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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 dissolv