrates in the 
soil itself as well as those added in commerical fertilizers are almost 
certain to be destroyed by denitrification. Subsequent experiments 
by many investigators demonstrated that under field conditions, deni- 
trification is a factor of slight moment; however, in the greenhouse, 
in the manure heap (under certain conditions) and in market gardening 
where manure is used at the rate of 45>359 kg. to 54,431 kg. (50 to 60 
tons) per acre, the danger of denitrification is real. 

ANALYTICAL AND SYNTHETICAL REACTIONS 

AMOUNT OF BACTERIAL SUBSTANCE IN THE SOIL. Various decom- 
position processes in the organic matter of the soil may be designated 
as analytical in that protein, carbohydrates and fats are split into more 
simple compounds. At the same time, the microorganisms concerned 
in the decomposition processes multiply very rapidly and fashion the 
complex compounds of their cell-substance out of the simple cleavage 
products in their medium. In other words, analytical and synthetical 
reactions proceed hand in hand in the soil. 

While it is not definitely known how large a proportion of the soil 
humus consists of the dead and living cells of microorganisms there 
is a mass of indirect evidence to show that these cells form a very con- 


398 MICROBIOLOGY OF SOIL 

siderable proportion of the total quantity of organic substances in the 
soil. For instance, it has been demonstrated that a large proportion of 
the dry matter of solid animal faeces may consist of bacterial cells. At 
various times and by different investigators the proportion of bacterial 
substance has been estimated at from 5 to 20 per cent or more of the 
total dry weight of faeces. A heavy application of barnyard manure 
may introduce, therefore, several hundred pounds of bacterial cells per 
acre of soil. Moreover, because of the extensive changes in the soil 
humus itself, as is evidenced by the rapid formation of nitrates, large 
masses of bacterial substances are constantly being formed and dis- 
integrated. 

AVAILABILITY OF BACTERIAL MATTER. Substances of microorganic 
origin are decomposed more or less rapidly, according to their com- 
position. The extent of transformation under favorable conditions is 
indicated by an experiment performed by Beyerinck and van Delden, 
in which 50 per cent of the nitrogen in Azobacter cells was transformed 
into nitrate in seven weeks. On the other hand, the humus of peat and 
muck soils, or that of worn-out soils, may contain microorganic residues 
of so inert a character as to yield but little available nitrogen to 
crops. 

TRANSFORMATION OF PEPTONE, AMMONIA AND NITRATE NITROGEN. 
The cleavage of protein compounds into peptones, amino-acids and 
ammonia, and the oxidation of the latter into nitrites and nitrates, may 
be properly included among analytical reactions. It should not be 
forgotten, however, that in the accompanying synthetical reactions the 
compounds just mentioned may be transformed back into complex 
proteins. It happens, thus, that large quantities of the available 
nitrogen compounds may be withdrawn from circulation by micro- 
organisms that use these as building material. Under extreme con- 
ditions microorganisms may become serious competitors of higher 
plants for available nitrogen food. 

Manure stored in heaps not infrequently deteriorates in quality, 
even when losses by leaching are excluded. This deterioration is largely 
due to the change of the water-soluble ammonia and amino-compounds 
into insoluble protein substances. While the extent of the change into 
protein compounds is variable it may range from less than a tenth of the 
water soluble material to more than three-quarters or four-fifths of it. 
Also in the soil the same processes take place, but not so intensively. A 


DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 399 

large number of species of molds and bacteria have been isolated and 
tested as to their ability to transform ammonia, amino- and nitrate 
nitrogen into protein compounds. Among the more recent investi- 
gations in this field those of Lemmermann and his associates testify that 
in three weeks 5 to 6 per cent of the nitrate added to the soil was changed 
into protein. In the presence of barnyard manure the proportion 
transformed was increased to 15 per cent. In the case of ammonium 
compounds the transformation may be even more far-reaching, amount- 
ing, at times, to more than 25 to 30 per cent of the material originally 
present. Generally speaking, molds will assimilate ammonia nitrogen 
more readily while bacteria and algae wilL assimilate nitrate nitrogen 
by preference. However, the preference of molds for ammonia nitrogen 
is often more apparent than real, because of the rapid formation of 
acid residues in culture media rich in certain ammonium compounds. 
Similarly, some species of bacteria will assimilate ammonia nitrogen 
in preference to nitrate nitrogen. 


CHAPTER III 

FIXATION OF ATMOSPHERIC NITROGEN 

THE SOURCE or NITROGEN IN SOILS 

EARLY THEORIES. When chemistry had made sufficient progress 
to allow the analysis of soils and plants it was recognized that nitrogen 
is always present in both. 'It was also recognized that the soil nitrogen 
is almost wholly confined to the surface portion and is evidently of 
atmospheric origin, since the unweathered, underlying rock is devoid 
of this constituent. The vast accumulations of nitrogen, known to 
exist in all arable soils, were ascribed, therefore, to the residues of many 
generations of plants; and the assumption seemed to be justified that 
the atmosphere, 79 per cent of whose bulk consists of nitrogen gas, is 
the direct source of this element to plants. It was not long, however, 
before plant physiologists demonstrated experimentally that nitrogen 
gas as such could not directly serve as food for plants. There thus 
arose one of the most interesting and, for a long time, one of the most 
puzzling problems in agricultural research. Among the earlier in- 
vestigators de Saussure believed, at the beginning of the nineteenth 
century, that nitrogen is taken up from the soil in combined form. 
Liebig in 1840 advanced his well-known "mineral theory" according 
to which plants secured their nitrogen from the air, in the form of 
ammonia. He assumed, thus, that plants cannot use elementary 
nitrogen, and that the supply of atmospheric nitrogen in the form of 
ammonia was great enough to meet the needs of growing vegetation. 
The latter view was not accepted by Lawes and Gilbert of the Roth- 
amsted Station in England. By a series of elaborate and carefully 
controlled experiments they demonstrated in 1858 that nitrogen in the 
elementary form cannot be used by plants. They further demonstrated 
that the amount of combined nitrogen brought down in the form of 
ammonia, nitrites and nitrates, by atmospheric precipitation was but 
slight when compared with the quantities annually removed by crops. 
Hence the problem as to the source and maintenance of combined 
nitrogen in the soil seemed to be more puzzling than ever. 

400 


FIXATION OF ATMOSPHERIC NITROGEN 401 

CHEMICAL AND BIOLOGICAL RELATIONS. The second and third 
quarters of the nineteenth century saw the birth of a number of theories 
dealing with this problem. It was suggested that nitrogen compounds 
may be formed in the soil by the oxidation of nitrogen to nitric acid. 
Compounds of iron, manganese and lime were supposed in some way 
to make such oxidation changes possible. It was likewise suggested 
that nascent hydrogen may be generated in the decomposition of organic 
matter in the soil, and reacting with elementary nitrogen, may give 
rise to ammonia. The various hypotheses were not supported by 
experimental proof; moreover, the situation was complicated by the 
knowledge, based on empirical observations, that crops of the legume 
family seemed to be more or less independent of the supply of combined 
nitrogen in the soil. Indeed, clovers and other legumes had, appar- 
ently, the ability to increase the content of combined nitrogen in the 
soil as was indicated by the experiments of Boussingault and of Lawes 
and Gilbert. Finally, the mystery was solved by the investigations 
of Berthelot and Hellriegel and Wilfarth who furnished the proof that 
elementary nitrogen may be utilized by plants when certain biological 
relations are met. These relations involve the presence and activities 
of microorganisms that by themselves, or in conjunction with higher 
plants, make available to growing vegetation the great store of 
atmospheric nitrogen. 

NON-SYMBIOTIC FIXATION OF NITROGEN 

HISTORICAL. Non-symbiotic nitrogen fixation, or Azofication, has 
already been defined as the production of nitrogen compounds out of 
atmospheric nitrogen by bacteria independently of higher plants. The 
part played by bacteria in this process was not recognized until 1885, 
when Berthelot published some of his data on the accumulation of com- 
bined nitrogen in uncropped soils. His results seemed to explain a 
number of scattered observations, made since the middle of the century, 
on the apparent increase of the nitrogen content of cultivated soils. 

While Berthelot's experiments proved that the nitrogen gains 
occurred only in unsterilized soils and were, therefore, due to micro- 
organisms, it remained for Winogradski to demonstrate, in 1893, that 
the formation of nitrogen compounds by certain types of bacteria 
may be accomplished in culture media nearly or quite devoid of com- 

26 


402 MICROBIOLOGY OF SOIL 

bined nitrogen. Soon after that he succeeded in isolating his organisms 
in pure culture, and described them as anaerobic bacilli allied to those 
of the butyric group. In 1901 our knowledge of Azobacteria was 
enriched by Beyerinck's discovery of a group of large, obligate aerobic 
bacteria that he designated as Azotobacter. Since that date it has been 
found that the ability to fix atmospheric nitrogen is possessed also by 
certain molds and by various species of bacteria. However, this ability 
is not only extremely variable, but is also very feeble as compared 
with that of the members of the two groups described by Winogradski 
and Beyerinck. These two groups may, therefore, be designated as 
including the nitrogen-fixing bacteria par excellence. 

ANAEROBIC SPECIES. The species isolated by Winogradski was 
named by him.B. (Clostridium) pasteurianus (Fig. 131). It was found to 


FIG. 131. B. (Clostridium') pasteurianus, a non-symbiotic nitrogen-fixing organism. 

(After Winogradski from Lipman.) 

grow readily under anaerobic conditions in culture solutions contain- 
ing dextrose and the necessary mineral salts, but no combined nitrogen. 
The products of growth included protein, butyric and acetic acids, 
carbon dioxide and hydrogen. In the presence of other bacteria B. 
(Clostridium} pasteurianus was found to develop also under aerobic 
conditions. Subsequently studies by Winogradski and other investi- 
gators showed that B. (Clostridium) pasteurianus, and varieties of this 
species are very widely distributed in cultivated soils. More recently 
Bredeman made a thorough and extended study of anaerobic Azo- 
bacteria and demonstrated their almost invariable presence in a large 
number of soil samples from Europe, Asia and America. In his opinion 
they correspond more or less closely to B. amylobacter described many 
years before by van Tieghem. 

AEROBIC SPECIES. A more or less pronounced power to fix atmos- 


FIXATION OF ATMOSPHERIC NITROGEN 403 

pheric nitrogen is apparently possessed by a considerable number of 
aerobic species. Lipman has demonstrated the fixation of small 
amounts of nitrogen by Ps. pyocyanea and Lohnis secured similar results 
with Bact. pneumonic?, B. lactis viscosus, B. radiobacter and B. 
prodigiosus. Gottheil has detected fixation by B. ruminatus and B. 
simplex; Pillai has described a nitrogen-fixing aerobic bacillus, B. 
malabarensis; Wester mann studied a similar organism that he named B. 
danicus; while Beyerinck and van Delden observed, some years earlier, 
that, certain strains of B. mesentericus could fix relatively large amounts 
of nitrogen. Similarly Ps. radicicola has been found to possess a slight, 
but nevertheless an appreciable power to fix elementary nitrogen in 
culture solutions or in the soil. 


FIG. 132. Azotobacter vinelandi, a non-symbiotic nitrogen-fixing organism. 

(After Lipman.) 

But while nitrogen fixation among aerobic soil bacteria is not as 
uncommon as was at one time supposed, this function is so feeble and 
so variable in most instances, as to be of negative, or, at best, of doubt- 
ful economic significance. On the other hand, the aerobic, Azotobacter, 
first described by Beyerinck in 190-1, may be regarded not only as pos- 
sessing a very pronounced ability to fix atmospheric nitrogen, but as 
playing a role of some moment in maintaining the supply of combined 
nitrogen in the soil. 

To the two species of Azotobacter, A. chroococcum and A. agilis 
described by Beyerinck and van Pelden, Lipman added A. mnelandii 


404 MICROBIOLOGY OF SOIL 

(Fig. 132), A . beyerincki and A . woodstownii, and Lohnis and Westermann, 
A. vitreum. Of these species A. chroococcum and A. beyerincki are most 
common and are widely distributed in cultivated soils of Europe and 
America, and probably also of the other continents. They are absent 
in acid soils deficient in humus, and most common in limestone regions 
and in irrigated soils rich in mineral salts. Their food requirements are 
covered by solutions containing potassium phosphate, magnesium 
sulphate, calcium chloride and ferric sulphate, and some organic 
nutrient, such as dextrose, saccharose, xylose, mannit, acetate, pro- 
pionate, butyrate, malate, ethyl alcohol, etc. An alkaline or neutral 
reaction and the presence of salts of iron are essential for 'the vigorous 
development of Azotobacter, while humates have been shown by 
Krzemieniewski to exert a stimulating influence on the growth of these 
organisms, even though not acting directly as a source of food and 
energy. As shown by Lipman and others, Azotobacter may gain an 
increased power of fixing atmospheric nitrogen in the presence of other 
organisms. It is resistant to drying, notwithstanding the fact that it 
produces no spores, and has been successfully isolated from soil samples 
that had been kept in a dry state for several years. For some reason 
it may be detected in the soil most readily in the fall and winter 
months. 

As to the nitrogen-fixation by fungi, it has been shown elsewhere 
that the evidence is, if anything, of a negative character. Some 
algae are able to fix atmospheric nitrogen, especially those that live 
symbiotically with azotobacter. 

ENERGY RELATIONS. In the fixation of nitrogen by bacteria the 
necessary energy for the process is furnished by the carbohydrates, 
organic acids, alcohols or other organic nutrients employed in the 
culture media. Since any given quantity of organic nutrient possesses 
a definite amount of potential energy the fixation of nitrogen is neces- 
sarily limited by the supply of such potential energy. This limitation 
was already recognized by Winogradski in his experiments with B. 
(Clostridium) pasteurianus. For every gram of dextrose used up there 
was produced, on the average, 2 to 3 mg. of combined nitrogen. In the 
experiments of Bredeman with B. amylobacter, and of Pringsheim with 
"Clostridium americanum r the amounts fixed were, at times, con- 
siderably larger. On the whole, however, it has been proved by a 
number of investigators that Azotobacter can fix much larger quantities 


FIXATION OF ATMOSPHERIC NITROGEN 405 

of nitrogen than the anaerobic bacilli. The extended investigations 
of Lipman showed that A . mnelandii has the ability to fix more nitrogen 
per unit of organic nutrient consumed than either A. chroococcum or 
A . beyerincki. Under favorable conditions A . mnelandii may at times 
fix 15 or even 20 mg. of nitrogen per g. of mannit used up. Krze- 
mieniewski found in experiments with A. chroococcum that additions 
of humates to the culture solutions increased the nitrogen fixed from a 
maximum of 2.4 mg. to a maximum of 14.9 mg. 

The practical bearing of the foregoing data lies in the fact that the 
fixation of nitrogen in cultivated soils is limited, among other things, by 
the energy available, that is, by the quantity of readily decomposable 
organic residues. An indication as to the extent of these is given by the 
amount of humus present; nevertheless, this must remain an indication 
merely, for most of the humus is too inert to serve as a source of energy 
to Azotobacter. From the data at present available different investi- 
gators have estimated the quantity of nitrogen fixed by Azotobacter 
at 6.8 kg. to 18 kg. (15 to 40 pounds) per acre, per annum. Assuming 
favorable conditions for fixation, so that 500 g. (i pound) of nitrogen 
could be fixed for every 50 kg.(ioo pounds) of carbohydrate consumed, 
it would still take an equivalent of 680 kg. to 1,814 kg. (1,500 to 4,000 
pounds) of sugar to produce this quantity of combined nitrogen. It may 
be noted in this connection that Azotobacter have been demonstrated 
to live in symbiosis with algae, obtaining thereby the necessary energy 
for their activities. This may explain, perhaps, the remarkable facts 
observed by Headden in Colorado, relating to the accumulation of such 
enormous quantities of nitrate in the soil as to destroy all vegetation. 
In some instances the nitrates were found to be present to the extent of 
90,718 kg. (100 tons), or more (per acre), to a depth of a few inches. If 
the accumulation of combined nitrogen was due to Azotobacter, as is 
claimed by Headden, and the bacterial residues oxidized by nitrifying 
bacteria to nitrates, it is difficult to account for the source of the 1,000 
or 2,000 tons of carbohydrates necessarily used up in the process of 
fixation, unless it could be proved that the energy was furnished by 
algae. 

SYMBIOTIC FIXATION 

HISTORICAL. Empirical observations extending well back into 
ancient agriculture have led to the recognition of the soil-enriching 


406 MICROBIOLOGY OF SOIL 

qualities of certain crops of the legume family. Columella mentions 
the fact that many Roman farmers regarded beans as possessing these 
qualities, but does not accept this belief for himself. On the other 
hand, he points out that luzerne (alfalfa), lupins and vetches improve 
the land and act as manure. He points out, also, that it was the 
practice of Roman farmers to plow under lupines in order to enrich the 
soil. In the centuries following the fall of Rome the use of legumes for 
soil improvement persisted to some extent in Italy, France and other 
countries; yet the practice was not followed consistently and the fer- 
tility of European soils was declining for lack of available nitrogen, 
and, to a large extent, also of phosphoric acid. The more general intro- 
duction of clover into Germany and England in the eighteenth century 
helped to restore the fertility of many farms, and led, ultimately, to the 
recognition of the peculiar place held by legumes in the maintenance 
of soil fertility. But while practical farmers knew of the soil-enriching 
power of legumes, and while they retained their belief in it even when 
it seemed contrary to scientific authority, they did not know the secret 
of this power. It remained for Hellriegel and Wilfarth to demonstrate 
in 1886, and more fully in 1888, that this power, already hinted at by 
the investigations of others, is the resultant of the combined activities 
of the plants and of bacteria that enter their roots, and produce there 
the well-known nodules or tubercles. They showed in no uncertain 
manner that legumes can improve the soil only in so far as they add 
nitrogen to it with the aid of the bacteria in the tubercles; in other 
words, legumes were shown to enter into a symbiotic relationship with 
certain bacteria and to acquire, thereby, the ability to fix atmospheric 
nitrogen. 

The presence of tubercles on the roots of leguminous plants was first 
recorded by Malpighi in 1687. He regarded them as root galls. The 
botanists who studied them in the first half of the nineteenth century 
classified them as modifications of normal roots or as pathological 
processes. In 1866 the Russian botanist Woronin found that the 
tubercles were filled with minute bodies resembling bacteria and con- 
cluded that they were pathological outgrowths. Some years later 
Frank, in 1879, not on ^y showed that tubercles are almost invariably 
present on the roots of legumes, but that their formation may be pre- 
vented by sterilizing the soil. Frank was thus in possession of facts 
that might have revealed to him the true nature of the root-tubercles. 


FIXATION OF ATMOSPHERIC NITROGEN 


407 


However, he later modified his belief in the origin of tubercles as due 
to outside infection, and accepted the interpretation of his pupil 
Brunchhorst who claimed that the bacteria-like bodies in the tubercles 
were merely reserve food materials. Because of their resemblance to 
bacteria Brunchhorst named them bacteroids. 

The studies of Marshall Ward, published in 1887, proved not merely 
that tubercle formation is due to outside infection, but that such infec- 
tion may be brought about at will by placing the roots of young plants 
in contact with pieces of old tubercles. Hellriegel in his preliminary 
communication of 1886 also showed that outside infection is necessary 
for the production of tubercles, and called attention to the true func- 


FIG. 133. Ps. radicicoia. i, From Melilotus alba; 2 and 3, from Medicago saliva. 
4, from Vicia mllosa. (After Harrison and Barlow from Lipman.} 

tion of the latter as laboratories wherein nitrogen compounds are 
manufactured out of elementary nitrogen. The true worth of Hell- 
riegel's investigations was brought out more clearly in another paper 
that he published jointly with Wilfarth in 1888. The authors showed 
that in sterilized soils legumes behaved precisely like non-legumes, and 
died ultimately of nitrogen hunger when not provided with nitrates or 
other suitable nitrogen compounds. On the other hand, when the 
sterilized soil was later infected with a few drops of leachings from fresh 
soil that had supported a normal growth of legumes, the starving plants 
recovered and grew vigorously. Under the same conditions non- 
legumes did not recover. The recovery of the starving legumes was 
found to be coincident with the formation of tubercles. 


4o8 


MICROBIOLOGY OF SOIL 


Hellriegel and Wilfarth's studies were soon confirmed by the inves- 
tigations of others. Wigand showed in 1887 that the tubercles con- 
tained within them true bacteria. In the following year Beyerinck 
reported the successful isolation of these bacteria on artificial media, 
and named the organism B. radicicola (Fig. 133). Prazmowski also 
isolated pure cultures of Ps. radicicola, and followed the entrance of 
the organisms into the root hairs of young plants, their passage through 
the cell-walls, and their transformation into bacteroids. These facts 
were all confirmed by other investigators, and it was further shown by 
Schloesing and Laurent that properly inoculated legumes not only can 
grow in soils devoid of combined nitrogen, but that when growing in 
such soils in a confined atmosphere they decrease the quantity of 
nitrogen gas surrounding them by transforming it into nitrogen com- 
pounds. It was, therefore, made clear by these investigations, and by 


FIG. 134. Sections through root tubercles, i, Cell from tubercle of Pisum 
saiivum, showing bacterial filament; 2 and 3, cells with bacterial filaments from 
tubercle of Trifolium pannonicum. (After Stefan from Lipman.) 

others not mentioned here for lack of space, that the belief of practical 
farmers in the soil enriching qualities of legumes was amply justified. 
It was shown, further, that the later experiments of Boussingault, as 
well as those of Lawes, Gilbert and Pugh failed to solve the problem 
because these investigators treated their soil so as to prevent the 
survival and subsequent entrance of Ps. radicicola, and deprived the 
leguminous plants of the ability to utilize atmospheric nitrogen. 

MODES OF ENTRANCE AND DEVELOPMENT. Tubercle bacteria con- 
sisting of small motile rods usually enter the legumes by way of the root- 
hairs. For this reason young tubercles, with but few exceptions, are 
found on young roots. The organisms multiply at the point of infection 
and penetrate into adjacent plant-tissue by means of a hypha-like 


FIXATION OF ATMOSPHERIC NITROGEN 409 

hollow thread or tube that seems to consist of a gelatinous material 
(Fig. 134) . The tubes branch out as they pass from cell to cell and carry 
the invading organisms with them. The bacteria which may be readily 
detected within the tubes and cells are the involution forms of Ps. 
radicle old and assume various irregular shapes. They are designated 
as bacteroids. Stefan has suggested that bacteroids may be produced 
within the tubes and, possibly, from the buds or swellings that appear 
on the tubes. While still young, the bacteroids are capable of dividing, 
but as they grow they swell up and finally degenerate. 

RESISTANCE, IMMUNITY AND PHYSIOLOGICAL EFFICIENCY.- -The 
invasion of legumes by Ps. radicicola and the acquisition by the plant, 
thanks to this invasion, of the power to fix elementary nitrogen are cited 
as a case of symbiosis. However, some writers would regard the pres- 
ence of Ps. radicicola in legume roots as a case of parasitism. According 
to them symbiosis presupposes the living together of two organisms 
with resulting benefit to both. In the present instance, however, 
conditions may arise when the host plant is injured, rather than bene- 
fited; and similarly, conditions may arise when the invading bacteria 
are suppressed by the plants. Making due allowance for the ob- 
jections raised we still find, nevertheless, that in the broad relation of 
the two groups of organisms there is an apparent benefit to both plants 
and bacteria. The former gain an adequate supply of nitrogen and 
the latter a supply of carbohydrates and of mineral salts. 

A more detailed study of this relation shows that the plants resist 
the entrance of bacteria. When an abundance of available nitrogen 
compounds is supplied tubercle formation may be largely or wholly 
suppressed. In that case the plants secure their nitrogen from the soil 
and are not only independent of the bacteria, but are strong enough to 
resist their entrance. It is further claimed by Hiltner that tubercle 
bacteria differ in their virulence, and that the more virulent the organ- 
isms, the more readily will they penetrate the root tissue. Moreover, 
he believes that when a plant is invaded by organisms of any degree of 
virulence, the host plant becomes immune to a large extent and can keep 
out all but the most virulent bacteria. The use of the term virulence, 
in this connection, has been objected to, since it is borrowed from 
animal pathology and is likely to be misleading. It is better to employ 
the term physiological efficiency as implying not only a more pro- 
nounced ability to enter the plant roots, but also to fix atmospheric 


410 MICROBIOLOGY OF SOIL 

nitrogen. It is conceivable that strains of Ps. radicicola may be de- 
veloped that would grow rapidly and yet possess but a feeble nitrogen- 
fixing power. In other words, they would possess a high vegetative 
power and a low physiological efficiency. 

MECHANISM OF FIXATION. It is generally believed that the fixation 
of nitrogen is accomplished by the bacteria within the tubercles. The 
claim, at one time, advanced by Stoklasa, that the fixation is accom- 
plished by the plants themselves with the aid of enzymes produced by 
the bacteria in their roots, has been disproved. It is known that the 
period of active nitrogen assimilation by the plants coincides with the 
appearance of the bacteroids in the tubercles, and it is supposed that 
the microorganisms fashion nitrogen compounds out of atmospheric 
nitrogen by using the carbohydrates and organic acids in the plant 
juices as a source of energy. The plants then seem to utilize the soluble 
nitrogen compounds that pass out of the bacterial cells. It is further 
supposed that bacteroid formation is an attempt on the part of the 
microorganisms to adjust themselves to the drain caused by the 
activities of the host plant. 

VARIATIONS AND SPECIALIZATION. Apparent differences in bacteria 
from different legumes were noted by Hellriegel. Some of his experi- 
ments indicated that bacteria from clovers could not produce tubercles 
on lupines and serradella. Analogous differences were found by 
Nobbe and his associates, nevertheless they were finally led to conclude 
that the root invasion of legumes is caused by a single species. How- 
ever, continued association with any particular legume accomplished 
in the end a certain modification, or specialization, as it were, of the 
microorganisms, and they were then no longer able to invade the roots 
of other legumes. Later, Hiltner and Stormer have been led to 
modify this view and have arranged the tubercle bacteria in two 
groups, possessing, according to them, well-defined morphological and 
physiological differences. One of these groups is included under the 
species '''Rhizobium radicicola'' and the other under " Rhizobium 
beyerinckii." The former comprises the organisms from lupines, serra- 
della and soy beans while the latter comprises all of the others. 

RELATION TO ENVIRONMENT. Nitrogen fixation by leguminous 
vegetation is readily influenced by soil conditions, particularly the 
supply of lime and of other basic substances; the supply of organic 
matter and the aeration of the soil. As to the first of these it is well 


FIXATION OF ATMOSPHERIC NITROGEN 


411 


known that all legumes, with the exception of lupines and serradella, 
are stimulated in their growth by generous applications of lime. 


FIG. 135. These two pea plants were grown in clean quartz sand to which had 
been added small quantities of all the necessary elements of plant food except 
nitrogen. The conditions were exactly identical except that plant A was without 
root nodules (see Fig. 136) and plant B had numerous nodules well developed (see 
Fig. 137). (Mich. Exp. Station.} 


The top dressing of lawns with lime, marl or wood ashes encourages 
the appearance of white clover; an adequate supply of lime makes 


412 


MICROBIOLOGY OF SOIL 


possible the successful growing of alfalfa in almost any soil, while the 
leguminous vegetation of limestone soils is proverbially vigorous. 
The favorable influence of lime is due to the direct action on the plants 
as well as on the bacteria in the soil. Similarly, the tubercle bacteria 
are favorably affected in their survival and multiplication by an 
abundant supply of organic matter. On the other hand, acid soils or 
those deficient in humus and inadequately aerated are but ill suited 
to the activities of Ps. radicicola. 


FIG. 136. Roots of Plant A without nodules (Fig. 135). 


SOIL INOCULATION* 

By soil inoculation is now understood the adoption of some 
artificial method for supplying suitable quantities of nitrogen-fixing 
organisms to soils deficient in these types. The first attempts at soil 
inoculation were made in 1886 by Hellriegel and Wilfarth during the 

* Prepared by S. F. Edwards. 


FIXATION OF ATMOSPHERIC NITROGEN 


413 


course of their studies on the cause of nitrogen accumulation by 
legumes. They found that when leguminous plants were grown in 
sterile sand, nodules were formed on the roots only after the addition 
of a small portion of aqueous extract of fertile soil, or an extract of 
crushed nodules, or in some cases (lupines and seradella) by soil itself 
from a field on which these crops had been grown. The first successful 
artificial production of nodules by the aid of pure cultures was made 


FIG. 137. Roots of plant B with nodules (Fig. 135). 

in 1889 by Prazmowski in the course of studies on the method of 
entrance of the organism to the root hairs of the host plant. 

The first inoculation experiments in a large way were those made in 
1887 at the Moor Soil Experiment Station, Bremen, Germany, where 
earth taken from fields that had borne luxuriant crops of various 
legumes was scattered over reclaimed heath or swamp soils upon which 
legumes had not previously grown, with the result that in every instance 
the yield on the inoculated portions of land was greater than on the 


414 MICROBIOLOGY OF SOIL 

uninoculated plots. After such favorable results, it was but a natural 
step to try the effect of similar applications of soil rich in the nodule- 
forming bacteria to ordinary cultivated soils of varying character. 
While results in some cases were eminently satisfactory, in others there 
was no increase in the vigor or amount of the crop as a result of the 
inoculation. 

METHODS OP SOIL INOCULATION. From these early experimental 
results there evolved two general methods of inoculation, namely, the 
application of soil from an already inoculated field, and the application 
of pure cultures of the nodule-forming bacteria to the seed before 
sowing. 

Inoculation with Legume-earth. The use of soil as inoculating 
material was tried by various experiment stations of the United States, 
with results not varying widely from those secured in the pioneer 
experimental work at Bremen. It was found in general that the 
commonly grown crops, such as the common clovers, peas and beans, 
made little or no increase as a result of inoculation with old legume-soil. 
With new crops, however, such as alfalfa and soy beans when they were 
first introduced, it was found impossible in many places to secure a 
successful stand until the fields on which these crops were to be grown 
had received a top-dressing of soil from land that had already grown 
the crop in question; and it became a common practice to inoculate 
soil in this manner before seeding with these new crops. It was early 
observed, however, that this method of soil transfer for inoculation 
purposes was not an unmixed benefit. Aside from the expense and 
difficulty of handling and transportation of soil, fungus and bacterial 
diseases, not only of legumes but of other crops, as well as the seeds 
of noxious weeds, were transmitted from one field to another and even 
from one section of country to another. It was to avoid this difficulty 
that the preparation of pure cultures was introduced. 

Inoculation with Pure Cultures. Nitragin. The first pure culture 
method was launched in 1896 by Nobbe and Hiltner, German investi- 
gators, who prepared cultures of the legume bacteria on nutrient gelatin 
and arranged with a firm of manufacturing chemists to place them on 
the market under the trade name of Nitragin. 

Dried Cultures. In the United States the matter of pure cultures 
was first taken up by the Department of Agriculture about 1902. 
Cultures of the nodule-forming bacteria were cultivated in nitrogen- 


FIXATION OF ATMOSPHERIC NITROGEN 415 

free culture media, dried on cotton and distributed to farmers with a 
small package of salts from which a culture solution was to be made 
by the farmer and applied to the seed. This method gave poor results, 
chiefly because the bacteria could not withstand the drying on cotton. 
Afterward the cultures were sent in a liquid condition with somewhat 
more satisfactory results. The dry cotton cultures were exploited 
for a time by a commercial firm under the name of Nitro-culture, and 
somewhat similar cultures were placed on the market in England under 
the name of Nitro-bacterine. Cultures of both kinds, however, were 
shown to be valueless, both by microbiological and by planting tests. 

Cultures on A gar. Very satisfactory results were secured from the 
use of pure cultures at the Ontario Agricultural College, Guelph, where 
Harrison and Barlow, in 1905, originated the method of growing the 
bacteria on a nitrogen-poor agar medium. By this method, the farmer 
has simply to apply the bacteria to the seed just before sowing. These 
cultures, used on all the common legumes, sown in all kinds of soil, 
gave favorable results in 65 per cent of cases in trials extending over a 
period of ten years. Similar agar cultures are now prepared by com- 
mercial firms who have adopted the method of Harrison and Barlow, 
and also by some of the U. S. Agricultural Experiment Stations. 

Cultures in Soil. *- -Temple has suggested that sterilized soil with 
the addition of a small amount of leguminous material furnished a 
very good medium for the propagation of legume bacteria and is suitable 
for their distribution. 

Attempts have been made to put on the market cultures containing 
so-called " fertilizing bacteria" good for "all crops," but the tests made 
with these cultures have thus far failed to bear out the claims made for 
them. The successful commercial exploitation of cultures containing 
strong cellulose and protein decomposing organisms, non-symbiotic 
nitrogen-fixing organisms, strong nitrifying organisms and other useful 
bacteria is still to be accomplished. 

Importance of Inoculation. Inoculation with pure cultures affords 
the farmer a rapid, easy, arid cheap method of supplying the bacteria 
essential for getting a successful stand of any legumes. Failure to secure 
a benefit from this method of inoculation may usually be attributed to 
unsuitable soil conditions rather than any inherent failing in the cul- 
tures used. No method of inoculation will compensate for poor 

* Prepared by Jacob G. Lipman. 


41 6 MICROBIOLOGY OF SOIL 

physical or chemical condition of the soil itself. The principle of using 
artificial cultures to be applied with the seed is sound, and if the cul- 
tures contain large numbers of virile bacteria, there is little reason 
why they should not prove of benefit when used under soil conditions 
that would seem to need inoculation. 

Azotobacter Cultures. Some experimental work has been done in 
the use of cultures of Azotobacter for soil inoculation. The results are 
contradictory, and more work needs to be done to prove the value 
of such cultures. 


CHAPTER IV 

CHANGES IN INORGANIC CONSTITUENTS 

WEATHERING PROCESS 

ORIGIN AND FORMATION OF SOIL. Rock surfaces exposed to the 
action of rain, sunshine and frost lose their fresh appearance, become 
pitted and uneven, and gradually crumble into larger and smaller frag- 
ments. In the course of time the layer of disintegrated material 
becomes deeper and its constituent particles smaller thanks to the 
uninterrupted process of subdivision. Finally, lichens, algae and 
bacteria make their appearance, the organic debris accumulates, and 
higher plants begin to find a suitable environment for their development, 
The rock has changed into soil. 

INFLUENCE OF BIOLOGICAL FACTORS. Soil-formation is not entirely 
a mechanical or chemical process. Even before the layer of weathered 
rock acquires any appreciable depth microscopical and macroscopical 
forms of life gain a foothold on the uneven surface. With the aid of 
sunlight they build organic compounds and make use of the combined or 
elementary nitrogen of the atmosphere. Their life activities result in 
the production of carbon dioxide and of varying organic and inorganic 
acids which in their turn react with the constituents of the rock particles. 
In this manner the biological activities become of utmost moment in 
the transformation and migration of mineral substances in nature. 
They assume an important role in - the circulation of calcium and mag- 
nesium, with the accompanying phenomena that find most striking 
expression in the formation of caves and canyons in limestone strata. 
They assume a no less important role in the circulation of sulphur; 
in the accumulation and removal of available potash compounds in 
the soil, as well as in the transformation of phosphorus and its migration 
from inorganic to organic compounds. 

LIME AND MAGNESIA 

REMOVAL AND REGENERATION OF CARBONATES. Lime and mag- 
nesia are present in soils in different combinations. They may occur 

27 417 


418 MICROBIOLOGY OF SOIL 

as silicates, carbonates, phosphates, humates, sulphates, etc. In 
humid climates the carbonates are being continually removed from 
weathered rock material, as is plainly shown by the composition of 
drainage waters. The losses become much greater in cultivated soils- 
thanks to the humus and the microorganisms present in them. The 
absolute amounts lost from year to year will depend on the proportion 
of lime and magnesia in the soil, the mechanical composition of the 
latter, its content of humus and the methods of tillage and fertilization. 
According to Hall the soils of the experiment fields at Rothamsted, 
containing about 3 per cent of calcium carbonate, are losing lime at the 
rate of 362 kg. to 453 kg. (800 to 1,000 pounds) per acre 'annually. In 
certain sections of Scotland where liming has been practised for a long 
time the farmers estimate the loss of lime from the land at 6 bushels 
per acre, annually; that is, approximately at the rate of 226 kg. to 
272 kg. (500 to 600 pounds). In New Jersey, New York, Pennsylvania 
and other eastern states farmers who use lime more or less regularly 
apply i ton of it at the beginning of each five-year rotation. This would 
provide for an annual loss of 181 kg. (400 pounds) per acre. The loss of 
lime and magnesia is increased under intensive methods of agriculture. 
When animal manures and green manures are employed, microbial 
activities are stimulated, the production of carbon dioxide is encouraged 
and the loss of the soluble calcium bicarbonate made greater. The 
removal of lime is hastened even to a more striking extent when 
ammonium salts are applied to the land. The resulting nitrification 
and loss of lime are illustrated by the following equation: 

(NH 4 ) 2 SO 4 +,2CaCO 3 + 4 O 2 = Ca(NO 3 ) 2 + CaSO 4 + 4H 2 O+ 2 CO 2 

As was already indicated, the loss of calcium and magnesium car- 
bonate from the soil is effected largely through the activities of bacteria 
and of other microorganisms. At the same time microorganic life is 
responsible for the restoration of varying amounts of carbonates. It 
has been demonstrated that, in the weathering of the complex silicates, 
carbonates and silicic acid may be formed in considerable quantities. 
In the presence of decaying organic matter and the consequent evolu- 
tion of carbon dioxide the formation of carbonates from silicates may 
be extensive enough to balance the losses. Similarly, calcium carbonate 
may be formed in the soil from humates and from the calcium salts of 
simpler organic acids. They may be formed, also, through the activi- 


CHANGES IN INORGANIC CONSTITUENTS 419 

ties of denitrifying and other reducing bacteria from the corresponding 
nitrates and sulphates. As pointed out by Nadson ammonium car- 
bonate produced in the decomposition of protein compounds may react 
with calcium sulphate as follows: 

(NH 4 ) 2 CO 3 + CaSO 4 = CaCO 3 + (NH 4 ) 2 SO 4 

Moreover, calcium sulphate may be reduced to sulphide and may react 
with carbon dioxide as follows: 

CaS + CO 2 + H 2 O = CaC0 3 + H 2 S 

Magnesium would be subject to similar reactions and Nadson has 
observed the formation of a mixture of calcium and magnesium car- 
bonates (corresponding to dolomite in composition) in media inoculated 
with a pure culture of B. (Proteus) vulgaris. 

LIME AS A BASE. The carbon dioxide generated in vast amounts in 
the life processes of most soil bacteria, the nitrous and nitric acids 
formed by the nitro-bacteria, the sulphuric acid produced in the 
oxidation of hydrogen sulphide and of sulphur by the so-called sulphur 
bacteria, and the great variety of organic acids formed in the decom- 
position of carbohydrates, fats and proteins all react with basic sub- 
stances in the soil. Of these basic substances calcium carbonate is by 
far the most prominent. Combining with the different acids it 
maintains a favorable reaction for microorganic life in the soil. 

The calcium salts thus formed are more or less soluble. In this 
manner enormous amounts of lime are a'nnually carried to the ocean 
as bicarbonate, and to an appreciable extent also as nitrate and 
sulphate. Thus soil bacteria help to furnish shell fish and other forms 
of marine life, the material necessary for the building of their skeletons. 
In the course of ages the latter become a portion of the solid land and as 
coral reefs, chalk cliffs and marl beds offer to microorganisms a new 
opportunity to start calcium carbonate on its migrations. 

EFFECT OF CALCIUM AND MAGNESIUM COMPOUNDS ON BACTERIAL 
ACTIVITIES. Being basic in character calcium and magnesium car- 
bonates are of great service in maintaining a suitable reaction in the 
soil. But somewhat apart from this service calcium and magnesium 
compounds seem to be particularly important for the growth of certain 
organisms. It has already been observed by Winogradski and Ome- 
lianski that magnesium carbonate is especially useful in facilitating the 


420 MICROBIOLOGY OF SOIL 

isolation and culture of nitrate bacteria. Heinze and others have 
noted the favorable action of calcium carbonate on the growth of 
Azotobacter, while the beneficial influence of calcium carbonate and sul- 
phate on the development of Ps. radicicola has been repeatedly observed 
by different investigators. 

Lipman and Burgess found that calcium carbonate stimulates 
nitrogen fixation by A. chroococcum in solution, but is without effect 
in soil. Magnesium carbonate is very toxic both in soil and in solution 
for cultures of A. chroococcum even in concentration of o.i per cent. 
Calcium carbonate exercises a protective action against the toxic 
properties of magnesium carbonate. 

PHOSPHORUS 

AVAILABILITY or PHOSPHATES. Phosphorus exists in the soil largely 
in the form of phosphates of calcium, magnesium, iron and aluminum. 
A small portion of it occurs in organic combination in lecithin, phytin 
and other compounds. The soil phosphates possess a very slight degree 
of solubility and often fail to become available rapidly enough to meet 
the demands of the growing crop. Fortunately the presence of 
carbon dioxide generated from decaying organic matter hastens the 
solution of the inert phosphates, thus: 

Ca 3 (PO 4 ) 2 + 2CO 2 + 2 H 2 O = Ca 2 H 2 (PO 4 ) 2 + Ca(HCO 3 ) 2 

For this reason a maximum supply of available phosphates may be 
secured by plants in the presence of readily decomposable organic 
matter. 

Apart from carbon dioxide as a means for making available inert 
phosphates, bacteria produce organic and inorganic acids that are of 
direct service. The influence of nitrous, nitric and sulphuric acids, all 
of them products of bacterial activity, is undoubtedly of some im- 
portance. The influence of lactic, acetic and butyric acids, as well as 
of the more complex humic acids, must be of considerable moment. 
For instance, in the decomposition of bone meal by B. mycoides, 
Stoklasa found that 23 per cent of the phosphoric acid had become 
soluble, whereas in similar uninoculated portions of bone meal only 3 
per cent of soluble phosphoric acid was found. The significance of 
organic acids produced by microorganisms is brought out even more 
strongly in the loss of phosphates from acid soils. 


CHANGES IN INOEGANIC CONSTITUENTS 421 

In so far as the organic phosphorus compounds are concerned bac- 
terial activities are important in that the processes of decay restore the 
phosphorus to circulation. Hence, it will be seen that microorganisms 
are directly concerned in the migration of phosphorus from the soil to 
the plant and from the plant back to the soil. 

RELATION OF PHOSPHORUS TO DECAY AND NITROGEN FIXATION.- 
Just as bacteria influence the transformation of phosphorus compounds 
in the soil, so phosphorus itself affects the growth and activities of 
bacteria. As one of the essential constituents of living cells it reacts 
on the growth of microorganisms and influences species relationships. 
There are undoubtedly species whose phosphorus requirement is greater 
than that of other species. Indeed, conditions may arise that favor the 
rapid assimilation of soluble phosphates by bacteria. In that case the 
microorganisms would act as competitors to the higher plants. Among 
the species favorably affected by an abundant supply of phosphates 
Azotobacter is quite prominent. Hence nitrogen fixation is in a meas- 
ure dependent upon a proper supply of phosphorus compounds. 

Fred and Hart have shown that the potassium ion does not mate- 
rially influence ammonification; soluble phosphates cause large increases 
in the number of bacteria, ammonification and carbon dioxide produc- 
tion. By applying soluble phosphates to the soil crop production is 
increased, and it is due, in part, to the promotion of bacterial activity. 
The increased bacterial activity results in a more rapid decomposition 
of the organic matter, thus making available for the growth of crops 
larger quantities of nitrogen and probably of minerals. 

SULPHUR 

SULPHUR COMPOUNDS IN THE SOIL. Sulphur occurs in the soil in 
the form of sulphates and in that of organic compounds. In ill- 
aerated soils the reduction products of sulphates, viz., sulphites, sul- 
phides and even elementary sulphur, may be present in small amounts 
as a transition stage. According to Berthelot and Andre the protein 
compounds of the soil humus are quantitatively more important than 
the sulphates. However, this is not true of arid and semi-arid soils 
in which sulphates represent a larger store of combined sulphur than 
is contained in organic substances. 

Sulphur-phosphate Composts. In the composting of sulphur, 
ground rock phosphate (floats) and soil certain soil bacteria oxidize 


422 MICROBIOLOGY OF SOIL 

the sulphur into sulphuric acid, which acts upon the insoluble rock 
phosphate and makes it soluble and available for higher plants. The 
best combination found at the New Jersey Agricultural Experiment 
Station consists of 100 parts of soil, 120 parts of sulphur and 400 parts 
of rock phosphate, inoculated with material from an old compost. 
The bacteria causing the oxidation of sulphur were isolated at the New 
Jersey Agricultural Experiment Station and were found to be short, 
non-motile, Gram-positive rods. They are obligate aerobes and are 
able to convert sulphur into sulphuric acid. 

SULPHUR BACTERIA. In the decomposition of protein compounds 
with a limited supply of air, hydrogen sulphide and mercaptans are 
evolved. The quantities of hydrogen sulphide produced may be 
large enough to become perceptible to the sense of smell, as happens in 
the putrefaction of eggs. At the bottom of seas, rivers, lakes and 
ponds (in canals, ditches, swamps, etc.) as well as in finer-grained soils 
the production of hydrogen sulphide goes on almost uninterruptedly 
owing to the activities of a great variety of bacteria. The hydrogen 
sulphide thus generated serves as a source of energy to a group of 
organisms known as sulphur bacteria. The oxidation of the hy- 
drogen sulphide by these bacteria may be expressed by the following 
equations: 

2 H 2 S + O 2 = 2 H 2 O + S 2 
S 2 + 2 O 2 = 2SO 2 

The sulphur dioxide produced is further changed into sulphuric acid 
in the presence of oxygen and water. In its turn the acid reacts with 
some base, usually calcium carbonate, resulting in the formation of 
calcium sulphate. Thus: 

SO 2 +0+H 2 O = 


We owe much of our knowledge concerning the sulphur bacteria to 
Winogradski. This investigator showed that in places where hydrogen 
sulphide is generated in considerable quantities sulphur bacteria grow 
vigorously and accumulate granules of sulphur within their cells. 
When the cells containing sulphur granules are removed to suitable 
media, in which no hydrogen sulphide is present, the sulphur seems 
to be gradually oxidized and disappears and the bacteria finally die of 


CHANGES IN INORGANIC CONSTITUENTS 423 

starvation. Thanks to the sulphur bacteria, the higher plants are 
enabled to utilize again the sulphur once locked up in plant and ani- 
mal tissues, and liberated thence by decay bacteria. The circulation 
of sulphur is thus made possible and the cycle is completed when the 
sulphates are again used by plants to build protein compounds. It 
may also be noted in this connection that "Thiobacillus denitrificans," 
described by Beyerinck, may also oxidize elementary sulphur. In 
this case, however, the oxygen is derived from nitrates instead of the 
atmosphere. Thus: 


6KNO 3 + 58 + 2CaCO 3 = 3K 2 SO 4 + 2CaSO 4 + 

SULPHOFICATION. Lint has found that under optimum temperature 
and moisture conditions, sulphur applied at the rate of 600 pounds 
per acre was almost completely oxidized within ten weeks. Boullanger 
and Dugardin in explaining the fertilizing action of sulphur on the 
basis of its effect on the supply of available nitrogen found that am- 
monification was increased by small amounts of sulphur, nitrogen- 
fixation was not affected and nitrification was depressed. It has been 
pointed out by Kossovitch, Brioux and Puerbet that the mechanism 
of sulphur fertilization is very complex and that the oxidation of free 
sulphur occurs entirely by bacterial and not by chemical means. 
Brown and Kellogg have recently advanced evidence to prove that 
soils have a definite sulphofying power which is determinable in the 
laboratory by a newly devised method. They claim that the process 
of sulphofication is mainly brought about by bacterial action, but 
probably there is also a small production of sulphates in soils due to 
chemical action. 

It has been observed that soils differentiated by various treatments, 
vary widely in sulphofying power, the presence of organic matter being 
responsible for an increase up to a certain point. Aeration and mois- 
ture must be optimum for favorable sulphofication while the addition 
of carbohydrates to soils depresses the process. 

SULPHATE REDUCTION. The fact that sulphates may be reduced to 
sulphides in the presence of organic matter has been known for many 
years. In compost heaps, and at the bottom of seas, lakes and rivers, 
the reduction of calcium sulphate is of common occurrence. Similarly, 
ferrous sulphate may be reduced in water-logged soils and in swamps 


424 MICROBIOLOGY OF SOIL 

and may give rise to deposits of bog iron. But while sulphate reduction 
is of common occurrence in certain localities, it has been shown by Bey- 
erinck and also by van Delden, that the reduction can be accomplished 
in artificial media by specific microorganisms. Two species isolated by 
these investigators have been named Sp. desulphur leans and Msp. 
cestuarii. When grown under anaerobic conditions in culture media 
supplied with combined nitrogen and organic nutrients these organisms 
were found capable of reducing sulphates. The oxygen withdrawn 
from the sulphates was used for the oxidation of organic matter in a 
manner analogous to that in nitrate reduction where ' the oxygen is 
derived from the nitrates. Apart from the two organisms that cause 
the specific reactions just noted, there are many common soil bacteria 
that may be responsible for sulphate reduction in a less direct manner. 
Nadson has observed that when the supply of oxygen is limited calcium 
sulphate may be reduced to sulphide by B. mycoides and by B. (Proteus) 
vulgaris. The calcium sulphide according to him may react with car- 
bon dioxide and water, giving rise to the formation of hydrogen sul- 
phide. Thus: 

CaS + CO 2 + H 2 O = CaCO 3 + H 2 S 

The hydrogen sulphide derived from sulphates or from proteins 
becomes a source of energy to the sulphur bacteria as already noted in 
the preceding pages. 

POTASSIUM 

THE TRANSFORMATION OF POTASSIUM COMPOUNDS IN THE SOIL.- 
Potassium occurs in the soil largely in the form of silicate minerals. 
Smaller amounts occur as nitrate, carbonate and in organic compounds. 
The portion present as silicates is often very large in clay-loam soils, 
amounting not infrequently to 22,679 kg. to 34,019 kg. (50,000 to 
75,000 pounds) per acre-foot. Unfortunately for the farmer, the grow- 
ing crops fail, in many cases, to secure sufficient quantities of available 
potash for their rapid development, notwithstanding these enormous 
stores of potassium compounds. However, when sufficient quantities 
of readily fermentable organic matter are present and the generation of 
carbon dioxide is rapid the silicates weather sufficiently fast to meet 
the demands of maximum harvests. The part played by carbon dioxide 


CHANGES IN INORGANIC CONSTITUENTS 425 

in the transformation of inert potash compounds may be illustrated by 
the following reaction: 

A1 2 O 3 K 2 O 6SiO 2 + CO 2 + 2H 2 O = A1 2 O 3 2SiO 2 2H 2 O + K 2 CO 3 + 4SiO 2 

Under actual conditions it is the aim of the farmer to stimulate 
bacteria] activities (and, therefore, the production of carbon dioxide) in 
his land by the use of animal manures or green manures and of com- 
mercial fertilizers. Apart from the influence of carbon dioxide avail- 
able potash compounds may likewise be formed on account of nitric, 
sulphuric, acetic, lactic, butyric and other acids produced by different 
soil bacteria. 

OTHER MINERAL CONSTITUENTS 

IRON. The investigations of Ehrenberg, Winogradski, Molisch, 
Adler, Ellis and others have accumulated a mass of data relating to the 
so-called iron bacteria. These organisms belong to the class of higher 
bacteria and recently forms, such as rod-shaped bacteria, have been 
isolated which have a marked ability to precipitate iron oxide out of 
solutions of iron salts. Winogradski believed that the reaction is a 
physiological one in that the microorganisms oxidize ferrous to ferric 
compounds, and utilize for their growth the energy thus made available. 
The investigations of Molisch, Adler and Ellis show, however, that the 
iron bacteria can exist very well without iron compounds and that the 
precipitation of iron oxide is due to mechanical rather than chemical 
influences. But whether physiological or mechanical the influence of 
these microorganisms is felt in the formation of bog iron, and in the 
filling up of iron pipes; in the latter instance much annoyance is occa- 
sionally experienced by those in charge of municipal water supplies. 

Compounds of iron are of considerable significance in the life 
processes of many bacterial species. For instance, it was shown by 
Lipman and after him by Koch, that Azotobacterwil]. not develop in cul- 
ture media devoid of iron compounds. In field practice small applica- 
tions of ferrous sulphate often seem to exert a favorable effect on crop 
growth, and there is reason to suspect that soil-microbial activities are 
of some moment in bringing about the results noted. 

ALUMINUM, MANGANESE, COPPER. Weathering processes and the 
relation of carbon dioxide to these processes have already been dis- 
cussed in connection with calcium and potassium compounds. To a 


426 MICROBIOLOGY OF SOIL 

great extent aluminum is affected by these reactions, for in the decompo- 
sition of feldspar, kaolinite is one of the important products formed. 
Hence, bacteria become a factor of considerable importance in the forma- 
tion of hydrated silicates of aluminum, at least, in the presence of 
organic matter. Moreover, it is recognized in the ceramic industries 
that after it is dug clay must undergo ripening in order to be suitable 
for certain purposes. The ripening process involves the activities of 
bacteria. Unfortunately very little is known about the reactions that 
occur in the ripening of clay. 

As to manganese and copper there is scarcely any experimental evi- 
dence available as to the part played by their compounds in the soil, 
particularly in so far as they affect microorganic life. To some extent, 
it is known that where Bordeaux mixture has been employed for spray- 
ing potatoes, cranberries, fruit trees, etc., plant growth is subsequently 
stimulated to a striking extent. In view of the very slight quantities of 
copper that are actually added to the soil by these sprays, it is possible 
that the effects noted are caused by stimulated or changed microbial 
activities. This view finds some support in the influence exerted by 
copper sulphate on the growth of algae in lakes, ponds, and shallow 
streams. 

It has also been reported that the decomposition of complex silicates 
has been effected from powdered minerals by nitrite bacteria. 


ANTAGONISM 

A subject which bids fair to become a fertile source of investigation 
is the application of certain biochemical laws, as established by Loeb 
and Osterhout in the animal and plant worlds respectively, to the 
effect of salts on the physiological efficiency of soil bacteria in pure and 
mixed cultures, as well as in the soil. C. B. Lip man has advanced in- 
formation concerning the antagonism between anions as related to 
nitrogen transformations in soils, with special reference to the reclama- 
tion of alkali lands. Antagonism exists to a more or less marked 
extent between anions of alkali salts (as for example between NaCl 
and Na 2 SO 4 , Na 2 CO 3 and Na 2 SO 4 and between NaCl and Na 2 CO 3 ) 
when the ammonifying or nitrifying powers of the soil are employed 
as criteria. The nitrogen-fixing flora, however, is not similarly 
affected, apparently offering greater resistance. The practical sug- 


CHANGES IN INORGANIC CONSTITUENTS 427 

gestion carried out of such data then, involves the addition of salts 
to the toxic salts already contained in a given soil, and thereby im- 
proving its ammonifying and nitrifying power. 

VARIABILITY IN SOIL FERTILITY INVESTIGATIONS 

Waynick has pointed out that the variations between different soil 
samples taken from a small area may be of such magnitudes as to throw 
doubt upon the validity of the experimental data obtained with one or 
a limited number of samples. A single sample of any soil is of little 
value as regards determinations which may be made upon it. A com- 
posite may be considered of value only after the probable error to which 
it is subject is known and this can only be determined by the use 
of a large number of individual samples. 


DIVISION IV 

MICROBIOLOGY OF MILK AND MILK PRODUCTS 


CHAPTER I* 

THE RELATION OF MICROORGANISMS TO MILK 

CHARACTER OF MILK 

The ideal milk is that which reaches the consumer in as nearly as 
possible the condition in which it leaves the udder of the healthy cow. 

The factors which determine the quality of commercial milk may be 
stated as follows: (a) Food value, (b) flavor and odor, (c) keeping 
quality, (d) cleanliness, (e) healthfulness. With the exception of the 
first, all of these qualities are in part or wholly dependent upon the 
microbial content of the milk. 

Fresh normal milk has a pleasant taste and aroma and is gener- 
ally liked as a food or drink; but unless properly cared for will not 
long remain in its normal condition. No article of human diet is 
more susceptible to undesirable changes, due to the delicate nature 
of the milk itself and to the conditions naturally surrounding its pro- 
duction and handling. The injurious changes which commonly occur 
in milk are of two kinds. 

ABSORBED TAINTS AND ODORS 

Milk is very quickly affected by odors of any sort. The foreign 
odor may be absorbed before the milk leaves the udder if the cow has 
eaten strong feeds, such as cabbage, onions, etc., or it may be absorbed 
after the milk is drawn from the cow. If milk is exposed to any 

* Prepared by W. A. Stocking with the exception of the paragraphs treating the acid-forming 
bacteria, prepare4 by E. G. Hastings. 

428 


THE RELATION OF MICROORGANISMS TO MILK 429 

strong odor, such as silage or foul air, resulting from lack of ventila- 
tion in the stable at milking time, these odors will be taken up by the 
milk with surprising rapidity. If placed in an ice chest with fresh 
strawberries or pineapple, or foods like cabbage or turnips, the milk 
will very quickly absorb the odor of these foods. The absorption 
of any foreign odor gives to milk a decidedly disagreeable taste. This 
is true even when the odor which is absorbed is pleasant in itself as 
in the case of strawberries or pineapples. When the "off" flavors are 
due to absorption they are strongest at the outset and become less 
pronounced as the milk becomes older, especially if it is subjected to 
some method of aeration. 

CHANGES DUE TO MICROORGANISMS 

While absorption of foreign odors is not uncommon, probably 
most of the undesirable flavors, found in milk when it reaches the 
consumer, are caused not by absorption but by the growth of 
microorganisms in the milk. In this class the changes are slight at 
first and increase with the age of the milk. Changes of this sort 
include the common phenomena of souring and curdling, the so- 
called sweet curdling, ropy or slimy milk, bitter flavors, gassy milk 
and a large variety of changes usually known as barny or cowy odors 
and flavors. If milk could be kept free from microorganisms, it 
might be kept for some time without showing perceptible changes in 
appearance or taste. No other food product will undergo fermenta- 
tion changes as rapidly as milk because it is an ideal culture medium 
for the growth of most kinds of microorganisms, especially bacteria 
and yeasts. Not only does milk contain the needed food elements but, 
being in liquid form, they are easily available for the use of micro- 
organisms. The proteins and milk sugar are most easily attacked 
and it is the breaking down of these which causes most of the changes 
in the milk. 

MICROBIAL CONTENT OF MILK 

The amount of care exercised in the production and handling is 
a most important factor in determining the bacterial contamina- 
tion of milk. On this basis milk may be roughly divided into three 
classes. 


430 


MICROBIOLOGY OF MILK AND MILK PRODUCTS 


COMMON MILK. When we recognize the extreme ease with which 
milk undergoes bacterial changes, we are not surprised to find that 
ordinary milk, when delivered to the consumer, contains relatively 
large numbers of bacteria. Age is one of the chief factors in de- 
termining the germ content of milk. We, therefore, expect to find 
the milk in large cities having a much higher germ content than in 
smaller cities and towns. The normal germ content of ordinary 
milk as it is found in the cities may be shown by the following 
tables. 

BACTERIA IN BOSTON MILK* 

Average taken from 2,394 Samples 

From June to September 

Per cent 
Below 100,000 bacteria per c.c 42.0 

Between 100,000 and 500,000 per c.c 29. 75 

Between 500,000 and 1,000,000 per c.c 9-75 

Between 1,000,000 and 5,000,000 per c.c 12.75 

Above 5,000,000 per c.c 5.0 

Uncountable plates 0.75 

BACTERIAL COUNTS OF CHICAGO (RAW) 

Date Number of Average Lowest Highest 

samples count count count 

January 64 1,067,000 27,000 5,500,000 

April 43 5,948,000 14,000 150,000,000 

July 183 12,548,000 8,000 190,000,000 

BACTERIA IN MILK OF CONNECTICUT CITIES J 

Bacterial count Number of samples 

Under 50,000 1,707 

50,000-100,000 130 

100,000-500.000 459 

500,000-1,000,000 98 

Over 1,000,000 73 

These figures give the results of 2,467 samples collected in seventy-five different 
towns in the State covering a period of one entire year. 

Goler gives the average bacterial count for 1,057 samples of market milk collected 
in Rochester during the year 1909 as 446,099 per c.c. Of these samples 1.79 per cent 
were above 5,000,000 and 38.4 per cent below 100,000. 

* Data given by Hill and Slack, 
t Data given by-'Tonney. 
t Data given by Conn. 


THE RELATION OF MICROORGANISMS TO MILK 431 

In Montclair, N. J., the average bacterial count for the year 1918, 
for the fifteen producers who delivered raw milk, was as follows: 

BACTERIAL COUNTS OF RAW MILK, MONTCLAIR, N. J., 1918 

Producer's Xo. Average Count 

1 6,OOO 

2 IO,5OO 

3 20,000 

4 37,000 

5 45,3oo 

6 47,000 

7 53,ooo 

8 65,500 

9 68,000 

10 75,ooo 

11 82,000 

12 82,OOO 

13 90,000 

14 171,000 

15 226,000 
Average 71,886 

In Ithaca, N. Y., samples taken for the year 1919 gave average 
bacterial counts by months as follows: 

BACTERIAL COUNTS OF MILK IN ITHACA, N. Y., 1919 

Month Average Count 

January ... 111,450 

February ... 145,990 

March 101,050 

April 93,46o 

May 123,320 

June 115,865 

July 66,525 

August 47,620 

September 151,260 

October 11,030 

November 27,120 

December 91,700 


' 43 2 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

The immense numbers of bacteria found in milk in the large cities 
are usually the result of the rapid growth of the Bad. lactis acidi group 
resulting from the age of the milk and the temperature at which it has 
been kept. Such milk may also contain large numbers of those sapro- 
phytic organisms which occur freely in nature and which may be 
abundant about the stables and milk-house. The number of this group 
depends largely upon the sanitary conditions of production and the 
initial contamination. In ordinary milk organisms of the Bact. lactis 
acidi type will constitute a very large percentage of those present when 
the milk reaches the city even before it shows any perceptible signs of 
souring. During the past few years great progress has been made in the 
production of clean milk and at present quite an important part of the 
general raw milk supply of our cities has a very much lower germ con- 
tent than it had a few years ago. 

SPECIAL MILKS. In this class may be considered those milks known 
as Selected, Inspected, or Guaranteed. As commonly used these terms 
mean milk which has been produced and handled with considerably 
more care than ordinary market milk but not with the extreme care 
required for certified milk. While these and similar terms do not always 
mean milk of the same grade in different places, they usually mean milk 
produced by herds which have been shown by the tuberculin test to be 
free from tuberculosis. Considerable care is exercised in all the opera- 
tions of handling the milk. The result is that these milks usually have 
a much lower germ content than the ordinary milk supply of the same 
city. Sometimes the germ content of such milk compares favorably 
with that of certified milk. These milks may contain various types of 
normal milk organisms but they should not contain any tubercle 
bacteria. 

CERTIFIED MILK. Certified milk means milk which has been pro- 
duced according to the regulations of and under the supervision of a 
medical milk commission. The stables and cows are kept extremely 
clean. No dust is allowed in the stable at milking time. The cow's 
flanks and udder are washed just before milking, the milkers wear white 
suits and wash their hands before milking each cow. Small-top pails 
are used and the milk is cooled as soon as drawn from the cow. The 
extreme care exercised in the production and handling of this milk has 
a very marked effect on the number of bacteria found in it. The follow- 
ing counts are typical of certified milk. 


THE RELATION OF MICROORGANISMS TO MILK 

BACTERIAL COUNTS OF CERTIFIED MILK IN DIFFERENT CITIES 
Boston, Oct. i, 1909 to Sept. 30, 1910* 


433 


Farm number 


Number samples 


Average bacteria count 


I 


17 


5,794 


2 


13 


4,176 


3 


30 


6,825 


4 


12 


1,475 


5 


7 


2,294 


New York City, Oct., 1909 to Sept., 1910! 


Farm number 


Average bacteria count 


I 


11,132 


2 


I0,5l6 


3 


8,504 


4 


16,193 


5 


2,863 


6 


11,246 


7 


23,705 


8 


5,370 


9 


15,062 


10 


459 


Chicago J 


Farm number 


Number samples Average bacteria count 

. 


I 


51 5,612 


2 


60 4,078 


3 


43 6,502 


4 


i? 2,553 


Brooklyn 

Moak gives the average of 321 counts for certified milk delivered in Brooklyn 
during the first six months of 1910 as 4,095 bacteria per c.c. The best average from 
any one farm was 561 bacteria per c.c. 

* Data given by Arms, 
t Data given by Park, 
t Data by Heinemann. 
28 


434 


MICROBIOLOGY OF MILK AND MILK PRODUCTS 


SOURCES OF MICROORGANISMS IN MILK 


The sources from which bacteria get into the milk have been the sub- 
ject of much investigation during the past few years, until now the chief 
sources of contamination are pretty well understood. These sources 
may be grouped in a general way under the following heads: 


FIG. 138.- 


- Vertical section of one quarter of udder showing teat, milk cistern, and 
larger milk ducts. (After Ward and Hopkins.} 


INTERIOR OF THE Cow's UDDER. Healthy Udders. Milk as it is- 
secreted by the normal udder of a healthy cow is probably free from 
bacteria. It is very difficult, however, to obtain milk from the udder 


THE RELATION OF MICROORGANISMS TO MILK 435 

which does not contain bacteria in greater or less numbers. This is due 
to the fact that immediately after secretion the milk becomes contami- 
nated by bacteria which exist in the interior of the udder. Early inves- 
tigators, notably de Freudenreich and Grotenfelt, believed that milk 
while in the udder was entirely free from microorganisms. Later inves- 
tigations, however, by Moore, Ward, Bolley, Hall and others, have 
shown that the healthy udder normally contains bacteria in appreciable 
numbers. It has been found that bacteria are present even in the upper 
portions of the udder in the small milk passages leading from the se- 
creting cells. These organisms, which normally exist in the milk pas- 
sages of the udder, gain entrance through the orifice in the end of the 
teat where they find suitable conditions for growth and, once inside, 
work up through the milk cistern to the larger milk ducts and finally 
though all parts of the udder (Fig. 138). The number of bacteria found 
in the udder varies widely in different cows as may be seen by the 
following figures: 

v 

BACTERIAL CONTENT OF ENTIRE MILK OF DIFFERENT Cows 

Cow No. i 850 bacteria per c.c. 

Cow No. 2 750 bacteria per c.c. 

Cow No. 3 25 bacteria per c.c. 

Cow No. 4 112 bacteria per c.c. 

Cow No. 5 70 bacteria per c.c. 

Cow No. 6 1)850 bacteria per c.c. 

If portions of milk are taken at different intervals during the process 
of milking in such a way that all external contamination is prevented, it 
will be found that the first few streams of " fore-milk" contain many 
more organisms than the milk drawn later. After the first ten or twelve 
streams the number of organisms will decrease quite rapidly, normally 
becoming less and less until the final strippings, when there is usually a 
marked increase. This condition indicates that the larger number of 
organisms exist in the milk cistern and larger milk ducts in the lower 
part of the udder and are therefore removed during the early part of the 
milking. The increase at the end of the milking is probably due to the 
greater manipulation, resulting in dislodging some of the organisms 
which have adhered to the walls of the milk passages. 

Not only does the number of organisms in different cows vary, but 
there is a marked difference in the different quarters of the same udder, 
as shown by the following figures. 


436 


MICROBIOLOGY OF MILK AND MILK PRODUCTS 
BACTERIA IN DIFFERENT QUARTERS OF Cow's UDDER* 


Right front 
quarter of 
udder 


Left front 
quarter of 
udder 


Right back 
quarter of 
udder 


Left back 
quarter of 
udder 


No. 
sam- 
ples 


Aver- 
age per 
c.c. 


No. 
sam- 
ples 


Aver- 
age per 
c.c. 


No. 
sam- 
ples 


Aver- 
age per 
c.c. 


No. 
sam- 
ples 


Aver- 
age per 
c.c. 


Herd of 190002 


79 
185 
46 


419 
199 
161 

249 


77 
174 
46 


378 

130 

107 

191 


80 

185 
46 


653 
636 

597, 
635 


80 

186 
46 


617 
698 
342 

625 


Herd of 191011 


Herd of A. G. L 


Averages 


Average germ content per c.c. in 316 samples from herd of 1900-02 518 

Average germ content per c.c. in 730 samples from herd of 1910-11 420 

Average germ content per c.c. in 184 samples from herd of A. G. L 320 

Average germ content per c.c. in 1,230 samples from 78 cows 428 

The number of organisms normally found in the udder is much 
smaller than would be expected when we consider the fact that ideal 
conditions of food and temperature are provided there for bacterial 
growth. The relatively small number of organisms is perhaps due to 
some germicidal action existing in the udder. Attempts to increase the 
germ content in the udder by injecting cultures of different species of 
saprophytic bacteria have failed to produce a continued increase, the 
injected organisms usually decreasing very rapidly in numbers until 
they disappear at the end of a few days. From the standpoint of 
ordinary market milk, the number of bacteria found in the healthy 
udder is so small that it is of little commercial importance. In dairies 
where a very small germ content is desired, however, this source of in- 
fection must be taken into account and in certain cases individual cows, 
which normally have a high bacteria content in the udder, can be dis- 
carded to advantage. 

It is evident that many species do not find the conditions in the 
udder suitable for their growth, since investigations have shown that 
comparatively few species exist for any length of time in the healthy 
udder. Certain types of micrococci .are the predominating forms with 
occasional cultures of other species. The Bact. lactis acidi type does 

* Harding and Wilson: Technical Bui. No. 27, N. Y. Agric- E*P. Sta., 1913. 


THE RELATION OF MICROORGANISMS TO MILK 


437 


not thrive in the udder. The types of organisms commonly found 
there do not seem to develop rapidly in the milk when it is held at low 
temperatures and fail to produce any appreciable changes in it during 
the normal life of market milk. 


FIG. 139. Colonies developing in agar plate held for ten seconds in position of 
milk pail after udder was brushed gently with the hand. 


Diseased Udders. If, however, the cow is suffering from disease 
in the udder, the bacterial condition may be quite different from that 
described above. In this case, the milk may be rilled with the specific 
bacteria before it leaves the udder. In cases of inflammatory trouble or 
tuberculosis in the udder the milk may contain very large numbers of or- 
ganisms, frequently many millions per c,c. at the time the milk is drawn. 


438 


MICROBIOLOGY OF MILK AND MILK PRODUCTS 


EXTERIOR OF Cow's BODY. The nature of the cow's coat and 
the condition under which she is normally kept favor the accumulation 
of dust and bacteria upon her body. Unless special care is taken to 
keep the cow's body free from dirt, the organisms which fall into the 
milk from this source at milking time will constitute one of the most 
important sources of contamination. The importance of this source 


FIG. 140. Colonies developing from cow-hairs planted in agar plate. 

of contamination may be recognized when we see what large numbers 
of microorganisms may be carried by small particles of dust or an 
individual cow hair. The amount of this source of contamination is 
indicated by the marked reduction in germ content resulting from the 
use of a small top pail (page 442). 

The importance of this source of contamination depends very 
largely upon the conditions under which the cows are kept and the care 
exercised in cleaning just previous to milking. In many of the certified 
milk dairies this source of contamination is reduced to a minimum and 
has little effect upon the milk. 


THE RELATION OF MICROORGANISMS TO MILK 


439 


ATMOSPHERE OF STABLE AND MILK HOUSE. --The atmosphere 
of the stable may be an important factor in influencing the bacterial 
content of fresh milk In well kept stables fairly free from dust this 
source of contamination is usually not important but in stables where 
the air is full of dust at tune of milking, the germ content of the milk 
may be appreciably increased from this source. In sanitary dairies 
this factor is fully recognized and every effort is made to prevent the 
presence of dust in the atmosphere at the time of milking. 


FIG. 141. Colonies developed from a bit of dust found in cow stable. Agar plate 

culture.' 

THE MILKER. Not infrequently the milker himself is a source of 
contamination. If his clothing and hands are dirty or if he brushes 
against the cow, the dust thus dislodged may carry into the milk 
large numbers of microorganisms. This is shown in the difference in 
the germ content of milk drawn by two men milking in the same barn 
under identical conditions. 


440 


MICROBIOLOGY OF MILK AND MILK PRODUCTS 


DIFFERENCE IN NUMBER OF BACTERIA IN MILK DRAWN BY MEN IN SAME STABLE 


Number of milkings 


Number of bacteria 
per c.c. 


Milker No. i 


IQ 


2.450 


Milker No. 2 


IQ 


17.100 


THE UTENSILS. If properly cared for, the dairy utensils should 
not add to the germ content of the milk. Not infrequently, however, 
they are faulty in construction. In open seams and other places the 
milk may accumulate and not be thoroughly washed out. Usually 
when utensils of this sort are used, the methods for washing and ster- 
ilizing are not sufficient and bacteria multiply in large numbers in the 
cracks and crevices and contaminate each new lot of milk put into 
them. Sometimes the utensils which are properly constructed may 
contaminate the milk because they have not been properly cleansed 
and sterilized. The possible effect of the utensils on the germ content 
of the milk put into them is shown by recent work done at the Illinois 
Agricultural Experiment Station.* It was found that when the uten- 
sils were properly washed and thoroughly steamed and dried they did 
not add many bacteria to the milk. On the other hand, when they 
were not well steamed and especially when allowed to stand wet for 
several hours they added very large numbers of bacteria to the milk. 
This is shown by the following table. 

AVERAGE NUMBER OF BACTERIA ADDED TO FIFTY LITERS OF MILK BY THE VARIOUS 
UNSTEAMED UTENSILS IN WHICH IT WAS HANDLED 


Source of bacteria 


Number of bacteria 
per cc. of milk 


Total number 
of bacteria 


Sources other than utensils 


=5,000 


2^o,OOO,OOO 


T. oails. 


4,63=; 


2,73i,7 c ;o,ooo 


O r 

i strainer 


7,3115 


36 <, 7^0,000 


i clarifier tank. . . 


8,038 


4OI,OOO,OOO 


i clarifier : 


IAI.^AO 


7,067,000,000 


i cooler 


C;O,QOO 


2,^4^,000,000 


i bottle-filler tank 


83,246 


4,162,300,000 


Total 


3^0,000 


I7,^23,7OO,OOO 


Total for utensils. 


34^,000 


/>O~O>/ ^^j^ 

I7.273.7OO.OOO 


/ > / O> / v - / ^ / j v ^ < -"~' 


* Illinois Bull. 204, 1918. 


THE RELATION OF MICROORGANISMS TO MILK 441 

These figures indicate that the utensils may play a much more impor- 
tant part in determining the total germ content of milk than was 
formerly supposed. The use of steam is the most efficient means of 
sterilizing all dairy utensils, but boiling water may give very satisfac- 
tory results if used at actual boiling temperature. If not used at the 
boiling temperature some of the resistant organisms will not be 
killed and will be left to inoculate the fresh milk. The ropy milk 
organism, B. lactis wscosus, often remains in the utensils from day to 
day in this way. 

WATER SUPPLY. Sometimes the water used for washing the dairy 
utensils is a serious source of contamination. Serious epidemics of 
disease have been traced to this source where the utensils were washed 
with water contaminated by typhoid or other disease organisms 
and were not sufficiently sterilized to kill those remaining in the uten- 
sils. Such dairy troubles as ropy milk and gassy milk may be caused 
by the water used for washing purposes. 


METHODS OF PREVENTING CONTAMINATION OF MILK 

INDIVIDUAL Cows. Normally the number of microorganisms 
found in the udder is not sufficient to be a serious source of contami- 
nation for market milk. There are, however, certain cows which 
have a much higher germ content than others, and where a very low 
count is desired in the milk, it may sometimes be advisable to elimi- 
nate such cows from the herd. 

CARE OF THE Cow's BODY. In order to reduce to the minimum the 
contamination from the cow's body, she should be kept as clean as 
possible. Dust should not be allowed to accumulate in her coat. 
It is well to keep the hair of the flank and udder clipped in order to 
prevent the accumulation of dust and also to facilitate the process of 
cleaning. The use of a damp cloth for wiping the flank and udder 
at milking time is a very efficient means of reducing this source of 
contamination. The beneficial effect of this method may be seen 
in the following table. 

Even when considerable care is taken to clean the surface of the 
cow's body, there will still be some organisms which may fall into the 
pail at milking time. This number can be very materially lessened 


442 


MICROBIOLOGY OF MILK AND MILK PRODUCTS 


EFFECT OF WIPING UDDER AND FLANK WITH A DAMP CLOTH AS SHOWN BY BACTERIAI 

COUNTS OF MILK 


Number of experiments 


Date 


Treatment 


Bacteria per c.c. 


I 


Apr. it 


Not wiped 


2,780 


2 


Apr. i< 


Wiped 
Not wiped 


530 
I;3IO 


3. . 


Apr. 1 6 


Wiped 
Not wiped 


310 
800 


May 28 


Wiped 
Not wiped 


754 
1,130 


Wiped 


590 


by reducing as far as possible the area through which dust can fall into 
the milk pail. This can be accomplished by the use of a milking 
pail with a small top. 

VALUE OF SMALL TOP PAIL IN REDUCING GERM CONTENT OF MILK 


Experiment 


Kind of pail 


Bacteria per c.c. of milk 


No. i 


Open 


15,500 


No. 2 


Small top 
Open 


7,750 
3,700 


No. 3. 


Small top 
Open 


I ; IOO 

30,000 


Small top 


4,700 


3 4 

FIG. 142. Some different styles of small top milking pails which 

are practical and efficient. 


THE RELATION OF MICROORGANISMS TO MILK 443 

Results of extended trials in different barns demonstrate the fact 
that approximately two- thirds of the organisms which would fall into an 
ordinary open pail are kept out by the use of a pail of the type shown in 
No. 3, figure 142. The following figures give average results of trials 
in three different barns. 

BACTERIAL COUNTS OBTAINED WITH OPEN AND SMALL TOP PAILS 

Barn Kind of pail Average bacterial count 

f Open 1,610 

No. i , 

Covered 280 

Open 6,000 

No. 2 r 

Covered 3,ooo 

No. 3 I? , 33>00 

( Covered 

AVOID DUST IN THE ATMOSPHERE. Many of the necessary 
operations of the cow stable stir up large quantities of dust and fill the 
air with microorganisms. It is astonishing to see how many bacteria 
can adhere to a small piece of hay or may be found in a gram of some 
of our common dairy feeds. When these materials are fed dry just 
previous to milking time, the atmosphere of the stable will be filled 
with organisms some of which may settle into the milk while it is ex- 
posed during the process of milking. The effect of this source of con- 
tamination in one stable may be seen by the following experiments: 

BACTERIAL CONTENT OF MILK AS AFFECTED BY FEEDING DRY HAY AND GRAIN 

Experiment Date Nature of sample Number bacteria per 


c.c. 


Before feeding 

No. i May 4 Ari , ,. 

( After feeding 


No. 2 May 1 7 

No. 3 May 18 


Before feeding 
After feeding 
Before feeding 
After feeding 


350 


2,900 
4,400 
4,100 
7,200 


In another stable* where the sanitary conditions were above the aver- 
age and where all the conditions were carefully controlled, the atmos- 
phere added from 7 to 937 germs to each c.c. of the milk, the number 
varying with the amount of dust in the air. 

* N. Y. (Geneva) Agr. Exp. Sta. Bull. 409. 


444 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

DAIRY UTENSILS. All utensils which are to be used in connection 
with milk should be so constructed that there are no cracks or crevices 
in which the milk can accumulate and from which it is not easily 
washed. A milk pail with an open seam may be the cause of serious 
trouble in the dairy. The dairy utensils should be simple in construc- 
tion, and so made that they can be thoroughly cleansed with ease 
and made of such material that they can be thoroughly sterilized 
either with water which is actually boiling or in steam. They should 
then be thoroughly dried and kept so till again needed for use. When 
moisture is left in cans and other utensils bacteria can grow rapidly 
and be the means of serious contamination when fresh milk is poured 
into them. 

THE MILKER. No food material requires greater care and cleanli- 
ness on the part of those handling it than does milk. All persons having 
to do with the handling of this delicate food product should constantly 
keep in mind that clean hands and clothing and extreme cleanliness in 
every operation is very necessary if milk of good quality is to be ob- 
tained. 

GROUPS OR TYPES or MICROORGANISMS FOUND IN MILK AND THEIR 

SOURCES 

In studying the types of bacteria found in milk, it is convenient 
to arrange them in groups based upon their action on the milk and 
their effect upon persons consuming it. There are certain types of 
organisms which are very troublesome to the milk dealer but which are 
not injurious to the consumer. Other species which may be of little or 
no significance from their action on the milk are of greatest significance 
from the standpoint of the consumer since most of the disease organisms 
which may be carried by milk have no appreciable action upon it. Still 
other forms are of but little importance to either the dealer or the con- 
sumer and others are troublesome to both. 

GENERAL SIGNIFICANCE OF ACID-FORMING BACTERIA. Of all the 
bacteria that find their way into milk, those that are able to ferment the 
milk sugar, producing from it different kinds and amounts of acids, find 
more favorable conditions for growth at ordinary temperatures, 15 to 
45, than do those belonging to other groups. Because of their greater 
rapidity of growth and because of the inhibiting effect of their by-prod- 


THE RELATION OF MICROORGANISMS TO MILK 445 

ucts upon the other groups of bacteria, the acid-forming types tend to 
predominate in milk and the specific change which they produce, the 
souring, is of such common occurrence that it is often looked upon as 
something inherent in milk. 

GROUPS OF ACID-FORMING BACTERIA.*- -The acid-forming bacteria 
that are constantly present in milk represent many kinds which differ in 
morphology, in cultural characteristics, and in their products of fermen- 
tation. They may be divided into four groups that vary greatly as far 
as their importance in the handling of milk is concerned. If milk is pro- 
duced under clean conditions and is kept at temperatures ranging from 
15 to 35, the acid fermentation will be almost wholly due to a group of 
bacteria closely allied to one of the pathogenic forms, Strept. pyo genes 
(Rosenbach). To representatives of this group, which is of the great- 
est importance in all phases of dairying, have been given various names 
by different investigator?. The most important organism of this group 
is one to which the name Bad. lactis acidi is applied. The group undoubt- 
edly includes a large number of organisms, all of which produce, how- 
ever, a similar change in milk. 

Second in importance is a group of organisms, of which the best 
known representatives are B. coll communis and Bact. lactis aerogenes. 
A large number of organisms of this group have been described and 
named. The most important characteristics of the representatives 
mentioned will, however, suffice to characterize the group. A third 
group is represented by Bact. bulgaricum and the rod-shaped organisms 
that were first studied in detail by de Freudenreich. A fourth group 
includes many acid-forming cocci, some of which exhibit proteolytic 
properties while others do not. Organisms of the third and fourth 
groups exert little or no effect in the normal acid fermentation of milk, 
although they are constantly present in varying numbers, as can be 
demonstrated by appropriate means, and are of importance in certain 
phases of dairy manufacturing. 

In any sample of milk the relative number of bacteria belonging to 
each of the first two groups is dependent upon the conditions surround- 
ing production, especially with reference to cleanliness. The bacteria 
belonging to the first group come largely from the milk utensils and are 
also found in the dust of the barn and on the coat of the animal. The 
source of the second group is largely the fecal matter that gains entrance 
to the milk, although they are also found in the upper layers of the soil 

* Prepared by E. G. Hastings. 


446 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

and on grain. They are introduced into the milk with the dirt. The 
cleaner the conditions of production, the smaller will be the number of 
these two groups of organisms found in fresh milk. 

The manufacture of the leading type of butter and of all kinds of 
cheese is dependent on the action of microorganisms, hence dairy manu- 
facturing should be classed as a true fermentation industry. In all 
such industries one of the factors determining the quality of the product 
is the type of microorganism employed to produce the desired fermen- 
tation, and the importance of insuring the presence of desirable organ- 
isms, and the exclusion of harmful kinds is well recognized. 

The most important properties of organisms employed in the fermen- 
tation industries are the physiological rather than the cultural or mor- 
phological, since the quality of the product is dependent on the by- 
products of the fermentation. Hence in characterizing the groups of 
acid-forming bacteria, the biochemistry of each group will be empha- 
sized rather than the cultural and morphological characteristics of the 
members of the group. 

Characteristics of the Bact. Lactis Acidi Group.* The organisms of 
this group are widely distributed in nature, as is shown by the constancy 
with which milk undergoes the characteristic fermentation produced by 
the members of the group. 

The cells are oval in form, about 0.6^1 to i/z in length, and 0.5^ in 
diameter. The shorter cells appear nearly spherical, which, together 
with the fact that chains of cells often occur, has led some to classify 
them among the cocci and Kruse has applied the name Strept. lacticus to 
a member of the group. In milk the cells are usually in twos, the outer 
ends of the two cells being pointed. None of the group is motile; spores 
are not formed and capsules are often noted. The members of the 
group are Gram-positive. 

The optimum temperature for growth lies between 30 and 35, the 
minimum growth temperature ranging from 10 to 12, while the maxi- 
mum is 4 2. They are to be classed as facultative aerobes. The growth 
on all culture media is marked by its meagerness; in the absence of a fer- 
mentable carbohydrate, no growth usually occurs; peptone favors the 
growth even in milk. In the case of freshly isolated cultures, the 
growth is almost invisible, on slopes of sugar agar appearing as small 
discrete colonies. On sugar agar plates the colonies are small, often 

* Prepared by E. G. Hastings. 


THE RELATION OF MICROORGANISMS TO MILK 447 

surrounded by a hazy zone, and always occur below the surface of the 
medium. In lactose-agar stab cultures growth occurs along the entire 
line of inoculation, but there is no surface growth. No liquefaction of 
gelatin occurs. In bouillon the medium is uniformly turbid or it re- 
mains clear with a slight sediment. On potato, growth is slight or is 
absent. Milk is usually curdled within twenty-four hours at the opti- 
mum temperature by members of the group, although some fail to cur- 
dle the milk, since the maximum amount of acid produced is not suffi- 
cient to cause this phenomenon. Still others cause curdling in the pres- 
ence of small amounts of acids, in which case a rennet-like enzyme may 
be present. No gas is produced in the fermentation of lactose, hence 
the curd formed in milk is perfectly homogeneous; it shows but little 
tendency to shrink and to express whey. In litmus milk the color is 
discharged from the entire mass of medium before curdling occurs, due 
to the reduction of the litmus to the colorless leuco-compound. Through 
the action of the oxygen of the air the litmus is slowly reoxidized and 
the pink layer, which immediately after curdling is but a few millimeters 
in depth, is slowly extended until the entire mass of curd has a uniform 
pink color. Saccharose, dextrose, maltose, and mannit are fermented. 
The maximum amount of acid produced by organisms that are most 
typical of the group is determined by the composition of the medium. 
It is often said that the organisms causing the normal souring of milk 
represent a group that can grow in a strongly acid medium. This is 
true as far as acid salts are concerned, but free acid totally inhibits 
growth. In a culture medium, which contains no substance that can 
combine with the acid formed and thus remove it from the sphere of 
action, no growth, or but very slight growth occurs. In sugar bouillon 
and in milk, the amount of acid formed is determined by the amount of 
substances in these liquids that can combine with the acid. In milk 
such compounds are the casein and some of the ash constituents, 
especially the phosphates. In normal milk, the maximum acidity 
attained ranges from 0.9 to 1.25 per cent calculated as lactic acid. If 
the content of neutralizing compounds per unit volume is varied by 
concentration, dilution, or by the addition of such substances as cal- 
cium phosphate, the maximum amount of acid produced by typical 
cultures will be changed. In sugar bouillon the maximum acidity 
produced rarely exceeds 0.25 per cent. 


44 8 


MICROBIOLOGY OF MILK AND MILK PRODUCTS 


The fermentation of lactose is usually expressed as follows: 

Ci 2 H 22 On + H 2 O = 


Thus 342 parts of lactose should yield 360 parts of lactic acid. The 
theoretical yield of lactic acid is never obtained, for the action of the 
organism on the carbohydrate is much more complex than is represented 
by the equation given. In the following table are given data obtained 
by a number of investigators. 

These data signify that other compounds than lactic acid are 
formed in the fermentation of lactose by these acid-forming bacteria. 
Acetic acid (CH 3 .COOH); formic acid (H.COOH); propionic acid 


[ Sugar content of 
milk, 
per cent. 


Sugar fermented, 
per cent. 


Lactic acid calcu- 
lated, 
per cent. 


Lactic acid found, 
per cent, of theo- 
retical 


4-54 


0.6o 


0.632 


89-56 


4.96 


0.56 


0.590 98-13 


4-94 


0.65 


0.684 97-89 


(C 2 H 5 .COOH); traces of alcohols, aldehydes and esters have been 
found. The lactic acid formed is the dextro modification. It is be- 
lieved that the fermentation is due to an enzyme, lactacidase, one of the 
intracellular enzymes that can be demonstrated only with difficulty. 

Milk fermented by members of this group has a mild acid taste, an 
agreeable odor, and the curd can be so finely divided by agitation as to 
produce almost as perfect an emulsion as in raw milk. The organisms 
are to be classed as desirable from the standpoint of the dairy manu- 
facturer, and the fermentation produced by them may be called a true 
lactic fermentation. 

Characteristics of the B. Coli- aero genes Group.* -This group includes 
a considerable variety of organisms, which differ in morphology, in cul- 
tural characteristics and undoubtedly in the character and amounts of 
their by-products. They are more distinctly bacilli than the members 
of the preceding group; are motile or non-motile; none produces spores 
and they are usually negative to Gram's stain. The optimum growth 
temperature, 35 to 40, is somewhat higher than for the preceding 

* Prepared by E. G. Hastings. 


THE RELATION OF MICROORGANISMS TO MILK 449 

group, the vegetation range being 15 to 45. They are to be classed as 
facultative anaerobes. 

The conditions for development are less narrow than for the Bact. 
lactis acidi group, growth occurring on all the ordinary culture media 
and in the absence of carbohydrates. Indol and hydrogen sulphide 
are often formed and nitrates are reduced. The growth is usually pro- 
fuse, the colonies large and surface growth occurring in stab cultures. 
Gelatin is not usually liquefied. 

Lactose, dextrose and saccharose are fermented, with the production 
of varying amounts of gas in which have been found carbon dioxide, 
hydrogen and methane. The maximum amount of acid produced in 
any culture medium is quite similar to that formed by the members of 
the previous group. The relative proportions between the non-volatile 
and volatile acids are far different, lactic acid comprising less than 30 
per cent of the total acid formed while volatile acids, such as acetic 
and formic, make up the remainder. Traces of succinic acid 
(C 2 H4(COOH) 2 ) and alcohol have also been found. The lactic acid is 
of the laevo-form. 

Milk is usually curdled, although some members of the group do not 
produce enough acid to cause curdling. The amount of gas produced 
varies widely. In the case of those forms that cause curdling, the 
presence of gas is made evident by rents in the curd. If consider ble 
gas is produced, the curd will be very spongy. When the acid formed is 
not sufficient to curdle the milk, the gas produced is likely to pass off 
unnoticed. The curd shrinks to a greater or less extent and thus 
becomes so firm that it is impossible to emulsify it again. The odor of 
the fermented milk is often offensive and the taste disagreeable and 
sharp. The organisms of this group are to be classed as undesirable 
and. the fermentation produced by them cannot correctly be called 
a lactic fermentation. 

Representatives of these two great groups of acid-forming 
bacteria are to be found in every sample of market milk in varying 
proportions. Both find in milk favorable conditions for growth, and 
the normal souring is produced conjointly by them, each producing 
its own specific products, the relative amounts of which are largely 
dependent on the number of each group that is originally introduced 
into the milk and on the temperature at which it is kept. The higher 

temperatures tend to favor the growth of members of the B, coli- 
29 


450 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

aero genes group over that of the Bad. lactis acidi group. The value of 
milk for butter and cheese is determined by the relative amounts of 
the products of the desirable and the undesirable acid-forming bacteria. 
The difference in taste and odor between milk fermented by pure 
cultures of Bact. lactis acidi, and that which has soured spontaneously, 
emphasizes the difference in the products of the fermentations produced 
by the two groups of acid-forming bacteria. 

Characteristics of the Bact. Bulgaricum Group.*- -The organisms of 
this group are to be classed as true lactic bacteria, since they produce 
almost exclusively lactic acid from the sugar fermented and only small 
quantities of other acids as formic, acetic, and propionic. They vary 
widely in form and size; but are usually large rods, 2/4 to 3ju long and 
o.5ju to ifj. wide. There is a tendency to form long threads. They 
are Gram-positive and when stained with methylene blue often show 
distinct granules in the cells; with Neisser's stain the appearance of 
some cultures is similar to that of the diphtheria bacterium. They 
are non-motile and do not form spores; capsules are seldom noted. The 
optimum growth temperature is from 40 to 50 and the minimum is 
asserted to be 25, although for many members of the group it must be 
much lower. 

The growth on all ordinary culture media is meager or is absent: 
the colonies are often microscopic in size and show radiating threads. 
Free acids do not inhibit development and the term acidophilous has 
been applied to the group. They grow slowly in milk, even at the 
optimum temperature, and curdling may not occur for several days; 
the curd is homogeneous and in litmus milk reduction occurs. The 
maximum amount of acid varies from 1.25 to 4.0 per cent. Some 
members of the group produce dextro-, others Isevo-acid, and racemic 
acid is formed in some cases. The curd may be easily broken by agita- 
tion, and through the solvent action of the acid is partially dissolved. 
The organisms do not liquefy gelatin, but the casein of milk is partially 
changed into soluble decomposition products, as was first shown by de 
Freudenreich, and later confirmed by Hastings. 

It has been supposed by many that this group was confined to 
and characteristic of certain of the fermented milks, especially those 
of eastern Europe and western Asia, such as Yogurt and Matzoon. 
Recent work has shown that this group is widely distributed in nature. 

* Prepared by E. G. Hastings. 


THE RELATION OF MICROORGANISMS TO MILK 451 

Representatives of this group are found constantly in milk and other 
dairy products. Their presence in milk can be demonstrated by 
placing a sample of milk in a corked bottle, and incubating at 37. The 
acidity of the milk increases rapidly at first, due to the growth of the 
members of the two previous groups. These ordinary acid-forming 
organisms are soon inhibited by the appearance of free acid, but the 
acidity of the milk nevertheless continues to increase slowly, and 
with this continued increase a change in flora is noted, the short, 
plump bacilli ceasing to predominate and long slender rods constantly 
increasing in numbers. The source of this group is undoubtedly 
the alimentary tract of the animal. 

Characteristics of the Coccus Group*- -This group is well represented 
by the bacteria which form the characteristic flora of the udder. They 
vary greatly in size and in other properties. They retain Gram's 
stain; many are chromogenic, the color ranging from a white to a 
deep orange. They grow slowly on all ordinary culture media, but 
the growth is not necessarily meager. Generally they are aerobic, 
although many grow under anaerobic conditions. Gelatin may be 
liquefied or not. Milk may or may not be curdled, the curd often 
resembling that formed by rennet-like enzymes. They produce no 
lactic acid, but only acetic, propionic, butyric and caproic acids, 
and hence cannot be classed as lactic bacteria. 

BACTERIA HAVING No APPRECIABLE EFFECT ON MILK. This 
group is made up of many different forms. They produce no changes, 
during the normal life of market milk, which can be detected either 
by the eye or the taste. They do not develop very rapidly in milk, 
and some species gradually disappear while others increase in numbers. 
Many of the organisms in this group are chromogenic, orange and 
lemon yellows being among the more common forms. They are 
mostly cocci and do not liquefy gelatin. From the standpoint of the 
commercial milkman these organisms are of little significance and this 
is probably also true from the standpoint of the consumer. 

THE CASEIN-DIGESTING OR PEPTONIZING BACTERIA. These organ- 
isms digest the casein either with or without coagulation. Many of 
them cause the milk to curdle. The reaction is alkaline. The curdling 
agent is a rennet-like enzyme. They liquefy gelatin. Most of the 
organisms of this group are rods of various shapes and sizes, some 

* Prepared by E. G. Hastings 


452 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

of them being the largest rods found in milk. Some are motile and 
some non-motile. Some representatives of this group produce little 
or no odor, but many of the species develop very strong putrefactive 
odors. Barny or cowy odors or other off-flavors sometimes found in 
milk and dairy products may be caused by the action of this type 
of bacteria. They are associated with filth and their presence in 
milk indicates insanitary conditions of production or handling. 

PATHOGENIC ORGANISMS. This group includes all those species 
which may gain access to milk, which are capable of causing specific 
diseases in human beings. They are of the greatest importance to the 
consumer. They do not appreciably affect the physical or chemical 
properties of the milk, or produce any changes in its appearance, 
flavor, or keeping quality which would indicate their presence. 
Some of them do not even develop in milk, as is the case with the Bad. 
tuberculosis. Others, as the diphtheria bacteria and typhoid fever 
bacilli, may grow in milk with great rapidity. This group also con- 
tains certain species which produce diarrhceal disorders, especially 
in infants and young children. Some of them are probably organisms 
which are also included in the peptonizing group. The specific 
pathogenic organisms, possibly with the exception of Bact. tubercu- 
losis, get into milk, either directly or indirectly, from human patients 
suffering with the particular disease. 

FACTORS INFLUENCING THE DEVELOPMENT OF MICROORGANISMS IN 

MILK 

The number of microorganisms found in fresh milk shows its bac- 
terial condition at that time ; but it gives little idea of the organisms 
which may be found in the same milk at later periods. There are 
many factors to be considered if we wish to study the development 
of the various types which get into ordinary milk. These factors 
may be considered briefly under the following heads: 

INITIAL CONTAMINATION. Fresh milk varies widely in the number 
of organisms which it contains as a result of the conditions under 
which it has been produced. There are differences not only in the 
numbers of organisms but also in the species which may be found in 
different samples of fresh milk. Both of these factors are important 
in the later changes which may take place. The effect of numer- 
ical initial contamination may be seen in the following tables where 


THE RELATION OF MICROORGANISMS TO MILK 


453 


EFFECT OF INITIAL CONTAMINATION ON DEVELOPMENT OF BACTERIA AND KEEPING 

QUALITY OF MILK 

Milk Having Moderately High Initial Contamination 
Bacteria per c.c. in fresh Bacteria I2 hours Bacteria 36 hours Hours to curdling 


187,000 


432,000 


45 


Milk Having Moderate Initial Contamination 


Bacteria per c.c. in fresh 
milk 


Bacteria 12 hours 


Bacteria 36 hours 


Hours to curdling 


3,000 


14,000 


149,650,000 


99 


Milk Having Small Initial Contamination 


Bacteria per c.c. in fresh 
milk 


Bacteria 12 hours 


Bacteria 36 hours 


Hours to curdling 


325 


1,712 


10,125,000 


121 


milk starting out with different numbers of organisms was kept under 
similar conditions until coagulation. Plate cultures made from these 
three samples show the relative development of the number of 
organisms. 

These samples were all kept at a constant temperature of 21 and 
the difference in the numbers of bacteria and the curdling time can 
therefore be fairly attributed to the difference in the initial contamina- 
tion of the three samples. All three of the samples showed a normal 
development of the lactic organisms, which constituted over 99 per 
cent of the total organisms present at the time of curdling. While 
this may be considered as showing the normal effect of the original 
contamination upon the milk, it is well to bear in mind the fact that 
there are many apparent exceptions due to some particular type of 
organism predominating and interfering with the normal development 
of the lactic types. 

STRAINING. The straining of milk is one of the most common 
operations in connection with its handling and is considered by most 
dairymen as one of the most essential from the standpoint of the qual- 


454 


MICROBIOLOGY OF MILK AND MILK PRODUCTS 


ity of the milk. If milk is strained through cheese cloth or wire 
gauze much of the insoluble dirt can be removed. This has led to the 
general belief that straining improves the sanitary and keeping quali- 
ties of the milk. 

The effect of straining on removal of insoluble dirt is shown by 
the following results of tests: 

DIRT REMOVED BY PASSING MILK THROUGH Two THICKNESSES OF FINE CLOTH 
(Weight of insoluble dirt given in milligrams per liter of milk) 


Experiment 


Before straining 


After straining Per cent removed 


No. i 


8. os 


4. 7O 


47 . ^ 


No. 2 


yj 
c cc 


A. Q^ 


T-/ o 

10 8 


No. 3. 


3 OO 

c re 


T- yo 
2 Q$ 


A.2 . 7 


No. 4. 


o o 
2 .41? 


yJ 
O 2O 


Ir* I 

QI 8 


No. 5 


*T J 

c .cx 


3 . IO 


38 6 


o ^o 


O 


O" 


It may be noticed that even after straining the milk contained 
appreciable quantities of insoluble dirt which had passed through 
the strainer cloth. The difference in per cent of dirt removed in 
different samples is due to the nature of the dirt itself. The coarser 
the dirt the greater the proportion that will be removed by straining. 

It is not true, however, that the keeping quality is necessarily 
improved by the simple process of straining. It depends largely upon 
the condition of the milk and the nature of the strainer. Not infre- 
quently passing milk through a strainer not only fails to improve its 
keeping quality but actually injures it. This has been shown by a 
number of investigators. The effect of straining upon the germ con- 
tent may be seen in the following figures where the milk was passed 
through a strainer composed of three thicknesses of fine cheese cloth 
supported by wire gauze. 

EFFECT OF STRAINING UPON BACTERIAL CONTENT OF MILK 


Experiment 


After straining, 
bacteria per c.c. 


No. i 


2 6OO 


3 600 


No. 2 


7 AOO 


6 ooo 


No. 3.. 


/ j'r'"""' 

I 2 8oO 


No. 4. 


8 800 


j' 5 


No. .;. 


2.800 


2.7OO 


THE RELATION OF MICROORGANISMS TO MILK 455 

The effect of straining upon the keeping quality is shown in the 
following experiments where the milk was strained through the same 
form of strainer mentioned above and the samples kept at constant 
temperature of 21 until coagulation. 

EFFECT OF STRAINING UPON KEEPING QUALITY OF MILK 


Not strained, Strained, 

hours to coagulation hours to coagulation 


Experiment No. i 42 

Experiment No. 2 

Experiment No. 3 35 


42 


Experiment No. 4.. 
Experiment No. 5.. 


89 54 


It will be seen that in no case was the keeping quality of these 
samples increased by the straining process while in some cases it 
was materially injured. 

Cotton filters are more efficient than cheese cloth and in some 
cases the keeping quality of the milk may be improved by this process. 

AERATION. This is the process of exposing the milk to the atmos- 
phere by allowing it to run over the surface of the aerator in a very 
thin film If milk has been produced under such conditions that it 
has absorbed foreign odors, this process may be of value in getting 
rid of the absorbed odors, but from the bacterial standpoint the process 
of aerating is not desirable, since it gives one more opportunity for 
the milk to become contaminated with organisms from the atmos- 
phere and from the aerator itself. It is possible to aerate milk under 
such conditions that the germ content will not be increased, but if 
aeration takes place in the cow stable or other place where the atmos- 
phere contains dust the number of organisms will be greater after 
aeration than before, the amount of increase being proportional 
to the sanitary conditions under which the aeration is done. It is 
even possible that the milk may absorb foreign odors during the proc- 
ess of aeration and be of poorer quality than it was before. It is thought 
by many that the process of aeration is necessary in order to get rid 
of the so-called animal odors commonly found in milk. These odors 
are, however, not normal to the milk but are absorbed from the foul 
air in the stables or other sources. This is shown by the fact that some 
of the very finest quality of certified milk is bottled while still con- 


45 6 


MICROBIOLOGY OF MILK AND MILK PRODUCTS 


taining the animal heat with the least possible exposure to the air, 
tightly sealed at once and plunged into ice water. Such milk contains 
no suggestion of animal odor. Aeration may be of value in removing 
undesirable odors from milk which is not produced under good 
sanitary conditions, if done in an atmosphere free from all dust and 
odors, but it is not necessary for milk of good quality. The common 
belief that aeration is valuable is probably due to the fact that most 
aerators are coolers as well, and the beneficial results are due to the 
cooling and not the aeration. 

CENTRIFUGAL SEPARATION. It is a common practice in some milk 
plants to pass the milk through a centrifugal separator or clarifier to 
remove any dirt which it may contain. This operation is effective 
for the removal of much of the insoluble dirt which may be in the milk, 
but it is of doubtful value from the standpoint of the bacterial content 
and the keeping quality of the milk. In spite of the fact that the 
separator slime is very rich in bacteria, the milk and cream as they 
come from the machine will normally show larger bacterial counts in 
agar and gelatin plates than will the milk before treatment, due of 
course to the breaking up of the bacterial groups. In some cases, 
however, there is an apparent decrease. The usual effect upon the 
germ content of passing milk through a separator or clarifier may be 
seen in the following tables : 

INFLUENCE OF PASSING MILK THROUGH A CENTRIFUGAL SEPARATOR UPON THE 
GERM CONTENT OF THE SKIM MILK AND CREAM 


Plate count in 
whole milk 


Plate count in 
skim milk 


Plate count in cream 


Sample No. i 


30 OOO 


6o.OOO 


7^,ooo 


Sample No. 2 


OVj^ 

44., OOO 


\jy,\s< 

76,000 


7QO.OOO 


Sample No. 3 


^6,OOO 


7^,000 


820,000 


Sample No. 4 


2OO,OOO 


336,000 


330,000 


OO^)^ 


JO^)^ 


EFFECT OF A CENTRIFUGAL CLARIFIER UPON THE GERM CONTENT OF MILK 


Sample 
number 


Plate count before 
clarifying 


Plate count after 
clarifying 


Numerical 
increase 


Percentage 
increase 


I 


6,OOO 


9,OOO 


3,000 


50 


2 


15,000 


22,000 


7,000 


46 


3 


6o,OOO 


156,000 


96,000 


1 60 


4 


133,000 


I97,OOO 


64,000 


48 


5 370,ooo 


643,000 


273,000 


73 


THE RELATION OF MICROORGANISMS TO MILK 


457 


Similar results have been reported by Bahlman,* by Mclnerney,f 
and by Sherman. J Some investigators, especially Hammer || and 
Marshall and Hood, have reported results showing that in some lots 
of milk the plate count from the clarified milk is less than in the original 
milk. This is shown in the following data given by the last named 
authors. 

BACTERIA IN COMMERCIAL MILK BEFORE AND AFTER CLARIFICATION 


Sample No. 


Number of bacteria 
in I cubic centi- 
meter of un- 
clarified milk 


Number of bacteria 
in I cubic centi- 
meter of 
clarified milk 


Per cent increase 


I 


250,000 


900,000 


260 


2 


100,000 


200,000 


IOO 


3 


75,000 


65,000 


-13 


4 


20,000 


50,000 


150 


5 


5,000 


12,000 


14 


6 


125,000 


7O,000 


-44 


7 


130,000 


400,000 


207 


8 


25,000 


48,000 


92 


9 


20,000 


35,ooo 


75 


10 


350,000 


250,000 


-28 


ii 


30,000 


40,000 


33 


12 


40,000 


50,000 


25 


13 


30,000 


20,000 


-33 


14 


10,000 


10,000 


15 


16,000 


33,000 


106 


In the case of the increased counts they do not mean that there is an 
actual increase in individual bacteria in these samples due to the action 
of the separator or clarifier. What it does mean is that the small 
clusters or groups of organisms, as they exist in the whole milk are 
thrown apart by the centrifugal force and therefore develop a larger 
number of individual colonies in the plate cultures in spite of the fact 
that large numbers of organisms are thrown out in the slime. 

* Bahlman, Clarence. Milk Clarifiers, Am. Jour, of Public Health, 1916, Vol. VI, No. 8, 
1916. 

t Mclnerney, T. J. Clarification of Milk. Cornell Agr. Exp. Sta. Bull. 389, April, 1917. 

J Sherman, James M. Bacteriological Tests of Milk Clarifier. Jour, of Diary Science, 
1917, Vol. I, No. 3, p. 272. 

|| Hammer, B. W. Studies on the Clarification of Milk. Iowa Agr. Exp. Sta. Bull. 28, 
1916. 

Marshall, C. E. and Hood, E. G. Clarification of Milk. Mass. Agr. Exp. Sta. Bull. 187, 
Nov., 1918. 


458 


MICROBIOLOGY OF MILK AND MILK PRODUCTS 


TEMPERATURE. The temperature at which milk is kept is one of 
the most important factors determining the development of its micro- 
bial content. Every one at all familiar with milk knows that it spoils 
very quickly if allowed to stand at warm temperatures. If, how- 
ever, the milk is held at temperatures of 10 or lower, its keeping 
quality is greatly increased. Most of the ordinary species of organisms 
which gain entrance to milk do not grow rapidly at temperatures 
of 10 or lower. There are, however, certain species which will 
grow with considerable rapidity at temperatures below 10. especially 
some of the spore-bearing non-acid forms. If the temperature of the 
milk is allowed to rise above 10, the growth of the common species 
increases rapidly. The influence of temperature upon the develop- 
ment of bacteria may be seen in the following experiment where 
a given lot of milk was thoroughly mixed and divided into seven 
portions, which were then held at the temperatures indicated for 
twelve hours, at the end of which time they were plated for the 
total germ content. 

EFFECT OF DIFFERENT TEMPERATURES UPON THE DEVELOPMENT OF BACTERIA 

IN MILK 


Temperature 
for 12 


maintained 
hours 


Plate count per c.c. at end 
of 12 hours 


Hours to curdling 
at 21 


c. 


F. 


4.5 


40 


4,000 


75 


7 


45 


9,OOO 


75 


10 


50 


l8,000 


72 


12.5 


55 


38,000 


49 


15.5 


60 


453,000 


43 


21 


70. 


8,800,000 


32 


26.5 


80 


55,300,000 


28 


The fresh milk showed a count of 5,000 per c.c. and curdled in 
fifty- two hours at a temperature of 21. The curdling time of these 
samples was determined by placing them at a constant temperature 
of 21 at the close of the twelve-hour period and holding them at this 
temperature until coagulation took place. The difference in time of 
curdling .therefore is due to the maintenance of the special tempera- 
ture for twelve hours only and not for the entire period up to the time 
of curdling. 


THE RELATION OF MICROORGANISMS TO MILK 459 

PASTEURIZATION. The term pasteurization is used to designate 
the process of heating milk to a temperature sufficient to destroy 
a portion of the bacteria, including the pathogens, and then cooling it 
to a temperature which will prevent the rapid development of the 
organisms that are left. The temperatures commonly used for this 
purpose vary from 60 to 85. The length of time the milk is exposed 
to the high temperature may also vary from a few seconds to thirty 
minutes, depending upon the method employed. The two chief 
purposes for the pasteurization of milk are to destroy any pathogenic 
organisms which the milk may contain and to increase its keeping 
quality. The purpose for which the pasteurization is done will deter- 
mine the method used. In commercial pasteurization, where the chief 
purpose is to destroy the lactic organisms and thus improve the keeping 
quality of the milk, the method used is that known as the "flash" or in- 
stantaneous method, where the milk is subjected to a high temperature 
for a few seconds only and then cooled. In this method of pasteuriza- 
tion varying degrees of efficiency are obtained, depending upon a 
number of factors, chiefly the bacterial condition of the milk to be 
pasteurized, the degree of heat and the length of the exposure and the 
temperature to which the milk is cooled. By this method, it is possible 
to destroy a large percentage of the organisms in the raw milk, and 
materially increase its keeping quality, but the temperature and time to 
which any particle of milk is exposed cannot be accurately controlled, 
and this method cannot be depended upon to kill all of the disease-pro- 
ducing organisms which may be in the milk. This method has been 
largely abandoned for the pasteurization of market milk. 

Under present conditions of the market milk business where the 
chief purpose of pasteurization is to render the milk free from disease- 
producing organisms, the so-called "holding' 1 method is employed. 
This consists in raising the temperature of the milk to about 60 
to 63 and holding it at this temperature for a period of twenty to 
thirty minutes. If this method is properly done, most of the organisms 
except certain spore forms should be killed and the milk at the end of 
the pasteurizing process contain only a small percentage of its original 
germ content. 

Formerly it was believed that heating milk to a high temperature 
killed all the lactic acid organisms, and favored the subsequent growth of 
other more undesirable species, but more recent studies on the bacterial 


460 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

flora of milk, pasteurized by the "holding" method, have shown that 
some strains of the lactic acid bacteria can survive the relatively lower 
temperatures used in this method, and that the later development of 
the different groups of bacteria is similar to that in raw milk of equal 
bacterial grade. 

Pasteurization at the temperatures used in the holding process does 
not seem to cause any injurious chemical changes in the milk constitu- 
ents, or affect its digestibility. 

Proper pasteurization gives a valuable means of rendering the milk 
supply for our cities reasonably free from pathogenic microorganisms, 
but, in order to insure this safety, the work must be carefully done, 
and all later contamination avoided. Preferably, the work should be 
done under expert, municipal supervision. Undoubtedly the ideal 
method is pasteurization in the sealed bottle which is to be delivered to 
the consumer, since this method reduces to the minimum the danger 
of subsequent contamination. 

Pasteurization must not be regarded as a substitute for care and 
cleanliness or a means of renovating old or dirty milk otherwise unfit 
for use, but rather as an additional means of protecting the consumer 
against disease-producing microorganisms in the milk supply. 

THE USE OF CHEMICALS. The addition of certain chemicals to milk 
will retard the growth of bacteria. The chemicals most commonly used 
for this purpose are calcium hypochlorite, borax and formalin. While 
the keeping quality of milk may be materially increased by the use of 
such chemicals, their use has been opposed by health authorities and is 
contrary to the Pure Food Laws. If milk is handled with any degree 
of care, there should be no need for the use of chemical preservatives. 
They are simply a means of counteracting the unsanitary conditions of 
the production and handling. The same results can be obtained by 
cleanliness in the production of the milk and the use of low temperatures 
for preventing the contamination and subsequent growth of the 
bacteria in the milk. The developments in the production of clean 
milk of the past few years have illustrated very clearly that the use of 
chemical preservatives is not necessary. 

NORMAL DEVELOPMENT OF MICROORGANISMS IN MILK 

The flora of any particular sample of fresh milk is determined by the 
conditions under which it is produced. In stables where extreme 
cleanliness is practised the flora may be practically limited to those 


THE RELATION OF MICROORGANISMS TO MILK 


461 


species which occur.in the udder of the cows, but under ordinary condi- 
tions there will be in addition to the normal udder types such others as 
may occur on the cow's body and in the dust and atmosphere of the 
stables. Market milk, therefore, when first obtained from the cow 
ordinarily contains a mixed flora, the different types present depending 
upon the sanitary conditions under which the milk is produced. 

The future development of this initial flora is largely dependent 
upon the temperature at which the milk is kept. If the milk is held at 
temperatures between 10 and 21 there will result what may be con- 
sidered as the normal development of milk fermentations. These 
changes may be divided for convenience into four periods or stages. 

FIRST STAGE. GERMICIDAL PERIOD. It has been shown by a num- 
ber of investigators that instead of an increase in the numbers of bacteria 
in fresh milk there is normally a decrease in the number during the 
first few hours after its production. The rapidity of this decrease and 
the length of time over which it extends seem to be determined largely 
by the temperature at which the milk is kept. The higher the tempera- 
ture the more rapidly the number of organisms decreases and the more 
quickly the end of the germicidal period is reached. If the tempera- 
tures are kept fairly low the rate of decrease is much slower but the de- 
cline will extend over a considerably longer period. This is shown by 
the following examples given by Hunziker. 

TABLE SHOWING THE GERMICIDAL ACTION IN Cow's MILK 


Name 
of 
cow 


Milk, 
warm 
and 
fresh 


Temp.* 
of 
milk 


After 

o 

hours 


After 
6 

hours 


After 
9 
hours 


After 

12 

hours 


After 

IS 

hours 


After 

v, 24 
hours 


After 
hours 


After 
hours 


f 


40 


i, 080 


1,220 


1,040 


1,020 


1,120 


1,360 


1,040 


400 


May 


1,212 \ 


55 


1,260 


1,400 


1,500 


1,462 


1. 160 


i, 080 


i COO 


T7 1 ACt 


( 


70 


1,000 


1,340 


i, 860 


3,46o 


3,460 


64,000 


800,000 


( 


40 


4,400 


4,260 


3,620 


3,700 


3,900 


4,000 


3,900 


3,840 


Ida 


5,120 i 


55 


3,900 


3,460 


2,980 


2,800 


2,920 


3,260 


3 220 


3 240 


t 


70 


3,S6o 


2,120 


1,880 


1,880 


1,240 


4,96o 


58,400 


f 


40 


1,170 


1,070 


1,120 


870 


1,120 


990 


1, 060 


1, 080 


Julia 


1.345 "I 


55 


1, 080 


990 


980 


1,400 


I, O80 


i 080 


3T TO 


68 800 


70 


1,000 


I.OOO 


1,200 


5,6oo 


17,720 


1,600,000 


The exact reason for this decline is at present not well understood. 
Some investigators believe that milk possesses a certain germicidal ac- 
tion or property which results in the destruction of a portion of the 
organisms found in the milk at the outset. 

* Fahrenheit. 


462 


MICROBIOLOGY OF MILK AND MILK PRODUCTS 


The work of other investigators seems to show that the so-called 
germicidal action is felt by certain species and not by others as is indi- 
cated by the following sample. 


Age of milk 


Total 
bacteria 


Acid 
bacteria 


Per cent, acid 
bacteria 


Liquefying 
bacteria 


Fresh 


12,^0 


I,2<O 


IO 


2OO 


3 hours.. 


I2,2^O 


2,ooo 


16 


2OO 


6 hours 


10,6^0 


2,2^O 


2? 


800 


9 hours 


^6,000 


2O, 2=50 


*o 
^6 


ceo 


12 hours 


114,2^0 


68,400 


O" 

60 


oo^ 
T.OOO 


t 


* }y^ 


This would seem to indicate that the decrease in number is due not so 
much to a definite germicidal property possessed by the milk as to the 
gradual dying out of certain species which for some reason do not find 
the milk a suitable environment for development, while other types, 
finding the milk suitable to their needs, develop uniformly from the 
start. 

Rosenau and McCoy found that the germicidal properties of milk 
were destroyed by boiling or by heating it above 80 and that lower 
temperatures destroyed it for certain organisms. These workers also 
found that there was marked agglutination of the organisms in raw milk 
and conclude that this accounts for the decreased number of colonies 
developing in plate cultures and that the germicidal action is therefore 
more apparent than real. 

SECOND STAGE. PERIOD FROM END OF GERMICIDAL ACTION TO 
TIME OF CURDLING. The period following immediately after the ger- 
micidal action is characterized by the rapid development of the lactic 
organisms. Under normal conditions this group develops much more 
rapidly than any other type. Not only do they increase rapidly in 
actual numbers but their percentage also rises rapidly. There may 
be a continual increase in numbers in the other species, but their growth 
is much less rapid than that of the Bact. lactis acidi type. As this period 
advances certain of the miscellaneous types may cease to grow entirely. 
During this time the gas-producing acid organisms of the B. coli and 
Bact. lactis aerogenes type may develop more or less rapidly, but if the 
milk is held at temperatures not much above 20, the Bact. lactis acidi 
type will develop much more rapidly, so that by the time the milk be- 
comes sour and curdles, this type will constitute 99 per cent approxi- 


THE RELATION OF MICROORGANISMS TO MILK 463 

mately of the total number in the milk. From the standpoint of the 
milk consumer milk ceases to be of value when the end of this period is 
reached, but there are further developments which are of importance in 
certain lines of dairy manufactures, notably cheese making. 

THIRD STAGE. PERIOD FROM TIME or CURDLING UNTIL ACIDITY 
is NEUTRALIZED. At the time milk curdles it contains enormous num- 
bers of the lactic bacteria. The number usually runs into the millions 
and may be even higher than one thousand million per c.c. By the 
time the coagulation takes place the acidity of the milk is so high that 
the growth of the lactic organisms is checked and from this time on 
their number decreases with more or less rapidity. 

During the period following the curdling certain other types of 
organisms which have remained more or less dormant in the milk during 
the earlier stages now begin to grow. The organisms especially impor- 
tant in this stage are Oidium lactis, certain species of molds, and yeasts. 
These organisms are able to grow in a highly acid medium, and as a 
result of their development the acid is decreased until the milk finally 
presents a neutral or alkaline condition resulting from the decomposi- 
tion of the proteins in the milk. 

FOURTH STAGE. FINAL DECOMPOSITION CHANGES. The reduction 
of the acidity affords favorable conditions for the growth of certain types 
of organisms which have remained in the milk during the earlier stages 
but have been practically dormant. In this fourth stage the conditions 
are suitable for the growth of the liquefying and peptonizing bacteria 
and they now grow rapidly, causing the decomposition of the casein. 
The changes resulting from this type of organisms are of special signifi- 
cance in cheese making and are discussed more fully in another chapter. 

ABNORMAL FERMENTATIONS IN MILK 

GASSY FERMENTATION. It frequently happens that instead of the 
normally rapid development of the Bad. lactis acidi type of organisms 
in the milk, other acid producers develop rapidly, with the production 
of more or less gas. The organisms most prominent in this type of fer- 
mentation are the B. coli communis and the Bact. lactis aero genes types. 
This group of organisms contains a number of varieties, some of which 
produce little or no gas while others develop large amounts. Their 
action in milk is usually accompanied by disagreeable odors and flavors. 
They grow readily in the presence of air and therefore develop abundant 
colonies on the surface of plate cultures. This distinguishes the mem- 


464 


MICROBIOLOGY OF MILK AND MILK PRODUCTS 


bers of this group quite clearly from those of the true lactic group which 
grow chiefly below the surface of the medium. The members of this 
group do not form spores, but certain varieties are quite resistant to 
heat and will oft times survive pasteurizing temperatures which com- 
pletely destroy the Bact. lactis acidi group. They grow most rapidly 
at high temperatures, between 20 and 37. 

SWEET CURDLING FERMENTATION. This phenomenon is caused by 
a variety of organisms which cause the milk to coagulate without the 
production of acid. The coagulation is brought about by a rennet-like 
enzyme produced by this type of bacteria. The resulting milk is 
either neutral or alkaline in reaction. Usually the coagulation of the 
milk is followed by the digestion of the casein as a result of another 
enzyme which is also produced by these bacteria. The coagulation 
caused by these organisms is slower than in the case of the acid 
formers and the curd is usually soft and mushy as compared with the 
curd formed in the normal acid fermentation. The members of this 

group get into the milk from and along with dust 
and dirt associated with unsanitary conditions. 
Some of the species produce spores and are not 
killed by the ordinary methods of pasteurization. 
This fact accounts for the occurrence of sweet 
curdling of pasteurized milk. This group of or- 
ganisms is unable to develop rapidly in the pres- 
ence of the lactic bacteria and for this reason we 
do not commonly get the sweet curdling of raw 
milk. The presence of these organisms is evi- 
dence of insanitary conditions. Frequently they 
develop very disagreeable flavors in the milk. 

ROPY OR SLIMY FERMENTATION. One of the 
most common milk infections causing trouble to 
the milk dealer is that which causes a ropy or 
slimy fermentation of milk. This is sometimes spoken of as stringy 
milk (Fig. 143). Several species of organisms are capable of pro- 
ducing this condition. These organisms grow most freely in the 
presence of an abundant supply of oxygen and for this reason the cream 
usually becomes slimy before any changes are apparent in the under- 
lying layers of milk. B. lactis mscosus is perhaps the most common 
species in this group. The slimy condition in the milk is supposed 


FIG. 143. Ropy 
cream lifted with a fork. 
(After Ward.} 


THE RELATION OF MICROORGANISMS TO MILK 


465 


to be the result of a very viscid capsule surrounding these organisms 
(Fig. 144). Representatives of this group are quite resistant to heat 
and frequently pass uninjured through the methods of cleansing and 
scalding used under ordinary dairy conditions. Because of this, 
dairy utensils once infected may become a constant source of infection. 


FIG. 144. Bacillus lactis viscosus from a milk culture. (After Ward.) 

This trouble can be effectively stopped by a thorough scalding of all 
utensils coming in contact with the milk. 

BITTER FERMENTATION. Bitter flavors in milk may be the result 
of bacterial changes after the milk has been drawn, or due to certain 
feeds which the cows have consumed. If the cows are allowed 
to eat certain kinds of vegetation, such as "rag weed" and certain 
other plants, they may impart a bitter taste to the milk, in which case 
the abnormal flavor will be apparent when the milk is fresh and usually 
becomes less pronounced as the milk becomes older, because of the 
volatile nature of the substances causing the bitterness. Most of the 
cases of bitter milk and cream, however, are due to the growth of 
certain types of bacteria in which case the bitterness increases in in- 
tensity with the age of the milk. Some of the species capable of 
producing bitter milk grow at quite low temperatures, which accounts 
for the fact that the most trouble with bitter flavors is found in milk 
and cream which has been held at low temperatures for some time. 

30 


466 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

ALCOHOLIC FERMENTATION.- -The bacteria as a group are not 
able to act on the milk sugar and produce alcohol, but it sometimes 
happens that yeasts get into the milk in sufficient numbers to ferment 
the milk sugar, producing appreciable amounts of alcohol. To the 
milk handler this trouble is not usually serious but the action of the 
yeasts is frequently of considerable importance in the cheese industry. 

OTHER FERMENTATIONS. It frequently happens that a consider- 
able variety of disagreeable flavors and odors develop in milk. These 
may be due to the direct absorption of odors from the foul stable 
atmosphere or strong-smelling feeds, such as silage; or they may be, 
and no doubt frequently are, the result of the growth of certain types 
of bacteria which have entered the milk from dirty surroundings. 
The growth of some of these organisms is frequently the cause of the 
so-called cowy and stable odors and flavors. 

COMMERCIAL SIGNIFICANCE OF MICROORGANISMS IN MILK 

RELATION OF DIRT CONTAMINATION TO GERM CONTENT. To the 
commercial milkman bacteria are of importance only as they influence 
the length of time the milk will keep in a salable condition. The 
consumers do not want milk that is sour or has unpleasant flavors 
and odors. In order to sell his milk, therefore, the milkman must 
avoid the presence of these undesirable conditions, and in proportion 
as he recognizes the relation between germ life and the quality of 
his product, will he pay attention to the presence and development 
of microorganisms in his milk. In like manner, the presence or ab- 
sence of dirt contamination is important from the commercial stand- 
point since it bears a relation to the bacterial count, and, therefore, 
affects the keeping properties of the milk. Under normal conditions 
there is a fairly direct relation between the amount of visible or soluble 
dirt and the number of bacteria found in any given lot of fresh milk. 
This relation may be shown by the following samples taken from four 
different milk producers: 


Producer 


Number of 
samples 


Average mg. 
dry dirt per liter 


Average number 
bacteria per 
c.c. 


Average hours 
to time of curdling 


A 


s 


Si. 5 


II ^,000 


I7C 


B 


16 


58.8 


27^,600 


78 


C 


21 


70. o 


428,600 


7C 


D. 


17 


71.0 


04.0,4.00 


68 


THE RELATION OF MICROORGANISMS TO MILK 467 

This relation does not always hold for the reason that a gram of 
one kind of dirt may contain infinitely more organisms than an equal 
amount of some other kind. The difference in the solubility of various 
forms of dirt always causes apparent discrepancies in this normal 
relation. In the majority of cases, however, the relation shown in 
the above examples will hold reasonably true in the case of fresh milk. 
There is also a general relation between the number of bacteria in 
fresh milk and the length of time it will keep before souring and curd- 
ling. In this case the relation is in inverse ratio, the smaller the initial 
contamination, the longer the keeping time, and vice versa. This re- 
lation is also shown in the table given above. There are many ir- 
regularities, however, in this relation because of differences in the 
flora of fresh milk. It may frequently happen that a sample of milk 
containing a relatively high number of organisms will not sour as 
quickly as another sample with a smaller original germ content. The 
associative action of the different species of organisms is an important 
factor here. In making comparisons of this sort, it is, of course, 
necessary that the different samples be held at the same temperatures. 

MILK AS A CARRIER OF DISEASE-PRODUCING ORGANISMS 

It is not the purpose of this chapter to discuss in detail the diseases 
which may be carried by milk, but a chapter on bacteriology of milk 
would be incomplete without a brief discussion of this important 
subject. 

From the standpoint of their relation to the health of the con- 
sumer the microorganisms in milk may be divided into three groups 
on the basis of whether they are beneficial, inert or injurious to health. 

Acid Forms. -The preservative properties of sour milk have been 
known since very ancient times. Its use as a preservative for meat, 
eggs and other perishable food products demonstrates the value of 
sour milk as a means of preventing decomposition. It has also been 
known for a long time that sour milk has a certain therapeutic value 
because of the action of the lactic bacteria in preventing harmful 
fermentations in the digestive tract. More recently the work of 
Metchnikoff has shown the usefulness of sour milk both for the treat- 
ment and prevention of intestinal disorders by inhibiting the develop- 
ment of the putrefactive bacteria in the digestive tract. In view of 
the value of sour milk for preventing certain forms of disease and its 


468 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

inhibiting action on certain undesirable organisms the Bad. lactis 
acidi type of bacteria must be regarded as beneficial organisms, and 
from the standpoint of the health of the consumer their presence in the 
milk is to be welcomed rather than discouraged. As the value of 
sour milk drinks becomes better known the importance of this group 
of milk bacteria will be more fully recognized. 

Neutral or Inert Forms. In ordinary milk there is a large class of 
bacteria which, so far as known, have no appreciable effect either 
upon the composition of the milk or the health of the persons consuming 
it. This group includes a number of species, many of them being 
coccus forms, some of them appearing in plate cultures as chromogenic 
colonies. They grow more or less freely in milk, depending upon the 
conditions, but they are usually held in check by the acid-forming bac- 
teria and do not constitute a very important part of the flora of normal 
milk. They are, therefore, of little significance from the practical 
standpoint except as they indicate the conditions under which the milk 
has been produced and handled. 

Injurious Organisms. The diseases which may be carried by milk 
are of two classes. 

Epidemic Diseases. --The, human diseases most commonly carried 
by milk are typhoid fever, diphtheria and scarlet fever and occasionally 
other diseases such as septic sore-throat, cholera and foot-and-mouth 
disease. The first three are by far the most important of this group. 
The outbreaks of typhoid fever which are traceable to milk occur 
most frequently. There is a large accumulation of data showing the 
occurrence of epidemics caused by infected milk. An epidemic caused 
by the milk supply has certain characteristics which distinguish it 
from epidemics resulting from other causes. A considerable number 
of cases of the particular disease will appear almost simultaneously 
and will be distributed along some particular milk route. Usually 
the epidemic stops as suddenly as it began except for a few secondary 
cases contracted from those first taken. The source of the disease 
organisms is a human patient suffering from the disease. The infection 
of the milk may be direct, as when a sick person handles a milk, 
or it may be indirect as when a person caring for a patient also works 
about the milk. In other cases it may be caused by contamination 
of the water used in washing the utensils or by cows wading in water 
of infected streams and getting the organisms on their body whence 


THE RELATION OF MICROORGANISMS TO MILK 


469 


they fall into the milk pail at milking time. The return of milk bottles 
from the sick room sometimes is the means of infecting the milk supply. 


600 
80 
60 
40 
20 

500 
80 
60 
40 
20 

400 
60 
60 
40 
20 

300 
80 
60 
40 
20 

200 
80 

60 
40 

20 
I QQ 


Jan. 


\ 


Feb. 


V 


^/ 


Mar 


A 


N 


Apr. 


-\ 


\ 


May 


Jun 


July. 


A 


Aug. 


5 


Sep 


t 


Oct. 


\ 


A 


\ 


T 


-\ 


NOV. 


\ 


V 


Dec. 


600 

80 

60 

40 

20 

500 
80 
60 
40 

20 

400 

80 

60 

40 

20' 

300 

80 

60 

40 

20 

200 

80 

60 

40 


20 

100 


FIG. 145. 

Unfortunately the specific organisms of these diseases grow readily 
in milk and a small infection is all that is necessary to render the 
milk dangerous by the time it reaches the consumer. 


470 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

Non-epidemic Diseases. There is another class of diseases which 
may be carried by milk which are not characterized by a sudden out- 
break, and for this reason are not so readily recognized as being asso- 
ciated with the milk supply. One of these diseases, namely tuber- 
culosis, is caused by the specific, well-known organism, Bad. tuberculo- 
sis, which may get into the milk from the udder of a tuberculous cow 
or by the organisms which have been given off from the digestive 
tract of the animal becoming scattered about the stable and finally 
getting into the milk with particles of dust and filth. In some 
cases the milk may become infected by persons having the disease 
being permitted to handle the milk. Fortunately for mankind Bact. 
tuberculosis does not multiply in milk. 

Regarding the danger of contracting tuberculosis from the use of 
milk there is at present some difference of opinion, but the con- 
sensus of opinion at the present time seems to be that there may 
not be very great danger for healthy adults, but that a considerable 
percentage of the cases of tuberculosis of children may be traced from 
the milk supply. Fortunately none of these specific disease organisms 
produce spores and the temperatures used in the process of pasteuriza- 
tion by the ''holding" method are sufficient to destroy any of the dis- 
ease bacteria known to be carried by milk. 

There is another class of disorders not so well defined as the above 
but which are nevertheless of great importance from the standpoint of 
public health, especially of young children and also to some extent of 
adults. This group includes such disorders as infantile diarrhcea, sum- 
mer complaint, cholera infantum and other disorders of the digestive 
tract. The organisms producing these troubles doubtless belong to the 
group of putrefactive bacteria which come from filth. Some of the gas 
producers and some of the peptonizers are probably responsible for 
these troubles. Shiga isolated from a large number of cases of infant 
diarrhoea a bacterium which he named Bact. dysenteries, but in general 
the specific organisms responsible for these intestinal troubles are not 
well known. Their importance, however, is shown by the relation of 
the germ content of milk to infant mortality (see Fig. 145). 

BACTERIOLOGICAL ANALYSIS OF MILK 

The development of our knowledge of the relation of bacteria to the 
wholesomeness of foods has led to a study of the bacterial content of 
milk as a means of determining its purity. The methods used for this 
purpose have followed quite closely those of the water bacteriologists. 


THE RELATION OF MICROORGANISMS TO MILK 471 

For many years, dairy bacteriologists have endeavored to determine the number 
of organisms in milk by plating it into nutrient agar or gelatin. By this method 
the number of colonies developing in the plates is assumed to represent the germ 
content of the milk. But even when the best methods are employed, the plate count 
represents only the approximate and not the exact number of bacteria in any lot of 
milk. It should also be borne in mind that such counts are always underestimates, 
because of the fact that not all species will develop in any given medium or incubation 
temperature. The careful worker can recognize certain types of bacteria in plain 
media, but the addition of blue litmus solution to either agar or gelatin, greatly 
assists in the differentiation of types and species. 

THE DIRECT MICROSCOPIC METHOD. The plating method is expensive because 
of the large amount of time and materials needed. It is not possible for one person 
to handle a large number of samples at one time. In routine work in the city labora- 
tories this labor has been a serious drawback to this method. In order to decrease 
the labor and give greater possibilities to the work Stewart devised a method by 
which the bacterial condition of milk can be studied by direct microscopic examina- 
tion. His purpose was to determine only the species present, but later Slack and 
still more recently Breed developed the method for determining the approximate 
numbers as well as the general species present in a given sample of milk. 

. LEUCOCYTES. The microscopic examination of milk sediment revealed the fact 
that frequently a sample would be found which showed the presence of leucocytes in 
greater or less numbers. The presence of these cells was regarded as important 
because it was assumed that they showed the presence of inflammation and pus for- 
mation in the udders of the cows producing the milk. 

Several methods have been used for determining the leucocyte content of milk. 
"The Smear Sediment" and "Blood Counter" are methods which more strictly 
belong to laboratory practices and will not be considered in this place. 

BACTERIOLOGICAL MILK STANDARDS 

The relation of the bacterial content of milk to its wholesomeness 
has led to the adoption of certain standa