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THE NEWER KNOWLEDGE 
OF BACTERIOLOGY 
AND IMMUNOLOGY 



THE UNIVERSITY OF CHICAGO PRESS 
CHICAGO, ILLINOIS 

THE BAKER & TAYLOR COMPANY 
NEW YORK. 

THE MACMILLAN COMPANY OF CANADA, LIMITED 
TORONTO 

THE CAMBRIDGE UNIVERSITY PRESS 

LONDON 

THE MARUZEN-KABUSHIKI-KAISHA 
TOKYO, OSAKA, KYOTO, FUKUOKA, SENDAI 

THE COMMERCIAL PRESS, LIMITED 

SHANGHAI 



THE NEWER KNOWLEDGE 
OF BACTERIOLOGY 
AND IMMUNOLOGY 



IL 



By 
EIGHTY-TWO CONTRIBUTORS 

Edited by 
EDWIN O. JORDAN and L S. FALK 

The University of Chicago 





THE UNIVERSITY OF CHICAGO PRESS 
CHICAGO • ILLINOIS 



COPYRIGHT 1928 BY THE UNIVERSITY OF CHICAGO 
ALL RIGHTS RESERVED. PUBLISHED APRIL I928 



COMPOSED AND PRINTED BY THE UNIVERSITY 
OF CHICAGO PRESS, CHICAGO, ILLINOIS, U.S.A. 



PREFACE 

This book was prepared in an attempt to make the latest results of investigation 
in various lines of bacteriology and immunology available for students and active 
workers. It does not purport in any sense to be a textbook, nor does it pretend to be 
a comprehensive survey of the whole field. Our object has been primarily to obtain 
authoritative critical reviews of topics in which at the present time interest is par- 
ticularly keen or investigation most active. It is our hope that the book will serve to 
promote research both by furnishing landmarks of progress and by affording sugges- 
tions on significant unsolved problems. 

With these ends in view, great latitude has been given to the individual contrib- 
utors, each of whom assumes direct responsibility for the material presented. While 
the editors have endeavored to avoid serious duplication and overlapping, they have 
in a number of cases intentionally asked for and included papers with opposing views 
and interpretations in order to set clearly before the reader the divergences of cur- 
rent opinion. 

This independence of treatment has even extended to such a matter as the no- 
menclature of microbic types and species. It was early found in our preliminary cor- 
respondence that many of our contributors had very strong convictions regarding 
nomenclatorial practice, and that their convictions were widely apart. Since we did 
not ourselves feel that the time had arrived for insistence upon a uniform and rigid 
bacterial nomenclature, we chose to give full rein to individual preference. While 
we are aware that this course is open to criticism, we believe that our decision will 
at least serve to bring into yet stronger relief the almost hopeless confusion and di- 
vergence of opinion into which classification and names of bacteria have fallen. 

We considered it an important feature of our undertaking that the individual arti- 
cles should come to hand as nearly as possible at the same time in order to insure 
promptness and timeliness of publication. We are greatly indebted to our contributors 
for their generally hearty response to this request. Many of them have been able to 
comply with this condition only at serious personal inconvenience and even sacrifice. 
In a few instances illness has interfered with the preparation of an intended manu- 
script. Several of our European correspondents who originally promised articles have 
failed to send anything. 

We are under particular obligation to the Board of Trustees of the University of 
Chicago and to Mr. Gordon J. Laing, director of the University Press, for aiding the 
publication of this volume with a special fund. Among the many individuals who 
have given us signal assistance, we wish to acknowledge special indebtedness to Mr. 
Donald P. Bean and Miss Anabel Ireland, of the University Press, and to Miss 
Theodora Piatt, of the Department of Hygiene and Bacteriology, for their efficient 
and untiring interest in seeing the book through the press. 

The Editors 

February 15, 1928 










^^. . %►• ^ ^ / 
CONTENTS "^ ^ A » • • J^/' 

CHAPTER ^^Ci^ O K/*^' PAGE 

I. The Newer Knowledge of the Morphology of Bacteria~T^ — T . i 
Herbert C. Ward 

II. The Chemical Structure of Bacteria 14 

Traugott Baumgdrtel 

III. Staining Reactions of Bacteria 19 

Johfi W. Churchman 

IV. Morphological Changes during the Growth of Bacteria ... 38 

Paul F. Clark 

V. Growth Curves of Bacteria 46 

R. E. Buchanan 

VI. The Rise and Fall of Bacterial Populations 58 

C.-E. A. Winslow 

VII. The Dissociative Aspects of Bacterial Behavior 84 

Philip Hadley 

VIII. Bacterial Associations 102 

W. L. Holman 

IX. Classification of Bacteria 120 

Roger G. Perkins 

X. Atoms, Ions, Salts, and Surfaces 136 

William D. Harkins 

XI. The Effect of the Surface Tension of the Menstruum upon Bac- 
teria and Toxins 179 

W. P. Larson 
XII. Oxidation-Reduction Potentials of Dye Systems and Their Sig- 
nificance in Bacteriology 188 

W. Mansfield Clark 

XIII. Anaerobiosis 198 

Ivan C. Hall 

XIV. Bacterial Oxidations and Reductions .211 

James Walter McLeod 

XV. Protein (Nitrogen) Metabolism of Bacteria 218 

Leo F. Rettger 

XVI. The Utilization of Carbohydrates by Bacteria 227 

Arthur Isaac Kendall 
XVII. Utilization of Aliphatic and Aromatic Compounds by Bacteria . 243 
S. A. Koser 

XVIII. Gas Metabolism of Bacteria 250 

M. H. Souk 

XIX. Enzymes of Bacteria 268 

Selman A . Waksman 

vii 



30 



^ 



viii CONTENTS 

CHAPTER PAGE 

XX. Synthetic Culture Media 279 

H. W. Schocnlein 

XXI. Determinations of Thermal Death-Time 285 

/. Russell Esty 
XXII. The Standardization of Disinfectants and Antiseptics . . . 301 
George F. Reddish 

XXIII. Nature, Distribution, AND Functions OF Soil Micro-organisms . 310 

Selman A . Wakstnan 

XXIV. Autotrophic Bacteria 322 

Robert L. Star key 
XXV. The Root-Nodule Bacteria of Leguminous Plants . . . -332 
• Edwin Broun Fred 

XXVI. Micro-organisms in Relation to Soil Fertility 341 

Jacob G. Lipman 
XXVII. The Role of Bacteria in the Treatment of Sewage . . . -351 
F. W. Mohlman 

XXVIII. Some Problems in Water Bacteriology 362 

John F. Norton 
XXIX. The Action of Ultra- Violet Light on Bacteria AND Their Products 371 
Johfi F. Norton 

XXX. Bacteria in Milk 378 

Robert S. Breed 

XXXI. Bacteria in Dairy Products 395 

L. A. Rogers 
XXXII. The Bacterial and Health Aspects of Pasteurization . . . 403 
Milton J. Rosenau 

XXXIII. Mechanical and Engineering Aspects of Pasteurization . . . 419 

George W. Putnam 

XXXIV. Contamination and Deterioration of Food 437 

Charles Thorn 

XXXV. The Bacteria of Food Poisoning 443 

Edwin 0. Jordan 

XXXVI. The Spirochetes 452 

Hideyo Noguchi 

XXXVII. Current Problems on Yeasts 498 

F. W. Tanner 

XXXVIII. The Aspergilli: A Typical Group of Molds 509 

Charles Thorn 

XXXIX. FiLTEElABLE VIRUSES 5^7 

Thomas M. Rivers 
XL. The Bacteriophage: Present Status of the Question of Its Nature 

AND Mode of Action 525 

J. Bronfenbrenner 

XLI. Filterability of Micro-organisms 557 

5". P. Kramer 

XLII. A Theory of Microbic Virulence 565 

/. S. Falk 



CONTENTS 

CHAPTER 

XLIII. Elective Localization of Bacteria in the Animal Body . 
Edward C. Rosenow 

XLIV. Bacteria in Relation to Plant Diseases 

George K. K. Link 

XLV. Communicable Diseases of Laboratory Animals 

K. F. Meyer 

XL VI. Bacteria of the Intestinal Tract 

Leo F. Rettger 

XLVII. Bacteria of the Respiratory Tract 

D. J. Davis 

XLVIII. Intestinal Protozoa of Man and Their Host-Parasite Relations 
Robert Hegner 

XLlX. The Immunological Bases for Different Types of Infection by the 

Blood Protozoa 

William H. Taliaferro 

L. Antigens and Their Specificity 

H. Gideon Wells 

LI. The Chemistry of Antigens 

Sara E. Branham 
LII. Antigenic Properties of the Bacterial Cell and Antibody Re- 
actions 

Hans Zinsser and J. Howard Mueller 

LIII. Heterophils Antigens and Antibodies 

C. G. Bull 

LIV. The' Physical Chemistry of Toxin and Antitoxin 

Mary E. Maver 
LV. The Preparation and Purification of Toxins, Toxoids, and Anti- 
toxins 

Edwin J. BanzhaJ 
LVI. The Titration of Toxins and Antitoxins by the Flocculation 

Method 

Stanhope Bayne-J ones 

LVII. Sublethal Intoxications with Bacterial Products . 
J. P. Simonds 

LVIIL The Mechanism of Agglutination 

Johi H. Northrop 
LIX. The Functional Role of Agglutinins .... 

G. Howard Bailey 
LX. Bacterial Agglutinins and Their Applications 
/. G. FitzGerald and Donald T. Eraser 

LXI. Precipitins and Their Applications 

H. M. Powell 
LXII. The Complement Fescation Reaction with Bacterlal Antigens 

Augustus Wadsworth 
LXIII. The Complement Fixation Test for Syphilis .... 
Ruth Gilbert 



IX 

PAGE 

576 
S90 
607 

639 

650 
660 

679 
702 
711 

721 
733 
739 

745 

759 
772 
782 
802 
811 
824 

831 
838 



X CONTENTS 

CHAPTER PAGE 

LXIV. The Kahn Reaction 848 

R. L. Kahn 

LXV. The Mechanism of Phagocytosis 861 

W. 0. Fenn 

LXVI. Phagocytes and Phagocytosis in Immunity 870 

W. B. Wherry 

LXVII. Local and Tissue Immunity 881 

Frederick P. Gay 

LXVIII. The Human Blood Groups 892 

K. Landsteiner 

LXIX. The Heredity of the Blood Groups 909 

Reuben Ottenberg and David Beres 
LXX. Antibacterial Sera 921 

F. M. Hunloon and R. H. Hutchison 

LXXI. The Use of Human Serum from Convalescent Cases in Prevention 

and Treatment of Disease 934 

William H. Park 
LXXII. Control and Standardization of Biological Products . . . 947 

G. W^ McCoy 

LXXIII. Anaphylaxis and Anaphylactoid Reactions 966 

Howard T. Karsner 

LXXIV. The Technique of Experimentation in Anaphylaxis .... 989 

W. H. Manwaring 
LXXV. Atopy 1004 

Arthur F. Coca 
LXXVI. Tuberculin and the Tuberculin Reaction 1016 

Esmond R. Long 

LXXVII. Origin of Antibodies 1035 

Katharine M. Howell 
LXXVIII. The Isolation of Substances with Immune Properties . . . 1049 
Arthur Locke and Edwin F. Hirsch 
LXXIX. Abderhalden's Dialysis Reaction and Theory of the So-called 

"Protective" Ferments 1056 

/. Bronfenbrenner 

LXXX. Venoms and Antivenins 1066 

Afranio do Amaral 
LXXXI. A Critique of the Ehrlich Theory, with an Outline of the Enzyme 

Theory of Antibody Formation 1078 

W. H. Manwaring 

LXXXII. Non-Specific Protein Therapy 1086 

William F. Petersen 

LXXXIII. Chemotherapy of Bacterial Diseases iioi 

John A. Kolmer 

Author Index 1135 

Subject Index 1167 



CHAPTER I 
THE NEWER KNOWLEDGE OF THE MORPHOLOGY OF BACTERIA 

HERBERT C. WARD 

School of Hygiene and Public Health, Johns Hopkins University 

For years bacteriology, dominated by the cellular doctrine of Virchow, has ac- 
cepted the thesis, more or less completely proved by Cohn and Koch, that each bacte- 
rial cell is derived from a previously existing cell of practically the same size and shape. 
Owing to the fact that a large number of highly diversified forms were included in the 
group of bacteria, considerable confusion existed before Koch devised his solid media 
and his plate-pouring methods. The proof that each type of cell, such as the spherical 
coccus or the elongated bacillus, came from a cell of the same type, a coccus or bacillus, 
was of enormous benefit in straightening out the confusion and in eventually reconcil- 
ing conflicting observations and opinions. While it was early recognized that the meth- 
ods by which the bacterial cells are derived from the pre-existing cells might differ in 
different species, two regular methods were definitely estabhshed: binary fission, 
where one cell divides transversely into two new cells which eventually attain the size 
and shape of the original; and spore formation, by which a single spore forms in a ba- 
cillus, this spore subsequently giving origin to a vegetative rod like the original rod be- 
fore sporulation begins. A more complicated cycle of development from conidia was 
established for certain species which were differentiated from the simple bacteria ex- 
hibiting transverse fission or spore formation as the "higher bacteria" and called 
"streptothrix" or "actinomyces." 

Exceptions to these regular methods of reproduction of the bacterial cells were 
frequently noted. With some species appearances were described which indicated 
that new cells might rise from old cells by a kind of branching of the cytoplasm, this 
phenomenon being described as "true branching" to distinguish it from the branching 
seen in certain plants related to the bacteria, where the cells divide transversely and 
eventually so crowd the sheath in which they are contained that this itself divides. 
Knoblike protrusions from the bacterial cells were noted occasionally, and the sug- 
gestion was made that these protrusions are in reality buds, capable of growth and de- 
velopment into adult forms. Large, irregular, distorted elements were described in old 
cultures, especially by Hueppe, who regarded them as true stages in the life-cycle of 
the bacteria and named them "arthrospores." Occasionally more than one spore was 
found in a single bacillus, and the idea naturally arose that spore formation may lead 
to an actual increase in the numbers of the cells. Such double spore formation was ad- 
mittedly very rare and generally doubted so that the thesis maintained by Kruse was 
usually accepted, to the effect that spores represent resistant stages of bacteria like 
the cysts of the protozoa which serve for the perpetuation of the species under adverse 
conditions, but not for multiplication. Finally, with the flexible spiral organisms, the 
spirochetes, differing from the ordinary bacteria in many of their characteristics, a pe- 



2 THE MORPHOLOGY OF BACTERIA 

culiar type of transverse division was early noted and later worked out in great detail 
by Gross who described it as "multiplication by incurvation." It is of interest in this 
connection to note that some years ago Gotschlich, in his studies on plasmolysis and 
plasmoptysis, came to the conclusion that bacterial cells may break up into small bits 
of protoplasm, resembling in no way the original cell in size or shape but serving as 
starting-points from which new cells germinate. Gotschlich admitted that no positive 
evidence exists to show such a kind of reproduction, suggesting it merely as a possibil- 
ity which cannot be excluded. 

During the past few years a number of observations have been made which indi- 
cate that many of our earlier ideas in regard to the morphology of the bacterial cells 
must be subjected to rigid scrutiny, and the new conceptions recently advanced will, 
if proved, modify our entire point of view with respect to these microscropical organ- 
isms. 

PLEOMORPHISM 

It has long been recognized that bacterial cells exhibit what we regard as their 
characteristic morphology under certain favorable conditions which we find in the 
laboratory, usually in young cultures and on media peculiarly favorable to their 
growth. Our effort, indeed, is to subject organisms to constant conditions of light, 
temperature, and nutrition and so maintain constancy of form. Certain species are 
peculiarly susceptible to environmental influences and prone to exhibit departures 
from their standard morphology. Thus the cholera vibrio and Vibrio proteus assume 
in old cultures the most bizarre shapes, swollen spheres, crescents, half-moons, and 
distorted cells with knoblike protuberances. Similar changes are seen when these 
species are cultivated on acid media. Transfer of these distorted forms to media of the 
proper alkaline reaction yields normal vibrios as evidence of their potential viability. 
Large spherical forms are common in old cultures of the micrococci and are sometimes 
seen in the streptococci. The plague bacillus is especially likely to produce enlarged 
distorted forms on certain kinds of media (salt agar), and their production is regarded 
as practically diagnostic. In the animal body also departures of bacteria from their 
characteristic morphology have been frequently noted. One can find in streptococcus 
infections of the throat large cocci (megacocci) bearing little resemblance to ordinary 
streptococci, yet cultures from such throats may yield only streptococci. With the 
plague bacillus the appearance of enlarged, distorted forms is not uncommon in both 
natural and artificial infections. 

Organisms exhibiting an atypical morphology are usually called "degeneration" 
or "involution" forms. A number of investigations bearing upon their origin have ap- 
peared within fairly recent years. Thus Wilson' has found that when Bacillus coli, 
Bacillus typhosus, Bacillus enteritidis of Gartner, the Friedliinder bacillus, and the 
plague bacillus are cultivated on media containing urine, a great diversity of forms re- 
sults. The organisms are large and distorted and filamentous elements are common. 
Hata^ has shown that the addition of magnesium chloride to agar upon which dysen- 
tery and plague bacilU are grown leads to marked variation in the morphology of 

' Wilson, W. J.: /. Path, b" Bad., ii, 394. 1906. 
'Hata, S.: Centralbl.f. Bakteriol., 46, 289. 1908. 



HERBERT C. WARD 3 

these organisms. Reed and Orr' attribute such morphological changes to the influence 
of the hydrogen-ion concentration in the media. Any acidity or alkalinity close to the 
limiting reaction for successful cultivation leads to the appearance of these aberrant 
forms. Changes in morphology cannot be attributed solely to the influence of the hy- 
drogen-ion concentration since Vay' had previously found that the addition of dyes 
like dahlia to the media leads to the production of long dye-stained threads by Bacillus 
typhosus and Bacillus paratyphosus. According to Henrici,^ who has studied the in- 
volution forms in Bacillus coli especially, variation in morphology seems to correlate 
with autolysis of dead cells. The rate of autolysis depends upon the degree of acidity 
or alkalinity of the medium. 

That bacteria may have different sizes at different stages has long been suspected. 
Evidence substantiating this view has been presented by Clark and Ruehl, and by 
Henrici. Clark and RuehP have noted that in general very young bacteria, in the 
first few hours of growth, are much larger than older organisms. This does not hold 
for all species, however, since these authors found that this enlargement does not occur 
with diphtheria and glanders bacilli. Henrici^ has found that morphological variations 
occur in the lag period of bacterial growth. The initiation of growth is marked by a 
transformation to embryonic or growing cells which are considerably larger than the 
cells produced during the phase of maximum development. 

The production of large, irregular involution forms regarded as characteristic of 
the plague bacillus has been found by Smillie^ in other representatives of the hemor- 
rhagic septicemia group. This work has not thus far been definitely confirmed. In- 
volution forms occur with such species as Bacillus suisepticus, but they are not as 
marked as are those in Bacillus pestis. 

Finally, a new point of view in regard to the branching of bacteria has been 
brought out by Gardner^ who believes that probably many pathogenic bacilli grow by 
three-point branching from Y-shaped forms. If Gardner's observations can be con- 
firmed they will give us a different conception of bacterial multiplication and may ex- 
plain many obscure points in morphology and physiology. 

FILTERABLE FORMS OF BACTERIA 

Under ordinary circumstances the passage of bacteria through the pores of filter 
material like the siliceous earth in Berkefeld candles and the unglazed porcelain in 
Chamberland bougies depends on the size of the pores and the size of the bacteria, al- 
though other factors of great importance may modify the results. The most important 
of these are the amount of pressure or suction applied to force the fluid through the 
filter mass, the composition of the fluid in which the bacteria are suspended, and its 
reaction. At the same time the type of the micro-organisms has an important influ- 
ence on their ability to pass through filter material, and it has long been recognized 

' Reed, G., and Orr, J. H.: J. Bad., 8, 103. 1923. 

*Vay, F. : Cenlralbl.f. Bakteriol., 55, 193. 1910. 

3 Henrici, A. T.: /. Infect. Dis., 39, 429. 1926. 

" Clark, P. F., and Ruehl, W. H.: /. BacL, 4, 615. 1919, 

s Henrici, A. T.: /. Infect. Dis., 38, 54. 1926. 

* Smillie, W. G.: ibid., 27, 378. 1920. ^ Gardner, A. D.: /. Path. 6* Bad., 28, 189. 1925. 



4 THE MORPHOLOGY OF BACTERIA 

that the flexible spirochetes can pass filters more easily than other forms of bacteria. 
On numerous occasions bacteriologists have found that the effluent from the finest fil- 
ters may yield species like the original on cultures, although organisms cannot be 
found on microscopic examination. At the same time the effluent from suspensions of 
pathogenic organisms may produce disease in animals although organisms cannot be 
found on examination, or obtained in cultures. Unexpected results of this character 
were usually attributed to defects in the filter material permitting a small number of 
viable organisms to pass, or to a prolonged time in the process of filtration which per- 
mitted motile organisms to penetrate the filter mass, or viable bacteria to grow through. 
It was eventually recognized that this type of explanation did not always suffice to 
explain the results and that other possibilities must be considered. 

Theoretically, the bacterial cell may at times develop into morphological minia- 
tures of the original but with dimensions only fractions of the standard, or it may pro- 
duce a kind of spore or seed within its waUs, visible, filterable and viable, but morpho- 
logically unrecognized except as granules of characteristic size, shape, and staining re- 
action. Finally, the bacterial cell as we see it ordinarily may be but one stage in a 
complicated life-cycle, another stage being represented by filterable units, invisible by 
present methods, but viable in cultures, or viable only in the human or the animal 
body. 

While Schaudinn in his investigations of protozoa and spirochetes anticipated 
theoreticaUy much of the work of more recent years and expressed his belief that cer- 
tain species produce units which can pass filters, Gotschlich, as mentioned above, was 
the first to emphasize the possibility of minute units among the simpler bacteria. In 
his most recent publication Gotschlich' accepts filterable forms of bacteria as demon- 
strated and states that they may be differentiated from the filterable and inanimate 
products of bacteria by the fact that they are viable, in some instances capable of 
growth and culture, and in other instances capable of producing a characteristic dis- 
ease picture in experimental animals. With bacteria obviously too large to pass 
through filters Gotschlich again emphasizes his earlier opinion that a fractional part 
of the cell may escape and a normal adult cell regenerate from this portion. A vast 
amount of work has been carried out to substantiate views of this nature, among 
which may be mentioned the extensive studies of life-cycles of bacteria by Almquist, 
Enderlein, Fuhrmann, Lohnis, Mellon, and others. Of especial importance in this im- 
mediate connection is the work on the tubercle bacillus. 

Much^ in 1907 found small gram positive granules in the tubercle bacillus forming 
an integral part of the cell and which can be recognized outside it by their peculiar ap- 
pearance. With material containing these granules and no regular tubercle bacilli. 
Much believed that he had produced tuberculous lesions in experimental animals from 
which the characteristic acid fast organisms could be obtained. A little later Fontes^ 
pointed out that the Much granules are able to pass through Berkefeld candles. He 
inoculated guinea pigs with filtered granules and found no lesions except in the spleen. 
With spleens from the first series of guinea pigs he inoculated another series, the ma- 

' Gotschlich, E.: Handb. d. path. Mikroorg., i, 33. 1927. 

» Much, H.: Beitr. 2. Klin. d. Tuherk., 8, 85. 1907. 

»Fontes, A.: Centralbl.f. Bakleriol., Abt. I, Ref., 51, 244. 1912. 



HERBERT C. WARD 5 

jority of which showed tuberculous lesions from which the tubercle bacillus was culti- 
vated. On the basis of these expermients Fontes concluded that the Much granules 
represent a filter-passing stage of the tubercle bacillus. Spengler' noted, in tuberculous 
sputum, granules somewhat like the Much granules in morphology but acid fast in 
their staining reactions. In cultures from this material Spengler found the same acid 
fast granules together with small acid fast bacilli. After some time the cultures re- 
vealed acid fast bacilli of the normal size. These minute bacillary types Spengler 
named "SpHtter" and regarded as filterable stages in the life of the tubercle bacillus. 
Splitter forms were more frequently noted by Spengler in bovine lesions than in hu- 
man, and this has subsequently been confirmed. 

A number of observers, such as Hauduroy and Vandremer^ and Valtis,^ have em- 
ployed tuberculous discharges filtered free of adult tubercle bacilli and injected them 
into animals. These animals later developed tuberculous lesions from which tubercle 
bacilli were obtained. Valtis was unable to get cultures from his filtrates but found 
multiple glandular enlargements in his inoculated guinea pigs. Many acid fast tuber- 
cle bacilli were present in the visceral and pneumonic lesions. These animals reacted 
to tuberculin just as did the animals inoculated with the unfiltered tuberculous ma- 
terial. Valtis concluded that the filtrates from the tuberculous material contained the 
tubercle bacillus in some exceedingly small form capable of passing his filters. His 
conclusions have recently been confirmed by Arloingt who employed tuberculous 
fluids from a variety of lesions and cultures of the tubercle bacillus. With carefully 
controlled filters the filtrates were found to be sterile. In animals inoculated with the 
filtrates the lymphatic glands were enlarged in a characteristic manner, and micro- 
scopic examination revealed numerous tubercle bacilli. Finally, De Potter^ made use 
of materials containing the avian type of the tubercle bacillus and controlled his fil- 
trations with the minute organisms which cause chicken cholera. His filtrates were 
sterile while his experimental animals developed tuberculous lesions from which the 
avian tubercle bacillus was cultivated. 

Observations similar to those made on the tubercle bacillus have been reported 
for a number of other organisms — for the dysentery bacillus by Hauduroy; for the 
typhoid bacillus by Almquist, Fijgin, and Bergstrand; and for Bacillus coli by Izar 
d'Herelle, and Tomaselli. The conception of filterable stages in these organisms does 
not differ materially from that in regard to the tubercle bacillus, but the experimental 
results are by no means as clear. For the present we can only state that very strong 
evidence has been presented in favor of filterable stages in the tubercle bacillus, but 
that more extensive confirmation of the work is necessary before the question can be 
regarded as settled. 

SPECIAL ORGANS OF REPRODUCTION 

Intimately associated with the question of filterable stages is that of the presence 
of special, complete morphological structures in the simpler bacteria analogous to the 
' Spengler, C: Ztschr.f. Hyg. u. Infektionskrankh., 49, 541. 1905. 
' Hauduroy, P., and Vandremer, A.: Compt. rend. Soc. de biol., 89, 1276. 1923. 
J Valtis, J.: ibid., 90, 74. 1924; Ann. de I'lnst. Pasteur, 38, 453. 1924. 
* Arloing, F.: Bidl. Acad, de med., Paris, 96, 301. 1926. 
5de Potter, F.: Compt. rend. Soc. de biol., 96, 138. 1927. 



6 THE MORPHOLOGY OF BACTERIA 

conidia of the actinomyces which are definitely reproductive elements and serve to 
perpetuate the species. It is debatable whether it is advisable to employ such an anal- 
ogy since Drechsler has pointed out that the actinomyces form a group closely related 
to, and possibly forming a subdivision of, the hyphomycetes. Knoblike protrusions 
have been observed in many species of bacteria, and certain authors have come to re- 
gard them as conidia, thereby claiming a complicated cycle of development for the 
ordinary simple bacteria. Almquist, Enderlein, and Fuhrmann have emphasized this 
phase of development. Thus Almquist' has found, in cultures of the typhoid bacillus, 
the dysentery bacillus, and the cholera vibrio, spherical or globular elements and 
giant cells, often with distinct internal structure, from which normal forms are regen- 
erated. The transformation of these aberrant forms to the normal has been observed 
directly under the microscope by Almquist. Similar observations have been made in 
this country by Lohnis, Mellon, and Bergstrand, with diphtheria bacilli and with 
streptococci. 

Large spherical bodies had previously been observed by Swellengrebel in the larger 
spirilla but were regarded by him as no longer capable of multiplication. The ability 
of these knoblike protrusions and spherical bodies to regenerate normal individuals 
may be regarded as practically settled in view of Almquist's observations, but the in- 
terpretation of these bodies as true conidia can hardly be accepted at the present 
time. Almquist and Lohnis have further described a kind of sexual multiplication, 
consisting of conjugation or amalgamation of two forms. Interesting as such observa- 
tions may be, real proof for sexual multiphcation in bacteria is still lacking. It should 
be remembered, however, that if bacteria continue to multiply indefinitely without 
some kind of change which represents conjugation they form an exception to the laws 
governing other free-living plants and animals. 

INTERNAL STRUCTURE 

The characteristics of the internal structure of the simpler bacteria have been the 
subject of investigation since the earliest days of bacteriology, with particular refer- 
ence to the question of nuclear material. Opinions have ranged from the view that the 
entire cell represents the nucleus of higher cells to the view that no true nuclear 
material is present in them. Chemical analysis has thrown much light upon the 
kinds of chemical substances present in the bacteria, and the chemical demonstration 
of nuclear material has stimulated bacteriologists to approach the question again and 
make use of the more refined staining methods devised by the cytologists. As a re- 
sult, a kind of diffuse nucleation was established for the bacteria, nuclear material 
(chromatin) being distributed through the bacterial cytoplasm. Certain authors like 
Nakanishi' and Dobell,^ however, have always claimed the presence of true mor- 
phological nuclei. Thus Nakanishi has described minute spherical bodies in Staphylo- 
coccus aureus, in the middle of the cells, which he regards as nuclei. Dobell found 
true morphological nuclei in a spiral organism which he named Paras pii ilium vejdov- 
ski, but which was not cultivated. There is still some doubt whether Dobell's organ- 

■ Almquist, E.: Ztschr.f. Ilyg. u. Infektionskrankh., 83, i. 1917. 

2 Nakanishi, K.: Centralbl.f. Bakleriol., 30, 145. 1901. 

3 Dobell, C. C: Arch.f. Proiist., 24, 907. 1912. 



HERBERT C. WARD 7 

ism should be included among the bacteria. Douglas and Distaso/ however, working 
with a capsulated bacillus easily cultivated by them from cases of respiratory in- 
fections, were able to stain successfully certain cellular uaiits closely resembling true 
nuclei. 

Recently Gutstein,^ by staining methods, has made out a differentiation of the 
bacterial cytoplasm into ectoplasm and endoplasm. Tn the endoplasm he finds a 
macronucleus and a micronucleus. The ectoplasm of the gram positive bacteria Gut- 
stein^ later has shown to contain a basic ground substance demonstrable by malachite 
green and tannin, and an acid body demonstrable by Victoria blue. This acid sub- 
stance Gutstein regards as a lipoid. Somewhat similar observations have been made 
by Churchman-' in this country in his valuable studies on the gram reaction. He has 
found that Bacillus anthracis consists of two distinct parts, a gram positive cortex 
and a gram negative medulla. The term "cortex" as used by Churchman is equivalent 
to ectoplasm and the term "medulla" to endoplasm. The cortex may be removed by 
exposure to acriviolet or gentian violet and sometimes by hydrolysis in distilled 
water. The material lost is probably protein in character since the Berkefeld filtrate 
of a suspension of Bacillus anthracis exposed to gentian violet gives a positive nin- 
hydrin test. Bessubetz^ believes that the bodies inside bacteria and demonstrable by 
the use of the Giemsa stain are true morphological nuclei. Similar conclusions have 
been reached by Schumacher*' on the basis of chemical reactions. 

In view of the recent investigations on the internal structure of the bacteria we 
can only say that while true morphological nuclei cannot be regarded as definitely 
proved, material giving the staining reactions of chromatin may be found in a great 
many different bacteria. In stained specimens this chromatin is sometimes agglomer- 
ated in masses which resemble the nuclei of plant and animal cells. 

CAPSULES 

Various new methods for the demonstration of capsules have been devised during 
the past few years and our ability to fix and stain these structures has been consider- 
ably enhanced. In addition to these newer staining methods a number of investiga- 
tions have been made in regard to the chemical composition of the capsular substance. 
It should be noted that these investigations relate to the capsules of the true encapsu- 
lated bacteria like the pneumococcus, the Friedlander bacillus, and to certain intes- 
tinal organisms like Bacillus coli and not to saprophytic bacteria. Such saprophytes 
as Bacillus suhtilis and Bacillus mesentericus under certain circumstances are provided 
with beautiful capsules, and it is certain tiiat a great many different species may have 
a capsular material deposited about them under environmental stimuli. With the or- 
ganisms mentioned above capsules form an integral part of the bacterial cell. With 
such species as the Friedlander bacillus Toenniessen^ has shown that the capsule is a 

' Douglas, S. R., and Distaso, A.: Ceniralbl.f. BakterioL, 63, i. 191 2. 

'Gutstein, M.: ibid., 95, 357. 1925. 3 Gutstein, M.: ibid., p. i. 1925. 

* Churchman, J. W.: Proc. Soc. Exper. Biol. iT Med., 24, 737. 1927. 

sBessubetz, S. K.: Ceniralbl.f. BakterioL, 96, 177. 1925. 

^Schumacher, J.: ibid., 97, 81. 1926. 

' Toenniessen, E.: ibid., 65, 23. 1912; ibid., 85, 225. 1921. 



8 THE MORPHOLOGY OF BACTERIA 

secretory product and made up of galaktan, a polysaccharide of galactose, and con- 
tains no protein. The investigations of Toenniessen were later extended by Kramar' 
who found that the capsule of the Friedlander bacillus consists of galaktan, that of 
Bacillus anthracis of a glycoprotein, and that of Bacillus radicicola of a dextran. The 
capsules of different bacteria thus differ among themselves radically in their chemical 
composition. 

Recently Avery, Heidelberger, and Goebel^ have approached the question of cap- 
sule formation in bacteria from still another angle. From the pneumococcus type II 
they have obtained the capsular substance and found it to be a nitrogen-free poly- 
saccharide. This substance was non-antigenic. From the bacterial cell they obtained 
a protein substance which was antigenic but had no type specificity. In order to get 
type-specific reactions in animals it was necessary to employ both the protein con- 
stituent of the cells and the capsular substance before separation of the cell mass into 
its constituents. A similar carbohydrate, a nitrogen-free polysaccharide, was obtained 
from a strain of the Friedlander bacillus. The carbohydrate obtained from the type 
II pneumococcus was the same as the substance found by Dochez and Avery^ in the 
blood and urine in pneumococcus infections in man. Julianelle,^ working with rabbits 
infected with a strain of the Friedlander bacillus, found specific substances in the urine 
and blood which were practically identical with those in the soluble portion of the or- 
ganisms. Finally, Smith^ has reported biochemical studies on certain encapsulated 
strains of Bacillus coli. She states that the capsule materials consist of 80 per cent of a 
special carbohydrate, a hexose, together with a small amount of glycuronic acid. In 
order to show that this carbohydrate really represents capsule substance, she employed 
strains of the encapsulated Bacillus coli deprived of their capsules through mutation 
cultivation (see Theobald Smith).'' On comparing the filtrates from the two organ- 
isms, she was able to show that the precipitin test was one hundred times less active 
specifically with the filtrate from the mutant than with the filtrate from the encapsu- 
lated strain. These observations on the kinds of substances which may be obtained 
from capsules are of fundamental importance and must, as they are further amplified, 
aid materially in the interpretation of the composition and especially the function of 
capsules.^ 

SPORES 

As already mentioned, the conception of Kruse that spores in the bacteria are 
resting bodies like the cysts of the protozoa has had wide acceptance. According to 
this conception, spores are produced under unfavorable conditions, especially as re- 
gards food supply, serve purely for the perpetuation of the species, and show no evi- 
dence of vital activity. A number of reports have recently been published which must 
change our interpretation of spore formation. Thus Ruehle^ has found that spores 

' Kramar, E.: Centralbl. f. BaclerioL, 87, 401. 1922. 

' Avery, O. T., Heidelberger, M., and Goebel, W. F.: /. Exper. Med., 42, 701, 709, 727. 1925. 

3 Dochez, A. R., and Avery, O. T.: ibid., 26, 477. 191 7. 

-I Julianelle, L. A.: ibid., 46, 113. 1926. 

s Smith, D. E.: ibid., p. 155. 1927. 'See chaptet lii in this volume. 

^ Smith, T.: ibid., p. 141. 1927- ' Ruehle, G. L. A.: J. Bad., 8, 487. 1923. 



HERBERT C. WARD 9 

show evidence of enzymic activity even when no evidence of germination is present. 
Oxidases as well as gelatinase can be demonstrated in spore material. Magoon' be- 
lieves that spores are not dormant under ordinary conditions but are sluggishly ac- 
tive. Resistance to heat is not fixed but variable, being influenced by age, tempera- 
ture, heredity, etc. This resistance to heat may be increased by selective action (Ma- 
goon).^ In Bacillus mycoides certain organisms surviving after heating seem to have 
greater resistance to heat than the original spores. Daranyi^ has recently pointed out 
that the ability of certain species like Bacillus anthracis, Bacillus subtilis, and Bacillus 
anthracoides to form spores depends in general upon the same optimum conditions which 
lead to good vegetative development of the bacteria. Spore formation is brought 
about primarily by colloidal reactions; the most important of these is a diminution in 
the water content of the organisms, resulting therefore in a shrinking of the colloids. 
Under natural conditions spore formation begins when the organisms grow older. 
This aging is primarily a loss in water on the part of the colloids (hysteresis). The 
lack of food material for the bacteria has a favorable influence on spore formation only 
in that the organisms get poorer in water. With artificial dehydration, Daranyi was 
able to bring about spore formation in well-developed young bacilli. 

Koser and McClelland^ have added interesting facts in regard to the fate of spores 
in the animal body. The spores of Clostridium tetani, CI. putrificmn, CI. chauvei, and 
CI. oedematis-maligni are capable of withstanding the deleterious influence of the body 
tissues and may be transported from the site of inoculation to different organs, where 
they remain latent. Aerobic bacteria do not apparently suffer the same fate. Such 
observations are of considerable importance in an explanation of the occurrence of 
tetanus after wounds in cases where the wound itself is originally uninfected; it is pos- 
sible that the latter infection is due to the presence of latent spores in other parts of 
the body. Finally, mention must be made of the newer methods of determining the 
heat resistance of spores. Esty and Williarass have estimated resistance by heating a 
large number of tubes containing spores and plotting curves from the results. The 
rate of destruction corresponds in general to the rate of destruction of bacteria by dis- 
infectants worked out by Chick, by Madsen and Nymen, and by Eijkman. 

MULTIPLICATION IN THE SPIROCHETES 

The flexible spirochetes differ considerably in their morphology from the simpler 
bacteria but not sufficiently to separate them from the group in which the first repre- 
sentative was placed originally by Ehrenberg. In at least three particulars is their 
morphology interesting to us at the present time — their motility, method of division, 
and the possession of granules which are occasionally extruded from the cell and 
which may be reproductive bodies. In regard to motility it has now been satisfactorily 
proved that the spirochetes possess no organs of locomotion like flagella, as was orig- 
inally maintained. Flagella-like structures may occasionally be found, attached to the 

' Magoon, C. A.: ibid., ii, 253. 1926. 

^Magoon, C. A.: /. Infect. Dis., 38, 429. 1926. 

3 Daranyi, J.: Centralbl.f. Bakteriol., Abt. II, 71, 353. 1927. 

* Koser, S. A., and McClelland, J. R.: /. Med. Research, 37, 259. 1917. 

5 Esty, J. R., and Williams, C. C: /. Infect. Dis., 34, 516. 1924. 



A 






t I ^ ^ A R Y- 

o 



V 



10 THE MORPHOLOGY OF BACTERIA 

outer rim of the organisms, but it has now been established that these are not flagella 
but bits of periplast torn off in fixation and staining. Under dark-field illumination no 
refraction of light can be detected like that seen with the ciliated bacteria. The crista 
present in some of the larger saprophytic spirochetes, the cristispiras, has been shown 
to lack independent motility. Motility in the spirochetes must therefore be attributed 
to streams of protoplasm passing through the bodies of the cells or to the presence of 
contractile elements within, like the myonemes of certain protozoa. No definite dem- 
onstration of myonemes has thus far been effected, although Noguchi has observed 
bodies which strongly suggest them. Tt is possible that streams of protoplasm do exist 
in these organisms and that their motility is due to changes of surface tension. A sat- 
isfactory explanation of motility is very much needed. 

It is now generally admitted that the simpler spirochetes (treponema, spironema, 
leptospira) multiply by transverse fission. It is true that appearances suggestive of 
longitudinal fission have often been noted, but these were explained as the separation 
of two organisms tightly coiled about each other. The chief evidence that spirochetes 
divide transversely and not longitudinally depends upon accurate measurements of 
the diameter of the cells, a method of study carried on especially by Shellack.' He as- 
sumed that the diameter of the newly formed cells must at some time be one-half of 
the diameter of the cells before division, if longitudinal division takes place. In careful 
measurements of certain spirochetes {Spironema duttoni, novyi, etc.) Shellack was 
never able to find organisms which were twice as thick as the thinnest forms. He was, 
however, always able to find organisms twice as long as the shortest forms. He there- 
fore concluded that the spirochetes always divide transversely and not longitudinally. 
Lange" has recently called attention to the fundamental misconception in our inter- 
pretation of measurements of all morphological types of bacteria. After equal binary 
fission of spherical organisms the diameter of the daughter-cells is not one-half that of 
the mother-cell, but equals the cube root of one-half the cube of the diameter of the 
mother-cell, or over three-quarters of it. With cylindrical organisms the daughter- 
cells are half as long as the mother-cells, after transverse division. After longitudinal 
division the diameter of the daughter-cells equals the square root of one-half the 
square of the diameter of the mother-cell, or a little less than three-quarters of it. 
Since the spirochetes are practically cylindrical organisms, the failure to find organisms 
whose diameter is one-half the diameter of the thickest cells cannot be regarded as ex- 
cluding longitudinal division. 

A large number of spirochetes, especially the pathogenic varieties, show minute 
spherical bodies in the interior of the cell, and these are occasionally extruded. These 
bodies have frequently been regarded as reproductive elements. This conception we 
owe primarily to Leishman who found numbers of granules in ticks infected with Spi- 
rochaeta duttoni but no spiral organisms. With these ticks he was able to convey spi- 
rochetal diseases experimentally. The matter was thoroughly investigated by Hindle^ 
and by Balfour.-* Hindle, studying ticks infected with Spirochaeta gallinarum, found 

' Shellack, C: Arb. a. d. kais. Gesamle., 27, 364. 1908. 

"Lange, L. B.: J. Bad., 14, 275. 1927. 

J Hindle, E.: J. Parasitol., 4, 463. Cambridge, 1911. 

* Balfour, A.: /. Trap. Med., 10, 153. 1907; Brit. M.J., 2, 1130. 1907. 



HERBERT C. WARD n 

granules and both small and large spirochetes representing every gradation in size 
from granules to normal adult spirochetes. While he did not observe the actual growth 
of granules into spirochetes, he concluded from the varying numbers of granules and 
spirochetes that the change must occur in this direction. Balfour described a special 
type of spirochete concerned with fowl spirochetosis which he named S pirochaeta 
granulosa penetrans, and which he believed entered the red-blood corpuscles of the 
fowl and broke up into granules that were responsible for a peculiar recrudescence of 
the disease known as the ''after-phase." There can be Uttle doubt that granules are 
produced in a number of both pathogenic and saprophytic spirochetes, and are shed 
from the cells under certain circumstances. That granules are reproductive elements 
cannot be regarded as settled since Button and Todd have shown that adult spiro- 
chetes can sometimes be found on prolonged search in infective ticks along with the 
granules. Filtrates from spirochetal material showing an abundance of granules may 
also reveal a few spirochetes which because of their peculiar boring motility are able 
to pass through the filter substance. Recently Meirowsky" has observed swollen 
bodies and coiled forms in Treponema pallidum in secondary syphilis and has suggested 
that they are stages in the life-history of the organism. Szilvasi and Feher^ have found 
Meirowsky's forms in both primary and secondary lesions in syphilis and agree with 
him in regarding them as infective stages of the treponema. It may be noted in this 
connection that Aristowsky and Holzer-' have described peculiar coiled, twisted forms 
in Spirochaeta ohermeieri and believe that this organism also passes through a defi- 
nite life-cycle. 

THE RICKETTSIAE 

Following the discovery of pleomorphic bacteria-like bodies in Mexican typhus 
(tabardillo) by Ricketts a number of similar organisms were observed in various dis- 
eases and in their insect vectors. For this group the term "Rickettsia" was proposed 
by Da Rocha-Lima'' and the type species named Rickettsia prowazeki in honor of 
Ricketts who discovered the first representative and von Prowazek who devoted 
many years to its investigation. The significance of this group of micro-organisms 
will be considered in chapter xxxix of this volume, but one or two points should be 
mentioned here in regard to their morphology. Hertig and Wolbach^ and Cowdry^ in 
this country have noted their wide distribution in insects and arachnids. According to 
Arkwright, Atkin, and Bacot,^ the rickettsiae are characterized by their minute size, 
usually being less than 0.5 /x in diameter; their pleomorphism from round, coccus-like 
bodies and diplococci to minute bacilli and threadlike forms; their resistance to or- 
dinary aniline stains; their loss of gram stain and affinity for Giemsa; their absence of 
motility; their resistance to cultivation on ordinary media; and their occurrence in very 
large numbers in the gut of blood-sucking insects. In some cases they may be found 

' Meirowsky, R.: Mimchen. med. Wchnschr., 60, 1870. 1913. 
'Szilvasi, J., and Feher, D.: Cenlralbl. f. BakterioL, 95, 436. 1925. 

3 Aristowsky, W., and Holtzer, R.: ibid., p. 175. 1925. 

4 Da Rocha-Lima: Miinchen. med. Wchnschr., 67, 1381. 1916. 

5 Hertig, M., and Wolbach, S. B.: J. Med. Research, 44, 329. 1924. 
^Cowdry, E. V.: J. Exper. Med., 37, 431. 1923. 

'Arkwright, J. A., Atkin, E. E., and Bacot, A.: /. ParasiloL, 13, 27. 1921. 



12 THE MORPHOLOGY OF BACTERIA 

in other organs. Later Wolbach, Todd, and Palfrey^ emphasized the specificity of the 
rickettsiae for certain insect hosts. Hertig and Wolbach further ampHfied the defini- 
tion of the rickettsiae, suggesting that the term should be limited to proved pathogen- 
ic organisms which are pleomorphic, non-motile, gram negative, stain rather lightly 
with the aniline dyes, and have a tendency to an intracellular habitat. 

The rickettsiae have been cultivated in only a few instances. Noller^ has been 
able to grow Rickettsia melophagi on blood agar inactivated by heating to 57° C, and 
Sellards^ recently has cultivated a rickettsia-like micro-organism from tsutsugamushi 
disease. This organism was pathogenic and was named by him Rickettsia nipponica. 
In consequence it is difficult to come to any definite conclusions as to their nature and 
proper classification. It is quite clear that they are not mitachondria (Cowdry),^ and 
in many of their characteristics they resemble the bacteria very closely. Their pleo- 
morphism is not greater than that of many bacterial species. In size they cor- 
respond to many of the smaller bacteria. They have no definite internal structure al- 
though a morphological nucleus has been claimed by Epstein^ for Rickettsia prowazeki. 
They stain badly by the ordinary aniline dyes but no more so than certain bacterial 
species like the glanders bacillus and the cholera vibrio. When cultivated they grow 
on media similar to those used for the majority of bacteria. For the present the rickett- 
siae should probably be included among the bacteria. In this connection the obser- 
vations of Cowdry and of Wolbach and Schlesinger are of great importance. Cowdry^ 
has described a rickettsia (Rickettsia ruminantium) in Amblyomma hebraeum, the 
bont tick, which transmits the virus of the disease of sheep, goats, and cattle known as 
"heart water." These organisms lay in the endothelial cells of the renal glomeruli and 
in the superficial gray matter of the cerebral cortex. They were uniform coccus-shaped 
bodies, 0.2-0.5 ix in diameter, sometimes in diplo formation. They stained deep clear 
blue by Giemsa, and easily by Lofifler's methylene blue and other aniline dyes. They 
were gram negative. Such organisms would naturally be included in the group of 
rickettsiae as above outlined. Cowdry^ further described in a number of ticks (Ar- 
gasidae and Ixodidae) certain non-pathogenic, gram negative organisms characterized 
by their resemblance to bacteria morphologically, their large size and their intracel- 
lular habitat. Obviously such organisms would not meet the exacting requirements 
laid down by Hertig and Wolbach for the rickettsiae. Size and pathogenicity are 
somewhat doubtful characteristics for classification, and it is difiicult to exclude from 
any group organisms which possess in the main the character of the group but are 
larger and endowed with pathogenic action. For the present the non-pathogenic 
organisms described by Cowdry should probably be regarded as rickettsiae and 
included with them in a larger group of pathogens and non-pathogens. 

• Wolbach, S. B., Todd, J. L., and Palfrey, F. W.: The Etiology and Pathology of Typhus. Cam- 
bridge, 1922. 

' NoUer, W.: Arch.f. Schijfs- n. Tropen-Hyg., 21, 53. 1917. 
^Sellards, A. W.: Am. J. Trop. Med., 3, 529. 1923. 
"Cowdry, E. V.: he. cil. 
'Epstein, H.: Centralbl.f. Bakteriol., 87, ^s^. 1922. 

* Cowdry, E. V.: /. Exper. Med., 42, 231, 253. 1925. 
T Ibid., 41, 817. 1925 



HERBERT C. WARD 13 

Finally, Wolbach and Schlesinger' have brought to cultivation the micro-organ- 
isms of Rocky Mountain spotted fever {Dermacentroxenus rickettsi) and typhus 
{Rickettsia prowazeki) in tissue-plasma cultures. These authors have shown that the 
parasite of Rocky Mountain fever shows a definite series of morphological changes 
in ticks, from coccus to bacillary forms. Such observations suggest a somewhat more 
complicated cycle of development than that accepted for the ordinary bacteria and 
bring further evidence in favor of the view that binary fission and spore formation are 
not the only methods by which the bacteria multiply. 

' Wolbach, S. B., and Schlesinger, M. J.: /. Med. Research, 44, 231. 1923-24. 



CHAPTER II 
THE CHEMICAL STRUCTURE OF BACTERIA^ 

TRAUGOTT BAUMGARTEL 
Technischen Hochschule, Munich, Germany- 
Like all plant and animal organisms, bacteria require ten elements — carbon, hy- 
drogen, oxygen, nitrogen, phosphorus, sulphur, potassium, calcium, magnesium, and 
iron — as indispensable building stones for their body substance. 

While bacteria take the metals (potassium, calcium, magnesium, and iron) as well 
as the metalloids (phosphorus and sulphur) mostly in the form of simple mineral salts 
and satisfy their need for hydrogen and oxygen chiefly from water, they are able, in 
the assimilation of carbon and nitrogen, to utilize numerous and widely diverse sources 
of nutrition. All substances — from complex natural compounds like proteins and poly- 
saccharides down to their simple cleavage and decomposition products and the very 
elements like nitrogen and hydrogen — can be utilized by the bacteria in their metab- 
olism and modified in many ways. For example, in their carbon nutrition they util- 
ize in this way complex plant as well as animal substances, proteins and their cleavage 
and decomposition products, carbohydrates, fats, alcohols, acids, carbohydrates, car- 
bon dioxide, carbon monoxide, and methane. Thus, the bacteria, in spite of all ad- 
vantages and choice of many food substances, show an unparalleled ability for ad- 
justment to the sources of food offered them. Furthermore, the entire process of nu- 
trition in the bacteria is in large part dependent on the hydrogen and oxygen content 
of the culture medium as well as on the reaction and temperature. 

For these reasons, the chemical composition of different kinds of bacteria varies 
markedly; even in one and the same kind of bacteria there are considerable variations, 
so that generally valid statements about the chemical composition of the body sub- 
stance of bacteria can be made only with due consideration of the breadth of their bi- 
ological variations. 

THE WATER CONTENT OF BACTERIA 

In order to determine the water content of bacteria by chemical analysis, the usual 
procedure is carefully to scrape off the colonies grown on solid medium, centrifugate 
the liquid growth promptly, and weigh the material thus obtained in its moist, living 
condition ("fresh weight") ; afterward, dry it at ioo°-iio° C. and weigh it again ("dry 
weight"). The difference between the fresh weight and the dry weight — expressed per 
loo gm. of fresh mass of culture — is of course the water content. In this way it has 
been determined that the water content of most kinds of bacteria is rather high. On 
the average it amounts to between 75 and 85 per cent of the fresh weight. There are 
rather wide fluctuations according to the kind of bacteria and the growth of the organ- 
ism chosen (whether on solid or liquid medium), and also according to the age of the 

' For a more extensive summary, cf . Baumgartel, T. : Grundriss der theoretischen Bakteriologie. 
Berlin, 1924. 

14 



TRAUGOTT BAUMGARTEL 15 

culture. It is clear that a particular kind of bacteria shows a different water content 
according to whether one is dealing with a young, fully developed culture in liquid 
culture medium or an old dying culture on solid medium. Especially rich in water are 
the mucus-producing kinds of bacteria. Species which store lasting reserve material 
like volutin, glycogen, or fat, and spores contain proportionately little water. 

THE DRY SUBSTANCE OF BACTERIA 

Differences in the water content of the bacterial cells lead to differences also in 
their dry substance, in which the proportions can vary markedly between the com- 
bustible ("organic") material and the non-combustible ("mineral") material. In gen- 
eral, a particular kind of bacteria when cultivated on liquid medium contains more 
water than when grown on solid medium. On the other hand, according to whether an 
increased content of mineral salts or of carbohydrates is put into the liquid or solid 
medium, sometimes the mineral part of the dry substance of bacterial growths is 
greater than usual, sometimes the organic part is greater. In view of the high varia- 
bility of the bacteria, no general statements can be made beyond this. So far as re- 
liable researches have shown, 70-97 per cent of a bacterium's dry weight is made up of 
organic substance, and correspondingly 30-3 per cent is mineral substance. 

THE ORGANIC DRY SUBSTANCE 

Doubtless the organic dry substance of bacteria consists of numerous compounds, 
still in part wholly unknown, which on account of their common properties belong 
chiefly to the proteins, carbohydrates, and fats. Like the water content of bacteria, 
the chemical composition of the organic dry substance also varies and is different in 
any one species according to the cultural conditions under which the colonies chosen 
for analysis were grown. For example, it has been found that cultures of Bact. pro- 
digiosum grown on potato contain almost 50 per cent more organic dry substance than 
the cultures raised on yellow turnip, and that the cultures of the same species kept on 
potato at 33° C. have more dry substance than similar cultures preserved at 16° C. 
It is also demonstrable that four-to-six-day-old cultures on potato show a larger quan- 
tity of organic dry substance than cultures thirteen to sixteen days old. 

Because in building their organic body substances the bacteria can adapt them- 
selves, within definite but rather wide limits, to the nutrient medium on which they 
are growing, there can be no generalization as to the results of chemical analysis 
on the qualitative and quantitative composition of the organic dry substance. Ac- 
cording to the findings in many studies, the protein content of the organic dry sub- 
stance of most bacteria varies somewhere between 40 and 70 per cent, the carbohy- 
drate content between 10 and 30 per cent, and the fat or lipoid between i and 10 per 
cent. In general, therefore, proteins make up the greater part of the bacterial body; 
then follow carbohydrates and fats or lipoidal substances. 

As is evident from precipitation and agglutination reactions, different kinds of 
bacteria grown on the same nutrient medium and under conditions precisely similar in 
other respects can be differentiated with respect to their proteins, so that with the aid 
of the serological reactions they can be identified without further difficulty. Chemical 
analysis, on the other hand, offers at present no method of distinguishing the different 



i6 THE CHEMICAL STRUCTURE OF BACTERIA 

kinds of bacterial proteins, for the methods are not sensitive enough and do not give 
unconditionally reliable results; nor do they permit generalization. At least worthy of 
mention, however, is the hydraulic-press method which produces from moist, living 
bacterial cultures, without deep-seated changes in the cell substance, protein-rich 
fluids. For example, in order to obtain in this way the protein material of the cholera 
vibrio, colonies of this organism are grown on nutrient agar; the thick, growing layer 
of vibrios is carefully lifted off by means of a platinum spatula; the colonies thus ob- 
tained are finely pulverized with diatomaceous earth and quartz sand in a mortar and 
through the addition of salt solution worked into a pulpy mass. This is thrown into a 
strong filter cloth and laid under the hydraulic press. By a pressure which is gradually 
increased to 400 or 500 atmospheres a press fluid is obtained which comes through the 
thick filter at first as a light, clear, protein-rich liquid, later changing its color in the 
air to yellow and brownish— the so-called "vibrio plasma." As the chemical investiga- 
tion shows, the plasma is largely precipitable with acetic acid in the cold; it does not 
dissolve with excess of acetic acid, i.e., it acts like a nucleoprotein. In other respects 
the expressed fluid gives the usual protein reactions. 

With the help of a number of color reactions the protein chemical differentiation 
of many kinds of bacteria can be carried out. The best known in this connection is the 
usual color method of Gram, which depends on the fact that salts of pararosanilin 
(for example, gentian violet and methyl violet) unite firmly with iodine and that 
certain kinds of bacteria take the color compounds thus formed very firmly, i.e., when 
treated with alcohol later, they do not so readily give up the color substance they have 
taken as do certain other bacteria. In consequence, bacteria may be separated into 
those "colored by Gram's method" (gram positive) and "not colored by Gram's 
method" (gram negative). The differential behavior of bacteria in the color test of 
Gram is due to an unexplained difference in the physico-chemical structure of the 
protoplasm.^ 

In analogous ways certain protein or protein-like cell constituents can be detected 
in the bacteria. Among these may be included the reserve material (volutin) stored 
up in many bacteria in the form of colorless and strongly light-refracting spheres, vis- 
ible under the microscope, the presence of which, for example, is utilized practically 
in the usual bacteriological tests for the diagnosis of diphtheria. Volutin is dissolved 
by warm water (30°) within two or three days. In water at 80° C. volutin is dissolved in 
five minutes. Volutin fixed with heat or alcohol (or formol) is insoluble in boiling 
water. A 5 per cent or saturated aqueous solution of sodium carbonate dissolves volu- 
tin in five minutes, as does also caustic potash. A freshly prepared solution of Javelle 
water dissolves volutin in five minutes. A 5 per cent sulphuric or hydrochloric acid 
solution dissolves volutin in from five to ten minutes; a 25 per cent nitric acid solution 
dissolves it at once; and a i per cent acetic or osmic acid as well as a 5 per cent car- 
bolic acid solution dissolves it slowly. On the other hand, alcohol, ether, chloroform, 
and carbon tetrachloride do not dissolve volutin. Potassium iodide-iodine solution, 
Millon's reagent, vanillin hydrochloride, and zinc chloride-iodine give no reaction 
with volutin; and trypsin and pepsin are also without effect. Moreover, dyestuffs also 
give a characteristic behavior with volutin. Methylene blue and carbol-fuchsin in 10 

' See chap, iii (by Dr. Churchman) in this volume. 



TRAUGOTT BAUMGARTEL 17 

per cent solution color volutin quite intensively, while eosin borax carmine and nigrosin 
do not stain it; safranin and Bismarck brown give a stronger color to volutin than to 
the cytoplasm. 

As with the protein materials, so with the carbohydrates of bacteria — little defi- 
nite investigation has as yet been done. Most of the work up to the present has con- 
cerned itself with the study of the carbohydrates in the composition of bacterial mem- 
branes and mucus. For example, the gelatin formed by Streptococcus mesenterioides 
has been analyzed. The purified masses of gelatin were extracted with 96 per cent al- 
cohol, then boiled for a rather long time with milk of lime and the thickened masses 
obtained in this way precipitated with carbonic acid. From the precipitated calcium 
carbonate the mucous solution was poured off, cleared with hydrochloric acid, and 
precipitated with alcohol. The substance thus obtained is called "dextran." It ap- 
pears that neutral lead acetate does not precipitate the concentrated dextran solution, 
while basic lead acetate produces a pastelike mass. Moreover, it happens that a heat- 
saturated solution of barium hydroxide precipitates from concentrated dextran solu- 
tion an oily substance, and Fehling's solution, without itself being reduced, precipi- 
tates a mucous substance. When boiled with dilute sulphuric acid, dextran goes over 
into dextrose slowly; when heated to 120° C, it goes over in a few hours. 

Here may also be mentioned the researches on the carbohydrate content of the 
pellicle which appears in cultures of Bad. xylinum. The leather-like colonies were 
first cleansed with water, then boiled with a 20 per cent caustic potash, washed with 
dilute hydrochloric acid or with water, and finally treated with bromine. In this way 
there were produced colorless, transparent, thin films which dissolved in ammoniacal 
copper oxide as well as in concentrated sulphuric acid and, by element analysis, 
showed the composition corresponding to the formula CcHioOj. When the film was 
dissolved in concentrated sulphuric acid and this solution — after preliminary dilution 
with water — boiled, neutralized with barium carbonate, and filtered, its reactions in 
regard to dextro-rotation and reduction power were like dextrose. 

The carbohydrates of the bacteria that are demonstrable by microchemistry are 
glycogen and iogen (granulose), which appear in the protoplasm of many cells as 
colorless, viscous-flowing masses. Glycogen and iogen differ microchemically in their 
behavior with iodine. If very dilute potassium iodide-iodine solution is added to bac- 
teria which contain glycogen and iogen, only the iogen wiU be colored at first (blue) ; 
with stronger iodine solution the glycogen will also take (a dark red-brown) color. If 
the carbohydrate of the bacteria is colored only red brown, then it consists solely of 
glycogen ; if, with a stronger iodine-KI solution it takes only a blue color, then only 
iogen is present. If carbohydrate-containing bacteria are boiled for five minutes in 
water, the carbohydrates are still demonstrable by means of iodine-KI solution. If 
pigmented carbohydrate-containing bacteria are boiled in water, they appear without 
color when examined on the warm stage of a microscope. On the other hand, they 
again show color when the iodine-KI solution is cold. If bacteria containing carbohy- 
drates are treated for three minutes with boiling concentrated sulphuric acid, the car- 
bohydrates are completely dissolved.' 

' For a discussion of polysaccharides ("specific soluble substances") from bacteria, of. chap, lii (by 
Drs. Zinsser and Mueller) in this volume. 



1 8 THE CHEMICAL STRUCTURE OF BACTERIA 

Fat in bacteria appears mostly in the form of strongly light-refracting drops which 
in the young cells are very finely distributed in great numbers in the protoplasm; with 
increasing age, however, the drops join to form larger drops which occasionally fill the 
whole cell interior. Analytical studies on the fat or lipoid content of bacteria have 
been frequently conducted. From these it appears that the amount and the composi- 
tion of the detectable fats, lipoids, and waxes vary for the several kinds of bacteria 
and are dependent upon the conditions of growth. Thorough investigations have been 
made most frequently with pure cultures of the tubercle bacillus, which has an un- 
usually high fat content. In a very detailed study of this fat, four-to-five-month-old 
glycerol-bouillon cultures of this organism were killed in the autoclave at iio° C, the 
colonies collected on filter paper and treated with hot water until all the ingredients 
of the nutrient medium were washed away. The material was then spread on porous 
earthenware plates and dried at 40° C. In order to measure the fat contents of the pul- 
verized bacterial masses, these were treated with various fat solvents in the Soxhlet 
extraction apparatus. 

For a more precise determination of the fat of the tubercle bacillus, the same bac- 
terial powder was extracted several times with chloroform, the different extract por- 
tions mixed, and, after distilling off the chloroform, dried at 100° C. In this way there 
was obtained a dark-brown, semi-solid mass with glassy wrinkles and with the odor 
typical for tubercle bacillus cultures, like good wax from linden or flower honey. The 
melting-point of this fatty substance was 46° C. Further chemical investigation showed 
that the fat of the tubercle bacillus is a completely homogeneous substance which dis- 
plays no resemblance to any other fat or wax. It seems to be rather a mixture com- 
posed of free fatty acids, neutral fats, fatty-acid esters, and higher alcohols (lecithin, 
cholesterin), and, in addition, a large quantity of extractives which are insoluble in 
water and which, when heated with alkalies, disintegrate in part to form products sol- 
uble in the water. 

The demonstration of bacterial fat is made by the usual microchemical methods. 
Glacial acetic acid and chloralhydrate dissolve fat; Javelle water does not dissolve it; 
osmic acid does not blacken it; iodine-potassium iodide solution colors it yellow brown. 
Caustic potash seems to saponify the fat. The behavior of the bacterial fat toward 
certain dyes is also notable. While the common stains (methylene blue, gentian violet, 
and fuchsin) do not color bacterial fat, staining succeeds with Sudan III, naphtol blue, 
and dimethylamidoazobenzol. The latter stains the fat yellow; Sudan III, red; and 
napthtol blue, a deep blue. 

THE MINERAL DRY SUBSTANCE 

Thorough investigations have been carried out on the quantitative analysis of the 
ingredients of the ash from many kinds of bacteria. It appears from these studies that 
the mineral dry substance of the bacterial body always forms only a small proportion 
of the cell substance, that it differs for the several kinds of bacteria, and that the 
amount and nature of the mineral substance in any particular species vary with the 
conditions of cultivation and the age of the culture.^ 

' For a review, cf. Falk, I. S.: Abslr. Bad., 7, 44. 1923. 



CHAPTER III 

STAINING REACTIONS OF BACTERIA^ 

JOHN W. CHURCHMAN 

Cornell University Medical College, New York 

CHEMISTRY OF DYES 

Bacteriological stains belong almost entirely to the group known as "aniline 
dyes." Since a number of these, however, are not derived from aniline and bear no 
direct relation to it, and since all are derivatives of the hydrocarbon, benzene (CeHo), 
"coal-tar dyes" is a better term. 

Coal-tar dyes are monacid salts of color bases or alkali salts of color acids. "Ba- 
sic," "acidic," "neutral" — the descriptive terms usually applied to them — are not 
particularly fortunate terms since the dyes are not necessarily bases or acids, and 
even if called "basic" may have an H-ion concentration on the acid side of neutrality. 
Basic dyes are usually encountered as dye salts of a colorless acid such as hydrochloric, 
sulphuric, oxalic, or acetic acid; acid dyes as sodium, potassium, calcium, or am- 
monium salts of dye acids. The terms "acidic" and "basic" as applied to dyes really 
refer to the affinity of the chromogenic radicle for acidic or basic groups as the case 
may be. The term "chromogenic radicle" leads directly to a consideration of the cur- 
rently accepted theory as to the molecular structure of the dyes. 

The basis of this structure is the benzene ring of Kekule from which — as is well 
known — an almost infinite number of derivatives can be formed. When the deriva- 
tives contain certain groups of elements known as "chromophores," these groups 
impart the property of color. Benzene derivatives containing chromophore radicles 
are known as "chromogens." Chromogens, although colored, are not dyes since they 
may have little or no affinity for fibers or tissues ; the color they impart to fibers or tis- 
sues is a superficial coat easily removed by mechanical processes — the color does not 
"take." In order that a chromogen become a dye the chromogen derivative must 
contain, in addition to the chromophore, auxiliary groups which are known as "aux- 
ochromes." These have themselves little or no color and are not the cause of the color 
of the dye; but they impart to the compound the property of electrolytic dissociation, 
•furnish it with salt-forming properties, and thus convert it into a dye. 

The formation of the yellow dye, picric acid, from the yellow chromogen, tri- 
nitro-benzene, by the addition of the auxochrome, hydroxyl ( — OH), illustrates 
present-day conceptions of the chemical structure of coal-tar dyes. When three H- 
atoms in the benzene ring are replaced with the chromophore (— NOj), tri-nitro- 
benzene results: O.N. .. .NO, 



NO. 
' The detailed history of staining in bacteriology may be found in Unna's articles, "Die Ent- 
wicklung der Bakterienfarbung," Centralbl.f. BakierioL, 3, 22-345. 1888. 

19 



20 STAINING REACTIONS OF BACTERIA 

This yellow substance, which is insoluble in water, is neither an acid nor a base. 
Since it cannot dissociate electrolytically, it is incapable of forming salts. It is not 
therefore a dye. If, however, one more H-atom in the benzene ring be replaced with 
the auxochrome hydroxyl (—OH), picric acid is formed: 




This yellow substance is an acid, capable of electrolytic dissociation and of form- 
ing salts with alkalies. It is a dye. 

Some auxochromes (e.g., the amine group, — NH) are basic; others (e.g., the hy- 
droxyl group, — OH) are acidic. The acidity or basicity of a dye — as these terms are 
used in the expressions "acidic" or "basic" dyes — is determined by the character of 
its auxochromes. According as the chromogen is united with acidic or basic groups, 
the dyes are known as "acidic" or "basic." If basic groups are united to an acid chro- 
mophore, the dye is more weakly basic than if the same basic groups were united with 
a basic chromophore. A dye retains its color only so long as its affinities for hydrogen 
are not completely satisfied. When they are satisfied, reduction occurs and colorless 
leukobodies are formed. 

The polychrome stains stand in a class somewhat by themselves. In principle, all 
depend upon a combination of eosin and methylene blue, these elements not only stain- 
ing as units, but acting together in combination. It is assumed that these compound 
dyes act on the protoplasm as follows: Certain parts of the cell have an affinity for the 
neutral stain and take it up as such. Others have an affinity for the basic dye and 
break up the neutral stain so as to obtain the basic portion of it or, if dissociation has 
taken place, take up the basic ion directly. Other parts of the cell, with an affinity for 
acid dyes, similarly combine with the acid portion of the stain. These three types of 
cell structures are known as "neutrophile," "basophile," and "oxyphile" elements, 
respectively.' Polychrome stains are used in bacteriology chiefly for the study of 
spirochetes, Vincent's spirilla, and protozoa; and for the demonstration of chromatin 
(Zettnow).* 

Two other types of substance besides dyes are used in staining, namely, mordants 
and decolorizers. Mordants are chemical substances which have the power of making 
dyes stain material which they would not stain otherwise. This method of staining, in 
which the presence of a third substance besides dye and material to be stained is re- 
quired, is called by Mann the "adjective or indirect" method in contradistinction to 
the "substantive or direct" method in which the chemical and physical natures of 
dye and material to be stained are so interrelated that the material acquires the color 

' Conn, H. J.: Biological Slains. Geneva, N.Y., 1925. 
^Zettnow: Zlschr.f. Hyg. u. Injektionskrankh., 30, i. 1889. 



JOHN W. CHURCHMAN 21 

without the addition of a mordant. Pure mordants have a strong chemical affinity 
both for the substrate and the dye, and are used where an anchorage of dye in sub- 
strate is desired. 

Decolorizers are used to withdraw stains from certain tissues or organisms or 
parts of an organism and thus by a process of "regressive staining" to differentiate 
them. "Regressive staining" is contrasted with the ordinary method of "progressive 
staining" in which the process is stopped when only those substances with great af- 
finity for the dye are stained. 

STRUCTURE OF THE CELL 

To appreciate the significance of staining phenomena and to be able to discuss the 
mechanism involved it is not only necessary to understand the structure of the dyes 
but also to be familiar with present-day conceptions of the structure of the living cell. 
The cell is not to be thought of as a mere random mixture of cell constituents. These 
constituents have a permanent spatial distribution and physical state. This special 
structural constitution or organization is responsible for the special peculiarities of 
chemical behavior. Present evidence indicates that the basic protoplasmic structure 
has a closer resemblance to an emulsion type of structure than to that of any other 
simple physical system. The reactions which cells undergo proceed most actively — 
although perhaps not exclusively — at the boundaries of protoplasmic phases; in other 
words, the surfaces of membranes, fibrils, granules, and other solid cell structures have 
an accelerating or catalytic influence on these reactions. Many features of the chem- 
ical organization and behavior of living protoplasm appear to depend on the presence 
of thin films (apparently consisting chiefly of lipoid material) by which its structural 
elements are bounded and inclosed. The entire cell is inclosed by a thin, semiperme- 
able film — the plasma membrane; the internal protoplasm is probably partitioned by 
similar films. Apparently the intracellular partitions undergo increase of permeability 
or break down at death so that many chemical reactions which are absent or inappre- 
ciable during life proceed rapidly in dead cells. The type of structure characteristic of 
living protoplasm appears to be one by which free diffusion is prevented or restricted. 

Various theories have been advanced to explain cell permeability, most of which, 
though not all, presuppose a plasma membrane which exhibits differential properties 
permitting some substances to enter the cell easily (alcohol, ether) ; others with diffi- 
culty (most salts, sugars, etc.); still others not at all (most colloids). While not uni- 
versally accepted, the membrane hypothesis seems to be the one with which the known 
facts are best in accord. In the theory of Overton this membrane is supposed to be 
impregnated with lipoids, probably not ordinary fat but a mixture of lecithin and 
cholesterol. Others (Ramsden, Loeb, Crozier) emphasize the importance of protein 
in the membrane, the first calling the structure a "haptogen membrane" ; and Nathan- 
sohn postulated a membrane composed of a mosaic of both lipoids and proteins. The 
size of the molecule doubtless plays some part in penetrating power; and if molecular 
weight be very high, penetration is prevented. But it must not be thought that be- 
low a certain point there is any correlation between molecular weight and difficulty of 
penetration; indeed, the reverse is often the case; fatty acids apparently enter cells 



22 STAINING REACTIONS OF BACTERIA 

with increasing ease as molecular weight increases, at least up to a certain poin*. 
Molecular weight alone has little to do with cell permeability except as a limiting 
factor.'^ 

MECHANISM OF STAINING 

The theory of the molecular structure of coal-tar dyes, which I have stated, while 
perhaps not certainly established in all details, accounts for all the known facts, and 
its adoption has been so fruitful that it is now generally accepted. About the actual 
mechanism of staining, on the other hand, there is by no means general agreement. 
Much of the discussion has been devoted to a debate as to whether the process is 
chemical or physical. This seems peculiarly futile since a rigorous definition of chem- 
ical and physical processes is difhcult if not impossible to formulate and since the two 
processes are ultimately one. Even the ardent adherents of one or the other theory 
are usually forced in the end to acknowledge the possible identity of the two. 

If a chemical process be defined as a reaction between two substances in which a 
new chemical substance is formed and a physical process as a reaction between two 
substances in which no new chemical substance is formed, it is clear that both proc- 
esses occur in staining. A dyestuff as a whole may enter into and be deposited upon a 
tissue or cell by a process which Michaelis describes as "insorption," in which case the 
coloring matter may be subsequently extracted by any chemically indifferent solvent. 
On the other hand, a dye may become chemically united to the cell protoplasm by the 
formation of a salt, and in such a case the color can be removed only by agents like 
free acids which are capable of decomposing salts. It is certain that Ehrlich's dia- 
grammatic conception of chemoceptors to account for selective staining can no longer 
be held. Many observations in recent years point to the fact that other properties of 
the molecule than its structural formula (upon which the Ehrlich theory laid so much 
stress) are concerned and are probably of greater importance. 

There is good reason to believe that chromophilic protoplasm, so far as it is di- 
rectly stainable (that is to say, without mordants), is amphophilic or amphoteric, i.e., 
it possesses basic (amido-) and acidic (carboxyl-) groups side by side in the molecule. 
It has therefore the structure of amido-carbonic acid. The chemical processes of 
staining go on best the more auxophoric groups there are in the dye and the greater 
the number and the adequacy of chromophilic groups in the material to be stained. 

The physical processes of staining consist of surface attraction, osmosis, diffusion, 
adsorption ; and the factors which promote these processes are proper size of the dye 
molecule and adequate pore-volume of the material to be stained. 

It cannot be said that a completely satisfactory description of the chemistry of 
bacterial staining can be given. This is perhaps not strange considering the enormous 
complexity of the chemical structure of dyes, the complicated structure of bacteria, 
the difficulties of chemical analysis of bacterial bodies which are constantly changing 
their composition during life, and the minuteness of the microchemical reactions on 
which we have to depend. 

One thing essential for the process of staining bacteria is water. Water-free, al- 
coholic solutions of dyes will not stain dehydrated bacteria, nor will water-free alcohol 

'Lillie, R. S.: General Cytology, p. 167. Chicago: University of Chicago Press, 1924; Jacobs. 
M. H.: ibid., p. 99. 1924. 



JOHN W. CHURCHMAN 23 

decolorize them. The demonstration that bacteria contain an abundance of nucleo- 
protein appears to explain their affinity for basic coal-tar dyes, and there are many 
grounds for the belief that— perhaps in the majority of cases — staining is due to a 
weak combination between nucleoprotein and basic dye, decoloration being due to 
dissociation of the dye-protein compound. Bacteria certainly behave in their staining 
reactions as if composed largely of protein. They were formerly thought to consist 
entirely of nucleus and cell wall without cytoplasm. Whether this be true or not, one 
other element in bacterial cells is of great — perhaps of prime — importance in many 
staining phenomena, namely, lipoids. Bacteria are known to contain fats and lipoids 
in varying amounts which, because of the marked effect on surface tension, would for 
purely physical reasons tend to become concentrated at the periphery of such a col- 
loidal system as the bacterial protoplasm. Whether or not there is a morphologically 
distinct limiting membrane, we can reasonably assume that the surface of the bac- 
terial cell is potentially lipoid. The presence of unsaturated fatty acids in the lipo- 
protein of the bacterial cell is thought by many to explain the mechanism of the gram 
stain. 

The amount of dye taken up by bacteria and the firmness with which it is held 
doubtless depends in large part on the H-ion concentration of bacterial protein. Since 
the pH of bacterial protein may change with age it might be expected that the stain- 
ing characteristics of bacteria would change, and it is of course well known that this 
does occur; the difference between the gram behavior of young and old cultures may 
be cited as an example. 

The chief physical factor of the staining process — emphasized by advocates of the 
physical hypothesis to explain the phenomena of dyeing, and which must in any event 
be taken into consideration — is the process of adsorption of the dye by the bacterial 
substance after it has passed through the wall membrane by osmosis. Adsorption is 
the property possessed by a solid body of attracting to itself by physical means from a 
surrounding solution certain compounds or ions present in that solution. In the case 
of the dyes it is assumed that, when once adsorbed, they remain in the stained tissue 
in solid solution. There are certain facts which point strongly to a physical explana- 
tion of staining. For example, there is no evidence of the formation of a new substance 
when tissue is stained; the colored tissue merely takes on one of the characteristics of 
the dye (color). It is usually possible to extract all or nearly all the dye by immersion 
in water or alcohol. Furthermore, tissue never removes all the stain from the dye solu- 
tion, no matter how dilute. These facts must be borne in mind, and physical processes 
must be thought of as playing an important role. But more and more evidence is ac- 
cumulating to indicate that strictly chemical processes are also concerned. 

THE GRAM STAIN 

Dyes are used in bacteriology for four purposes: (i) to make organisms visible, 
(2) to display their structure, (3) to reveal their chemical nature, and (4) to influence 
their growth. It is well known that most bacteria are stained easily and a few with 
difficulty, and that certain parts of the bacterial cell (the spores, capsules, and flagel- 
la) are not stained at all by ordinary methods. However, with the exception of the 
acid fast group, the vast majority of bacteria behave very much alike toward simple 



24 STAINING REACTIONS OF BACTERIA 

stains, without mordants. A few instances of elective staining have been described — 
as, for example, the affinity of picric acid and neutral red for cholera vibrios and 
kresylechtiviolet for gonococci; but, on the whole, there is so little difference in the 
behavior of most bacteria to dyes that elective staining methods have not proved of 
striking value. To this statement there are of course two notable exceptions — the 
method of Gram and the method of Ziehl-Neelsen. 

The method of Gram illustrates well the fact that the progress which results from 
a new observation is often in a different direction from that in which the observer was 
at the time searching. Gram was attempting to develop a method for staining micro- 
organisms in tissue. The differential staining which he noticed proved to be of great 
value for the general identification and classification of bacteria. 

The ability to retain dye when stained by the method of Gram is not a property of 
living cells in general but is almost entirely confined to yeasts and bacteria. All tissue 
elements appear to be decolorizable save perhaps keratohyalin. Henrici found that 
sections of vegetable tissue contained no gram positive elements; and in animal tissue 
— while the nucleus retains the stain somewhat longer than the cytoplasm — all the 
elements may be ultimately decolorized. Molds stain irregularly, isolated granules in 
the mycelia retaining the stain, while large areas do not stain at all. Protozoa, spiro- 
chetes, and malarial parasites are gram negative. It is well known, of course, that the 
differentiation of bacteria into gram positive and gram negative is not hard and fast, 
that the gram characteristic of a given organism, like any of its other characteristics, 
may change with age or be disturbed by variations in environment and in other ways. 
But within its well-established limitations the method of Gram is an exact one. It is 
clear that the differential behavior of gram positive and gram negative bacteria must 
ultimately depend upon difference in chemical or physical characteristics (or both) of 
the bacteria themselves. A number of such differences have been proved to exist, and 
for certain others there is strong, if not conclusive, evidence. Of these diiferences, the 
principal ones are shown in Table I. 

It can be objected that the facts in nature are not quite so clear cut as a table of 
this sort suggests. Not all gram positive organisms are equally gram positive, and it 
has been shown that their gram behavior may in different groups of gram positive 
organisms rest on an entirely different anatomical basis. They therefore differ among 
themselves and cannot all be classified together. The same thing is doubtless true of 
the gram negative organisms. Such a statement, for example, as the one that gram 
positive organisms are as a rule much more susceptible to the bacteriostatic action of 
basic triphenyl-methane dyes than gram negative' is open to a criticism of this kind, 
and the criticism has in fact been made.^ Nevertheless, the statement as originally 
enunciated is correct: with about lo per cent of exceptions, gram positive aerobes, 
whether spore bearing or not, are extremely susceptible to the bacteriostatic effect of 
these dyes and, with about lo per cent exceptions, gram negative aerobes are resistant. 
The original statement of this parallelism was extended by Smith, Eisenberg, Simon, 
and Wood^ to include about sixty dyes, and the work of these and other investigators 

' Churchman, J. W. : loc. cit. 

= Stearns, A. E., and E. W.: /. Bad., g, 493. 1924. 

3 Simon, C. E., and Wood, M. A.: Am. J. M. Sc, 147, 247, 524. 1914. 



JOHN W. CHURCHMAN 



25 



TABLE I 



Gram.Positive Organisms 



Gram-Negative Organisms 



1. Killed bacteria not digested by trypsin 
or pepsin (Kantorowiecz) * 

2. Only a few are digested by gastric juice 
(Burgers) t 

3. Resistant to alkalies; not dissolved by i 
per cent KOH (Kruse,t Smith§) 

4. Lipoids resistant to fat solvents (Jobling 
and Petersen) 1 1 

5. Degree of dispersion of nucleoproteins 
high (Hottinger)^ 

6. Sensitive to weak electrolytes (dyes) 
Smith, § Burke**) 

7. Optimum growth at relatively high pH 
(Burke)** 

8. Resistant to non-electrolytes (Smith) § 
Q. Limited iso-electric staining range cen- 
tered about pH 2-3 (Stearns and 
Stearns) ft 

10. For the most part (about 10 per cent 
exceptions) very susceptible to bacterio- 
static effect of triphenyl-methane dyes 
(Churchman) ft 

11. May produce spores 

12. In the living state, more readily per- 
meated by dyes (Benians)§§ 

13. May be acid fast 

14. More susceptible to quinine and hydro- 
cuprein derivatives (Traube);^|| 

15. More susceptible to iodine (Traube)ll|| 

16. More susceptible to mesohematin, mag- 
nesium, and manganese derivatives of 
mesoporphyrin (Kammerer)TfTf 

17. More readily adsorbed because of surface 
lipins (Eisenberg)*** 

18. Adsorb halogens less strongly (Breinl) fff 

19. Unaffected by toluol provided no 
emulsoid be added (Benians)ttt 

* Munchen. med. Wchnschr., 56, 897. 1909. 

\ Zlschr.f. Hyg. u. Infeklionshrankh., 70, 119. 1911. 

X MUnchen. med. Wchnschr. 57, 685. 1910. 

^Am. J. Hyg., 2, 607. 1922. 

II /. Ex per. Med., 20. 456. 1914. 

H Cenlralbl.f. Bakkriol., 76, 367. 1916. 

**/. Bad., 7, 159. 1922. 

tt Ibid., g, 463, 479, 491. 1925; 10, 13. 1925. 

ttJ. Exper. Med., 16, 221. 1912. 

§§ /. Palh. &* Bad., 17, 199. 191 2. 

nil Zlschr.f. Immunitdtsforsch. u. exper. Therap., 29, 2861. 

'ill Arch. f. exper. Palh. u. Pharmakol., 88, 247. 1920. 

*** Centralbl. f. BaklerioL, 54, 145. 1912; ibid., 81, 72. 191 

ttt Zlschr.f. Immunildtsforsch. u. exper. Therap., 29, 343. 

XXX Zlschr.f. Chemolherapie, 2, 28. 1913. 



1. Killed bacteria digested by trypsin and 
pepsin 

2. Majority digested by gastric juice 

3. Dissolved by i per cent KOH 

4. Lipoids less resistant to fat solvents 

5. Degree of dispersion of nucleoproteins 
low 

6. Resistant to weak electrolytes 

7. Optimum growth at relatively low pH 

8. Less resistant to non-electrolytes 

9. Wider iso-electric staining range cen- 
tered about pH 5 

ID. For the most part less susceptible to 
bacteriostatic effect of triphenyl-meth- 
ane dyes 

11. Never (?) produce spores 

12. Less permeable to dyes, in living state 

13. Never acid fast 

14. Less susceptible to quinine and hydro- 
cuprein derivatives 

15. Less susceptible to iodine 

16. Less susceptible to these substances 



17. Less readily adsorbed 

18. Adsorb halogens more readily 

19. Killed by toluol 



26 STAINING REACTIONS OF BACTERIA 

TABLE l— Continued 
Gram-Positive Organisms Gram-Negative Organisms 

20. Less readily subject to auto- and sero- 20. More readily subject to auto- and 
lysis (Benians)§§§ serolysis 

21. Less likely to form demonstrable anti- 21. More likely to form antibodies 
bodies in infected host (Benians)§§§ 

22. Less susceptible to quinine (Graham- 22. More susceptible to quinine 
Smith)1f111f 

§§§y. Path, b" Bad., 23, 411. 1919-20. 
HHH/. H>'5-. 18, 1. 1919. 

suggests that perhaps the author's figures for the exceptions in each group are too high 
This clear-cut but not all-inclusive parallelism between gram behavior and triphenyl- 
methane behavior is striking, in spite of the fact that an exceptional gram positive 
organism (like the streptococcus) may be resistant and an exceptional gram negative 
one (like M. neisseri) may be susceptible; and in spite also of the fact that within each 
group there are quantitative differences in susceptibility to dyes. 

All the relations stated in the table are doubtless subject to similar qualifications 
which may be summarized as follows: 

1. The division between gram positives and gram negatives is not absolute. Other 
factors enter in. For instance, the anaerobes form a class by themselves, and, although 
usually gram positive, may have many characteristics not shared by other gram pos- 
itives. They are really not included in the table. Again, organisms like the acid fast 
have peculiarities of structure which separate them chemically from others. 

2. The gram positives differ among themselves, and gram positivity does not al- 
ways depend, in the various bacterial species, on the same mechanism. This point has 
been established by the observations of Deussen,' and more recently by the author,^ 
who has shown that two strongly gram positive organisms like B. anthracis and M. 
fretidenreichi differ markedly as to the stability — and probably as to the underlying 
cause — of their gram positivity. 

3. A very interesting and important fact has been established by a number of ob- 
servers, that there is a group of organisms which — though gram negative — are inter- 
mediate between the two groups in most of their other characteristics. M. neisseri, for 
example, though gram negative is dye sensitive, falls w'ith the gram positives in opti- 
mum H-ion test, is quite resistant to lysis by KOH as compared with other gram neg- 
atives, and shows no cytolysis in peptic-digestion tests. There is some evidence (from 
peptic-digestion tests) that vibrios and spirilla also occupy in some respects a mid- 
position between the two groups. 

4. The presence of spores also somewhat complicates the situation. The author^ 
has shown that certain dyes (acriflavine, acid fuchsin) exhibit a reverse selective bac- 
teriostatic action when tests are made between gram positive spore-bearers and the 
ordinary gram negative bacilli. 

'Deussen, E.: Ztschr.f. Hyg. u. Infektionskrankh., 85, 235. 1918. 

'Churchman, J. W.: Slain Technology, 2, No. i. 1927.. 

3 Churchman, J. W.: J. Expcr. Med., 37, i. 1923; ibid., 38, i. 1923. 



JOHN W. CHURCHMAN 27 

5. Not even all strains of a given species necessarily behave exactly alike toward 
dyes. The existence of "strains-within-a-species variants," as measured by the bac- 
teriostatic effect of gentian violet, has been described; and from a given pure culture 
of B. coli two strains have been isolated — one dye sensitive and the other not — which 
were identical in all cultural, morphological, and tinctorial respects ("strain-within-a- 
strain variant").' 

The facts are that the properties of the two groups stated in the foregoing table 
are probably to be regarded as "independent variables in the sense that any one of 
them may be possessed by a particular strain of bacteria independent of the other 
properties of that group." None the less, as Smith has stated, "this almost clear sep- 
aration of the families of bacteria on the basis of biochemical reactions cannot be 
without significance." 

It is inevitable that the dilemma as to the physical or chemical nature of staining 
which we have encountered in discussing the general mechanism of the staining proc- 
ess must also be faced in discussing the method of Gram. On the whole, the tendency 
is to emphasize the chemical rather than the physical factors of this reaction, and 
many observers (e.g., Deussen)^ regard the process as purely chemical. That physical 
structure is also involved seems none the less certain. Benians^ has shown that bac- 
terial disintegration induced by mechanical measures upsets the gram behavior; 
Churchman has shown that certain organisms (e.g., B. anthracis) are gram positive 
only in the cortex, which may be removed by chemical means, exposing a gram nega- 
tive medulla ; and that some sort of bacterial membrane plays a physical part in the 
process is taken for granted in most of the theories which attempt to account for the 
phenomenon. Benians has been the chief exponent of a purely physical explanation of 
the gram stain and has advanced perhaps the most convincing evidence for this view. 
From experiments in which bacteria were studied after disintegration by crushing, 
Benians^ drew the following conclusions: 

1. The gram positive property is inherent in the physical structure of the bacte- 
rial cell, and — since mordants are not essential — is not conferred on it by the mordant. 

2. Nothing in the nature of chemical fixation of the compound dye to bacterial 
substance occurs. 

3. The effect of the mordant is to dissociate dye from its adsorption compound 
with the tissues, forming with it a large, compound molecular body which in alcoholic 
solution does not easily pass out of the gram positive bacteria. This conception of the 
role of the mordant supplanted Benians' earlier idea that it acted by preventing al- 
cohol from entering the cell. 

4. Capacity for retaining the compound dye is chiefly dependent on the structure 
and integrity of the limiting membrane. 

5. The essential character of the gram positive cell membrane is that it does not 

' Churchman, J. W., and Michael, W. H.: ibid., 16, 822. 191 2; Churchman, J. W.: ibid., 33, 
569. 1921. 

^Deussen, E.: loc. cit. 

3 Benians, T. H. C: J. Path. & BaQi., 17, 199. 1912. 

''Benians, T. H. C: ibid., 23, 411. 1919-20. 



28 STAINING REACTIONS OF BACTERIA 

readily permit the contained large, compound iodine molecule in alcoholic solution to 
pass through it. 

6. Gram negative bacteria are of two types as regards cell membrane: (a) (Rep- 
resented by M. neisseri) allow attachment and probable permeation of the dye ; but 
from them iodine-dye precipitate is readily washed out by the decolorizer. These or- 
ganisms approximate the gram positives so far as they contain the dye, but the ab- 
sence of the necessary specific cell membrane does not permit the retention of the dye 
when alcohol is applied, (b) (Represented by B. coli) allow no penetration of the dye 
but only peripheral adsorption. 

7. Not all bacteria which fail to retain the stain are similar in structure. The ap- 
parently permeable gram negatives (M. neisseri) probably have more in common 
with the gram positives than with the gram negatives. 

Burke corroborated Benians' conclusions by demonstrating a reverse gram re- 
action.' He found that an alcoholic solution of the iodine-dye complex stained the 
gram negatives but not the gram positives, so that gram positives are characterized 
by permitting the entrance of water-soluble dye but not the egress of the alcohol- 
soluble iodine-dye complex. The opposite is true of gram negatives. 

Brudny,^ on the other hand, considered the gram positives more permeable to io- 
dine so that in these organisms a deeper iodine-dye precipitation occurs which is less 
accessible to the decolorizers. Other observers have laid stress on the chemical con- 
stituents of the bacterial surface. Eisenberg found that ether extraction of staphy- 
lococcus reverses its gram behavior, concluding that the lipoid-protein compounds on 
the surface were the important factor; while Dreyer, Scott, and Walker were able to 
turn B. coli into gram positive organisms by treating them with lecithin. 

Stearns and Stearns^ called attention to the fact that bacteria exhibit the ampho- 
lytic character common to protein and tend to retain acid stains when in acid solu- 
tion and basic stains when in alkaline solution. They found that gram positive bac- 
teria can be rendered gram negative by increasing acidity, and that the reverse effect 
is produced by alkalies ; at the iso-electric point there is little staining, and the so-called 
"iso-electric range" is generally wider with gram negative than with gram positive 
bacteria. Mordanting, they suggest, is due to a mild oxidation which increases acidity 
and hence the affinity for basic dyes. They suggest further that gram positivity de- 
pends on the presence in the compound bacterial lipoproteins of unsaturated fatty 
acids which are partially oxidized by the mordant, intensifying the acid properties and 
increasing affinity for basic dyes. The presence of unsaturated fatty acids has been 
invoked by other observers to explain the phenomenon, but in a different way. Thus 
Jobling and Petersen'' suggested that gram positivity depended on a high fatty acid 
content and a high affinity for iodine; and Tamura found that the lipoid extract from 
bacteria contains the element responsible for retention of the dye. Hottinger,^ on the 

'Burke, V.: loc. cit. 

= Brudny, V.: CcnlralU. f. Bakleriol., 21, 62. 1908. 

3 Stearns, A. E., and E. W.: op. ciL, 9, 463, 479, 491. 1925; 10, 13. 1925. 

■t Jobling, J. W., and Petersen, W. H.: loc. cit. 

sHottinger, R.: loc. oil. 



JOHN W. CHURCHMAN 29 

other hand, thought gram positivity is solely dependent on the degree of dispersal of 
the nucleo-proteins: in gram negatives the stained nucleoprotein forms a colloid of 
high dispersion ; in gram positives it forms an emulsoid ; when gram positives become 
negative the dispersion is increased. Deussen' concludes his exhaustive study of the 
subject by stating that the gram reaction depends on the chemical nature of the cell 
contents which undergo — according to their chemical structure — a greater or lesser 
degree of hydrolytic splitting of the molecule. The reaction belongs in the complica- 
ted field of nuclein combinations. It is chemical and not physical. 

It has been for a long time believed on the basis of statements attributed to Unna^ 
that gram positivity depends on the formation of a peculiar iodine-dye-protein com- 
pound and that only para-rosanilin dyes could be used for this purpose. That the 
chemical process involved may not be quite so simple as this was shown by Eisenberg 
who reported that the reaction could be produced with deeply colored dyes of the 
acid class as well as with basic dyes and that the use of mordants is not necessary. 
None the less, para-rosanilin dyes are the best for the purpose, and much the most 
clear-cut results occur when mordants are used. 

Bacteria were for a long time treated as though their constitution were constant, 
and characteristics were assigned to them which — by implication at least — they were 
supposed to exhibit always and under all conditions. That they are, as a matter of 
fact, biological units showing all the changes of metabolism, that they carry on res- 
piratory processes, that they undergo dissociation, that they exhibit polymorphism — 
these things are now well known. It is clear, therefore, that many of their character- 
istics are constantly changing and that many of them may be made to change more or 
less at will by alterations in the environment. Reversibility of the gram reaction by 
acids (Deussen)^ and by coal-tar dyes (Churchman)^ has already been referred to; and 
it must again be emphasized that the gram reaction of a given organism is constant 
only when the conditions of examination are held constant. It must also be borne in 
mind that gram positive organisms differ among themselves as to the stability of 
their gram positivity. Neide,4 for example, used the term Gramdauer to indicate that 
since a number of factors influenced decolorization by alcohol (among them the con- 
centration and temperature of the alcohol used) gram positive organisms differed 
among themselves as to the length of time they could retain stain when exposed to 
decolorizers. Henrici^ showed that in yeast cells the cytoplasm is not homogeneous as 
regards gram positivity, certain granules appearing in the decolorizing cell which hold 
the dye longer than others. The author has recently made very clear the fact that not 
all gram positive organisms are equally stable in their gram positivity.* Certain of 
these organisms (e.g., M. freudenreichi) are extraordinarily stable in this respect; 
others (e.g., B. anthracis), though equally gram positive, are less stable. Exposure of 

' Deussen, E. : loc. cit. 

'Unna, P. G.: Monatschr.f. prakt. Dermatol., Supplement No. 6. 1887. 

3 Churchman, J. W.: Stain Technology, 2, No. i. 1927. 

<Neide, E.: Centralbl.f. BakterioL, 35, 508. 1904. 

sHenrici, A. T.: /. Med. Research, 30, 409. 1914. 

^ Churchman, J. W.: Stain Technology, 2, No. i. 1927. 



30 STAINING REACTIONS OF BACTERIA 

the latter, for example, to certain coal-tar dyes completely reverses their gram reaction 
although similar treatment is without effect on the gram reaction of the former. More- 
over, thorough modifications of the gram technique in which time of exposure to dye and 
mordant is greatly shortened and time of exposure to decolorizer greatly prolonged de- 
colorize B. anthracis in large part but are without effect on M.freudenreichi (see Plate 
I, (c), facing p. 36). In specimens of B. anthracis stained by such a modification, beauti- 
ful partial decolorizations occur in which a gram negative central rod is to be seen shin- 
ing through among remnants of undissolved gram positive material which cling to the 
surface of the bacteria in the form of lumps, plaques, or dots (see Plate I). I have 
also called attention to the fact that the gram negative forms of B. anthracis were 
much smaller in diameter than the gram positive forms. Filar micrometer measure- 
ments of about three hundred organisms showed a reduction in diameter of 0.5145 fx 
or 42 per cent. Evidence was adduced to indicate that the difference between the 
gram stability of an organism like B. anthracis and that of an organism like M.freu- 
denreichi depends on the fact that the latter is composed of an easily destructible gram 
positive cortex and a gram negative medulla, an anatomical structure which is not 
possessed by an organism like M. freiidenreichi. 

A critical review of all the known facts leads Smith' to sum up our knowledge of 
the mechanism of the gram stain as follows : Gram positivity is due to some property 
of the bacterial cell which permits the ingress of the water-soluble dye but does not 
permit the egress of the alcohol-soluble iodine-dye compound. A gram negative cell 
is not readily penetrated by the water-soluble dye but is easily penetrated by the 
alcohol-soluble iodine-dye complex. In both cases permeabihty is conditioned by the 
physical state of the components of the intact cell membrane. When gram positives 
become gram negative, what is lost is a particular condition or physical distribution of 
the constituents of the cell membrane. 

The exact technique which Gram himself advised is not now generally used. Im- 
mediately after its introduction, modifications of the technique were suggested, identi- 
cal in general principle with the original method though differing in details. With 
these every bacteriologist is familiar. In a recent study of this subject involving the 
examination of nearly fifteen thousand smears, I tried out all the better-known modi- 
fications and came to the definite conclusion that Burke's modification' is in every 
way superior to all others. In this method the stain (methyl violet) is alkalinized on 
the slide by the addition of sodium carbonate, the mordant is used in strong solution, 
and acetone-ether, a powerful decolorizer, is employed. The colors in the final result 
contrast sharply — the pink of the counter-stain (safranine O) with the bluish black of 
the gram positive forms — and one is never in doubt in which group to place a given 
organism. The results are constant and clear cut; that is to say, they are constant and 
clear cut if the conditions of experiment which are known to modify gram behavior be 
kept in mind and controlled. For it must be emphasized that the method of Gram is 
an exact one, and irregular results are due, not to the defects of the method but to 
failure to keep all the conditions of the examination constant. For this reason, since 
all of the factors are now known, the great variability in results which were obtained 
in the early work — before these factors were understood — has largely disappeared. 
' Smith, H.: loc. cit. » Burke, V.: /. Bad., 7, 159. 1922. 



JOHN W. CHURCHMAN 31 

STAINING OF ACID FAST BACTERIA 

The phenomenon of acid fastness was first observed in 1881 by Neisser' in his ap- 
plication of Weigert's staining methods to Hansen's leprosy bacillus. Bienstock and 
Gottstein in 1886^ had called attention to the relation between this phenomenon and 
a high lipin oontent, but it was Hammerschlag^ in 1888 who first discovered the high 
fatty and lipin content. Aronson in 1898 showed that a large part of the ether and al- 
cohol extractable substance was not true fat but a waxy substance. Although acid 
fastness is not confined to the so-called "acid-fast bacteria" — since spores, the eggs of 
certain tenia, etc., also possess the property — the phenomenon is of interest in bac- 
teriology, chiefly as a characteristic of B. tuberculosis, B. leprae, B. smegmatis, and the 
related avirulent species. Of the latter, about forty or fifty strains have been isolated, 
many of them doubtless identical; there is evidence that the acid fastness of these is 
associated with a certain common antigenic character, and all are rich in lipins. B. 
tuberculosis differs from the saprophytes in the possession of a higher content of waxy 
lipins. An analysis of B. leprae (Gurd and Denis)'' showed the following percentage 
composition: 

Per Cent 

Fat, fatty acids, and cholesterol 34. 7 

Lecithin 1.7 

An explanation of the mechanism of acid fastness presents difficulties similar to 
those encountered in attempting to account for gram staining behavior. The peculiar 
chemical constitution of acid fast organisms suggested that their distinguishing stain- 
ing characteristics were due to their fat content. This idea obtained generally and 
for a long while, although there was disagreement as to whether waxes, alcohols (par- 
ticularly the "mykol" of Tamura)^, fatty acids, or lipoid proteins were the factors ac- 
tually concerned. As in the case of the gram stain, however, the mechanism appears 
to be by no means a simple one, and both chemical and physical factors are doubtless 
involved. Tubercle bacilli are impermeable to the very fat soluble dyes which readily 
stain their isolated fats (Sudan III, scarlet R, janus green, etc.).^ On the other hand, 
basic fuchsin (which is only slightly fat soluble), eosin and methylene blue (which are 
not fat soluble at all), stain the individual organisms deeply in a relatively short time. 
Corper^ showed that tubercle bacilli within tubercles do not become stained when fat 
soluble dyes are injected into the tuberculous animals, and Sherman^ has called at- 
tention to the well-known but often overlooked fact that an apparent staining by fat 
soluble dyes may really be due to the staining of extra-bacillary substances and not to 
penetration of the organisms. Such facts as these make the role of the fatty sub- 

' Neisser, M.: Virchow's Archiv., 54, 514. 1881. 

* Bienstock, B., and Gottstein, A.: Forlsch. d. Med., Nos. 6 and S. 1886. 
3 Hammerschlag, A.: Cenlralhl. f. klin. Med. No. i. 1891. 

* Gurd, F. B., and Denis, W.: /. Exper. Med., 14, 606. 1911. 
sTamura, S.: Ztschr.f. phys. Chemie, 89, 289. 1914. 
^Sherman, H.: /. Infect. Dis., 12, 249. 1913. 

7 Corper, H. J.: ibid., 11, 373. 1912. ' 



32 STAINING REACTIONS OF BACTERIA 

stances somewhat uncertain, to the extent at least that they can hardly be the sole 
factors concerned in acid fastness. Just as in the case of the gram stain, bacillary in- 
tegrity is essential for acid fastness. It was pointed out by Koch and later by Benians, 
and confirmed by others, that disintegrated tubercle bacilli lose the property, from 
which it would appear that the fatty constituents of the tubercle bacillas are not per 
se the cause of the staining reaction characteristic of the organism. 

Like gram positivity, acid fastness is not an absolutely constant characteristic but 
is influenced by factors like age of culture and conditions of environment, and may be 
entirely upset by treatment with chemical agents. It appears established that very 
young tubercle bacilli may not stain at all by Ziehl's method (Krylow),' and from the 
work of Wherry, Mellon, and many other observers there is strong evidence that 
acid fast organisms go through a varied life-cycle during which their morphology is 
profoundly altered and their staining characteristics modified. Bienstock and Gott- 
stein were able to make ordinary bacteria acid fast by artificial Einfettimg through 
growth on butter agar. Wherry^ was able to render acid fast saprophytes non-acid 
fast by continual growth under conditions unfavorable to the synthesis of fats. 
Ritchie^ has shown that tubercle bacilli treated with boiling xylene, boiling toluene, 
boiling benzene, or Aronson's mixture (ether, alcohol, and HCl) lose their acid fast- 
ness; and Browning and Gulbransen^ have shown that the property is rapidly re- 
moved by treatment with CHCI3 to which minute amounts of HCl have been added 
together with a small amount of C2H5OH to yield a permanent mixture. These obser- 
vations support the view that the loss of the acid fast property is due to a dissociation 
of the constituents of the bacillus which is readily effected by the combined action of 
small amounts of HCl along with lipoid solvent like CHCI3. 

Any amount of extraction of tubercle bacilli with simple fat solvents leaves them 
acid-fast, probably due to the fact that they continue to contain a small amount of 
lipin. But if this last trace of lipin be removed by treatment with acid, acid fastness 
disappears (Long).s 

It is not easy to harmonize completely the observations which have just been 
cited and to present an entirely satisfactory theory of acid fastness. Three facts seem 
to be established (Long) : (i) Disintegration of the cell destroys acid fastness. (2) Ex- 
traction of the cell body with fat solvents until no more lipin is removed leaves the 
bacilli acid fast provided they have not been mechanically disintegrated (as by tritu- 
ration) during the process. (3) The wax of the tubercle bacillus is acid fast.^ It re- 
mains to reconcile the two chief conflicting explanations: that the phenomenon is due 
to the presence of an impermeable membrane and that it is due to the content of 
acid fast wax. Both factors may — as in the case of the gram stain — be of importance. 
Acid fastness appears ultimately to depend on the small amount of lipin held as an 
emulsion in the protein substrate and not extractable by fat solvents. But since it is 

'Krylow, D. O.: Zlschr.f. Hyg. u. Infeklionskrankh., 70, 135. 1911-12. 

'Wherry, W. B.: /. Infect. Dis., 13, 144. 1913. 

3 Ritchie, W. T.: /. Path, b- Bad., 10, 334. 1905. 

■• Browning, C. H., and Gulbransen, R.: ibid., 27, 326. 1924. 

swells, H. G., DeWitt, L. M., and Long, E. R.: Chemistry of Tuberculosis. 1923. 



JOHN W. CHURCHMAN 33 

influenced by physical disintegration of the cell wall something more than a chemical 
process appears to be concerned. Perhaps degree of fineness of emulsification of the 
acid fast constituent plays a part. 

MUCH GRANULES 

Acid fast organisms are also gram positive. But the two characteristics do not 
necessarily appear at the same stage of bacterial development, and one of them may 
be upset by processes which do not disturb the other. Krylow,' for example, showed 
that very young tubercle bacilli stain neither by the method of Ziehl nor by that of 
Gram, and that they become gram positive before they become acid fast; Wherry^ 
showed that by growing on suitable media the organisms may be made to lose their 
acid fastness while remaining gram positive ; and Aronson^ showed that the gram pos- 
itivity of B. tuberculosis could be reversed by treatment with trichlor ethylene. 

In 1907 Much, in examining the tuberculous lesions of a group of animals which 
had died of the disease, was unable to find organisms which stained by the method of 
Ziehl but did find granular forms which stained by that of Gram.'' He concluded that 
he was dealing with a cycle of B. tuberculosis other than the one usually encountered, 
and stated that it appeared probable that the method of Ziehl stains substances of the 
tubercle virus other than those stained by the method of Gram. It is therefore pos- 
sible to obtain positive results with the method of Gram in cases in which examination 
by the method of Ziehl is negative. His findings were as follows: (i) There is a gran- 
ular form of tubercle virus not stainable by Ziehl. (2) This granular form is virulent. 
(3) It occurs in tuberculous organs either as the only stainable manifestation of the 
etiological factor of the disease or in company with a fine rod which is also not stain- 
able by Ziehl. 

Krylow,' in reviewing Much's findings, came to the conclusion that gram positive 
material appears in B. tuberculosis much earlier than acid fast material, and this ma- 
terial tends to concentrate in granules. Acid fast material, on the other hand, is 
spread diffusely throughout the bacterial body. Hence in young cultures a modified 
method of Gram will demonstrate the Much granules. 

The significance of the Much granules is open to debate. Much's findings have 
been repeatedly confirmed, and there seems little doubt that the bodies he described 
are actually to be found in many tuberculous lesions. They may, however, be de- 
generation products (Rosenblat). In any event, demonstration of their presence is 
almost without diagnostic value since other bacilli form granules of similar appear- 
ance. 

STAINING ELEMENTS 01" BACTERIAL STRUCTURE 

Certain structural portions of some types of bacteria may, under certain condi- 
tions of growth, be demonstrable by suitable methods of staining. Here belong the 
spore, the capsule, flagella, polar bodies, metachromatic granules (the Babes-Ernst 

'Krylow, D. O.: loc. cil. 

^^ Wherry, W. B.: loc. cit. 

3 Aronson, H.: Berl. klin. Wchnschr., 35, 484. 1898; 47, 1617. 1910. 

''Much, H.: Bcilr. z. Klin. d. Tuberk., 8, 85. 1907. 



34 STAINING REACTIONS OF BACTERIA 

granules), and chromatin. The methods for staining these substances are well known 
and a discussion of their biological significance will be found in chapter i of this vol- 
ume. 

STAINING OF BACTERIA IN TISSUES, ETC. 

That the immediate environment of bacteria may profoundly influence their be- 
havior toward dyes is well known. Serum, for example, may interfere to some extent 
with the bacteriostatic action of triphenyl-methanes; the presence of alkalies enhances 
the staining powers of basic dyes, etc. For these reasons, bacteria in tissues, in milk 
soil, sputum, feces, pus, do not necessarily behave toward stains as they do when 
examined in smears from cultures, and special precautions are necessary in examin- 
ing these materials to overcome the difficulties presented by the environment. These 
difficulties are particularly great in examining tissues for bacteria, because the elab- 
orate processes of fixation, hardening, and clearing may profoundly alter the bacteria 
before they are stained. Recent studies have thrown no particular light on the 
problems involved, and the classical methods of staining are still in use. 

STAINS IN MEDIA 

That certain aniline dyes have the power of affecting bacterial viability has long 
been known, and their antiseptic properties have been recorded since the days of 
Koch. Recent observations make it clear that "bacteriostatic properties" better de- 
scribes the power of many dyes to interfere with the reproductive mechanism (gen- 
esistasis. Churchman)' without necessarily killing or even interfering with their other 
properties. Genesistatic potency is more striking and is more definitely established 
than bactericidal power. Probably the most important feature of the bacteriostasis 
produced by dyes is its highly selective character. This feature was translated into a 
selective cultural method of great practical value by Conradi and Drigalski^ when they 
introduced a medium for the selective cultivation of B. typhosus, by Petroff in his in- 
troduction of a gentian-violet medium for the selective cultivation of B. tuberculosis, 
by Krumwiede in his brilliant-green medium, etc. Of a great deal of interest and some 
theoretical importance as regards the microchemistry of bacteria is the parallelism — 
marked but not all inclusive — which exists between the selective bacteriostasis of 
triphenyl-methanes and the gram reaction. Premonitions of this parallelism are 
to be found in the observations of Stilling,^ of Dreyer, Kriegler, and Walker,"! and 
of others; but it was first established as a general relationship by the author^ in 191 2 
and later confirmed by Simon and Wood* and others. The mechanism of this selective 
bacteriostasis is still obscure. The curious fact that it does not entirely parallel se- 
lective bactericidal properties has been emphasized by Churchman^ and by Burke.* 

■ Churchman, J. W.: Proc. Nat. Acad. Sci., 9, 78. 1923. 

^ Conradi, H., and von Drigalski, W.: Zlschr.f. Hyg. u. Infcktionskrankh., 39, 283. 1902. 

3 Stilling, J.: Anilinfarbstoffe als Antiseptica, ii.s.w. Strassburg, 1890. 

^ Smith, H. W.: Am. J. Hyg., 2, 607. 1922. 

5 Churchman, J. W.: J. Exper. Med., 16, 221. 1912. 

^ Simon, C. E., and Wood, M. A.: loc. cit. 

1 Churchman, J. W.: /. Expcr. Med., 37, 543. 1923. 

* Burke, V., and Skinner, C. E.: ibid., 39, 613. 1924. 



JOHN W. CHURCHMAN 35 

That certain dyes (acid fuchsin) may exhibit the property in a reverse sense has also 
been established. These facts are perhaps dependent on the presence of spores in 
certain bacteria. 

Whatever the explanation, it is sufficiently striking that so hardy an organism as 
B. subtilis is entirely unable to develop in the presence of a minute amount of gentian 
violet (1-750,000) or that a vigorous and virulent M. aureus, when stained with this 
dye, may be injected into susceptible experimental animals without any untoward 
effects to the host; while, on the other hand, a sporeless organism like B. prodigiosus — 
easily killed by slight amounts of heat — will grow vigorously in the presence of this 
dye in dilutions of 50,000, or less, and even though deeply stained, when transplanted 
to agar will grow apparently as well as the untreated controls. That the parallelism 
between the gram reaction and selective bacteriostasis although striking is not com- 
plete, has already been brought out. Reasons have also been advanced why the 
tempting explanation cannot be accepted that the parallehsm depends on the greater 
ease with which triphenyl-methane dyes penetrate gram positive organisms than 
gram negative ones. It is quite possible that in these selective reactions some of the 
dyes may actually stimulate the growth of the organisms which they fail to inhibit. 

VITAL STAINING 

Much of what has thus far been said applies to bacteria which have been killed 
during the process of fixation. Since it is well known that profound chemical changes 
may set in rapidly when protoplasm dies and that disturbance of the cell membrane, 
as shown particularly by the work of Chambers,' may induce immediate alterations 
in the cell, care must be taken not to assume too hastily that the phenomena observed 
in stained specimens represent the facts of the living cell. Such an experiment as that 
of Benians, in which the bacterial membrane is deliberately ruptured before exami- 
nation, is perhaps open to criticism on the ground that the treatment provides a differ- 
ent substance for observation from that present in the living cell. Nevertheless, the 
examination of bacteria stained in vivo has brought out no facts which are at variance 
with what has been learned from the staining of fixed smears, Ernst,^ Nakanishi,-' 
Amato,^ and Pappenheim^ have more particularly studied this subject. Methy- 
lene blue, brilliant kresyl blue, and toluidin blue have been the dyes chiefly em- 
ployed. 

A special value of the method of vital staining resides in the fact that what is 
seen may be regarded as characteristic of the living cell and not an artefact, such as 
might be produced by staining fixed smears. Large spirilla. Vibrio cholerae, B. typho- 
sus, and B. coli have been chiefly studied, and particular attention has been paid to 
the vital staining of the spore. There is little doubt that some difference exists be- 
tween the accessibility of dead and living bacteria to certain chemical agents. Ny- 

' Chambers, R.: General Cytology, p. 237. 1Q24. 

^ Ernst, P.: Ztschr. f. Hyg. u. Infeklionskrankh., 4, 25. 1888. 

^Nakanishi, K.: Mimchen. med. Wchnschr., No. 6. 1900. 

'•Amato, A.: Centralbl. f. Bakkriol., 48, 2,85. 1908. 

s Pappenheim, A.: Monatschr. f. prakt. Dermatol., 37, 429. 1903. 



36 STAINING REACTIONS OF BACTERIA 

feldt/ for example, has shown this to be the case for silver nitrate. Many observers 
have referred to the fact that stained bacilli may remain actively motile, and they 
have therefore assumed that the bacteria had been actually stained while alive. This 
raises the question of the possibility of staining living cells without injuring them. So 
far as the nucleus is concerned, it has long been held that this structure cannot be 
stained during life although there is a great deal of evidence contrariwise (e.g.. 
Churchman).^ That bacteria which are stained, at least in the sense that they appear 
colored (although it might be objected that the stain was only adhering to the surface 
and had not penetrated the cell), may grow well has been proved by the experiments 
of the author.^ I have shown that gram negative organisms deeply stained with gen- 
tian violet grow luxuriantly when transferred to agar, and by single-cell transplanta- 
tion have proved that this growth is not due to bacteria which happen to have escaped 
the stain. Experiments tend to confirm the theory that the penetration of a basic dye 
into living cells depends on the fact that the dye is dissociated and that, in the form of 
the free base which predominates at higher pH values, it penetrates very readily, 
while in the form of salt its penetration may be so slight as to be negligible. Irwin,'' 
on the basis of experiments with Valonia and Nitella flexilis, has warned against the 
danger of assuming that the dye which appears to have entered a living cell from a 
solution is actually the dye present in that solution rather than one of its lower 
homologues. 

Many observers have noted that the gram positive organisms appear to take up 
dyes in vivo more readily than the gram negative ones. From this fact it might be 
argued that their greater sensitivity to the staining and bacteriostatic effect of tri- 
phenyl-methane dyes was due to their increased permeability. But gram negative 
and dye-sensitive strains of B. enteritidis and B. coli have been observed which were 

EXPLANATION OF PLATE I 

Photographs, through color screens, of camera lucida drawings in color. The gram positive 
elements have photographed black; gram negative elements, gray. Magnification X3200. (a). 
Smear from a suspension of a" 4-hour culture of B. anthracis (American Type Culture Collection 
No. 10) to which acriviolet has been added. Smear made after 45-minutes exposure to the dye. The 
organism, sharply gram positive at the beginning of the experiment, has become about 75 per cent 
gram negative. The gram negative forms are about 40 per cent smaller in diameter than the gram 
positive. Thesporeisentirely contained within the gram negative portion, {h). Smears from a 4-hour 
culture of B. anthracis which has been stained by a modification of Burke's method in which the 
period of exposure to dye and mordant has been greatly shortened and the period of exposure to 
decolorizer greatly lengthened (10 sec, 10 sec, i min.). A large variety of partial decolorizations is 
seen, the gram positive material (cortex) being in some cases entirely removed, in other cases per- 
sisting as plaques, or lumps, or dots among which the gram negative medulla shines through. Notice 
the terminal caps of gram positive material at a, b, and c. (c). Smear of a mixture of M.frcudenrcichi 
and B. anthracis stained by a modification of Burke's method (stain 5 sec, iodine 5 sec, decolorizer 
5 min.). Almost all the individuals of B. anthracis have been completely decolorized; M.frcuden- 
rcichi has been unaffected. (Photograph made from colored drawings appearing in article by the 
author, J. Exp. Med., 56, 1007. 1927.) 

' Nyfeldt, A.: Nord. med. Arhv., 50, 184. 1917. 

2 Churchman, J. W., and Russell, D. G.: Proc. Soc. Exper. Biol. 6" Med., xi, 120-24. 1914. 

3 Churchman, J. W., and Kahn, M. C: /. Expcr. Med., 33, 583. 192 1. 
•« Irwin, M.: Proc. Soc. Exper. Biol, b' Med., 24, 425. 1927. 



PLATE I 




.■#^ 



.-€ 



\ 




(See Explanation on Page 36) 



JOHN W. CHURCHMAN 37 

not easily permeated by the dyes (Churchman).' Furthermore, when gram positives 
become gram negative (e.g., streptococcus), they do not become dye sensitive. For 
these and other reasons it is not Hkely that sensitivity is dependent directly on those 
factors which determine permeabihty to the reagents of Gram's stain. These two 
properties are independent variables. In view of their usual association, it may be as- 
sumed that they are indirectly associated through some biochemical factor not yet 
recognized. 

SILVER IMPREGNATION 

The method of van Ermengem^ for the demonstration of flagella suggests pos- 
sibilities for the study of the microchemistry of bacteria by methods allied to those of 
staining. Van Ermengem applied to the study of bacteria the methods of certain 
photographic processes. The fixed smears were immersed for one to three seconds in 
a 0.5-1 per cent solution of silver nitrate, and bacterial structure (particularly the 
flagella) were in this way rendered clearly visible. The phenomenon was regarded as 
one not simply of silver precipitation but as due to a true chemical combination 
pointing to a different chemical structure between flagella (which appear gray black) 
and bacterial bodies (which appear orange or dark brown). Furthermore, flagella are 
less easily decolorized by immersion in gold solutions than bacterial bodies. This also 
points to a stronger affinity of their substance for silver. 

' Churchman, J. W., and Michael, W. H.: /. Exper. Med., 16, 822. 191 2; ibid., 33, 569. 1921. 

'van Ermengem, E.: Travaux du laboraloire d' hygiene et de bacteriologie de VUniversite de 
Gaud, i, No. 3. 1892. 



CHAPTER IV 

MORPHOLOGICAL CHANGES DURING THE 
GROWTH OF BACTERIA 

PAUL F. CLARK 

University of Wisconsin 

In spite of the efforts of investigators for more than half a century, three general 
conceptions of the morphology of bacteria still exist. The first assumes that bacteria 
are simple in form and structure. According to this theory, the size and shape of each 
species are fixed, varying only within narrow limits, and the organisms multiply only 
by transverse fission into two daughter-cells of similar size. A second conception at 
the other extreme has been emphasized especially during the last decade. Several ob- 
servers have described complicated life-cycles in bacteria including reproduction by 
budding, and complex mitoses following conjugation; marked changes in form have 
been reported in presumably pure cultures, even to the extent of the metamorphosis 
of a diphtheroid into a streptococcus and of filterable viruses into spore-formers. The 
third position, as one might expect, follows a middle path with the acceptance of a 
considerable degree of pleomorphism, especially in certain genera such as Corynebac- 
teria and Azotobacter, but with a skepticism concerning the demonstration of proved 
life-cycles. This conception also includes an emphasis on the importance of observing 
the changes in form and size during the growth of the organism. 

The first-mentioned idea of bacterial morphology exists now chiefly on the pages 
of the briefer textbooks and in the minds of beginning students. Everyone who has 
examined ordinary stained preparations of root-nodule bacteria, diphtheria bacilli, 
streptococci, or members of the Spirillaceae grown on a variety of media will have 
noted the occurrence of aberrant morphological types to such a degree that the fixed 
morphology concept must be discarded. Furthermore, some of these forms appear in 
young cultures (two to eight hours) as well as in those which have been incubated for 
several days, so that they cannot be dismissed as involution or degeneration forms. 

Although this idea of the fixity of bacterial form has been largely discarded in its 
more precise interpretation, systematic bacteriology is, nevertheless, largely based on 
this conception; and, in routine, we find it possible, though at times difficult, to work on 
this foundation. The usual procedures tend to emphasize uniformity; the standard- 
ized artificial media select those organisms best fitted to grow under these saprophytic 
conditions; the crude drying and flaming methods of fixation and the gross overstain- 
ing effectively conceal any fine differences in structure; the diurnal rotation of the 
earth gives us a standard time for the examination of cultures based on our convenience 
rather than on any physiological cycles in the development of bacteria. Furthermore, 
bacteria are so fascinatingly varied in their functions and apparently so alisurdly simple 
in morphology — what they do is so vastly more important biologically than what 
their form and structure may be — that bacteriologists quite generally have become 

38 



PAUL F. CLARK 39 

physiologists, and have tended to minimize the importance of morphological studies. 
And if unusual forms are seen, what can be easier than to assume that these are 
mutants, involution forms, or contaminations? 

In spite of this tendency to stress uniformity, the variability of bacteria has forced 
its way through our methods so that a number of men have been led to make careful 
cytological observations and eventually to take a position diametrically opposed to 
the concept of fixed morphology. The work of Nakanishi,' Schaudinn,^ Grimme,^ 
Mencl,"* GuilliermondjS Ambroz,** Dobell,' Lohnis,* Hort,' Mellon,'" Bergstrand," En- 
derlein," Kirchensteins,''^ Thornton and Gangulee,''' and Cunningham and Jenkins,'^ 
to name only a few, has been especially noteworthy. It has given foundation for the 
opinion in the minds of some bacteriologists that bacteria are not simple structureless 
cells, but fungi with formed nuclei, some of them showing mitotic division and com- 
plex life-cycles. Since this conception is philosophically attractive and would co-or- 
dinate bacteria more closely with the rest of the biological world, it is easier for some 
to accept the evidence as adequate, while others feel that this attractive prospect is 
an additional reason for maintaining a position of unusually strict skepticism. 

The existence of a formed nucleus even in the simpler bacteria (Eubacteriales) has 
been demonstrated beyond doubt especially by Nakanishi,'* Dobell,'? Guilliermond,'* 
Meyer,'' Mencl,^" and Kirchensteins.^' By different technique, with material from 
a variety of sources, in organisms from young and old cultures, in both the simpler 
and the higher bacteria, spore-formers and non-spore-formers, nuclei have been dem- 
onstrated. The form of the nucleus is variable not only in different species, but also 
in different stages of the development of one species. In cocci the nucleus is usually 
more or less spherical, and in fission begins to divide before the division of the cyto- 
plasm. In rods and spirilla the nucleus is more variable and may be seen in the form 

' Nakanishi, K.: Munchen. -wed. Wchnschr., 47, 187. 1900; Centralbl.f. BakterioL, Abt. I, 30, 97, 
145, 193, 225. 1901. 

^ Schaudinn, F.: Arch. Prolistenk., 2, 421. 1903. 

3 Grimrae, A.: Centralbl.f. BakterioL, Abt. I, 32, i. 1902. 

'• Mencl, E.: Arch. Prolistenk., 8, 259. 1907. 

5 Guilliermond, A.: Bull. Inst. Pasteur, s, 273, 321. 1907. 

*Ambroz, A.: Centralbl.f. BakterioL, Abt. I, 51, 193. 1909. 

'Dobell, C. C: Quart. J. Micr. Sc, 56, 395. 1910-11. 

'Lohnis, F.: Mem. Nat. Acad. Sci., 16, i. 1921. 

9}iort,E.C.: Proc. Roy. Soc.,'B, 8g, 468. 1917; 5n7. M. /., i, 571, 664. 1917; j&i(/., 2,377. 1917; 
J. Roy. Micr. Soc, 365. 191 7; J. Hyg., 18, 361. 1920; ibid., p. 380. 1920. 
'"Mellon, R. R.: Am. J. M. Sc, 159, 874. 1920. 
" Bergstrand, H.: /. Bad., 8, 365. 1923. 
'^ Enderlein, G.: Bakterienzyklogenie. Berlin, 1925. 

'3 Kirchensteins, A.: Structure interieure et Mode de Develop pement des Bacteries. Riga, 1922. 
"•Thornton, H. G., and Gangulee, N.: Proc. Roy. Soc, B, 99, 427. 1926. 
'5 Cunningham, A., and Jenkins, H.: /. Agric Sc, Part I, 17, 109. 1927. 
'^ Nakanishi, K.: loc cit. ^^ Dobell, C. C.: loc. cit. '* Guilliermond, A.: loc cit. 

'"Meyer, A.: C&ntralbl.f. BakterioL, Abt. II, 6, 339. 1900. 
'0 Mencl, E.: loc. cit. ^' Kirchensteins, iK.: loc. cit. 




40 MORPHOLOGICAL CHANGES DURING GROWTH 

of discrete granules (chromidia), irregular filaments, branched rods, or one or more 
large masses of nuclear substance. Even different stages of mitosis have been de- 
scribed by Mencl', Guilliermond^, and others. 

Beyond this point it is difficult to analyze the large number of observations which 
various authors have presented as evidence of the existence among the bacteria of the 
more complex methods of reproduction commonly found among the Protista. Many 
authors describe various budlike structures, large bizarre forms, minute deeply stain- 
ing bodies, and imperfectly stained masses, and have read into these observations cer- 
tain sequences which, it seems to me, have not been adequately proved. The diffi- 
culty of demonstrating genetic descent with organisms so minute is apparent. 
Since, however, the purity of the culture is so completely essential for evaluation of 
these observations, should we not insist that some method of single-cell isolation be 
employed as a basis for work of this sort, even though successful transplants can be 
obtained in a very small percentage of the attempts? Most of the work on "life- 
cycles" in bacteria has not been carried out with this foundation, and many of the 
observations are inadequate as experimental proof of developmental cycles, however 
much the notion may appeal philosophically. A few papers presenting observations 
in the field of soil bacteriology cause the scales to tip rather sharply toward the "life- 
cycle" side. Notable recent contributions are those of Thornton and Gangulee^ with 
Bacillus radicicola and Cunningham and Jenkins^ with B. amylobacter . Unfortunate- 
ly, neither of these papers is based on single-cell isolations. 

That bacteria, even among the Eubacteriales, do at times reproduce by means 
other than equal fission seems to me to be definitely proved. Some of the best ob- 
servations in this connection have been made by Hort,^ who has studied a number of 
the common pathogens, watching the development of the organisms by means of a 
combination of single-cell technique and warm stage growth. He has demonstrated 
that, under adverse conditions, some strains of the colon-typhoid group reproduce by 
budding, by branched Y-shaped forms, and by the production of large aberrant forms in 
which the fragmented chromatin appears as deeply staining granules. These granules 
are subsequently extruded and are small enough (0.1-0.2 micron in diameter) to pass 
through the coarser bacterial filters. He watched the subsequent development of 
these "gonidial bodies" into typical baciUi. He avoided the assumption of any defi- 
nite life-cycle, but emphasized his demonstration that methods of reproduction other 
than binary fission occur among bacteria, and that the irregular bodies cannot be 
considered involution forms since he has shown that they reproduce actively. 

The essential details of Hort's observations were confirmed by a specially ap- 
pointed committee consisting of Leishman, Adami, Farmer, and Harvey.^ 

With a somewhat similar technique Gardner^ has also described reproduction at 
each of the three growing points from Y-shaped organisms in six members of the colon- 

' Mencl, E.: loc. cil. " Guilliermond, A., loc. cil. 

3 Thornton, H. G., and Gangulee, N.: loc. cit. 

^ Cunningham, A., and Jenkins, H.: loc. cit. 

sHort, E. C.: see various works cited previously. 

^Leishman, W. B., Adami, J. G., Farmer, J. B., and Harvey, D.: /. II yg., 18, 380. 1920. 

' Gardner, A. D.: /. Falh. &° Bad., 28, 189. 1925, 



PAUL F. CLARK 41 

typhoid group and V. cholerae. He found that this is a common occurrence in the 
stage of rejuvenation of cultures of pathogenic bacteria, but found no evidence that 
these Y-forms are part of a complex life-cycle. 

The fact that so many of the common pathogens have, in recent years, been passed 
through infusorial earth and porcelain filters especially after anaerobic growth or 
through the influence of bacteriophage is additional evidence in favor of the occur- 
rence of minute forms which under suitable conditions are capable of growing into 
typical organisms. Apparently some nuclear reorganization does occur, but that con- 
jugation is an essential precursor does not seem to have been proved. 

Filterability is admittedly not the best basis for judgment as to the size of particles, 
since electro-physical phenomena play such a large part in the results (Kramer,' 
Mudd)-. But when filtrations are carried out under more or less standardized condi- 
tions with silica filters and bacteria suspended in physiological saline, the positive 
results obtained by a number of observers certainly tend to indicate the occurrence 
among our common pathogens of filterable bodies smaller than the type forms com- 
monly recognized. 

Uninterrupted study of the developing organisms by some hanging-block method 
(Hill,^ OrskoVji Hort,^ Gardner'^) or by moving pictures (Bayne- Jones, Bronfenbren- 
ner)7 will continue to be essential to the growth of knowledge of the morphology and 
development of bacteria. Li such studies, however, individual cells quickly become 
lost in the "log jam" of the dividing bacteria so that clear observation is difficult. An- 
other method which has given some information in regard to the morphological 
changes during the growth of bacteria is the examination of smears removed at fre- 
quent intervals from cultures growing under uniform conditions, the measurement of 
large numbers of the individual bacteria, and the reconstruction of the life-story large- 
ly by statistical methods. The common notion of the "normal" morphology of bac- 
teria is based upon the inspection of material from original habitats or from cultures 
which have been growing approximately twenty-four hours or some multiple of that. 
The writer has pointed out the general failure to appreciate this time factor and the 
resulting errors in our conception of the morphology of the common bacteria. Clark 
and RuehP have shown that even on the ordinary standardized media bacteria pass 
through striking morphological changes which are coincident with the different growth 
phases described by Lane-Claypon' in the life-history of a bacterial culture. Later, 
Henrici"* confirmed these findings and worked out many points more completely. 

'Kramer, S. P.: /. General Physiol., g, 811. 1926. 

= Mudd, S.: J. Bad., 8, 459. 1923. 

3 Hill, H. W.: J. Med. Research, 7, 202. 1902. 

''Orskov, J.: /. BacL, 7, 537. 1922. 

s Hort, E. C: see various works cited previously. * Gardner, A. D.: loc cit. 

7 Bayne- Jones, S., and Tuttle, C: /. Bad., 14, 157. 1927; Bronfenbrenner, J.: Meeting Soc. 
Path. 6* Bad. April, 1927. 

« Clark, P. F., and Ruehl, W. H.: J. Bad., 4, 615. 1919. 

9 Lane-Cla>TDon, J. E.: J. Hyg., 9, 239. 1909. 

"Henrici, A. T.: Proc. Soc. Exper. Biol. &= Med., 19, 132. 1921; 20, 179, 293. 1922-23; 21, 215, 
343, 345- 1923-24; 22, 197. 1924; Science, 61, 644. 1925; /. Infed. Dis., 37, 75. 1925. 



42 MORPHOLOGICAL CHANGES DURING GROWTH 

During the early latent period, when growth is slow or completely lacking, no apparent 
morphological change is seen. Following this, during the logarithmic period when 
maximum reproduction occurs, the cells from the young cultures of many genera of 
bacteria attain their maximum size, two to six times larger than the cells from the 
twenty-four-hour parent-cultures. After four to eight hours the cultures pass gradu- 
ally into the stationary period, in which the number of organisms remains relatively 
constant. During this progression to the stationary period, the bacteria become 
gradually smaller in size until, by the time the cultures are eighteen to twenty-four- 
hours old, the classical textbook picture is presented. Cultures older than twenty- 
four and forty-eight hours present more and more of the so-called "involution forms," 
irregular staining, in many instances bizarre forms, and organisms averaging smaller 
than those found in the twenty-four-hour cultures predominate. 

Clark and RuehP studied by this method seventy strains in all, including cultures 
of the following species: 

COCCACEAE Strains 

Staphylococcus aureus 5 

Staphylococcus albus 2 

Streptococcus hemolyticus 6 

Diplococcus pneumoniae 5 (including types I, II, and III) 

Neisseria gonorrheae 

Neisseria catarrhalis 2 

Neisseria intracellularis 

Neisseria mucosis 
Spirillaceae 

Vibrio comma 

Vibrio metchnikovi 

Vibrio schuylkilliensis 
Bacteriaceae 

Proteus vulgaris 

Escherichia coli 

Escherichia communior 

Eberthella typhi 5 

Eberthella dysenteriae 

Eberthella paradysenteriae 

Salmonella paratyphi 

Salmonella schottmiilleri 

Salmonella suipestifer 

Encapsulatus pneumoniae 2 

Pseudomonas pyocyaneus 

Serratia marcescens 

Pasteurella avicida 

Hemophilus influenzae 

Hemophilus pertussis 

Bacillus anthracis 

Bacillus subtilis 

Bacillus megatherium 

' Clark, P. F., and Ruehl, W. H.: loc. cit. 



PAUL F. CLARK 43 

Bacillus vulgatus 
Bacillus mycoidcs 
Mycobacteriaceae 
Mycobacterium leprae 
Mycobacterium smegmatis 
Mycobacterium phlci 

Coryncbacterium diphthcriae 4 

Corynebacterium hofmanii 
Coryncbacterium xerosis 
Corynebacterium hodgkinii 
PJeiferella mallei 

Henrici' studied in detail Vibrio comma, Escherichia coli, Bacillus megatherium. 
Bacillus cohaerens, and an unidentified member of Corynebacterium. 

The increase in size during the "youth" of the cultures occurs in all the organisms 
studied except the Corynebacteria and B. mallei. In these bacilli, more especially in 
the first named, exactly the opposite progression occurs; the individuals from the 
younger cultures (two to six hours) are the smallest, averaging in C. diphtheriae less 
than half the size of the rods from a twenty-four-hour culture. The minimum size is 
reached during the period of rapid reproduction ; during the phase of slow growth the 
size increases, reaching the maximum during the resting period. As the individual or- 
ganisms become smaller, the metachromatic granules disappear and the bacteria stain 
uniformly and deeply with Loffier's methylene blue. Not infrequently at this stage 
members of this genus form coccoidal chains. The peculiar pleomorphism, irregular 
staining, and metachromatic bars and granules, so characteristic of the diphtheria 
group, reappear as the cells increase in size again. 

The method employed in these studies hardly needs further description save to 
add that Henrici' developed an admirable adaptation of the negative staining method 
of Benians^ to distinguish between the living and dead bacteria in smears. This made 
it possible to correlate precisely the changes in morphology with the rapidity of cell 
division in the bacterial cultures. 

Members of the colon-typhoid group will serve as a basis for the more detailed con- 
sideration of the typical mode of progression. A large majority of the bacteria in 
a four-hour culture of B. typhosus are 4-6 micra long and 0.7-0.8 micron wide, so 
large that they resemble the vegetative cells of the common spore-formers rather than 
the usual picture of B. typhosus based on the examination of twenty-four-hour growth. 
The increase in size is greater in length than in breadth, so that the larger cells are 
relatively more slender. Chain formation occurs commonly at this stage even in those 
species which in older cultures are characterized by discrete organisms. The bacteria 
stain more intensely, and the outline is more sharply defined. The time when the 
maximum average size occurs varies somewhat in different strains of the same or re- 
lated species. This is dependent largely upon the duration of the period of "lag," 
which according to Chesney^ is an expression of injury to the bacterial cell. By plot- 

' Henrici, A. T.: see various works cited previously. 
'Benians, T. H. C: Brit. M.J., 2, 722. 1916. 
3 Chesney, A. M.: /. Exper. Med., 24, 387. 1916. 



44 MORPHOLOGICAL CHANGES DURING GROWTH 

ting the projected image of the bacterium divided by the length squared, Henrici' has 
obtained an index of the variation in form. The coefificient of variation in length of 
cells, as well as in the area-length index, is increased during the period of increased 
size. 

The various members of the Spirillaceae, because of their marked pleomorphism, 
offer especially interesting opportunities for the use of this method. The three species 
studied follow the usual series of changes in size. During rapid growth, the new cells 
are long and plump and relatively straight save that where several organisms have 
remained attached they show definite spirals. As reproduction becomes slower, the 
individuals become more slender, more curved, and distinctly granular in staining. 
During the period of decline unusual forms are observed — various bulging or budding 
organisms and coccoid bodies both large and small. Henrici points out that "these 
latter types are the forms which Lohnis and others have described as extraordinary 
reproductive cells but that the trend of the growth curve would indicate that this is 
not the case." 

The three members of the Mycobacteria studied need no especial comment save 
to point out that the "senile" forms are distinctly granular with many coccoid bodies 
present. 

Even the cocci pass through similar cyclical changes in morphology, although the 
proportional difference between the diameter of the young and the senescent forms is 
less than with most of the rods and spirilla. The four- to six-hour cultures contain 
many deeply staining cocci approximately twice the diameter of those usually seen. 
In studying B. megatherium, Henrici' has pointed out a number of factors which 
affect the rate of progression through the period of maximum reproduction and con- 
sequently the onset of the phase of decline. If the volume and constituents of the media 
are constant and if varying numbers of the bacilli are inoculated, the fewer the cells 
introduced the longer is the period of maximum reproduction and the greater is the 
maximum size of the organisms. With the same seedings, if the nutrient ingredients 
of the media are varied, then the richer the media the longer is the period of logarith- 
mic growth and the greater is the maximum size attained. When transplantations 
are made during the period of increasing size, the organisms in the subculture con- 
tinue to increase in size, progressing even beyond the maximum reached by the parent- 
culture. Transplantations made immediately after the parent-culture has returned 
to the original size show no evidence of lag; the subcultures increase rapidly in size 
again. After two or more hours in the stationary phase, however, subcultures show 
an appreciable lag and do not progress beyond the curve of the parent-culture. Al- 
though other organisms have not been studied with reference to these points, pre- 
sumably they will follow the same laws. 

It is interesting to note that spore formation begins toward the end of the ac- 
tive growth period, so that the factors which lengthen the phase of positive accelera- 
tion in growth delay spore formation. 

It would appear, then, that even apparently simple bacteria growing under stand- 
ardized conditions admirably fitted to suppress variation and to increase uniformity 
pass through a series of cyclical changes which indicate a progression from youth 
' Henrici, A. T.: see various works cited previously. 



PAUL F. CLARK 45 

through maturity to old age. These three phases show marked differences in meta- 
boHc and reproductive rate, and correlated changes in size and structure. Sherman 
and Albus' have shown that other physiological correlations exist, in that young bac- 
teria are destroyed by brief exposures to cold and 2 per cent sodium chloride while 
cells from older cultures are not injured. Differences in agglutinability have been 
known for many years. There is no evidence that conjugation occurs, but there is 
ample evidence that, as Child^ has shown in higher forms, youth and rejuvenescence 
may occur apart from sexual processes. 

Observations such as the foregoing suggest an interesting correlation between the 
growth of bacterial cells and that of other living organisms. Protoplasmic changes, 
similar in kind to those cellular changes we associate with youth, maturity, and senil- 
ity in metazoan forms, occur even in these supposedly "immortal" single cells. Def- 
inite changes in gross morphology and in finer structure are apparent. A logical as- 
sumption that bacteria, in common with their more highly organized relatives, regain 
their youth and vigor of reproduction by the injury and stimulus of conjugation is 
not borne out by these observations. It must be borne in mind, however, that these 
observations were all made under conditions admirably suited for growth. Just as 
numbers of species of fungi have for years been classified with the Fungi imperfecti 
but later, by more complete studies under varying conditions of growth, have been 
shown to have definite sexual reproduction and, consequently, have been removed 
from this family, so it would seem to be highly probable that more detailed study will 
remove bacteria from their unique position and link them more closely with the rest 
of the biological world. 

'Sherman, J. M., and Albus, W. R.: J. Bad., 8, 127. 1923; 9, 303. 1924. 
=> Child, C. M.: Senescence and Rejuvenescence. 1915. 



CHAPTER V 



GROWTH CURVES OF BACTERIA 

R. E. BUCHANAN 

Iowa State College 

The following discussion of growth rates of bacteria and their graphical representa- 
tion in growth curves will be concerned solely with rates of increase. 

CHARACTERISTICS OF GROWTH CURVES 

The general characteristics of growth curves may be developed through considera- 
tion of the changes in numbers of bacteria which follow inoculation into a medium 

suitable for growth. For the purpose 
of preliminary discussion it is advan- 
tageous to assume that the inoculum 
consists of bacterial spores, for such 
material will permit of the maximum 
opportunity for differentiation of 
stages or phases of growth. 

Examination of such a culture at 
suitable intervals will show that an 
appreciable time elapses before any 
increase in numbers occurs, i.e., some 
time is required before any of the 
spores germinate and vegetative cells 
develop and divide. This may be 
termed the "initial stationary phase." 
It is scarcely to be anticipated that 
all the spores will germinate at the 
same instant. However, after cell 
division has been initiated it will pro- 
ceed with a considerable degree of 
regularity. Finally, all the viable 
spores will have germinated, and the 
culture will have completed its second or "lag phase." For a time thereafter the 
numbers of cells will increase more and more rapidly, with the rate of growth per cell 
remaining nearly uniform. This is the third or "logarithmic phase," during which 
there is a geometrical increase in cells with time. Conditions eventually become less 
favorable, and the rate of growth decreases. This is the phase of "negative growth 
acceleration." Finally the bacteria cease to multiply, and the "maximum stationary 
phase" is instituted. It is thus possible under favorable conditions to differentiate 
some five different growth phases. 

46 




a 10 jz J4 
Time in Hours 



20 22 2^ 



Fig. I. — Growth curve with various phases 

a-h. Initial stationary phase 

h-c. Lag phase 

c-d. Logarithmic phase 

d-e. Phase of negative acceleration 

e-/. Maximum stationary phase 



R. E. BUCHANAN 



47 



These facts and relationships may be shown graphically in several ways. The 
standard growth curve such as that noted above may be graphed (Fig. i) by plotting 
numbers of bacteria against time. In- 
spection of the graph shows the ex- 
istence of some four readily differen- 
tiable phases; the distinction between 
the lag phase and the logarithmic phase 
is not easily made by examination of 
this type of curve. 

A second method of representing 
increase in numbers is to plot the total 
increase in numbers of bacteria in each 
equal interval of time against time, 
thus developing a "rate curve." Such 
a curve corresponding to the growth 
curve of Figure i is given in Figure 2. 

A third type of graph may also be 
used to illustrate growth rates: one in 
which the successive rates of increase 
per cell (or, conversely, the generation 




Fig. 2. — Rate 
curve of Fig. i. 



Time in Hours 
curve corresponding to growth 



time) may be plotted against time. 
Curves such as those in Figure 3 may 
be thus secured. In this graph the 
identification of the five growth phases 
is more readily accomplished than in 
Figures i and 2. 

A still clearer differentiation of the 
various growth phases is to be secured 
by a fourth type of graph in which the 
logarithms of the numbers of bacteria 
are plotted against time, as in Figure 
4. During the initial stationary phase 
(a-6) a straight line with o slope is 
developed, during the lag phase (&-c) 
a curved line, during the logarithmic 
phase a straight line {c-d) with posi- 
tive slope; during the phase of nega- 
tive acceleration a curved line {d~e), 
and finally during the maximum 
stationary phase (e-/) a straight line 
of o slope. 

A study of the typical growth curve 
(Fig. i) shows it to be more or less 
S-shaped. Its exact form in each case will depend upon the type of organism, its 
immediately antecedent history, and the various environmental influences. 





1 








V ' 






\ 




e 


y 


1 




c 




d 






c 


A 


_d 






a 


.y 








^-^e 


/ 



13 20 JZ2 2^ 



'? c e JO 12 14 I 

Time in Houns 

Fig. 3 

A . Graph of rate of growth per cell 

B. Graph of generation times 
a-h Initial stationary phase 
h-c Lag phase 

c-i Logarithmic phase 

d~e Phase of negative acceleration 

c~j Maximum stationary phase 



GROWTH CURVES OF BACTERIA 



Two principal types of explanation or interpretation have been used for the form 
assumed by bacterial growth curves. The first of these (a succession of growth phases) 
is the one developed above. In its essentials it was apparently first outlined by 
Lane-Claypon/ and later expanded by the writer.^ A somewhat different method 
of attack was suggested by McKendrick and Pai,^ and emphasized among others by 
Robertson^ and Lotka\ These authors conclude that the sigmoid shape of the growth 
curve is evidence of the resemblance of growth to the phenomenon of autocatalysis 

(or, in the terminology suggested by 
Ostwald, "autocatakinesis"). While 
these two interpretations are not 
essentially antagonistic, they represent 
somewhat different points of view, 
and require separate treatment. 

GROWTH CURVES CONSIDERED AS A SUC- 
CESSION OF GROWTH PHASES 

It was noted above that in some 
cases at least as many as five different 
growth phases may be observed in a 
culture of bacteria. They will be con- 
sidered in order. 




flnne in Hours 



Fig. 4. — Growth curve graphed as logarithms of 
numbers of bacteria against time. 

a-h Initial stationary phase 

h-c Lag phase 

c-i Logarithmic phase 

d-e Phase of negative acceleration 

e-j Maximum stationary phase 



INITIAL STATIONARY PHASE 



During this phase there is no in-, 
crease in numbers. In the illustration 
above its existence was ascribed to the 
time required for spores to germinate. 
Experience shows, however, that this 
phase is sometimes evident in a bac- 
terial transfer although the organism is one which does not sporulate. It is apparent 
that bacterial cells from old cultures (in the maximum stationary and later phases) 
possess some of the same inertia and slowness to develop under favorable environ- 
ment usually regarded as characteristic of spores. They may be regarded as in 
some respects the physiological, though not the morphological, equivalents of endo- 
spores. In some cases this phase may be prolonged for days or weeks. Such prolonga- 
tion has been noted particularly by Esty and Meyer^ in heated cultures of the bacil- 
lus of botulism. This phase is not found when the inoculum contains any considerable 
proportion of actively multiplying bacteria. 

' Lane-Claypon, Janet E.: J . Hyg., 9, 239. 1909. 

" Buchanan, R. E.: /. Infect. Dis., 23, 109. 1918. 

3 McKendrick, A. G., and Pai, M. Kesave: Proc. Roy. Soc, Edinburgh, 31, 649. 1911. 

■t Robertson, T. B.: /. Physiol., 56, 404. 1922. 

sLotka, Alfred J.: Elements of Physical Biology. 1925. 

' Esty, J. R., and Meyer, K. F.: J. Infect. Dis., 31, 650. 1922. 



R. E. BUCHANAN 49 

LAG PHASE 

During this phase the average rate of growth per cell is increasing to the maxi- 
mum characteristic of the succeeding (logarithmic) phase. Some authors do not dif- 
ferentiate between this phase and the preceding, terming the two together the "lag 
phase." 

It is evident that during this period the number of bacteria present is a function 
of the time; and several attempts, both empirical and theoretical, have been made to 
formulate the mathematical relationships. In the analysis of certain data Ledingham 
and Penfold' found that a graph of the logarithms of the logarithms of the numbers 
of bacteria and the logarithms of the time is a straight line. This leads to the formu- 
lation: 

h=Beki' (i) 

in which 

J = Number of bacteria after time i 

5 = Initial number of bacteria 

k and 5 = Constants which require evaluation for each 

particular set of experiments 
e = Base of natural logarithms 

These authors found the value of s to vary from 1.56 to 2.7. 

LOGARITHMIC PHASE 

During this phase the generation time is a constant, as is also the rate of growth 
per cell. This phase is the one most susceptible to simple mathematical analysis, and 
is of major importance in the study of the effect of environment upon bacteria. 

Methods for estimating the generation time and number of generations during 
this phase were apparently first developed by Buchner, Longard, and Riedlin.^ If it 
be assumed that the cells are multiplying regularly by binary fission, and 

jB = Initial number of bacteria 

6= Number of bacteria after time t 

«= Number of generations in time / 

g=Length of one generation, i.e., time required for 
the bacteria to double in numbers 

then, 

b=B2» (2) 

and, since « = - , ^ • 

^ b=Bis (3) 

^^log^-log5 (^) 



log 2 

/ log 2 



log ^— log B 



(5) 



' Ledingham, J. C. G., and Penfold, W. J.: /. Hyg., 14, 242. 1914. 

* Buchner, H., Longard, K., and Riedlin, G.: Centralbl.f. BaklerioL, 2, i. 1887. 



5° 



GROWTH CURVES OF BACTERIA 



If the numbers of cells present at any two times during the period of logarithmic 
growth are determined, the values of n and g may be readily derived. Nomograms for 
such determination have been developed by the writer/ If the numbers of bacteria 
are plotted against time a curve will be developed whose equation is (3) (see Fig. 5). 

Equation (5) may also be written 
in the form, 




log&=— ^+Iog5 



(6) 



This indicates that if the logarithms 
of the numbers of bacteria are plotted 
against time a straight line will be 



developed, with slope 



log 



and with 



Time 

Fig. 5. — A. Growth curve during logarithmic 
growth phase. B. Plot of logarithms of numbers of 
bacteria against time. 



the intercept on the y-axis at log B 
(Fig. 5). This is a convenient criterion 
for determining whether or not a cul- 
ture is in the logarithmic phase. 

For some purposes it is advisable 
to determine the rate of growth per 
cell. If the cells are increasing regu- 
larly in geometrical progression, the 
rate of increase in the number of cells 

-,- ) is constantly proportional to the 
at J 

number of cells, i.e., 
db 



dt 



= kb 



(7) 



in which k is the proportionality constant termed the "velocity coefficient" of the rate 
of growth. Since 

db 

k=dt 

b 

the velocity coefficient is the rate of growth per cell. Integration of (7) gives the rela- 
tionship 

In Z) = ^/+ Constant of integration 



When i = o, the constant of integration is equal to l)i B, and 

In b = kl+ln B 

1, h ^ 



(8) 
(9) 



' Buchanan, R. E.: Iowa Stale College J . Sc, i, 63. 1926. 



R. E. BUCHANAN 51 

Equation (8) is in the form of an equation of a straight line. It follows, therefore, that 
if the logarithms (to base e) of the numbers of bacteria are plotted against time, a 
straight line will be developed with slope k (rate of growth per cell) and intercept on 
the y-a,xis at In B. It follows that 

b = Bck^. (10) 

This is another form of equation (3), the equation of the logarithmic growth curve. 
It is sometimes convenient to evaluate k (rate of growth per cell) in terms of g 
(generation time) . Since the generation time is inversely proportional to the rate of 
growth per cell, • 

g=J- (II) 

The value of the proportionality constant C may be determined from equations (5) 
and (9). 

g=^ (12) 

and 

C=ln 2 = 2.307 log,o2 = 0.692 

PHASE OF NEGATIVE GROWTH ACCELERATION 

This phase succeeds the logarithmic phase when conditions become progressively 
more unfavorable to growth, due either to decrease in concentration of nutrients or 
to the accumulation of toxic products. Mathematical analysis of this phase has been 
attempted, but the adequacy is questionable. 

MAXIMUM STATIONARY PHASE 

This is reached when the cells cease to increase in numbers. A count at this time 
gives the maximum crop yield. The rate of growth is o. 

SPECIAL MODIFICATIONS OF GROWTH CURVES 

In some cases growth curves are found to be more complex than the type indicated 
above. They may, for example, exhibit more than one logarithmic phase. One may 
find cultures of organisms which both produce CO. and are stimulated by it. A small 
seeding of such an organism (in the logarithmic growth phase) would for a time show 
a constant rate of growth per cell, later the CO2 would increase to a point where its 
stimulating action would be manifest, and the value of k would increase with increase 
in concentration of CO.. Eventually saturation with CO2 would occur and the rate 
of growth per cell would again become constant. Similarly, more than one of certain 
other growth phases may be manifested. 

GROWTH CURVES INTERPRETED AS AUTOCATAKINETIC PHENOMENA 

McKendrick and Pai (loc. cit) and later Robertson {he. cit.) and Lotka {loc. cit.) 
have suggested that the entire growth curve shows marked resemblance to a curve 
of autocatalysis. The relationship may be derived as follows: 

It has previously been shown that under constant environmental conditions the 
rate of increase of bacteria is constantly proportional to the number of bacteria pres- 



52 GROWTH CURVES OF BACTERIA 

ent. It may be assumed that the rate of increase is also proportional to the concen- 
tration of the available nutrients, or to that of some single nutrient which acts as a 
limiting factor. If concentrations of cells and of nutrients are the only two factors 
governing the increase, the rate will be jointly proportional to the two. A convenient 
method of estimating the amount of available nutrient is to determine the total max- 
imum number of bacteria which may be produced in the culture. The difference be- 
tween the number of bacteria present at any instant and the maximum number of 
bacteria which may be developed is proportional to the available remaining nutrients. 
If /3 = maximum bacterial count, then 



Upon integration, 
When t = o,b = B, and 



j^=Kb{^-h) (13) 

ln-^^=K t+C (14) 

Equation (14) shows a straight-line relationship between In ~ — r and time, the 

straight line having a slope, K. This relationship may be used to determine whether 
in any case the growth curve resembles that of autocatalysis, or whether (according 
to Ostwald) the growth curve is autocatakinetic. 

The equation of an autocatakinetic growth curve may be derived from equation 

(14): 

A convenient evaluation of C may be made by taking /i as the time which has elapsed 

to the instant when b = -, i.e., until the number of bacteria has reached one-half the 
2 

maximum. 

C=-Ki^ 



and 



^w«ir5=^' (^-^') 



i^r^+FKins- <"' 



A curve of this type is illustrated in Figure 6. It will be found to be symmetrical, and 
asymptotic to the lines 6 = and 6 = /(3. The point of inflection occurs at - after 
time ti. The y-axis is cut at b = B. 



R. E. BUCHANAN 



53 




Data of bacterial growth as studied by McKendrick and I'ai and by Lotka have 
been found to conform measurably well to such autocatakinetic curves. Other cases 
may be cited, however, in which the agreement is not good. This is particularly true 
when there is manifested a prolonged initial stationary phase or lag phase. Then, too, 
the rate of growth per cell may not be directly proportional to the concentration of 
the available nutrients. Other factors may also alter the form of the growth curve, 
and in consequence it may be unsymmetrical. The equations and relations of such 
curves are more complex, and are not subject to ready analysis. Even if the data se- 
cured are found to iit symmetrical curves, comparisons between curves secured under 
varying environmental conditions are /? 
not as readily made as between cor- 
responding (particularly the logarith- 
mic) growth phases. 

EFFECT OF AGE OF PARENT-CULTURE 
UPON FORM OF GROWTH CURVES 

The work of Lane-Claypon {loc. 
cit.) and others has shown quite clearly 
that the phase of growth of the culture 
from which a transfer is made influ- 
ences markedly the form of the growth 
curve in the daughter-culture. In most 
cases the following results will be 
secured: (i) Transfers from the initial 
stationary phase will show a continua- 
tion of this phase, followed by lag phase, etc., in normal sequence. (2) Transfers 
from the lag phase will usually show a continuation of this phase, followed by the 
logarithmic phase, etc., in normal sequence. (3) Transfers from the logarithmic phase 
usually show a continuation of the logarithmic phase. In some cases allelocatalysis 
(see below) may cause the culture to show an initial lag phase. (4) Transfers from the 
phase of negative growth acceleration will usually show a lag phase. (5) Transfers 
from the maximum stationary phase may show an initial stationary phase or a lag 
phase. 

EFFECT OF SIZE OF INOCULUM UPON THE FORM OF THE GROWTH CURVE 

It has been shown by Robertson^ that growth in a subculture of certain organisms, 
particularly protozoa, is stimulated by the presence of other cells of the same type. 
Single-cell transplants to hanging drops exhibit a much slower initial rate of growth 
per cell than do seedings of a larger number. This phenomenon of mutual or self- 
stimulation he terms "allelocatalysis." Wliile in general results with bacteria do not 
show this effect under the usual conditions of culture, there is evidence that with some 
forms, as the pneumococcus, single-cell isolations are very difficult. The work of 
Valley and Rettger^ and others seems to indicate that many organisms grow very 

' Robertson, T. B.: Biocliem. J ., 15, 595. 1915. 

^ Valley, George, and Rettger, Leo F.: J . Bad., 11, 78. 1926. 



Time 
Fig. 6. — An autocatakinetic growth curve 



54 GROWTH CURVES OF BACTERIA 

slowly or not at all until a certain minimum concentration of carbon dioxide is pres- 
ent. This would lead to the development of a definite lag phase which would be much 
longer with small than with large seedings. Slator has shown that when very large 
inocula of yeast are used there may be no logarithmic phase. 

EFFECT OF CONCENTRATION OF CONSTITUENTS OF SUBSTRATE 
UPON GROWTH CURVES 

The various constituents of the culture medium may act either as accelerators or 
inhibitors of growth. The effect upon rates of increase is in general a function of the 
concentration. The exact relationship may be most satisfactorily evaluated usually 
by comparison of the rates of increase per cell {k) in different concentrations during 
the logarithmic growth phase. In chemical reactions generally it is found that the 
velocity coefficient of the rate of the reaction varies directly as some constant power 
of the concentration of the reactant. It is frequently advisable, as a first approxima- 
tion, to test the hypothesis that a similar relationship holds between rate of increase 
and concentration of a nutrient or inhibiting agent. This would give the relationship 



Integration yields 
in which 



f^=KC^b (i8) 

In b^KCH+ln B (19) 

/ir = Constant 

C= Concentration of chemical 

«= Constant 

It is apparent that the velocity coefficient (rate of increase per cell, k) is evaluated as 

k=KC^ (20) 

A determination of the validity of this relationship with varying concentrations may 
be made by plotting the logarithms of the velocity coefficients {k) against the log- 
arithms of the concentrations. Since 

log ^ = w log C+log i? (21) 

a straight line should be developed with slope n. If increase in concentration increases 
the growth rate, n will be positive; if it inhibits, n will be negative. 

EFFECT OF TEMPERATURE UPON THE FORM OF GROWTH CURVES 

The effect of temperature changes upon rates of increase is usually best evaluated 
by comparisons of the velocity coefficients {k) during logarithmic growth. It is cus- 
tomary to designate the ratio between the velocity constants at the higher and at the 
lower temperature as the temperature quotient (Q). The temperature interval for 
which determinations are usually made is 10° C. For this interxal the quotient is 
commonly designated as ()i„. 

It is frequently desirable to determine whether temperature effects upon growth 



R. E. BUCHANAN 55 

rates resemble the effects upon rates of chemical reactions. It is commonly found that 
chemical reactions are accelerated by rise in temperature, and in ranges of o°-ioo° C. 
frequently doubled or trebled in rate by each increase of io°. This tendency to double 
or treble the rate has come to be termed the "R.G.T." {Reaktionsgeschwindigkeit 
Temperatur) rule. Studies upon rates of bacterial growth in certain ranges have 
shown values of Qw frequently equal to two or three. 

It has been shown that in chemical reactions the value of Qw tends to decrease 
with rise in temperature. This relationship has been developed by van't Hoff and 
Arrhenius into the generalization : The rate of change in the logarithm of the velocity 
coefficient of a chemical reaction with temperature is inversely proportional to the 
square of the absolute temperature, i.e., 

dink _ A . , 

It was also shown that the constant^ may be substituted by-^ , in which ju is a con- 

K 

stant characteristic of the reaction (thermal increment) and R the gas constant 

(numerically equal to 2). Integration of equation (22) yields 

lnk = -j,+C (23) 

Conformity of a reaction to the relationship of equation (23) may be determined by 
plotting the values of In k against the reciprocal of the absolute temperature; agree- 
ment is manifest by the development of a straight line, with slope - . 

It is of interest to determine whether growth rates (and growth curves) of micro- 
organisms are similarly related to temperature. The work of Crozier ef al.^ indicates 
that results of value may be secured by studies of the values of thermal increments 
(m). It is contended that growth rates are controlled by rates of chemical reactions, 
and changes in the latter due to temperature changes should produce corresponding 
changes in the former. Since the growth is probably the resultant, in many cases at 
least, of a catenary series of reactions, the rate of growth would be controlled by the 
slowest rate. Changes in temperature may therefore modify the rate of growth in the 
same manner as they modify the rate of the slowest reaction. 

From equation (23) the following relations are evident: 

k. H-ffi) 

Q,o=eT'T. (25) 

in which ^2 and kj represent the velocity coefficients at the higher and lower tempera- 
tures (absolute) T2 and Ti, respectively. 

' Crozier, W. J., el al.: J. General Physiol., 7, i8g. 1924. 



56 



GROWTH CURVES OF BACTERIA 




.OOBee .0036I .OO3S<0 .O03S/ .00346 .003-1-I .00336 .0033I 



0032S 



Reclproca/ of Absolute lemperafure. 



Fig. 7 



i 
















































; 
















































; 
























; 










)^-"''^ 














-, 








-^^ 
























r-^ 
















i 




























< 




















; 
























; 




/ 




















\ 
























■ 
























( 
















































1 
























1 
























J 
























\ 1 

























Reciprocal of /Absolute Temperature 

Tig. 8 



R. E. BUCHANAN 57 

The size and constancy of the thermal increment ^ may be determined either by 
substitution of values in equation (24) or by plotting the logarithm (base e) of k against 
the reciprocal of the absolute temperature. Such a graph is given in Figure 7 for 
the effect of temperature upon the growth of a bacterial culture between 0° and 30° C. 
It will be noted that the points apparently determine in this case two intersecting 
straight lines. Crozier and others interpret a finding of this type as indicating a 
change at a certain temperature from one basic reaction in the catenary series to an- 
other as governing the growth rates. Graphs of this type are made most conveniently 
by using a semilog paper in which the abscissae are indicated as temperatures centi- 
grade but are spaced in proportion to the value of the corresponding reciprocal of the 
absolute temperature. The data of Figure 7 are plotted on this type of co-ordinate 
paper in Figure 8. 

It is evident that if values of the thermal increment are known, and the relation- 
ships outlined above hold, it is possible to predict the form which growth curves will 
assume at different temperatures. 



CHAPTER VI 
THE RISE AND FALL OF BACTERIAL POPULATIONS 

C.-E. A. WINSLOW 
Yale School of Medicine 

I. THE LIFE-CURVE OF A BACTERIAL POPULATION 

In a study of the distribution of bacteria in their various natural habitats the bac- 
teriologist is inevitably impressed with a sense of a relatively stable adjustment be- 
tween a specific environment and the numbers and kinds of bacteria which will gener- 
ally be found therein. The botanist knows that on a certain kind of soil in a certain 
climate such-and-such trees and shrubs will be present, about so many to the acre. So, 
in our microscopical realm, we find that uncultivated sandy soils may yield 100,000 
bacteria per gram while garden soils show 1,500,000. A given river in a dry summer 
will contain from 1,000-2,000 bacteria per cubic centimeter while a lake will contain 
only 50-150, and the deep waters of the ocean or those of a driven well will show only 
5-10. From day to day, and from year to year, both the numbers and kinds of mi- 
crobes in a given habitat will remain extraordinarily stable — provided that the en- 
vironmental conditions themselves remain approximately constant. 

If, on the other hand, the conditions of the habitat change, or if a section of the 
bacterial population be transferred to a new environment, the balance is upset. A 
new and active struggle for existence is initiated, such as has occurred among the 
higher forms of life when a glacial epoch has changed the climate of a continent. With 
our short-lived forms of life, capable of completing a whole cycle of evolution in 
twenty-four hours, we can trace the course of such a struggle in a fashion which 
should be the envy of the ecologist; and as we do so, we find a remarkable degree of 
constancy underlying even the phenomena of change which characterize such a period 
of adaptation. 

The curve which marks the rise and fall of a bacterial population in a new environ- 
ment is illustrated graphically and schematically in Figure i ; and it may be claimed 
that this curve is a widely representative one for all conditions, with the limitation 
that according as the environment is more or less favorable the subdivisions of the 
curve may be relatively increased or decreased or even suppressed entirely. 

2. THE PHASE OF ADJUSTMENT 

The first phase in the cycle of a bacterial population (if the environment be not 
too severe) is what may be called the "phase of adjustment." In a medium which is 
highly favorable this phase will be indicated, as in the solid line AB of Figure i, by 
a relatively slow increase, the period of lag or dormancy, as described by Rahn (1906), 
Barber (1908), Lane-Claypon (1909), Coplans (1909), Penfold (1914), Chesney (1916), 
and Sherman and Albus (1924). 

The first careful studies of the rate of bacterial multiplication and the first de- 

S8 



C.-E. A. WINSLOW 59 

scription of the lag phase in a favorable medium were due to Miiller (1895) who intro- 
duced the now-familiar formula for generation time: 



log 6— logo 



where G= minutes per generation, r = elapsed time in minutes, a = initial number of 
bacteria, and 6 = final number of bacteria. 

He showed that G increased with the age of the primary culture used for inocu- 
lating the medium in which generation time was measured. Thus typhoid bacilli inoc- 
ulated from a 2.5-3-hour mother-culture gave a generation time of 40 minutes while 




Fig. I. — Ideal curve of a bacterial population cycle. Ordinates = numbers; abscissae = elapsed 
time. 

A-B. Phase of adjustment D-E. Phase of decrease 

B-C. Phase of increase E-F. Phase of readjustment 

C-D. Phase of crisis 

those from a 6j-hour culture completed a generation in 80-85 minutes, and those from 
a 14-16-hour culture in over 160 minutes. He attributed the slower generation in 
media inoculated from older cultures to what is now called a "lag effect." Hehewerth 
(1901) confirmed these results. It was Rahn (1906), however, who first studied the 
preliminary lag period intensively, using B. fliwrescens in broth. 

He inoculated from (I) 20-hour broth, from (II) 20-hour agar, and from (III) 4- 
month broth, and obtained such results as are shown in Table I. 

If the medium be less favorable, the phase of adjustment will be marked by a de- 
crease followed by an.increase. This latter phenomenon was noted at least as early as 
1894 (Fuller, 1895) in studies made at the Lawrence Experiment Station of the Massa- 
chusetts State Board of Health which showed that a bottled sample of sewage orig- 
inally containing 1,190,000 bacteria per cubic centimeter fell off to 1,085,000 after 2.5 
hours, then rose steadily to a maximum of 23,100,000 after 25.5 hours, and then fell 



6o 



THE RISE AND FALL OF BACTERIAL POPULATIONS 



steadily to 2,341,000 after 8 days. Whipple (1901) noted the same effect — an initial 
fall, followed by a rise, as a universal phenomenon in portions of natural waters stored 
in sample bottles under various conditions. This type of reaction, indicated by the 
dotted line AB in Figure i, does not appear to be substantially different from the lag 
phase in a richer culture medium and, indeed, it seems probable that whether the ob- 
served net effect be an increase or a decrease there is going on during this period a 
multiplication of some cells and a death of others, the relative rate of these two proc- 
esses determining the end-result. The fact that the bacterial count of milk shows an 
initial decrease was pointed out by Fokker (1890), and a long controversy has been 
carried on in regard to its cause (excellently summarized by Heinemann, 1919). Many 
investigators claim that freshly drawn cow's milk possesses special germicidal proper- 
ties due to the presence of agglutinins and other bactericidal substances, the presence 
of living phagocytes, or the restraining action of lecithin. Heating the milk destroys 
this "bactericidal power," but it must always be remembered that heating also changes 
the nutritive qualities of a medium and may therefore make it more favorable. In 
general, the phenomenon seems to be a special case of the lag period in a medium 





TABLE I 
Generation Time in 


Minutes 




Hours 


I 


n 


III 


0-^ 








^-6 


1,083 

113 
60 


1,309 
77 
63 


997 
212 


6—12 


12-24 


62 







more or less unfavorable to certain types of bacteria which have gained access to the 
milk. 

Ledingham and Tenfold (1914) and Slator (1917) have attempted to formulate 
mathematical expressions for the multiplication rate even in the highly variable lag 
period of the population cycle; but if the conclusion be justified that the phase of ad- 
justment may, according to circumstances, be characterized by a decrease followed by 
an increase or by a gradually accelerating increase, the futility of any attempt at 
mathematical analysis will be apparent. 

The inflection and the slope of the curve of the bacterial population will vary with 
three general factors: the type of bacteria involved, the medium into which they are 
introduced, and the temperature. The first two factors may in a sense be reduced to 
one — the suitability of the particular medium for the particular bacteria in question. 
If the medium be entirely inadequate, the bacteria simply die off and the phase of ad- 
justment merges into the phase of decrease. On the other hand, as I shall point out, 
if the medium be ideal and the bacteria in the right condition, the phase of adjustment 
may be reduced to proportions which are not measurable, and the logarithmic in- 
crease will begin at once. There may be an infinite number of gradations between 
these two extremes, giving a longer or a shorter phase of adjustment. Coplans (1909), 
for example, found that in transferring from peptone water to peptone water the lag 
period lasted about i hour while in transfer from one dulcitol medium to another it 



C.-E. A. WINSLOW 6i 

was longer. In fresh unsterilized milk the lag lasted 6 hours, but was apparently abol- 
ished by previous heating of the milk. Cohen and Clark (191 9) note that the onset of 
the period of maximal increase for Bad. coli was 2-4 hours in peptone phosphate broth 
atpH values between 6.1 and 8.1 but was increased to 3-5 hours at pH values below 
5.5, and to 10-12 hours at pH 8.9 (37° C). 

A high temperature, of course, decreases the length of the phase of adjustment. 
According to Whipple's data for water bacteria stored in sample bottles, the period of 
lag averaged about 8 hours at 20^-24° C. and 17 hours at 12° C. Lane-Claypon (1909) 
reports a lag period in culture media varying from i hour at 42° to 6 hours at 20° C. 

An extreme case of lag may probably be found in the fact that both spores and 
vegetative cells occasionally show an exceedingly slow development in entirely favor- 
able culture media. Thus Burke, Sprague, and Barnes inoculated broth and agar 
tubes with approximately one cell of Bad. coli per tube. While 85 per cent of the 473 
tubes which gave growth did so within 2 days and 97 per cent within 6 days, there 
were 10 tubes which developed only on the fourteenth day, 4 only on the fifteenth, 
and 4 only on the sixteenth. Spores of B. subtilis remained dormant under similar 
conditions for 39 days and spores of B. megatherium for 90 days. 

The fundamental causes of the lag phenomenon have been exhaustively discussed, 
particularly by Rahn (1906), Tenfold (1914), Chesney (1916), and Buchanan (1918). 
The phenomenon must be considered in the light of the observation of Miiller (1895) 
and Hehewerth 1901) that the rate of increase of bacteria in a given medium bears a 
generally inverse relation to the age of the mother-culture from which this medium 
was inoculated. Hehewerth found that the generation time for Bad, coli in broth, 
when transferred from a young broth culture, was 21-27 minutes while when 
transferred from an older broth culture it was 43 minutes. It is of cardinal sig- 
nificance to note that lag disappears entirely if transfer is made to an identical medi- 
um while the mother-culture is in its phase of logarithmic increase (Tenfold, Barber, 
Chesney). Furthermore, Tenfold shows that if the growth in a culture be stopped by 
chilling for a very short time the growth recommences at a normal rate when the tem- 
perature is raised; while more prolonged chilling and subsequent increase of tempera- 
ture is followed by a lag. 

The occurrence of lag cannot in general be due to the presence of inhibitory sub- 
stances carried over from the mother-culture (as might be concluded from Tenfold's 
finding that centrifugalized cultures showed decreased lag) since we note the same 
phenomenon in a sample of water transferred from a lake to a sample bottle. Further- 
more, Tenfold and Chesney show that lag in a secondary culture does not increase 
with the progress in the mother-culture of the logarithmic phase, and that while it 
does grow more marked with passage from the logarithmic phase to the phase of 
crisis there is no further increase with later aging of the mother-culture. 

Lag is therefore primarily associated with the biological condition of the cells 
which are transferred to a new medium (or placed under new environmental condi- 
tions, as when a water sample is collected). Under certain conditions this may be as- 
sociated with definite prior injury to the cells. Sturges (19 19) found that the develop- 
ment of colonies on plates seeded from sewage disinfected with copper or sulphurous 
acid was very much retarded (although the fact that chlorine-disinfected sewage ex- 



62 THE RISE AND FALL OF BACTERIAL POPULATIONS 

hibits no such phenomenon suggests that the disinfectants were perhaps carried over 
to the plates in antiseptic concentration). Allen (1923) and others have ascribed sim- 
ilar slow growths of bacteria in milk after pasteurization as due to attenuation by 
"temperature shock." 

Chesney (191 6) believed this to be the main factor in the lag phenomenon in gen- 
eral, and he cites a striking case in which the generation time of pneumococci was 
slowed down in a filtrate from an old broth culture of the same organism. That the 
shock theory cannot be of general application, however, is made clear by the fact that 
similar phenomena occur in bottled-water samples. 

The second possibility which suggests itself is in a sense the converse of the idea 
that lag is due to a state of injury produced by an earlier environment. It involves the 
conception that, among the cells carried over from any environment A to another en- 
vironment B, some find themselves ill adapted to the latter and that a process of natu- 
ral selection must ensue until the less adapted cells are weeded out. Such a condition 
must apparently be assumed when the lag period is characterized by an actual de- 
crease in number such as occurs in samples of water, milk, or sewage, or in soU to which 
an antiseptic has been added. 

When a pure culture of a single species is transferred to a medium identical with 
that in which it is already living there must be something still more fundamental in- 
volved. We may conclude from the work of Penfold and Barber that an initial period 
of slow development is an essential necessity whenever bacteria pass to a new en- 
vironment from one in which they are not multiplying rapidly. Bacteria in active 
multiplication appear to be in a different biological state from bacteria in other phases 
of the population cycle, and it takes time to effect this change of state. 

We can, however, perhaps go a little further and visualize certain more concrete 
conceptions of what this difference in state may mean. The work of Sherman and Al- 
bus (1922 and 1924) indicates that cells during the early lag period are less sensitive 
to slightly toxic salts than are cells in the late lag and logarithmic phase, and they find 
that the sensitiveness to salts appears slightly before multiplication sets in at its most 
rapid rate. This suggests the possibility that permeability phenomena may play a 
part. On the whole, however, among the numerous possibilities Penfold's explanation 
that maximum growth presupposes the existence in the cells of intermediate bodies in 
the synthesis of protoplasm — intermediate bodies which diffuse out and are lost when 
growth is checked — seems on the whole most plausible. On such a hypothesis cells in 
the "resting stage" of an old culture or in a lake water or in any stable environment 
would be cells lacking these "intermediate bodies" while the "rejuvenated" cells of 
the logarithmic growth phase would be rich in them. 

It is also possible that such "intermediate bodies" may be transferred in the form 
of dead as well as of living bacterial cells, or in solution in the surrounding menstruum. 
This would explain the finding of Chesney that cells transferred from a mother-cul- 
ture during the phase of increase and washed free from the medium by centrifugation 
show a lag when placed in a new culture, while the cells left in the mother-culture still 
continue to grow at a logarithmic rate. Such a supposition would also be in accord 
with the conclusion of Rahn (1906) that maximal multiplication coincides with the 
presence of heat-stable, non-filterable substances "formed by the bacteria," and with 



C.-E. A. WINSLOW 63 

the observation of Penfold (1914) that generation time decreases with an increase in 
inoculum. Rettger (1918) suggests that lag in culture media may be decreased or 
eliminated by supplying "satisfactory substitutes for the intermediate bodies in the 
form of amino acids and perhaps amines of simple composition, and also certain 
growth-accessory substances." 

3. THE PHASE OF INCREASE 

When the phase of adjustment (whether this involves selection of more readily 
viable cells or the development within the cells of intermediate products favorable to 
rapid growth, or both) has been completed in a favorable medium, there next follows 
a period of rapid and regular increase. During this phase it has been shown by Clark 
and Ruehl (1919) that the average size of cell is greatly increased as compared with 
that which is dominant in an older culture. According to Henrici (1921, 1922, 1923, 
1924), the large cells appear toward the end of the lag phase and the beginning of the 
phase of logarithmic increase, the average size returning to normal as the phase of in- 
crease proceeds. In a highly unfavorable medium, this phase will of course be entirely 
suppressed; but it is a very common phenomenon, by no means limited to the rich 
culture media of our laboratories. It was perhaps first exhaustively studied by the 
early water bacteriologists in the case of samples of natural waters which had been 
placed in a new environment by the mere fact that they had been collected in a sam- 
ple bottle in the laboratory. Thus Leone as early as 1886 records the following results 
for Munich water simply stored in flasks without the addition of any foreign material : 

Storage Period in Days Numbers per Cc 

o 5 

1 100 

2 10 , 500 

3 67,000 

4 315,000 

5 Over 500 , 000 

Miquel (1891) gives the striking curves reproduced in Figure 2 for a series of spring 
waters stored in flasks at 29°-3o° C. 

In the phase of logarithmic increase we are dealing with a very simple relationship 
due to the fact that binary fission carried on at a regular rate leads to a progressive 
logarithmic increase. In other words (Ledingham and Penfold, 1914) : 

t=K log b/B, 

when / = time, & = final number, and 5= initial number. 

The actual figures obtained for generation times under certain more or less typical 
conditions are indicated in Table II. The studies of multiplication in soil, feces, and 
bottled waters did not include counts made at sufficiently frequent intervals to be 
quite certain that only the phase of logarithmic growth was included, but they are 
cited in the table as representing the most rapid increases (under this condition) with 
which the writer is familiar. 

It appears from all the more careful work upon this subject that under the most 



64 



THE RISE AND FALL OF BACTERIAL POPULATIONS 



NUMBER OF 
BACTERIA IN 
I.C.C. 



1,000,000 



900^000 



600,000 



700,000 



6oo>ooo 



500,000 



400,000 



^00,000 



2 00,000 



100,000 



(\ 














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DMUI5 — — -—--.- 
ST. LAURENT 








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DAYS 



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Fig. 2. — Multiplication of bacteria in bottled samples of certain spring waters (Miquel 
[Frankland, 1894]). 



C.-E. A. WINSLOW 



65 



favorable conditions of medium and temperature the cells of bacteria may divide once 
in 17-20 minutes, while under less favorable conditions the rate of multiplication 
may be slackened to any desired degree. 

TABLE II 

Typical Generation Times during the Phase of Logarithmic Increase 



Observer 

Conn, 1918 

Jordan, 1926 

Frankland, 1894 

Whipple, 1901 

Harrison and Vanderleck, 1909. . 
Buchner, Langard and Riedlin 

1887 , 

Barker, 1908 



Type of Organism 



Soil bacteria 
Bad. coli 
Water bacteria 
Water bacteria 
Bact. coli 

V. cholerae 
Bact. coll 



Medium 



Manured soil 


20 


Feces 


20 


Bottled sample 


20 


Bottled sample 


24 


Milk 


37 


Broth 


37 


Broth 


37 



Temperature, "C 



Generation Time, 
Minutes 



840 
720 
196 
IIS 

40 

19-40 
17 



The most obvious of the environmental conditions which determine the genera- 
tion time — and hence the K in the curve for rate of logarithmic increase — is the tem- 
perature (see Ward, 1895). The results of certain of the most exhaustive studies on 
this point are cited in Table III. 



^ TABLE III 

Generation Time at Various Temperatures in Minutes 



Observer . 



Organism . 
Medium. . 



Whipple 

Water bacteria 
Bottled sample 



Harrison and 

Vanderleck 
Bad. coli 
Milk 



Buchanan 

Bad. coli 
Broth 



Lane-Claypon 

Bact. coli 
Broth 



Temperature, ° C. 



864 



15- 
17- 
20. 

25- 

30- 
31- 
34- 
35- 
37- 
40. 
42. 



720 
'386' 



4S0 



143 
'56 



45 



180 



76 

43 
29 



17 



52 



32 
28 



19 



If we plot the logarithms of these generation times against temperature, we obtain 
a series of straight lines, as was pointed out by Lane-Claypon (1909). The increase in 
rate of growth is approximately doubled for a 10° C. rise in temperature, a relationship 
which Snyder (1908, 1911) has shown is as generally characteristic of biological proc- 
esses as of chemical reactions. 

We may next consider briefly some of the other factors which determine the rate 
of logarithmic increase — a problem by no means easily elucidated. It is easy enough 
to understand why bacteria increase when inoculated into a rich, sterile culture me- 



66 



THE RISE AND FALL OF BACTERIAL POPULATIONS 



dium but that bacteria in more or less stable equilibrium in the water of a spring or 
well should increase many thousand fold (see Fig. 2) merely because a portion of 
the water in which they are living is placed in a sample bottle (even without any in- 
crease in temperature, as Whipple's data show) is far more difficult to explain. And 
the fact reminds us how subtle are the factors which constitute a bacterial environ- 
ment. 

Similarly, it is of interest to note the observation of Jordan (1926) that fresh hu- 
man feces (containing already 75,000,000 bacteria per gram) show an enormous fur- 
ther increase on storage, sometimes reaching several hundred times the original figure 
after a few days. This increase is mainly due to multiplication of Bad. coli and occurs 
at 10° and 20° as well as at 37°C. It is suggested by Jordan that the multiplication 
may be due to the loss of specific inhibitory influences present in the lower intestine. 

Miquel attributed the rapid growth in a new medium to the absence of toxic 
products of prior bacterial growth and found that boiling destroyed the "toxicity" of 

TABLE IV 

Bacterial Content of Normal and of Toluene-treated 
Soil at Various Temperatures 



Temperature, °C 



5- 1 
20. 

30- 
40. 

50- 



Bacteria, Millions per Gram 



Untreated Soil 



5 Days 



27 Days 



58 Days 



Toluened Soil 



5 Days 



8 
50 
43 
12 

2 



27 Days 



27 

30 

24 

4 



58 Days 



28 

30 

31 
6 



water rendered unsuitable for growth by such earlier development. When we transfer 
from an old culture to a new sterile tube of the same medium in the laboratory, this 
factor must play a major part, since, as I shall point out in discussing the phase of 
crisis, growth is often undoubtedly checked in old cultures by the accumulation of 
acids or other toxic products. Cohen and Clark (191 9) found for Bad. coli in broth a 
generation time of 27 minutes at pH 5.0 while at pH 8.9 the time increased to 46 min- 
utes and in the acid range at pH 4.6 there was a decrease instead of an increase. For 
the multiplication following collection of a water sample such an explanation seems, 
however, clearly inadmissible, and the favorable effect of boiling is probably due 
chiefly to the alteration which heat produces in certain of the foodstuffs which are 
present. 

The work of Russell and Hutchinson (1913), Hutchinson and MacLennan (1914), 
and Buddin (1914) from the Rothamstead Experiment Station and the similar studies 
of Truffant and Bezssanoff (1922) in France have given us some important data in re- 
gard to the multiplication of bacteria in soils. They found that the treatment of a soil 
by moderate heat or by mild antiseptics caused a marked secondary increase in bac- 
terial numbers and crop fertility. Thus Table IV (from Russell and Hutchinson, 



C.-E. A. WINSLOW 



67 



1913) shows that in an untreated soil bacterial numbers remained constant, unaffected 
by temperature variations between 5° and 40° C. ; while in a soil to which a slight 
amount of toluene had been added marked increases occurred, at 20° and 30° and, 
more slowly, at 5°-i2° C. 

Table V (from Hutchinson and MacLennan, 1914) is of special interest as demon- 
strating the influence of lime upon a highly acid soil. It will be noted that o.i per cent 
CaO produced a very slight stimulating action. The next three concentrations (0.2, 
0.3, and 0.4 per cent) caused progressively increasing stimulation with a maximum 
count on the ninetieth day. A strength of 0.5 per cent was somewhat less effective but 
its influence was more prolonged, while a concentration of i.o per cent proved toxic at 
first, with a later stimulation giving a higher count after the two-hundredth day than 
was shown by any other sample. 

In this particular instance the effect of the lime upon the reaction of the soil no 
doubt played an important part in stimulating bacterial multiplication. In general, 



TABLE V 
Bacterial Content of Acid Soil Treated with Varying Amounts of Lime 



Days 

Control 

-|-o.i per cent CaO . . 

.2 per cent CaO . . 

.3 per cent CaO . . 

.4 per cent CaO . . 

0.5 per cent CaO . . 

-fi.o per cent CaO . . 







Millions 


of Bacteria 


ler Gram of Dry Soil 




10 


50 


90 


120 


150 


200 


232 


5 


5 


4 


4 


4 


4 


5 


6 


II 


II 


9 


10 


4 


5 


23 


78 


82 


68 


73 


29 


19 


97 


170 


170 


132 


133 


106 


77 


151 


387 


906 


345 


178 


154 


87 


270 


281 


470 


260 


247 


180 


200 


2 


0.2 


6 


7 


II 


176 


329 



310 

5 
3 
13 
63 
71 
77 
337 



however, Russell and Hutchinson attributed the effect of heat and antiseptics upon 
soils to the destruction of predatory protozoa. It seems quite possible that the in- 
fluence of the treatment upon available foodstuffs (whether derived from the bodies 
of protozoa or from other sources) may have been an even more important factor, as 
in the case of Miquel's experiments cited above. 

In many other instances, it is certainly to the availability of the food supply, 
rather than to the absence of inhibiting substances, that we must attribute the initi- 
ation of the logarithmic growth phase. The amount of food required by the bacteria 
may of course be exceedingly minute, particularly in the case of prototrophic water 
forms. Kohn (1906) determined the minimal nutrient requirements for certain of 
these types and found that they could develop in the presence of 198X 10 — '" to 198X 
10 — ^^ per cent of glucose, 66X10—*^ to 66X10 — ^9 per cent ammonium phosphate. 
With more fastidious organisms, however, a much ampler and more diversified diet is 
necessary for maximal growth. Thus Tenfold and Norris (191 2) found that the maxi- 
mum generation time of Bait, typhosum in i per cent peptone at 37° C. was 40 minutes, 
but that when the peptone in the medium was reduced below 0.4 per cent the genera- 
tion time increased and rose quite regularly with decrease in peptone content down to 
0.2 per cent. In a o.i per cent peptone medium the generation time could be cut in 



68 THE RISE AND FALL OF BACTERIAL POPULATIONS 

half by the addition of 0.17 per cent glucose. The literature is full of comparative 
studies of media for water and milk analysis or for the isolation of specific organisms 
which bear upon this point. As an example of such studies we need only cite the recent 
work on media for milk analysis which has indicated the widely different results ob- 
tained with various brands of peptone (Shrader, 1926). Davis and Ferry (1919) note 
that both the growth and the toxin production of the diphtheria bacillus in a beef- 
infusion medium are dependent on the particular types of amino acids present and 
suggest that other accessory factors, perhaps of the nature of vitamines, are also es- 
sential. The presence of growth hormones was held to be necessary for meningococci 
and gonococci by Lloyd (191 6) and Cole and Lloyd (19 17); and Wildiers (1901) 
claimed that a hypothetical substance called "bios" was necessary for the fullest 
growth of yeast in a synthetic medium. 

Devereux and Tanner (1924) and Werkman (1927) have recently reviewed va- 
rious aspects of this subject and conclude that the evidence as to the influence of true 
vitamins or growth-promoting substances (other than those of a nutrient character) 
is very doubtful.' 

In addition to those nutrient materials which are directly essential for the up- 
building of bacterial protoplasm the rate of multiplication of bacteria is also governed, 
like many other biological processes, by the regulative action of mineral salts. This 
subject has been admirably reviewed by Falk (1923), and we need only point out here 
that Hotchkiss (1923) and others have shown that a wide variety of salts stimulate 
bacterial growth in low concentration and inhibit it in a higher concentration. Even 
such toxic salts as HgCla may stimulate growth when present in a dilution of one- 
millionth of a molar concentration (and inhibit it entirely in a concentration of one- 
hundred-thousandth molar) while NaCl and KCl stimulate in .25-M concentration 
and inhibit in 2-M concentration (under the conditions of the Hotchkiss study). It 
seems possible that the multiplication of water bacteria in a sample bottle may in part 
be due to the stimulant action of minute traces of salts dissolved from the glass during 
sterilization, since Kohn (1906) has shown that the increase is most marked in bottles 
of the more soluble types of glass. 

Finally, the dissolved gases in a medium affect bacterial multiplication in far- 
reaching ways which we are as yet far from comprehending. Wolffhiigel and Riedel 
(1886) found that multiplication of bacteria in a flask stoppered with cotton was 
greater than in one closed with a rubber stopper, and Whipple (1901) observed that it 
was greater when a bottle was only partly filled than when it was filled more nearly to 
the top. Curiously, however, agitation and artificial aeration seemed unfavorable to 
growth in WTiipple's experiments. One of the most important contributions to bacte- 
riology in recent years has been the demonstration by Valley and Rettger (1927) that 
a small percentage of carbon dioxide is essential to bacterial growth and that when 
this gas is entirely removed growth ceases completely. 

4. THE PHASE OF CRISIS 

After a lapse of time, varying with the nature of the organism, the medium, and 
the temperature, the period of logarithmic increase draws to a close, and after an in- 
termediate phase of crisis a phase of decrease supervenes. This is of course a phenom- 

' Cf. chapter xxxvii in this vohime. 



C.-E. A. WINSLOW 69 

enon familiar to us in our culture media and in such substrata as soil or milk. It was 
noted in water by Cramer as early as 1885 in the following very clear example. Lake 
Zurich water stored for a period of seventy days: 

Storage Period, Days Numbers per Cc 

o 143 

1 12,457 

3 328,543 

8 233,452 

17 17.436 

70 2 , 500 

A much less marked but fundamentally similar cycle is given by Conn (1918) for 
the change of bacterial numbers in freshly manured soil. 

Storage Period, Days Numbers per Gram 

o 35, 000 , 000 

I 120, 000 , 000 

2 100 , 000 , 000 

3 145 ,000,000 

4 150, 000 , 000 

6 120, 000 , 000 

9 70 , 000 , 000 

16 70 , 000 , 000 

60 75 , 000 , 000 

120 23,000,000 

Jordan (1926), in one of his experiments on stored feces, noted that, taking the 
initial number of bacteria present as 100, the relative number rose during storage to 
between 2,000 and 3,000 on the fifth to the ninth day and then fell to about 1,000 on 
the eleventh day and to less than 400 on the twenty-first day. For colon-group or- 
ganisms under similar conditions he gave the following results: 

COLON BACILLI IN FECES STORED AT ROOM TEMPERATURES 
Relative Numbers Considering Initial Number as 100 

Storage Period, Days Relative Numbers 

3 7 , 000 

5 3.730 

7 3 , 600 

9 2 , 400 

II 930 

14 530 

16 24 

18 26 

21 24 

23 14 

The curves in Figure 3 plotted from averaged results given by Prescott and Baker 
(1904) for the numbers of colon bacilli and streptococci developing in glucose broth 
inoculated with polluted water and incubated at 37° C. illustrate the difference in the 



70 



THE RISE AND FALL OF BACTERIAL POPULATIONS 



rate of growth of different species in the same medium, the colon bacilH rising more 
rapidly at first and the streptococci becoming dominant in the later stages of the cycle. 
Reed and Reynolds (1916) give interesting data as to the period of maximum growth 
for various types of bacteria inoculated into milk in pure culture, the periods at 37° C 
varying from i day ior Bad. lactis-acidi and Sarcina lutea to 21 days for M. citrictis 
and 42 days for Oidium lad is. 

In media which are unfavorable the phase of crisis may set in very early. Chick 
(191 2) found that the usual relations as to lag period, logarithmic multiplication, and 



2,ooo 










t 










B. 




I 








l»800 
l,600 
1,400 
l,200 
1,000 

8 00 
600 






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400 
200 








i 


/ 














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DAYS-*-© 7 II 16-17 23 Z7 39-40 52 63 

Fig. 3. — Multiplication of Bad. coli and streptococci in glucose broth culture into which they 
were simultaneously inoculated. (Average of results reported by Prescott and Baker, 1904.) 

influence of temperature held for the growth of Bad. coli in normal rabbit serum — the 
only difference being that the period of increase is very brief and leads only to a 
doubling of bacterial numbers, after which a rapid decline sets in, due to the bacteri- 
cidal effect of the serum. In rich culture media the initial growth will be rapid, but 
here too the phase of crisis will usually set in within 24 hours as a result of the forma- 
tion of inhibitive waste i)roducts. In milk containing mixed cultures of many species 
of bacteria, the bacteria present may continue to increase for days and may reach 
enormous figures (several Inllions per cubic centimeter, for example, in samples held 
at 15.5° C. by Ayers, Cook, andClemmer, 1918). In most studies on milk bacteriology, 
indeed, the existence of a period of crisis and subsequent decline has not been appar- 
ent since observations have usually not been continued for a long enough period to 



C.-E. A. WINSLOW 71 

pass the critical point. In the important contribution of Ayers and Johnson (1910), 
however, many individual samples show the beginning of a decline after 4 or 5 days 
even when held at 10° C. 

So far as the temperature effect is concerned we may note that Lane-Claypon 
(1909) found the end of the period of logarithmic increase for Bact. coli in broth to oc- 
cur after i\ hours at 37° C, after 8-8^ hours at 30° C, 12-15 hours at 25° C, and 
20-24 hours at 20° C. In the work of Reed and Reynolds (1916) on the growth of 
pure cultures of thirteen different species of micro-organisms in milk the average 
period of maximum growth was 7 days at 35° C. and 20 days at 13° C. 

The explanation of the onset of the phase of crisis must obviously be sought in the 
changed composition of the medium due to the growth of the bacterial population 
during the phase of increase. It is clear that there is no essential and invariable life- 
cycle involved since the results of Penfold and of Barber indicate that transfer of cells 
from a culture in the early phase of increase to a new tube of the same medium yields 
a constant and continuous development at a logarithmic rate. The change in the 
medium which makes it unsuitable for further growth might theoretically be either an 
exhaustion of essential foodstuffs or the formation of toxic waste products, and both 
processes may no doubt play a part under certain conditions. In spring-water samples 
containing but little organic matter it seems very probable that the exhaustion of food 
may play a predominant role (though even Miquel stressed the conception of toxic 
products). In rich culture media the formation of waste products is probably the fac- 
tor of major importance. In milk and other sugar media the production of acid is 
often by itself entirely adequate to explain cessation of growth. Thus Heinemann 
(1915) found that a series of pathogenic bacteria were all destroyed when the acidity 
of milk reached 0.45 per cent and that even Bact. coli died out when the milk reached 
an acidity of 0.6 per cent, while Bact. coli itself increased the acidity of milk to 0.5 per 
cent. Cohen and Clark (1919) found the limiting pH values for growth in peptone 
broth to vary from 4.4 for Bact. aerogenes to 5.5 for Bact. alcaligenes; but they pointed 
out the extreme complexity of the phenomena involved, as shown by the fact that 
while fermentative activity seems to cease in a sugar medium at about the pH con- 
centration known to limit growth, in previously adjusted media growth in the sugar 
medium seems to stop at a point corresponding to concentration of the acetic acid 
formed rather than the pH. Furthermore, in a broth medium free from sugar growth 
ceases much sooner than in the sugar medium and with a total concentration of cells 
only one-fifth as great as that which is found in the presence of sugar. 

In dealing with the phase of crisis, as in the case of the phase of adjustment, it 
does not seem particularly profitable to refine our methods of analysis too far until 
more detailed experimental data are available. If we assume that the cycle of a bac- 
terial population starts from and returns to a stable state there are really but two 
fundamental processes involved, an increase from the original stable condition and a 
subsequent decrease to a second level, with phases of transition before the increase, 
between the increase and the decrease, and before the period of ultimate stability. 
Clearly, as a result of various factors, such as food supply, toxic products, and temper- 
ature, the slope of the curve during the phase of crisis may take any form from a sharp 
tooth to a flat plateau. 



72 THE RISE AND FALL OF BACTERIAL POPULATIONS 

5. THE PHASE OF DECREASE 

From the earliest days of bacteriology it has been noted that the decrease in bac- 
terial numbers under the influence of an unfavorable environment (such as is present 
for one reason or another on the descending side of the curve of the population cycle) 
followed a gradual and more or less orderly course. This was at first attributed to a 
process of natural selection, the surviving organisms being assumed to be of a specifi- 
cally more resistant character. With the work of Koch (1881), Paul and Kronig (1896), 
Kronig and Paul (1897), Ikeda (1897), Madsen and Nyman (1907), and Chick (1908, 
1 9 10) on the action of chemical disinfectants the mortality curve was given a new in- 
terpretation as an expression of a more fundamental chemical phenomenon. As sum- 
marized by Phelps (191 1), these researches have shown that "the rate of dying, 
whether under the influence of heat, cold or chemical poison, is unfailingly found to 
follow the logarithmic curve of the velocity law, if the temperature be constant." 

The general slope of the mortality curve during the period of rapid decline is 
therefore the same as that of the curve for the increase of a bacterial population dur- 
ing its period of rapid multiplication. In the phase of increase the logarithm of the 
number of new cells formed from a single initial cell in a given time is proportional to 
the lapse of time ; in the phase of decrease the logarithm of the proportion of the cells 
present which perish in a given interval is proportional to the length of that interval. 
In other words, increase and decrease alike bear a direct relation to the number of 
cells present at the beginning of a unit period and a logarithmic relation to any time 
period of greater duration. 

The formula for the rate of decrease is, therefore, in the form used by Chick (1908), 
as follows: 



t2-h "= n 

where /i is the initial and (2 the final time and n^ and «. the corresponding numbers of 
bacteria present. Phelps, taking the elapsed time (as /) instead of the initial and final 
time readings (/j — ^i) and B as the initial and b the final number of bacteria, expresses 
the formula as: 

log J = Kt 

which is of course the formula for a monomolecular reaction.^ 

The results presented by Chick (1908) in regard to the regularity of the process of 
disinfection were very striking and seemed to justify her conclusion that "a very com- 
plete analogy exists between a chemical reaction and the process of disinfection, one 
reagent being represented by the disinfectant, and the second by the protoplasm of 

' This formula is written by Falk and Winslow, 

o.434/v.= i// log —-^ 

where a = 7i, of Chick and a—x = niOi Chick because A'i= i/l \ogc ^^ and K=\/t log,o — - when 



C.-E. A. WINSLOW 73 

the bacterium." Later Miss Chick (1910) showed that the death of bacteria when 
dried or exposed to sunhght, or even when killed by moderate heat in water, proceeded 
in general in accord with the logarithmic law. Cohen (1922) confirmed these general 
conclusions for the much more gradual death-curve of colon bacilli in water. In all of 
these latter cases, of course, the reacting agents which cause death must be within 
the bacterial cells themselves. 

This simple concept of the disinfection process has been vigorously challenged by 
a considerable group of workers such as Loeb and Northrop (1917), Brooks (1918), 
Peters (1920), and Smith (1921) who attribute the form of the mortality curve to bio- 
logical differences in the resistance of the individual bacterial cells. 

Miss Chick herself invoked this conception of individual-cell variation to explain 
irregularities in her mortality curves for non-spore-forming organisms such as para- 
t>phoid bacilli and staphylococci. When very young cultures of these organisms were 
used she obtained constant values of K, but with older cultures the rate of disinfection 
fell off in the later stages of the process. Even Cohen's curves in many instances ex- 
hibit a tendency to flatten out toward the end of the periods studied, and Falk and 
Winslow (1926) present results on the death of Bad. coli in dilute salt solutions 
which suggest an initial rise in the value of K with the passage of time, followed by a 
subsequent gradual fall. 

It appears certain, however, from a review of all the available literature that when 
a bacterial population of a reasonably homogeneous character (spores or young vege- 
tative cells) is subjected to an unfavorable environment — whether the unfavorable 
condition be a chemical disinfectant, heat, sunlight, or merely storage in a dried con- 
dition or in an aqueous medium where growth cannot occur — there is a period during 
which the death of the bacterial cells follows a fairly regular logarithmic rate. This 
period of regular decrease may be preceded by a brief period of slower decline, repre- 
senting a sort of lag or adjustment to the unfavorable environment, and seems general- 
ly to be followed by a final period of still slower decline. The latter may not occur 
when death is due to strong disinfectants such as were used by Miss Chick and 
did not appear in Cohen's studies because they were not prolonged for a sufficient 
period. 

It seems, however, entirely unnecessary to postulate biological variations in the 
cells of the bacteria to account for these deviations from the logarithmic curve. As 
Falk and Winslow (1926) have pointed out, it is much simpler to assume that the 
lethal reactions which go on in bacterial cells dying in an unfavorable environment 
proceed in accord with a bimolecular reaction or reactions of a still higher order rather 
than in accord with the monomolecular formula. There is no reason to assume that 
the decomposition of a single chemical compound is always the determining cause of 
death. It is shown in the paper cited that the values of K may often be best explained 
on such an assumption. Just as the lag period in bacterial growth involves the as- 
sumption of catenary reactions, so do the variations at the beginning and end of the 
curve of bacterial mortality. To obtain the values which most nearly approximate a 
logarithmic curve and represent the period when the lethal process most nearly sim- 
ulates a monomolecular reaction, Phelps has suggested that K should be determined 
for the middle portion of the curve — say from a reduction of 75 to one of 25 per cent. 



74 THE RISE AND FALL OF BACTERIAL POPULATIONS 

It is a curious and interesting fact that disinfection by sodium hydroxide follows 
an entirely different law from that observed in other cases, K increasing progressively 
with the time of exposure (Levine, Buchanan and Lease, 1927). 

It may be of interest, in spite of the various factors involved in the problem, to 
consider some of the absolute values of K which have been observed in certain specific 
instances and to note their significance in terms of percentage reduction. 

At one extreme stand such results as those obtained by Jordan (1926) for the 
secondary reduction of Bad. coli in stored feces. These give (from the third to the 
twenty-third day) a K per hour of .006. The values which I have cited for the reduc- 
tion of the numbers of a certain strain of colon bacilli in water (Winslow and Cohen) 
give a A' per hour of .01 for the third to the tenth day, corresponding to a reduction in 
numbers of 2.5 per cent per hour. The reduction of Bad. typhosum in ice for the first 
three days (Sedgwick and Winslow) is of the same order {K = .02) and involves a re- 
duction of 5 per cent per hour. For Bad. coli dried in sand (Winslow and Abramson) 
K = .o6 for the ninth to the forty-eighth hour (a reduction of 13 per cent per hour). 
The mortality of anthrax spores exposed to 5 per cent phenol at 20° C. is about the same 
(Chick, 1908). Falk and Winslow's studies give a A'- value of about .1 per hour for 
dilute salt solutions (20 per cent per hour), and Cohen's data for acid (pH 8) give a K- 
value of i.o (90 per cent reduction per hour). Chick's analysis of Clark and Gage's 
results on the disinfectant action of sunlight give a A of 2.6, which would involve a 
reduction of 95 per cent per hour. The death of paratyphoid bacilli exposed to 6 per 
cent phenol is more rapid still, with a A of about 20, according to Chick's data (99.4 
per cent reduction per hour) ; while hot water (54° C.) gives a A of over 60.0. This in- 
volves a reduction of 99.75 per cent per hour. 

The factors which govern the rate of the mortality of bacteria are essentially the 
same as those which govern the rate of their multiplication, though of course most of 
them operate in an inverse sense. When bacteria die out in water or when stored in a 
dried condition it is the normal katabolic processes of the cell (in the absence of com- 
pensating anabolism) which must control the process. It seems doubtful whether 
drying in itself exerts any specific harmful effect, and (if rapid and complete) it may 
even slow down vital processes and thus prolong life. Ficker (1898), however, found 
that alternate drying and moistening accelerated the lethal process. It is of interest to 
note that Paul, Birstein, and Reuss (1910) found the disinfection constant in drying 
proportional to the square root of the oxygen concentration of the atmosphere, fol- 
lowing the law which obtains in the slow oxidation of phosphorus. The whole subject 
of the longevity of micro-organisms under the influence of desiccation has been well 
reviewed by Giltner and Langworthy (19 16). 

The presence of external toxic substances may of course be one important factor 
in the death of bacteria in water or in soil. Thus Jordan, Russell, and Zeit (1904) found 
that typhoid bacilli inclosed in collodion sacs survived much longer when the sacs 
were suspended in pure water than when they were suspended in polluted water. In 
the presence of known chemical disinfectants this factor of direct toxicity is of course 
the determining feature. The value found for A may therefore vary within the widest 
possible limits, depending on the toxicity of the particular disinfectant studied; and 
the study of the relation between toxicity and chemical composition (as worked out, 



C.-E. A. WINSLOW 75 

for example, by Schaffer andTilley [1927] for the various members of the alcohol and 
phenol group) is a fascinating one. 

Chick (1908) showed that within certain limits the efficiency of a chemical disin- 
fectant bears a logarithmic relation to its concentration, the expression 

log 7^ 



In to L'oto 

remaining constant in value when to and /„ are the time periods necessary for disinfec- 
tion corresponding to concentrations Co and C„. Furthermore, it is important to note 
that disinfectants not only vary in their efficiency at a given concentration but also 
vary in the degree to which their toxicity increases with increasing concentration. 
The ordinary carbolic acid coefficient (representing the ratio of the concentration of a 
given disinfectant to the concentration of carbolic acid which will sterilize in a given 
time) therefore gives a very incomplete idea of the true relationships. Thus Chick 
(1908) found that mercuric chloride may have a carbolic acid coefficient of 13 at one 
concentration and of 550 at another. 

Phelps (191 1 ) therefore suggests that values of K (K and K') should be deter- 
mined at two different concentrations (C and C). If we express the K of our previous 
formula (p. 72) by KC" (to allow for this concentration factor) we get 

log ^=KCH 
and 

log ^=K'C'H 
Therefore 

n=Iog-^ '^^^S'C 

From the two determinations of K we can then compute n or the concentration 
coefficient of the particular disinfectant studied. This figure n shows by what power 
of 2 the efficiency is increased if the concentration be doubled. For anthrax spores in 
mercuric chloride at 20° C. it is 1.08. 

The reaction of the medium is a special case of chemical toxicity which has been 
studied with particular care. Such a phenomenon occurs in nature in streams re- 
ceiving acid wastes, and the disinfectant action of carbon dioxide is due to the same 
cause (Koser and Skinner, 1922). As pointed out above, carbon dioxide, aside from 
its effect upon reaction, is beneficial and indeed essential to bacterial life. 

Winslow and Lochridge (1906) reported twenty years ago that the toxic effect of 
mineral acids was in large measure due to dissociated hydrogen. Cohen (1922) has 
given us one of the most careful recent studies of this problem, and we may cite one of 
his summary tables (Table VI) indicating the effect of hydrogen-ion concentration 
upon the death-rate of Bad. typhosum. 

The effect of cations other than hydrogen upon bacterial growth and death has 
been studied by numerous observers (see review by Falk, 1923). Those who have 
worked on the increase of bacterial populations in favorable media, from Richet 



76 



THE RISE AND FALL OF BACTERIAL POPULATIONS 



(1892) to Hotchkiss (1923), have found that practically all cations may show either a 
stimulating or an inhibiting influence, depending on their concentration, and observers 

TABLE VI 

Average Velocity Constants for the Death of Bad. lyphosnm 
AT Different pH Values, 20° C. 

PH 3-8 S-o 5.4 6.4 7.1 7.6 8.7 9.5 

K 1055 00134 o.oiio 0.0138 0.0437 o. HOC 0.2134 0.2855 

Relative iir*95. 5 1.2 i.o 1.5 4.8 10. o 22.4 31.4 

* Taking K at pH 5.4 as unity. 

like Winslow and Falk (1923) who have studied the death-rate of bacteria in unfavor- 
able media find that in the same way small amounts of cations favor survival while 

Kl 
+1.5 



+1.0 



♦0.5 



-0.5 













CaCU 
/ 














/ 


/ 


NaCI 








**— 




v 




/ 

























0.001 o.oi o.f 1.0 

MOLAR CONCENTRATION OF SALT 



1.0 



Fig. 4. — Relation between viability of Bad. coli and salt concentration (Falk and Winslow, 
1926). 

large amounts increase the mortality. Falk and Winslow (1926) pointed out the sig- 
nificant fact that if values of K for the effect of NaCl and CaCl2 upon Bad. coli in 
water be plotted against concentration one obtains a reasonably smooth curve for 
both salts, passing from positive values (indicating toxicity) to negative values (indi- 
cating a preservative effect) (see Fig. 4). It will obviously be difficult to harmonize 
such a phenomenon with any simple chemical assumptions. 

It is also important to remember that the net effect of various chemical and physi- 
cal factors, acting simultaneously, may be an exceedingly complex one. Thus Chick 
(1910) showed that very minute excesses of acid or alkali might enormously accel- 
erate the rate of disinfection by hot water; and the same phenomenon is of great prac- 
tical importance in connection with heat sterilization. On the other hand, a lethal 
factor may equally well be neutralized by a favorable one. Sometimes the effect is 
relatively simple, as in the reduction of the disinfectant power of mercuric chloride 
due to the presence of organic matter (Chick and Martin, 1908), a fact which makes 



C.-E. A. WINSLOW 



77 



this substance wholly unsuitable for the disinfection of feces, or in the interference of 
organic matter in water with the action of chlorin. Under other conditions more sub- 
tle reactions are involved, as in the results recently reported by Winslow and Brooke 
(1927). These observers found that several different types of bacteria die out almost 
immediately when washed free from culture medium and resuspended in distilled 
water. Salt and sugar solutions will not check the mortality, so we are not deahng 
merely with a question of osmosis; but the cells can be protected by the presence of 
peptone or meat extract, serving as "protective colloids" as indicated in Table VII. 

TABLE VII 
Viability of B. cereus 





Percentage Surviving 




Broth 


Peptone 


Meat Extract 


Ringer-Locke 
Solution 


Before centrifugation 


100 
56 
71 


100 
43 

53 


100 
51 

57 




After centrifugation 


3 

I 


One hour later 



A concentration of .005 per cent peptone or of .003 per cent meat extract will 
protect the cells; but a concentration of .0005 per cent peptone or of .0003 per cent 
meat extract fails to do so. 

Finally, we must consider the influence of temperature upon the course of the 
curve of decreasing numbers of bacteria. Since the processes of death, like those of 
life, are essentially chemical in nature, it is of course obvious that an increase of tem- 
perature will favor the lethal process when lethal factors are dominant just as it favors 
the growth process when growth factors are dominant. The classic work of Houston 

TABLE VIII 

Effect of Temperature on Survival or Typhoid 
Bacilli in Water (Houston) 



Temperature ° C 


Percentage of Typhoid 
Bacilli Surviving 
after One Week 


Period of Final 
Disappearance of 
Bacilli (in Weeks) 





46.00 

14- 00 

0.07 

0.04 


9 

7 
5 
4 


s 

10 

18 





(1911) on the survival of typhoid bacilli in water (illustrated in Table VIII), for ex- 
ample, seems at first sight puzzling, since we find an organism whose optunum for 
growth lies at 37° C. dying more rapidly at that temperature than in cooler waters. 
The explanation lies in the fact that typhoid bacilli in water lack the conditions 
essential for anabolism; only katabolism can go on, and katabolism is increased by 
a rise in temperature. 

The effect of high temperature in increasing the eflEiciency of chemical disinfect- 
ants was noted by Koch (1881) in the earliest studies of disinfection, and was first 



78 



THE RISE AND FALL OF BACTERIAL POPULATIONS 



carefully studied by Madsen and Nyman (1907), Chick (1908), and Paul (1909). 
These investigators found that the reaction velocity of disinfection increased with a 
rise in temperature, according to the formula of Arrhenius, the function 



T-T, 



log 



remaining constant where ti and ^2 represent times taken for disinfection and Tj, and 
T2 represent absolute temperatures. 

As the temperature at which such a reaction proceeds increases, the velocity of 
the reaction increases in geometrical progression. If K' and K are the constants of the 
reaction at the temperatures T' and T, respectively, and d is the temperature coeffi- 
cient (Phelps), 

K ^ 

By determining the reaction velocity of the lethal process for bacteria at two 
temperatures 10° C. apart, we may obtain this temperature coefhcient from the 
formula 

For anthrax spores exposed to 0.5 per cent mercuric chloride 6 = 1.17. I^i general, 
the mean velocity of disinfection with metallic salts increases two- to four-fold for a 
10° rise in temperature (centigrade), while with other disinfectants the increase may be 
considerably greater. 

The effect of temperature upon the natural death of bacteria in water is essen- 
tially the same, although the absolute value of the coefficient seems to be somewhat 
lower. Table IX, from Cohen (1922), illustrates this phenomenon. 

TABLE IX 

Velocity Coefficients for Death of Bact. typhosum and 
Bad. coli at pH 3.5 at Different Teiiper.\tures 





Bad. typhosum 


Bad 


. coli 


Ratio of K 
FOR Bad. 


Temperature, ° C 


K 


Increase 
for 10° 


K 


Increase 
for 10° 


typhosum to 

K FOR Bad. 

coli 




1. 186 

1-919 

2.928 

5-176 




0.0176 

• 0373 

•1654 

0.6214 




67 
51 
18 


10 


1.62 
1-53 
1-77 


2.12 
4-36 

3 76 


20 


IQ 


8 







It will be noted that the temperature coefficient for Bact. coli is much lower than for 
Bact. typhosum but rises much more rapidly for a 10° C. increase. 

A peculiarly interesting contribution was made by Chick (1910) in the demon- 
stration that the death of bacteria in hot water follows the same general time relation, 
although of course with an enormously high time factor. In the case of Bact. 
typhosum the coefficient was 1.635 per 1° C. The whole phenomenon of cell death 



C.-E. A. WINSLOW 79 

under the influence of heat is explained by Chick and Martin (1910) as a heat 
coagulation consisting in a reaction between protein and water which is highly accel- 
erated by heat. From the practical standpoint of food preservation, Bigelow (1921) 
has shown that the logarithmic relationship between sterilizing time and temperature 
holds for the destruction of both spores and vegetative cells by high heat. 

Phelps (191 1) has given a very valuable analysis of the variations in the effective- 
ness of a given disinfectant with respect to concentration, time, and temperature, and 
has shown how its constants can all be fixed by determining its efficiency in two dif- 
ferent dilutions at the same temperature and at two different temperatures in the 
same dilution (three determinations in all) ; the values for any other set of conditions 
can then be obtained from the formulas: 

log^ = KCH 
and 

when (as before) jB= initial number of bacteria present, 5 = final number of bacteria 
present, / = elapsed time, C = concentration of disinfectant. A' = velocity constant cal- 
culated at temperature of experiment, irr° = same at any temperature T°, Kio" — 
same at 20°, w = concentration exponent, and = temperature coefficient. 

6. THE PHASE OF READJUSTMENT 

Toward the close of the phase of decrease the rate of mortality slackens and the 
curve passes imperceptibly into the final phase of readjustment. In the work of 
Chick (1908) on the effect of chemical disinfectants upon vegetative cells, and in the 
studies by Falk and Winslow (1926) on the death-rate of bacteria in dilute salt solu- 
tions, it even appears, as we have seen, that the value of K falls progressively through- 
out the phase of decrease (see Fig. 4) so that there is no clear distinction between the 
two processes. In other instances, however, it is often possible to observe two fairly 
distinct periods, one of rapid and one of slow decline. Thus, Winslow and Cohen (1918) 
in their studies of the life of Bact. coli in water observed values of K ranging from .004 
to .020 for the first ten days while from the tenth to the sixtieth day the values varied 
only between .001 and .002. 

The mortality curves given by Sedgwick and Winslow (1902) for the life of ty- 
phoid bacilli in ice and in dry earth, by Winslow and Abramson (191 2) for colon bacilli 
in water, all show a falling rate of mortality toward the close of the cycle of decrease. 
Values for K calculated from these data are cited below, obtained by merely sub- 
tracting the log of the number present after a given time interval from the log of the 
number present at the beginning of that interval and dividing by the elapsed time. 
(It should be noted that in computing these values the reduction in numbers for each 
period are computed on the basis of the number present at the beginning of that pe- 
riod instead of following the usual procedure of computing K on the basis of reduction 
from the beginning of the whole experiment. The latter method naturally tends to 
obscure any differences which may occur.) 

The very gradual rate of decrease during the phase of readjustment may end in 



8o 



THE RISE AND FALL OF BACTERIAL POPULATIONS 



either of two different ways. In a moderately favorable medium, such as water, the 
cycle usually ends with a new level of stability {EF in Fig. i) on which the number of 
bacteria may remain reasonably constant for an indefinite period. Miquel describes 
an experiment in which a bottle of Seine River water containing originally 4,800 bac- 
teria per cubic centimeter was stored for nine years and showed 220 bacteria per cubic 
centimeter at the end of that time. 

Sometimes, as is shown in Figure 2, there may be one or more secondary waves of 
increase before the final level of stability is reached. 

In his study of stored feces Jordan (1926) found that after the initial multiplica- 
tion and subsequent decrease a level was reached which remained more or less con- 
stant for long periods. The total number of bacteria present after many weeks may be 
as high as, or higher than, the number initially present. Bact. coli, however, ultimate- 
ly disappears from the fecal flora under such conditions. In a less favorable medium, 

TABLE X 
Values of K for Various Periods of Certain Mortality Curves 



Observer 


Organism 


Medium 


Temp. 


Period 


K per Hour 


Sedgwick-Winslow 


Bad. typh. 
Bact. typh. 

Bact. coli 

Bact. coli 


Dry earth 
Ice 

Dry sand 

Water 


0° 

— I 

20 

10-20 


/First 3 days 
\ 3-14 days 
("First 3 hours 
■{First 3 days 
[3-14 days 
fFirst 9 hours 
\ 9-48 hours 
[48-216 hours 
[First 3 days 
J3-10 days 
[10-60 days 


0.04 


Sedgwick-Winslow 

Winslow- Abramson 

Winslow-Cohen 


.004 
.08 
.02 
.002 

•03 

.06 

.006 

.004 

.on 




o.ooi 



on the other hand, the phase of readjustment ultimately ends in complete sterility, as 
in disinfection with high heat or strong chemicals {EF' in Fig. i). 

In an intermediate case — where conditions are neither sufficiently favorable to 
permit of the balanced growth and death which maintains a constant stable level nor 
sufiiciently unfavorable to lead to rapid and complete extinction — a small proportion 
of the original bacterial population may persist for a very long period. Thus Parkes 
(1903) was able to isolate typhoid bacilli from blankets soiled with feces after more 
than six months. Konradi (1904) reports Bact. typhosimi as surviving in water after 
seventeen months. Robertson (1898) isolated the same organism from moistened soil 
after eleven months. Studies by numerous observers on the drying of typhoid, diph- 
theria, and tubercle bacilli (summarized by Chapin, 191 2) have shown that all these 
organisms may survive drying for several months. (Table X.) 

Winslow and Kligler (1912) found over 51,000 colon bacilli and 42,500 acid- 
forming streptococci per gram of dust from city streets and 940 colon bacilli 
and 22,040 acid-forming streptococci per gram of house dust. Winslow and Sanjiyan 
(1924) conducted an extensive study of the distribution of the acid-forming strepto- 
cocci, presumably indices of pollution from mouth spray, on objects and surfaces of 
various kinds. Objects, such as eating utensils, directly exposed to mouth contan? 



C.-E. A. WINSLOW 8i 

ination showed these organisms in 62 per cent of the cases studied while objects re- 
cently handled (door handles, push buttons, etc.) showed them in 42 per cent of the 
cases. On locations such as walls six feet above the ground, the undersides of chairs 
and tables and the like, these organisms were isolated in 10 per cent of the cases. 

From the standpoint of epidemiology, it is essential to note that the pathogenic 
bacteria which survive for such long periods as those noted above are so few in number 
that their presence is of little or no practical significance for disease transmission. 
Houston (1908), for example, was able to isolate typhoid bacilli from water after nine 
weeks; but 99.9 per cent of the bacteria originally present had perished after one week. 
Nothing is of course more certain than the fact that transmission of disease germs oc- 
curs in the vast majority of instances only through the rather direct and immediate 
transfer of fresh body discharges. 

In the earlier days of bacteriology it was customary to refer to the few pathogenic 
bacteria which survive after long periods of time in water or earth as representing a 
"resistant minority." It is quite possible that in certain instances a selection of more 
resistant variants may in fact take place. Sedgwick and Winslow (1902) showed that 
individual strains of typhoid bacteria differ markedly in their ability to survive in 
ice; and Ayers and Johnson (191 5) found that various strains of colon bacilli show 
great differences in their ability to resist various pasteurization temperatures. There 
is, however, no direct evidence that the cells which die out toward the end of the cycle 
of bacterial population derived from a single strain are intrinsically more resistant 
than those which perish earlier; and it seems probable that the curve for such a cycle 
is mainly determined by a series of catenary reactions following the ordinary laws of 
more simple chemical processes. 

BIBLIOGRAPHY 
Allen, P. W.: J. Bad., 8, 555. 1923. 

Ayers, S. H., Cook, L. B., and Clemmer, P. W.: U.S. Bureau of Animal Industry, U.S. De- 
partment of Agriculture, Bull. 642. 
Ayers, S. H., and Johnson, W. T.: ibid., Bull. 126. 
Ayers, S. H., and Johnson, W. T.: /. Agric. Research, 3, 401. 1915. 
Barber, M. A.: /. Infect. Dis., 5, 379. 1908. 
Bigelow, W. D.: ibid., 29, 528. 1921. 
Brooks, S. C: J. General Physiol., i, 61. 1918. 
Buchanan, R. E.: J. Infect. Dis., 23, 109. 1918. 

Buchner, H., Langard, K., and Riedlin, G.: Centralbl.f. Bakteriol., 2, i. 1887. 
Buddin, W.: /. Agric. 5c., 6, 417. 1914. 

Burke, V., Sprague, A., and Barnes, L.: /. Infect. Dis., 36, 555. 1925. 
Chapin, C. V.: Sources and Modes of Infection. New York, 1912. 
Chesney, A. M.: /. Exper. Med., 24, 387. 1916. 
Chick, H.: /. Hyg., 8, 92. 1908. 
Chick, H.: ibid., 10, 237. 1910. 
Chick, H.: ibid., 12, 414. 191 2. 
Chick, H., and Martin, C. J.: ibid., 8, 654. 1908. 
Chick, H., and Martin, C. J.: /. Physiol., 40, 404. 1910. 
Clark, P. F., and Ruehl, W. H.: /. Bad., 4, 615. 1919. 
Cohen, B.: ibid., 7, 183. 1922. 



82 THE RISE AND FALL OF BACTERIAL POPULATIONS 

Cohen, B., and Clark, W. M.: ibid., 4, 409. 1919. 

Cole, S. W., and Lloyd, D.: /. Path. Bad., 21, 267. 1917. 

Conn, H. J.: Tech. Bull. 64, N.Y. Agric. Exper. Sta. Geneva, N.Y., 1918. 

Coplans, M.: /. Path. Bad., 14, i. 1909. 

Cramer, W.: Die Wasserversorgung von Zurich und ihr Zusammenhang mil der Typhusepi- 

demie des Jahres 1884. Zurich, 1885. 
Davis, L., and Ferry, N. S.: /. Bad., 4, 217. 1919. 
Devereux, E. D., and Tanner, F. W.: ibid., 14, 317. 1927. 
Falk, I. S.: Abstr. Bad., 7, 33, 87, 133. 1923. 
Falk, I. S., and Winslow, C.-E. A.: J. Bad., 11, i. 1926. 
Ficker, M.: Ztschr. f. Hyg. u. Infektionskrankh., 29, i. 1898, 
Fokker, A. P.: ibid., 9, 41. 1890. 

Frankland, P.: Proc. Roy. Soc, London (1884-85,) 38, 379-93. 1885. 
Frankland, Mr. and Mrs. Percy: Micro-Organisms in Water. London, 1894. 
Fuller, G. W.: Ann. Rep., State Board of Health of Massachusetts for iSg4, 26, 461. 1895. 
Giltner, W., and Langworthy, H. V.: J. Agric. Research, 5, 927. 1916. 
Hadley, P. B.: /. Infect. Dis., 40, i. 1927. 

Harrison, F. C, and Vanderleck, J.: Rev. gen. du lait, 7, No. 15. 1909 
Hehewerth, F. H.: Arch. f. Hyg., 39, 321. 1901. 
Heinemann, P. G.: /. Infect. Dis., 16, 479. 1915. 
Heinemann, P. G. : Milk. Philadelphia and London, 1919. 
Henrici, A. T.: Proc. Soc. Exper. Biol. &° Med., 19, 132. 1921. 
Henrici, A. T.: ibid., 20, 179. 1922. 
Henrici, A. T.: ibid., 21, 215. 1923. 
Henrici, A. T.: ibid., p. 343. 1924. 
Hotchkiss, M.: /. Bad., 8, 141. 1923. 

Houston, A. C: First Rept. on Research Work, Metropolitan Water Board. London, 1908. 
Houston, A. C: Seventh Rept. on Research Work, Metropolitan Water Board. London, 191 1. 
Hutchinson, H. B., and MacLennan, K.: J. Agric. Sc, 6, 302. 1914. 
Ikeda, K.: Ztschr. f. Hyg. u. Infektionskrankh., 25, 95. 1897. 
Jordan, E. O.: /. Infect. Dis., 38, 306. 1926. 
Jordan, E. O., Russell, H. L., and Zeit, F. R.: ibid., i, 641. 1904. 
Koch, R.: Mitt. a. d. Kaiserlich. Gesundheitsamte, i, i. 1881. 
Kohn, E.: Centralbl. f. Bakteriol., Abt. II, 15, 690. 1906. 
Konradi, D.: ibid., Abt. I, Orig., 36, 203. 1904. 
Koser, S. A., and Skinner, W. W.: /. Bad., 7, in. 1922. 
Kronig, B., and Paul, T.: Ztschr. f. Hyg. u. Infektionskrankh., 25, i. 1897. 
Lane-Claypon, J. E.: /. Hyg., 9, 239. 1909. 
Ledingham, J. C. G., and Penfold, W. J.: ibid., 14, 242. 1914. 
Leone, C: Arch.f. Hyg., 4, 168. 1886. 

Levine, M., Buchanan, J. H., and Lease, G.: Iowa State Coll. J. of Sci., 1, 379. 1927. 
Lloyd, D.: J. Path. Bad., 21, 113. 1916. 
Loeb, J., and Northrop, J. H.: J. Biol. Chem., 32, 103. 1917. 
Madsen, T., and Nyman, M.: Ztschr. f. Hyg. u. Infektionskrankh., 57, 38S. 1907. 
Massachusetts: Tivcnty-sixth Ann. Rept., State Board of Health of Massachusetts for 1S94, 

1895. 

Miquel, P.: Rev. d'hyg., 9, 737. 1887. 

Miquel, P.: Manuel pratique d' analyse baderiologique des eaux. Paris, 1891. 

Muller, M.: Ztschr. f. Hyg. u. Infektionskrankh., 20, 245. 1895. 



C.-E. A. WINSLOW 83 

Parkes, L. C: Practitioner, 71, 297. 1903. 

Parsons, L. B., and Sturges, W. S.: /. Bad., 14, 181, 193, 201. 1927. 

Paul, T.: Biochcni. Ztschr., 18, i. 1909. 

Paul, T., Birstein, G., and Reuss, A.: ibid., 25, 367. 1910. 

Paul, T., and Kronig, B.: Ztschr. f. phys. Cheniie, 21, 414. 1896. 

Penfold, W. J.: /. Hyg., 14, 215. 1914. 

Penfold, W. J., and Norris, D.: ihid., 12, 527. 1912. 

Peters, R. A.: J. Physiol., 54, 260. 1920. 

Phelps, E. B.: /. Infect. Dis., 8, 27. 1911. 

Prescott, S. C, and Baker, S. K.: ibid., i, 193. 1904. 

Prescott, S. C, and Winslow, C.-E. A.: Elements of Water Bacteriology (4th ed.). New 

York, 1924. 
Rahn, 0.: Centralbl. f. BakterioL, Abt. II, 16, 417. 1906. 
Rahn, O.: Tech. Bull. 5, Mich. Agric. Coll. Exper. Sta. 1910. 
Raju, V. G.: J. Hyg., 21, 130. 1922. 

Reed, H. S., and Reynolds, R. R.: Tech. Bull. 10, Va. Agric. Exper. Sta. 1916. 
Rettger, L. F.: /. Bad., 3, 103. 1918. 

Richet, C.: Compt. rend. Acad. d. sc, 114, 1494. Paris, 1892. 
Robertson, J.: Brit. M. J., i, 69. 1898. 

Russell, E. J., and Hutchinson, H. B.: J. Agric. Sc, 5, 152. 1913. 
Schaffer, J. M., and Tilley, F. W.: /. Bad., 14, 259. 1924. 

Sedgwick, W. T., and Winslow, C.-E. A.: Mem. Am. Acad. Arts &' Sc, 12, 471. 1902. 
Sherman, J. M., and Albus, W. R.: /. Bad., 8, 127. 1922. 
Sherman, J. M., and Albus, W. R.: ibid., 9, 303. 1924. 

Shrader, J. H.: Fifteenth Ann. Kept., Int. Assoc. Dairy b' Milk Inspectors, p. 208. 1926. 
Slator, A.: /. Hyg., 16, 100. 1917. 
Smith, J. H.: Ann. Appl. Biol., 8, 27. 1921. 
Snyder, C. D.: Am. J. Physiol., 22, 309. 1908. 
Snyder, C. D.: ibid., 28, 167. 191 1. 
Sturges, W. S.: /. Bad., 4, 157. 1919. 
Truifant, G., and Bezssanoff, N.: Science du sol, i, 3. 1922. 
Valley, G., and Rettger, L. F.: J. Bad., 14, loi. 1927. 
Ward, A. R.: Proc. Roy. Soc, 58, 265. 1895. 
Werkman, C. H.: /. Bad., 14, 335. 1927. 
Whipple, G. C: Tech. Quart., 14, 21. 1901. 
Wildiers, E.: La Cellule, 18, 313. 1901. 

Winslow, C.-E. A., and Abramson, F.: Proc. Soc Exper. Biol, b" Med., 9, 107. 1912. 
Winslow, C.-E. A., and Brooke, O. R.: J. Bad., 13, 235. 1927. 
Winslow, C.-E. A., and Cohen, B.: /. Infect. Dis., 23, 82. 1918. 
Winslow, C-E. A., and Falk, I. S.: /. Bad., 8, 215 and 237. 1923. 
Winslow, C.-E. A., and Kligler, I. J.: Am. J. Pub. Health, 2, 663. 191 2. 
Winslow, C.-E. A., and Lochridge, E. E.: J. Infect. Dis., 3, 547. 1906. 
Winslow, C.-E. A., and Sanjiyan, D. H.: /. Bad., 9, 559. 1924. 
Wolffhiigel, G., and Riedel, O.: Arb. a. d. Kaiserlich. Gesundheitsamte, i, 463. 1886. 



CHAPTER VII 
THE DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR 

PHILIP HADLEY 
University of Michigan 

INTRODUCTION 

Two of the most remarkable circumstances relating to the development of bac- 
teriology during the past half-century are: first, that bacteriologists have been so long 
content to conduct their experiments and to formulate their views in terms of the old 
monomorphic hypothesis regarding the nature of bacteria and of bacterial reproduc- 
tion; second, that they have been so active in evolving inadequate schemes for class- 
ification before they knew exactly what it was they had to classify. Looking back on 
the road over which we have traveled, it is impossible to estimate the loss sustained 
by bacteriology, especially in latter years, through the repressive and misguiding in- 
fluence of the strict monomorphic conceptions, or to appreciate the often serious bio- 
logical blunders that are to be laid at its door. In times of festival and jubilee we are 
accustomed to congratulate ourselves on the significant conquests of modern bacteri- 
ological science; and they have, it is true, been considerable. But they should have 
been greater; and they would have been far greater today if the science, a half- 
century ago, had not become impaled on a false biological conception which has never 
ceased to influence unfavorably both bacteriological thought and practice. And 
which, it may be added, even today represents a malicious dogma, accepted by tradi- 
tion, if not actually embraced by perhaps the majority of bacteriologists. 

The doctrine of monomorphism has descended to us from the early conceptions of 
the nature of bacteria maintained by Cohn, Koch, and others of the early school. 
Under its influence, in the earliest and most plastic days of the science, there were set 
up strict notions of "normal" bacterial cell types, "normal" colony forms, and "nor- 
mal" cultures. Whatever departed from the expected normality was at once relegated 
to the field of contaminations; or to the weird category of "involution forms," "de- 
generation forms," or pathological elements possessing neither viability, interest, nor 
significance. This monomorphic conception found its natural and fundamental sup- 
port in the assumed mode of reproduction characteristic of the fission-fungi. The dic- 
tum was then laid down that "the mode of reproduction of bacteria is by simple 
fission" — a view which has descended through two generations of bacteriologists and 
through numerous generations of textbooks, even to the year 1927. 

Although some early opposition to these views arose, it was probably unfortunate for 
the beginnings of the science that the first attempts toward modification were made by such 
extremists as Niigeli and his associates in the Munich group. The extreme plurimorphism 
which they so eagerly championed through years of bitter controversy was too radical to be 
accepted graciously as an antidote to strict monomorphism. Kor this reason Niigeli gained 
few permanent supporters; and, with the hnal collapse of his views, all views, even those 

84 



PHILIP HADLEY 85 

espousing a more temperate plurimorphism suffered. The interpretations of the Berlin school 
triumphed — and to such an extent as to become later the dogma of "normal" colony and 
culture types that has endured, with hardly a respite, even to the present day. 

There did arise, however, at a later date some slight reaction to the monomorphic trend 
of the science. In the later eighties Gruber and his pupils were demonstrating examples of 
common and often curiously persistent variability in bacterial cultures. These observations 
were at variance to the demands of the Berlin group, although the instances concerned also 
fell far short of conforming to the extreme variability earlier pictured by Niigeli.' The same 
was true of the depictions of variability presented in the splendid series of contributions of 
Eisenberg.^ "Pathological variants," "involution forms," and "degeneration forms" were 
for a long time quite adequate to dispose of all such insignificant, though still somewhat 
bothersome, departures from the "normal" type. In 1906 and 1907, however, the variation- 
ists were fortunate enough to secure a more logical and trustworthy outlet for their explana- 
tions of "abnormal" forms of cells or cultures. This was found in the observations of Neisser' 
and of Massini-* on B. coli mutahile, whose pictures of variation they believed involved the 
phenomenon of mutation. In this way the De Vriesian term, together with many of its con- 
notations, was first introduced into bacteriology. This event was the starting-point for nu- 
merous observations and studies on mutating bacterial forms; and the discovery of bacterial 
"mutants" has continued without appreciable interruption up to the present day. Seldom, 
however, has the De Vriesian term been used advisedly. Its employment has merely provided 
a dignified and logical escape from the increasingly unacceptable "involution" hypothesis, 
as also from the necessity of offering any other more valid explanation of the phenomena 
concerned. The general result has been to bring into bacteriology many of the terms em- 
p'oyed in genetics — "plain variations," "impressed variations," "hereditary variations," 
''clones," "biotypes," and "pure lines." But the conceptions apparently supported by the 
somewhat lavish use of these terms have usually lacked concreteness; and in few instances 
have the appellations been either appropriate or logical. For the most part, notions of bac- 
terial heredity among bacteriologists have been marked by extreme haziness and uncer- 
tainty. 

Beginning about 1907, however, there began to arise among the variationists two groups: 
first, the larger group of strict variationists who saw in their culture modifications merely a 
transient, but sometimes permanent (hereditary), departure from the otherwise monomor- 
phic type; second, the cyclical variationists, hardly numerous enough to term a group, and 
represented by such workers as Fiihrmann,' who believed that they could detect a certain 
order and direction in the culture modifications. Later supporters in this group have been 
rare — six only who, since the year 1907, have combatted to the best of their ability the false 
but always overwhelming views of bacterial type-stability. These investigators merit nam- 
ing at this point in our story; they are I'lihrmann, Hort, Almquist, Lohnis, Enderlein, and 
Mellon. To these workers particularly may be given the credit for directing the current of 
bacteriological thought into new channels. 

'v. Nageli, C: Untcrsuchungcn iiber die nielere Filze iind ihren Beziehiing zu den Infektions- 
krankheiten itnd der Gcsiindhcitspjlcge. 1877. 

^ See Bibliography in monograph on microbic dissociation by Hadlcy, Philip: J. Infect. Dis., 40, 
I. 1927. 

i Neisser, M.: Centralbl.f. BakterioL, Abt. I, Orig., 38, 98. 1906. 

'•Massini, R.: Arch.f. Ilyg., 61, 250. 1907. 

sFiihrmann, F.: Verli. d..ges. deiitsch. Natiirf. u. Arize, p. 278. 1906. 



86 DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR 

THE DISSOCIATIVE REACTION 

As I have pointed out in a previous publication/ extreme instability of bacterial 
types has become recognized in recent years as a commonly observed phenomenon. 
But its significance has been vastly underestimated and its cause or causes a matter 
of uncertainty. Although descriptions of bacterial "variants" and "mutants" have 
appeared with increasing frequency in the literature of the past thirty years, it is seldom 
that they have been regarded as possessing significance outside of that referable to the 
Darwinian or De Vriesian conceptions. It has, indeed, been only in quite recent times 
that the striking orderliness and persistence with which these variants are found to 
appear in difi'erent bacterial species and groups have enabled us to relate them to a 
definite law of variation, widely operative in the bacterial world. Indeed, these ob- 
servations have led us to the view that, within each bacterial species, there are con- 
stantly occurring certain transformations relating to cell morphology, colonial form, 
biochemical, serological, and immunological characteristics and to virulence; more- 
over, that these transformations are neither random variations from a "normal" type 
in the old Darwinian sense nor sudden saltations characteristic of mutations; but that 
they represent modifications which occur with a certain degree of precision in many 
different species when confronted with similar changes in environment. 

It is only within recent years that serious attempts have been made to correlate 
any of the different characters of the variants, such as virulence with colony form or 
serological reaction with cell type. Indeed, it has been the common view that such 
correlations were seldom possible; or, at least, not sufficiently constant to be of impor- 
tance. We know today, however, that such correlations are the rule rather than the 
exception, although they may be partly obscured at times, for a variety of reasons; 
moreover, that these correlations possess supreme importance for bacteriology, pathol- 
ogy, and medicine. 

The results of many observations have thus served to indicate that pure line cul- 
tures, of many bacterial species to say the least, are composed of cells all of which are 
by no means identical. From the same pure line strain may arise, depending on the 
manner of cultivation and on other environmental conditions, substrains possessing 
little resemblance either to each other or to the parent-strain. These diflferent culture 
types may be spoken of conveniently as "dissociated forms" or as "dissociants"; and 
the phenomenon involved in their production has been termed "microbic dissociation." 
The terms Umwandlung, Keimiimwandlimg, and Umformung (of bacterial species), 
employed by certain German and Swedish investigators, may be regarded as referring 
to the same phenomenon. Microbic dissociation thus becomes established as a new 
and highly significant field in the wide province of bacteriology. 

But it will be clear that the reactions characteristic of the dissociative phenomenon 
are, in a sense, superficial. Behind microbic dissociation there must exist a biological 
mechanism; and this mechanism, we shall come to see, concerns microbic heredity. 
Through this medium, therefore, dissociation is intimately related to important studies 
which have been conducted by a small group of investigators dealing with the so- 
called "life-cycles" of bacteria. 

' Hadley, Philip: loc. cii. 



PHILIP HADLEY 87 

One of the first to suggest that bacterial reproduction was characterized by phenomena 
more complex than those appertaining to simple fission, and something even more diversified 
than simple back-and-forth variation between two or three variants, was Fiihrmann' who 
in 1907, proposed his Eiitwicklungscydus. This conception of a cyclical development in bac- 
teria was further indicated in a work by Hort^ in England in 1916, by Lohnis and Smiths in 
the United States, and by Enderlein^ in Berlin, also in 1916. It was at this time that the 
latter worker introduced into bacteriological terminology the term "cyclogeny" {Cydogenie) , 
implying the cycle through which the microbe passes in leading up to the highest cytological 
state (Kulmi)ianle) and returning to its basal state (Mychil). Many of the papers by Alm- 
quists and Mellon^ on microbic heredity have dealt with the problem of life-cycles among 
bacteria; and in recent years this term, often used rather loosely to indicate certain obscure 
cellular transformations, has occurred in the literature with considerable frequency. 

While it seems probable that the time will eventually arrive when we can speak intelli- 
gently regarding definitely cyclical aspects of bacterial reproduction, and even though at the 
present moment we can often detect a certain direction in the serial transformations observed, 
it has seemed to me that our present knowledge of cyclical development in its details is still 
too slight to justify a common use of this term as describing the transformations thus far ob- 
served. Until more definite knowledge of the distinctly cyclical development of some one 
species is at hand, it is perhaps more appropriate to employ a term suggesting merely cultural 
transformations, often, it is true, apparently directive, but not overemphasizing the cyclical 
feature. For such a term, "microbic dissociation" will for the present suffice. The ultimate 
realities on which it depends are those involving the mechanics of microbic heredity. Micro- 
bic dissociation might therefore be defined as embracing those distinctly transformatory 
processes occurring in bacterial cultures, in vitro or in vivo, through which there arise one or 
more new culture forms which differ from the mother-type, and which (i) may persist for a 
variable time in an apparently stable state, or (2) may become transformed into still another 
culture type, or (3) may "revert" to the original form. 

The studies that bear on the problems of microbic dissociation may be grouped under 
two headings: (i) those random observations on culture variations which, even by the 
authors themselves, were not recognized at the time when the work was conducted as related 
to the fundamental problem of microbic heredity; (2) those later and more concise studies 
definitely directed upon the meaning of bacterial variation, its causes and effects. Regarding 
the first category, little need be said except that there exist in the bacteriological literature 
of the past thirty years or more numerous isolated citations which, by virtue of our present 
knowledge of the dissociative process, we are able to translate into the terms of microbic dis- 
sociation. Some of these I have brought together in a previous publication.' The second cat- 
egory of studies mentioned above includes those which consciously attack the problem of 
bacterial variation. These, in turn, are divisible into two groups, each differing from the 
other in its mode of approach to the fundamental problem. These involve (i) the C3^tologi- 
cal approach and (2) the cultural approach. In the latter group may also conveniently be 
included the biochemical and serological characteristics of the variants. 

' Fiihrmann, F.: loc. cil. ' Hort, E. C: /. Roy. Micr. Soc, p. 11. 1926. 

3 Lohnis, F. and Smith, E. R.: Jour. Agr. Res., 6, 675. 1916. See also Lohnis: ibid. 23, 401. 
1923; also Men. Nal. Acad. Sc., 16, 252. 1921. 

■•Enderlein, G.: Sitzungsb. ges. naturf. Frennde. Berlin, 1916. 

5 Almquist, E.: Cenlralbl. f. BaklerioL, Abt. I, Orig., 60, 167. 191 1; Biologische Forschiing tiber 
die Bakterien. Stockholm, 1925. 

* See Hadley, Philip: loc. cit. ^ See ibid. 



88 DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR 

THE CYTOLOGIC AL APPROACH 

The cytological approach to the problem of microbic dissociation has centered 
mainly on the variable morphology of the bacterial cells and their nuclear appara- 
tus. It has followed (especially in the morphological aspects) the lines laid down in 
mycology at a much earlier date. Here the attempt has been made to discover, in the 
peculiar bacterial elements often observed in cultures, the groundwork for an inter- 
pretation of microbic heredity, involving modes of reproduction quite different from 
simple fission. Most of the earlier studies dealt with the microscopical cell changes, 
but without special reference to nuclear behavior. Later studies, and particularly 
those of Almquist, Enderlein, and Mellon, have concerned themselves as well with 
alterations in the nuclear apparatus. In view of the importance of the work dealing 
with the nuclear changes, a few words must be said regarding later conceptions of th; 
nuclear structure and the "chromatin" of the bacterial cell. The following represents 
some of the essential features of Enderlein's' view which I believe we may accept as 
reflecting the best knowledge now available regarding this important organelle. 

The nuclear unit (Mych) of the microbic cell possesses a spherical or oval form and, in 
the coccus, often attaches itself to the inner wall of the cell, against which it may sometimes 
be flattened. In cocci there is but one nuclear body while in all other forms of bacteria there 
are two or more. The diameter varies between o.i and 0.25 /x. It contains no chromatin 
and, with weak fuchsin, stains hardly any stronger than the cytoplasm of the cell. With 
methylene blue it may remain practically unstained. 

The nuclear body is observable only when the cell containing it holds but little food sub- 
stance in reserve. The latter commonly exists in the form of ultramicroscopic granules, the 
trophoconia, and this substance represents the actual "chromatic" material of the cell. It 
stains strongly because of its high content of nucleic acid and nucleo-proteins. The failure of 
a cell to stain well with methylene blue is due to the absence of the food-reserve substance. 
When the trophoconia bodies are abundant, their substance forms a dense aggregation about 
the nuclear body, and the element so formed stains readily; this is the trophosome. If only a 
light and thin layer of reserve substance clusters about the nucleus, this body becomes the 
trophosomelle, which is smaller and more delicate. Neither of these bodies (trophosome or 
trophosomelle) is the actual nucleus, however; they merely contain the nucleus as a central 
granule. Some of the granular bodies earlier described for bacteria, such as the Much gran- 
ules, the sporogenous granules of Ernst, and the metachromatic granules of Babes, are ac- 
tually trophosomes or trophosomelles. Other granules appear to be c|uite different struc- 
tures — sometimes the gonidia. They are all easily observable and often gram positive. If the 
trophosome is situated at the end of a rod form, it is a "telotrophosome"; if at other points 
in the cell, it is an "ascotrophosome." Moreover, to continue Enderlein's somewhat elabo- 
rate but necessary terminology, if a cell is free from reserve substance (trophoconia), it is 
an "atrophite." If it is merely poor in reserve substance, it is a "metatrophite"; if rich in 
reserve, it is a "pliotrophite." 

When the reserve substance in a cell has been used up, as in bacteria that have been 
starved (as in distilled water), the last remnant clings tenaciously about the nuclear body. 
Large amounts of food-reserve substance, which may conceal not only the nucleus but also 
the trophosomes, may be removed from the cell by alcohol. Under these conditions, when 
properly stained, it is observed that, in coccus forms, only a single point takes the stain. In 
rod forms, on the other hand, two or several such bodies take the stain. These are often at 

' Endcdein, G.: Bak'ericn-Cydogcnie. Bcriin, 1925. 



PHILIP HADLEY 89 

the poles of the cell and represent the true bacterial nucleus or nuclei. After cell division the 
heavily staining reserve substance soon appears in the daughter-cells. 

The first important departure from simple fission is gonidia formation. This form 
of reproduction was first recognized and named by Cohn' in 1872 in his study of 
Crenothrix, but the phenomenon was not carried over to the lower forms of bacteria. 
Gonidia were also recognized Ijy Lancaster in 1873, and they were noted the same year 
in B. lactis by Joseph Lister. They were also indicated in V . choJerae and in V . proteus 
[Vibrio Jiuklcr- prior) by Finkler and Prior in 1885. Out of the gonidial bodies, which 
were often regarded as spores, there were seen to arise extremely minute microspiral 
forms which, through further development, eventually attained normal size. Since 
these early days, gonidia have doubtless been seen many times without recognition. 
They have been definitely reported by Jones, ^ Lohnis,' Almcjuist," Mellon, s Enderlein,*" 
Tunnicliff,^ and others. Enderlein, who has been able to recognize them in many bac- 
terial species, regards them as the most common seed form (Fruchtform) of bacteria, 
being homologous with the spores of the fungi (conidia, ascospores). Morphologically 
and actually, according to Enderlein, they represent the true bacterial spore, which is 
not true of those elements usually termed "spores" by bacteriologists. 

Some of the so-called Much granules are regarded as gonidia, others as trophosomes. In 
1870 Cohn differentiated gonidia into the large ("macrogonidia") and the small ("micro- 
gonidia"). In the tubercle bacillus the microgonidia are not acid fast but may be gram posi- 
tive. Enderlein mentions several sorts of gonidia, named according to their point of origin 
in the cell. If they arise at the end of a rod, they are termed "telogonidia"; if throughout the 
whole length of a rod, they are termed "ascogonidia." Apparently compared with such high- 
er forms as Crenothrix, bacteria produce only small numbers of gonidia. That the micro- 
gonidia may pass Chamberland filters seems to have been demonstrated by Lourens^ for the 
bacillus of swinepest in 1907, by Almquist' for B. typhosus in 1911, by Miehe^ for a number of 
bacterial species in 1923, and by Mellon^ for B. fusiformis in 1926. In other instances of the 
discovery of filtrable forms of bacteria there is slight basis for an opinion as to the exact nature 
of the filtrable unit; although, in the experiments of Fontes,^ who in 1910 was the first to 
demonstrate the filtrability of the tubercle bacillus, the description of his cultures suggests 
that the filtrable forms were microgonidia whose presence, among other small granular 
bodies, he was unable to recognize. The telogonidia have been recognized in spirochetes 
(Treponema and Leptospira) and may be concerned with the filtrability of organisms of this 
class, as was first demonstrated by Novy and Knapp^ for the relapsing-fever spirochete in 
1906, although it also appears to be true that fine spiral forms also may pass the Berkefeld 
filter. According to Enderlein, the filtrable forms of bacteria comprise, not the gonidia 
alone, but also the gonites, next to be described. 

Our knowledge of the bacterial reproductive elements known as the "gonites" is 
limited to the results of Enderlein's'" studies, and particularly with reference to the 
cholera vibrio. His observations, which are highly suggestive, but which naturally de- 
mand extended confirmation, are presented forthwith. 

' See Hadley, Philip: loc. cit. ''Enderlein, G.: loc. cil. 

^ Jones, D. H.: /. Bad., 5, 325. 1920. '' Tunnicliff, R.: J . Infect. Dis., 36, 430. 1925. 

3 Lohnis, F.: loc. cit. ^ See Hadley, Philip: loc. cit. 

'• Almquist, E.: loc. cit. ' Almquist, E.: loc. cit. 

5 See Hadley, Philip : loc. cit. '» Enderlein, G.: Bakterien-Cyclogenie. Berlin, 1925. 



go DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR 

If the gonidia are maintained under conditions involving lack of nutriment, as, for in- 
stance, in aged cultures (thus preventing their entrance into a higher cyclostage), or if they 
are submitted to the influence of prolonged warming at 37° C, they become transformed into 
new and smaller elements, the gonites. The number of these forms increases with the gradual 
decrease of the gonidia. In cholera cultures left standing in the laboratory for a month or 
more, one finds that a great number of gonites have been produced. They are all extremely 
small, carry a much reduced amount of cytoplasm, and the smallest are known as the 
"microgonites." If the original culture is placed in sunlight, the same result occurs in a 
much shorter time — sometimes within a few days. The gonite is unable further to repro- 
duce itself as such. When cultures that have entered completely the gonite stage are trans- 
planted to agar, no growth occurs. Such cultures appear to be destitute of living cells. If, 
however, such a gonite culture is transplanted to broth, the gonites undergo further de- 
velopment, within a period of five to seven hours, into two new forms — namely, the spermite 
($) and the oite (+). In a liquid medium copulation occurs, and the fertilized cell is capable 
of soon regenerating the original cell type. Here, then, we have a strict sexual form of re- 
production. Enderlein has followed the details especially in the cholera vibrio. 

With further reference to the foregoing considerations, it may be said that the re- 
productive significance of the gonidia in bacterial reproduction is now beyond a matter 
of doubt. These bodies have been observed repeatedly, and their subsequent develop- 
ment into the original cell type followed by several competent investigators. As for 
their further transformation into the gonites, and the subsequent transformation of 
these into the sex cells — although this phenomenon eventually may be found to occur, 
and to underlie a true sexual form of reproduction in the bacteria — in so important a 
matter one is justified in postponing a conclusion until the striking observations of 
Enderlein can be confirmed in the cholera vibrio and extended to other species. It may 
be remarked here, however, that many of Enderlein's cytological observations have al- 
ready been confirmed by Schumacher/ And in this case the confirmation is the more 
valuable since Schumacher was not acquainted with the work of Enderlein at the time 
of his own studies. 

The cytological aspects of microbic heredity have also been furthered in recent years by 
the valuable researches of Mellon in a series of contributions extending over many years. 
Among other matters of importance, Mellon's investigations have dealt especially with a 
mode of reproduction involving conjugation and zygospore formation, following many of the 
details of isogamic conjugation in higher forms. These zygospore-like bodies are probably 
identical with similar forms pictured less clearly by many earlier workers; perhaps with the 
Pettenkofer bodies described more recently by Kuhn.^ Mellon conceives that the origin of 
the zygospore is through the fusion of adjacent cells of a filament, sometimes indirectly by 
means of a peduncle. The bodies seem to be formed equally among the small and the large 
rodlike elements in the culture. They are often small, but in certain diphtheroids, as ob- 
served by Massini and by Mellon,^ and in B. diphthcriae (Park No. 8), as observed by my- 
self,-* they may attain a diameter of 6-7 /i. By favorable staining they usually reveal a 

' Schumacher, Josef.: Centralbl.f. Bakleriol., Abt. I, Orig., 97, 81. 1926. 

^Kuhn, Philaethes: Centralbl.f. Bakleriol., Abt. I, Orig., 93, 280.* 1924. See also: Arch. f. 
Schijfs- u. Tropen Hyg., 30, 133. 1926. 

3 Massini, R.: Arch.f. Hyg., 61, 250. 1907; Mellon, R. R.: /. Bad., 2, 81. 1917. 

4 Hadley, Philip: loc. cit. 



PHILIP HADLEY 91 

cluster of nucleus-like granules. Mellon has also recorded the liberation of large numbers of 
minute and apparently motile granules from the "giant coccus" forms. This has also been 
reported by Kuhn/ who has presented beautiful micro-photographs of these cells (Petten- 
kofer bodies) both before and after the liberation of the minute granular bodies. From his 
work it appears that these small forms often fail to stain by the ordinary methods, but that 
the Giemsa stain is especially favorable. It may be added at this point that Kuhn sees a 
relation between the presence of these bodies and the ability of the culture containing them 
to generate the bacteriophage; and this is quite in harmony with the theory of transmissible 
autolysis which I have proposed in an earlier paper. 

Regarding the development of the zygospores, Mellon has observed that in some cases 
they appear to undergo a double segmentation and yield a large coccus form like that often 
encountered in dissociating cu tures of B. diphthcriae. In such instances Mellon regards the 
zygospores as transition Anlagen for the development of a new form of culture, the exact 
nature of which will be determined by the environment surrounding the germinating zygo- 
spores. 

Finally, with reference to the hereditary mechanism of bacteria, there should be 
mentioned the formation of symplastic structures such as those first described by 
Jones^ in 1913 and 1920 for Azotobacter, and confirmed by both Lohnis-* and Ender- 
lein.'' Briefly, the reaction involves the fusion of a mass of bacteria into a single group 
in which the cell boundaries are lost and a union of nuclear elements occurs. From 
such symplastic structures arise new individual cells. 

THE CULTURAL APPROACH 

As early as 1888 observations dealing with several different and more or less per- 
manent culture types arising from pure cultures had been described by Firtsch^ for 
V. proteus, and in 1895 Dyar^ gave a report on changes of a somewhat similar nature 
for B. lactis erythrogenes. In later years other investigators presented other instances 
in which striking departures arose from the long-assumed "normal" and constant 
type. Many of these instances we owe to the splendid researches of Eisenberg.'^ It re- 
mained for Baerthlein,* however, in 1918 to point out the frequent occurence of such 
variations, their cultural, biochemical, and — to a limited extent — serological reactions. 
The primary basis for Baerthlein's important study was colony variation, the signifi- 
cance of which had been clearly seen by Firtsch in the case of a single species, Baerth- 
lein showed that plating from old laboratory cultures commonly resulted in the ap- 
pearance of colony forms quite unlike that of the original culture. Sometimes only 
one or two colony variants were encountered; at other times, four or five of them. But 
the most important and significant feature of Baerthlein's study was his demonstra- 
tion that, commonly associated with colony variation, were variations in other char- 
acteristics — morphological, biochemical, and serological. 

' Kuhn, Philaethes: loc. cit. 

^ Jones, D. H.: loc. cit. 

3 Lohnis, F. : loc. cit. 

'' Enderlein, G.: Bakterien-Cyclogenie. Berlin, 1925. 

5 Firtsch, G.: Arch.f.Hyg.,8,s(>g. 1888. 

^Dyar, H. G.: Ann. N. Y. Acad. Med., 8, 322. 1895. 

' See Hadley, Philip: loc. cit. ^ Baerthlein, K.: ibid., 81, 369. 1918. 



92 DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR 

It is doubtful if Baerthlein appreciated the full significance of his observations. 
For some time at least he believed that his various culture types represented merely 
"mutations," such as had been described earlier (especially in secondary colony forma- 
tion) by Neisser,' Massini,^ Miiller,^ Tenfold,^ Thaysen/ Burri/ Eisenberg/ Leding- 
ham," and many others. It thus remained for Arkwright/ in 1921, to grasp more fully 
the significance of Baerthlein's work. Among members of the colon-typhoid-dysentery 
group Arkwright noted particularly two colony forms which occurred in each species 
with marked persistency. One was round, regular, opacjue, and characterized by a 
smooth, glistening surface; the other was flat, irregular, translucent, and showed a 
rough or sandpaper-like surface. The former was termed the "S" type (smooth), the 
latter the "R" type (rough). The S type culture, on aging, transformed readily into 
the R. The latter, however, held to its new characteristics with considerable tenacity. 
While the S type culture grew in broth with a homogeneous clouding, the R type gave 
an agglutinative or sedimentary form of growth. These two culture forms were ob- 
served by Arkwright in B. coli, B. typhosus, and B. dysenteriae. His splendid and far- 
seeing work marks the beginning of a new epoch in the study of bacterial variation. 

In 1921 De Kruifs also made, independently, a contribution of fundamental im- 
portance dealing with dissociation in the rabbit Pasteurella type. Bad. lepisepticum. 
In this species he observed two forms of culture, differing from each other in colony 
form, manner of growth in broth, serological and immunological reactions, and par- 
ticularly in virulence. De Kruif designated these two types "D" and "G," respectively. 
As we can now see, his D form was analogous to Arkwright's S, while his G form was 
analogous to Arkwright's R. In De Kruif's experience, while the D type culture was 
highly virulent for rabbits, the G form possessed little, if any, virulence. Moreover, 
while the D form in the killed state was of little value as an immunizing agent, the G 
form, living, when injected into rabbits even in small doses, produced immunity to 
large amounts of virulent culture. Even one dose secured these results. 

Since the important works of Arkwright and De Kruif in 1921, the same line of study 
has been carried into many other fields: to the streptococci by Cowan*; to the pneumococcus 
by Griffith^, also later by Reimann^ and by Amoss^; to B. typhosus and B. enteritidis b\' Ark- 
wright and Goyle,9 and by Goyle'" alone; to the Salmonella forms by White," and by Topley 
and Ayrton"; to B. cholerae suis by Orcutt"; to the cholera vibrio by Balteanu'^; to FriedLind- 
er's bacillus by Julianelle'^ ; and to B. coli quite recently by Dulaney.'^ De Kruif's work has 

' Neisser, M.: loc. cit. ^ Massini, R.: loc. cit. ^ Hadley, Philip: loc. cit. 

'> Arkwright, J. A.: /. Path. &° Bad., 24, 36. 1921. 
sde Kruif, P.: J. E.xper. Med., 33, 773. 1921; also 35, 631. 1922. 
' Cowan, Mary: Brit. J . Exper. Path., 3, 187. 1922. 

^ Griffith, F.: Public Health and Medical Subjects, Ministry of Health, Rep. 18. London, 1923. 
* See Hadley, Philip: loc. cit. 

' Arkwright, J. A., and Goyle, A. N.: Brit. J. Exper. Path., 5, 104. 1924. 
'0 Goyle, A. N.: /. Path, c^ Bact., 29, 149. 1926. 

" White, P. B.: Special Rep., Med. Research Council, No. 91. London, 1925. 
" Balteanu, I.: /. Path, b' Bact., 29, 251. 1926. 
■3 Julianelle, L. A.: /. Exper. Med., 44, 683. 1926; also p. 735. 
'■* Personal communication. 



PHILIP HADLEY 93 

been followed by Webster into lines of considerable interest. The study of Theobald Smith 
and Gladys Bryant' on a "mutating" form of B. coli may also be mentioned as an instance 
dealing with the dissociative reaction, although the authors did not emphasize the relation. 
Gratia at an earlier date had made somewhat similar, though less detailed, observations on 
the same species. The chief results of all of these studies have been to demonstrate with ever 
increasing clearness the new characteristics possessed by the recognized variants; also to 
validate many earlier observations of a similar nature, but manifestly concerned with the 
same phenomenon. In addition, the more recent studies, besides depicting the S and R forms, 
have presented evidence for the existence of the third significant culture type, the O form 
(intermetliate), also recognized by earlier workers, lying as a transitional form between S 
andR. 

COLONIAL, CULTURAL, AND MORPHOLOGICAL ASPECTS 
OF MICROBIC DISSOCIATION 

As has already been pointed out, microbic dissociation manifests itself in varia- 
tions in colony form, in cultural growth, in comparative cytology, in cell morphology, 
in biochemical reactions, in immunological reactions, and in virulence. In the present 
section we shall consider some of the details relating to the first three of these points, 
with the attempt to indicate that the variations and correlated characters observed 
are not the result of chance, but depend on certain laws governing the transformations 
in widely separated bacterial species.^ 

1 COLONIAL ASPECTS 

As Firtsch^ clearly suggested by his remarkable study of variation in V. protcus 
in 1888, colonial variation is the most fundamental, consistent, and clearly observed 
phenomenon in dissociative variation. Each bacterial species possesses, not one "nor- 
mal" colony form, but a variety, each of which one must be able to recognize before 
he can affirm that he knows the "species." Each of these forms is determined by the 
stage of cyclogeny attained by the individual cells that comprise the colony structure. 
The time is now past when similarity in colony form must be taken as evidence of the 
close relationship of the organisms contained; or when dissimilar colony types must 
be regarded as indicating unrelated species. The fact of the matter is, that the de- 
gree of variation in colony form in one and the same species may be, and usually is, 
greater than the degree of variation in colony form of equivalent cyclostages of clearly 
distinct species. The diverse colony forms observed within pure lines of B. subtilis 
(Soule),4 of B. anthracis (Preisz,^ Wagner, Nungester*), of hemolytic streptococci 
(Cowan),'? of S.fecalis (Faith Hadley),^ of the meningococcus and gonococcus (Atkin),^ 

' Smith, T., and Bryant, Gladys: /. Exper. Med., 46, 133. 1927. 

^ It is impossible to consider the biochemical, serological and immunological aspects within the 
limits of this chapter. 

3 Firtsch, G.: loc. cit. 

'•Soule, M. S.: Jour. Inject. Dis. 1928, No. 2. 

5 Preisz, H.: Centralbl.f. Bakteriol., Abt. I, Grig., 35, 280. 1904; also 53, 510. ign. 

'Nungester, W.: Proc. Soc. Exper. Biol, b' Med., 24, 959. 1927. 

'Cowan, Mary: loc. cit. 

* Personal communication. 

' Atkin, E. E.: loc. cit. 



94 DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR 

of the pneumococcus (Griffith,' Reimann,^ Amoss^), of B. diphtheriae (Corbett and 
Phillips),^ of Sp. finkler-prior (Firtsch)," and of V. cholerae (Eisenberg,^ BalteanuO 
differ from one another to such a degree that, on morphological grounds, all of the 
variants would be regarded as contaminations — indeed, often have been so regarded 
and treated accordingly. As one illustration of this, and as Soule^has already pointed 
out, it may be noted that the R type colony of B. subtilis on plates is almost indistin- 
guishable from the R type colony (Medusa-head type) of B. anthracis. On the other 
hand, one may conclude from the studies of Nungester^ that the S type anthrax 
colony is one that few investigators have ever seen, or at least recognized; and one 
which would commonly be taken as a contamination, so different is it from the common 
Medusa-head type. It is thus a rather curious fact that, while it is the S colony form 
of B. subtilis that has come to be regarded by bacteriologists as the "normal," it is 
the R type colony of B. anthracis which, during the fifty years of study that this 
species has received, has become established as the "normal" form of culture. 

In most bacterial species for which knowledge is available, the colony of the S type is 
smaller and more delicate than the other forms. It is round, even, usually opaque (even when 
young), and, in certain species such as those of the intestinal group, the streptococcus, the 
pneumococcus, the proteus, the pneumobacillus, and others, presents a glistening luster or 
"moist" appearance. In addition, a distinct fluorescent effect by transmitted light is usually 
seen (intestinal group, pneumobacillus, proteus, and others). The co'ony consistency is 
commonly soft or butyrous. Such colonies, after four or five days' growth on rich and some- 
what alkaline agar, often show pale or translucent, wedge-shaped invaginations where disso- 
ciation into the O or R types is under way. Culturing from these "blue" areas will give cul- 
tures of a quite different type from the original, but usually not well stabilized at this stage. 
Repeated culturing, accompanied by colony selection, will increase the stability of the (com- 
monly obtained) R form. 

The R type colony presents, in most species, a quite different appearance from the S 
form. It is usually larger, irregular in shape, uneven; when young it is thin and translucent, 
and reveals a distinctly rough surface, or sometimes merely a dull luster as in B. subtilis 
(Soule). The fluorescent effect is invariably lacking. Old colonies, however, may become as 
opaque as those of the S form. The consistency of the R colonies is sometimes similar to that 
of the S form, but often, and when they are well stabilized (as in the "extreme" R), they may 
be hard or even brittle, so that they may be pushed about over the agar surface. These have 
been observed in the pneumococcus (Griffith) and in the streptococcus (Cowan). One curious 
feature of the R type colonies of several species (B. diphtheriae, B. mallei, B. proteus, V. 
cholerae, V. proteus, B. mesentericus, meningococcus, 5. fecalis, and probably other species) 
is that they may take on a yellow or brown chromogenesis. Apparently a similar phenom- 
enon occurs in the fungus of blastomycosis (Mellon).* I have also observed brownish colo- 
nies arising in the dissociation of M. citrcus. 

Regarding the colonial features of the type O cultures, first clearly pictured by Firtsch 
in 1888 for V. proteus, we have less knowledge. In general, the O colonies are larger than the 
S, round, even, smooth, glistening, but more fleshy and convex, simulating the colonies of 

' Griffith, F.: loc. cit. 

2 See Hadley, Philip: loc. cit. «> Soule, M. S.: loc. cit. 

3 Corbett, L., and Phillips, G.: /. Path. &' Bad., 4, 193. 1897. 

-• Firtsch, G.: loc. cit. ' Nungester, W.: loc. cit. 

5 Balteanu, I.: loc. cit. * See Hadley, Philip: loc. cit. 



PHILIP HADLEY 95 

B. aerogenes. They often manifest a mucoid consistency. Cultures of this sort commonly ap- 
pear in members of the colon-typhoid-dysentery group; also in B. anthracis, S.fecalis, and 
probably other species. In B. anthracis some of these colonies are slimy and readily coalesce 
on the agar plate. Similar colonies have been reported for various species among cultures 
resistant to the bacteriophage. In B. proteus the equivalent oMhe S form yields a spreading 
growth while the growth of the O type is restricted. In several species, such as pneumococ- 
cus, streptococcus, meningococcus, and gonococcus, the O type has not been described 
clearly. In general, it is highly unstable and may sometimes transform with great rapidity 
into the R. Such cultures have been termed "suicide cultures." They are very difficult to 
maintain on agar, and it is sometimes impossible to cultivate them in broth. It is reason- 
able to believe that the O colony form is at some time present in cultures of all bacterial 
species, but may pass unobserved. Certain members of the intermediates, such as those 
representing the Pettenkofer bodies of Kuhn, may play an important role in the phenom- 
enon of the bacteriophage, as I have pointed out elsewhere.' 

Although it often appears that the three chief colony types (S, O, and R), but particu- 
larly S and R, are clear cut in their essential features, and usually quite stable on appropriate 
medium, in other cases there may be observed distinct intergradations; and these may take 
on various aspects. For example, the S type colony may show, about its edge, either at a 
single point or about its entire circumference, an outcropping of the R type culture. Inter- 
esting examples of this have been described by Soule for B. subtilis, and observed by Nun- 
gester for B. anthracis; also by Faith Hadley for S. Jecalis.^ The fairly smooth and circum- 
scribed S colony, possessing (subtilis) the shallow fringe of filaments extending outward from 
the border ("bayonet-front" effect), sends out longer outgrowths which soon begin to curl 
under and to give a marginal appearance which simulates that of the R type anthrax colony 
(Soule). This outgrowth is bluish and translucent by transmitted light and rough by re- 
flected light. The result of this reaction is to yield an S type colony imprisoned within a ring 
of R type culture, the breadth of which varies with conditions which cannot be considered 
here. The type S anthrax colony may undergo transformations similar to those mentioned 
above (Nungester). Such outgrowths have been termed the "halo" or "regeneration fringe." 
In some cases, as in B. proteus for example, the halo may be made up of O type rather than 
S type culture. Indeed, there may be a series of alternate dissociations and recoveries, fol- 
lowing each other at intervals of a few hours, in the growth of the colony, the final effect 
being the production of the typical "ring growth" characteristic of B. proteus. 

But the O type colony also may send out such regeneration fringes; and in this case the 
nature of the fringe growth seems to depend on the degree of stability of the O colony. If it 
is in the early intermediate state, it usually forms a halo of normal S culture. If it is in the 
late intermediate state, it is more likely to send out a fringe of R type culture. Under these 
conditions, culturing from the center or from the edge of the colony will yield cultures of two 
different forms. The natural destiny of the type O culture is apparently to attain the R; and 
it usually accomplishes this in the course of time. It is often difficult to maintain the inter- 
mediate form in this culture state. The O form of B. proteus seems to be unusually stable, but 
here the R form has not been recognized with certainty. It may be added here that it has 
not been observed clearly that the type R culture gives regeneration fringes. It may, how- 
ever, as we shall see later, produce clusters of S type secondary colonies which usually appear 
on the free edges of growth; and these are analogous to the fringe, since it can be observed 
that it is by the coalescence of numerous colonies appearing at the margin of growth that the 
distinct fringes are produced. 

' See Hadley, Philip: loc. cit. Also Arch, of Path, and Lab. Med., 1928. In press. 

^ Personal communication. 



96 DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR 

Another manner in which dissociative reactions may reveal themselves in cultures 
is, as just intimated, the generation of secondary or "daughter-colonies" occurring in 
a background of primary culture. Instances of this phenomenon were given added 
significance in 1906 and 1907 through the observations of Neisser and Massini on B. 
coll mutabile, and similar observations were soon made on many forms. All these cases, 
manifesting the spontaneous origin of new culture types within the old culture mass, 
were quickly seized upon as indications of true mutations among the bacteria; and 
this false conception persists in the minds of many bacteriologists, even at the present 
day. 

Secondary colonies may be few or numerous ; sometimes one only, or again there 
may be several hundred discrete bodies. As the number increases, howexer, they 
blend more and more into the mass of mother culture and thus lose their colonial iden- 
tity — at least macroscopically. Microscopically they may still be traced as "granula- 
tions" of varying size, until the culture mass becomes a fine mosaic of the two or more 
culture elements. Sometimes the secondary colonies arise near the surface and form 
the well-known "papillae." Again they lie deeply imbedded in the culture, or even 
in the medium itself, as in the case of S.fecalis, and do not register on the contour of 
the primary colony. In some species (S.fecalis — Faith Hadley)' both forms of second- 
ary colony may be observed, and in this case they appear as entirely different culture 
types. 

The most common forms of secondary colony relate to centers of O or R type 
culture arising in a background of S culture. On the other hand, it appears from the 
older work of Preisz- and Pesch-' on B. anthracis, and from the more recent studies of 
Anna Dulaney on B. coli, that secondary, S type colonies may arise in R type cul- 
tures. In Dulaney's case they were generated particularly at the free margins of the 
type R colonies after a prolonged growth. Tertiary colonies arising within the second- 
ary have also been occasionally observed, particularly by Preisz^ for B. anthracis. The 
whole subject of secondary and tertiary colony formation may possess additional in- 
terest in its bearing upon the nature of the bacteriophage reaction, and with special 
reference to the homogamic theory which I have briefly outlined in a previous publi- 
cation.'' Here it was suggested that the resistant colonies which d'Herelle, Bordet, and 
others have termed "secondaries" (arising in the lytic sites on agar, or in culture fil- 
trates) may, in reality, be tertiary colonies arising after the disappearance of the sec- 
ondaries. In this case the characteristic lytic plaques would be regarded as the sites 
of disappearance of colonies of the secondary type, which itself might represent one of 
the intermediate forms of culture. 

For the present we may leave the subject of the secondary colonies with the con- 
clusion that these formations are of considerable significance as indicating that, hidden 
in the mass of mother culture, regardless of the type concerned, there may exist cer- 
tain centers where small or large groups of organisms, quite different from the mother 
culture in form and physiology, have arisen and where they are carrying on their inde- 

' Personal communication. 

'Preisz, H.: Cenlralbl. f. Bak'.eriol, Abt. I, Orig., 35, 280. 1904; also 53, 510. 1911. 

3 See Hadley, Philip: loc. cil. ■* Personal communication. s Preisz, H.: loc. cit. 

^ See Hadle}', Philip: loc. cil. See also: Arch, oj Path, and Lab. Med. 1928. In press. 



PHILIP HADLEY 



97 



pendent activities. When removed and purified by plating methods, they afford new 
forms of culture which sometimes manifest considerable permanence in their newly 
acquired characters. It is these forms that have erroneously been regarded as mu- 
tants. 

CHANGES IN CELL MORPHOLOGY 

Closely associated with the specific colony type, and in all probability determin- 
ing, within limits, its characteristics, are to be observed some fairly distinct morpho- 
logical characteristics of the cells and their organelles. Microscopic study of the type S 
culture, which usually passes under the appellation of "normal culture," although ex- 
ceptions have been noted, usually reveals a preponderance of those cell forms which 
are regarded as characteristic of the "species" in question. The type R culture, on 
the other hand, is likely to present a different picture; but this, in turn, varies with 
the species. In the members of the intestinal group the well-stabilized R type cell is 
most often coccoid, and a similar shortening is characteristic of the R forms of the 
diphtheria bacillus, the plague bacillus, probably of the tubercle bacillus and of several 
other species. In other cases, as in B. subtilis, B. anthracis, and perhaps in all the spore- 
formers (none of which, with the exceptions noted, has been carefully studied), the 
R type are much more elongated than the S type cells and are sometimes distinctly 
filamentous. 

The nature of the cell population of the O t^-pe cultures is not so clearly recog- 
nized. But it may be affirmed that, in comparison with the S and R, it is highly di- 
verse. It is particularly in cultures of the O type that one finds amassed those peculiar 
cell bodies which, for many years, have been termed "involution forms." They con- 
tain long, swollen rods, filaments (often fungoid in nature), giant coccoid bodies, 
apparently identical with Kuhn's Pettenkofer bodies, zygospore-like bodies, and often 
numerous minute granules, the exact nature of which is perhaps still in doubt, although 
it seems probable that some of them arise from the zygospores. The whole picture is 
extremely bizarre but is fairly constant for the intermediate type cultures of many 
bacterial species. Such forms have been produced by Kuhn and others by growth on 
media containing traces of lithium chloride. But it is sufficient to convince us that, 
under the cover of these bacterial monstrosities, are proceeding reproductive events of 
which we have, as yet, slight cognizance. Of one point, however, we may be assured. 
These peculiar forms are not "pathological" nor evidences of degeneration. They have 
been termed "involution forms"; but, as Mellon has suggested, "evolution forms" 
would be more appropriate. The details of their production and reproductive be- 
havior constitute at present one of the most important problems in microbic dissocia- 
tion. It seems possible that in their action is hidden the problem of the filtrable forms 
of bacteria, if not of the filtrable viruses; also perhaps the mystery of the bacterio- 
phage. 

Just as the morphology of the bacterial cell is correlated with the type of culture, 
so also are correlated motility, capsule formation, and perhaps spore formation. The 
time is past when we can state with discretion that such and such a bacterial "species" 
is motile, for both motility and flagellar equipment depend on the cyclostage. Up to 
the present time, observations seem to indicate that, if an organism shows motility, 
it belongs to the S type as opposed to the O or R, which are commonly non-motile. Ark- 



98 DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR 

wright,' however, has presented certain exceptions. Of this fact we now have evidence 
in members of the intestinal group, in B. siihtilis, B. proteus, and several other species. 
Motility of a culture therefore loses all significance for species differentiation in sys- 
tematic bacteriology, unless we can succeed in recoghizing the cyclostage with which 
we are dealing. 

With reference to bacterial capsules, the same situation exists as for motility. So 
far as we know at present, the capsule is the property of the organism of the S type, 
the R form being destitute. With reference to the anthrax bacillus, which presents 
certain other anomalies, the situation is not yet clear. The correlation is, however, 
now recognized for B. coli, the pneumococcus, Friedlander's bacillus, M. tetragenus 
and some Pasteurella forms. In none of these species does the presence or absenc.' 
of capsules possess significance for systematic bacteriology unless we can recogniz.^ 
the type of culture under examination. 

In addition to the foregoing, microbic dissociation manifests itself in important 
biochemical and serological differences in the dissociated cultures; also, in a striking 
manner with certain problems relating to virulence and immunity. These interesting 
aspects of the subject cannot be discussed within the limits of the present con- 
tribution.^ 

THE INCITANTS TO MICROBIC DISSOCIATION 
AND THE PROBLEM OF REVERSION 

Although dissociative reactions must be regarded as occurring to some degree in 
all bacterial cultures, probably beginning in the earliest hours of colony life, and al- 
though in some cases they attain spontaneously such a magnitude that they attract 
the notice of the alert investigator, fortunately, in the study of this phenomenon, we 
are not dependent upon cultural material showing such spontaneous transformations. 
The reaction may easily be "forced" as a result of bringing to bear on the young, grow- 
ing culture certain extraneous influences; and the nature of these influences may be 
diverse. 

Probably the first influence to attract attention was aging, as first pointed out by Firtsch 
for the spirillum of Finkler-Prior in 1888. Most of Baerthlein's colony variations, as also 
those of Eisenberg, were consequent to aging in broth or on agar, and to the use of alkaline 
media. Under these conditions, one may often discover in the transition from the S to the R 
forms the presence of the intermediate or transitional O, as clearly depicted by Firtsch, 
Eisenberg, and many others. Other conditions of cultural growth favoring the reaction, with 
the subsequent generation of the O or R forms of culture, include the following: changes in 
temperature, various food substances, starvation, the physical state of the medium (solid 
or liquid), the presence or absence of oxygen, the presence of antiseptic substances or dyes, 
the reaction of the medium, the volume of the medium, microbic associations, passage through 
animals, the influence of various kinds of normal animal blood or tissues, normal sera or 
ascitic fluid, specific immune blood or sera, body excretions or secretions, and finall\' the 
metabolic growth products of the same or other bacterial species. To these may be added 
the influence of the bacteriophage which, in last analysis, is the reagent par excellence for 
enforcing dissociation upon the sensitive, or even on the partially resistant, culture. Indeed, 
only one other influence approaches it in degree or speed of action — and that is homologous 
immune serum, as amply demonstrated by many experiments. 

' See Hadley, Philip: Joe. cit. ^ See chap, xlii 



PHILIP HADLEY 99 

Of the influence of these various incitants, only a few brief statements may here 
be made. First, dissociation cannot occur unless growth occurs; cultures in a state of 
suspended growth, while still alive, remain fixed in the type in which growth last oc- 
curred. This is not necessarily true of old cultures in which growth still takes place 
slowly. This circumstance reminds us of the conditions limiting the action and re- 
generation of the bacteriophage. Dissociation, moreover, occurs most freely in liquid 
media, and most actively at a reaction point of pH 7,8-8,0. This, it may be noted, is also 
the optimum reaction for bacterial autolysis in most species. In cultures frequently 
transferred on favorable solid media, dissociation is more restricted and may even ap- 
pear to be absent. When dissociation has occurred, and the R type has once been pro- 
duced, this type of culture is more stable than the S, and much more stable than the 
0, both on solid and in liquid media. The presence of phenol, pancreatin or lithium 
chloride, and other chemical substances, favors the transformation of the S type to 
the O type culture in some species. 

The influence of blood serum is of special interest. Sometimes a normal serum of cer- 
tain species will force the reaction. This is likely to concern the serum of animals which 
are not susceptible to the organism, or sera supposed to be germicidal. As mentioned 
above, however, the strongest dissociation-furthering power is possessed by homologous 
immune serum. This can be demonstrated by growing the S type culture for a few 
generations in broth containing about 10 per cent of the immune serum, the result being 
the formation of the R type culture. This has been demonstrated for the pneumococ- 
cus, the streptococcus, B. subtilis, Friedlander's bacillus, B. typhosus, B. paratyphosus 
B (SouleO, B, coli (Dulaney'), and some other forms. There is some evidence that 
the same reaction occurs in vivo as well as in vitro; and this suggests the possibility 
that the chief mechanism of protection of the bacteriotropic antibodies may repre- 
sent merely the enforcement in the body of a dissociation of the invading organisms, 
comparable to that observed in the culture tube. This possibility, which was first 
approached by the researches of Griffith on the dissociation of the pneumococcus in 
1923, 1 have considered at greater length in an earlier publication. With reference to 
the effects of immune sera on the R and S culture types, it should be added here that 
it has been shown for B. subtilis by Soule,^ for B. coli by Dulaney, for the pneumococ- 
cus more recently by Avery, and for B. paratyphosus B by Soule, that, when the R 
type culture is grown in serum immune to this culture form, a retransformation is 
enforced to the original S, 

When one observes that the dissociative reaction can be precipitated by such di- 
verse substances or conditions of growth as those mentioned above, the question of the 
actual cause of dissociation seems to be as far removed as at the beginning. But the 
situation may be simplified if we can discover a common factor among all of these in- 
fluences. It seems safe to say that there exists such a common factor, and that it may 
be defined as any condition or substance that is antagonistic to the continued growth 
of the so-called "normal" culture type. That this factor may not be the same for all 
bacterial species is a view that we may well accept, but that the same influence will 
operate in the same manner on different members of the same group of bacteria seems 
well demonstrated by numerous observations. 

' Personal communication, * Soule, M. S,: loc. cil. 



loo DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR 

Regarding the permanence of the dissociates, with their newly acquired char- 
acters — it has of ten been stated that they are irreversible; and, for this reason, they 
have often been described in the literature as mutations. This interpretation has con- 
cerned the O forms much less often than the R, for the former are notably unstable. 
Even the R types represent a series in which the degree of stability is very variable. 
It seems to depend, in part, upon the length of time that the R culture has been prop- 
agated continuously under the conditions that gave rise to it. The longer the stimulus 
is applied, the more stable the type seems to become. There are, however, some ex- 
ceptions to this. Some R type pneumococcus cultures are apparently irreversible; and 
the same is true of some R type Friedlander strains, according to Julianelle.' The R 
form of B. paratyphosus, B. typhosus, B. sicipestifer, B. dysenteriae, Bad. lepisepticum, 
B. diphtheriae, B. subtilis, and B. anthracis have been found highly stable, and have 
sometimes been reported as permanent variations. But Jordan,^ by a special method 
of cultivation, succeeded in causing the reversion of his R type of B. paratyphosus B; 
end Soule, by growth of his R type of B. subtilis in an R immune serum, was finally 
able to cause a return to the S type of culture. Later he was able to cause the reversion 
of an R type of B. paratyphosus B by similar measures. Mellon^ has emphasized the 
stabihty of variants of his diphtheroid forms, and I have been unable to effect the 
retransformation of an R type of B. pyocyaneus even after four years, although I have 
not tried the action of R immune serum. Apparently, the R forms of culture may 
remain stable for many years, but I believe there is no justification for the conclusion 
that they represent hereditary variations or mutations in the strict meaning of the 
term. It is more likely to mean that we have not yet been able to discover the 
adequate means for causing their return to the original form of culture. 

BIOLOGICAL SIGNIFICANCE OF DISSOCIATION 

In concluding this chapter the question arises. What is the deeper meaning of these 
phenomena concerned with the separation of bacterial cultures into distinct compo- 
nents whose nature and behavior we have now briefly reviewed? It can mean only 
one thing: that those living cells that we have commonly regarded in past years as 
among the simplest of plant forms, and characterized by a correspondingly simple re- 
productive apparatus, possess in reality a highly complex genetic mechanism, which 
enables them to reveal, in cultures, pictures of morphological and physiological di- 
versity with which our old and limited notions of "reproduction by simple fission" are 
utterly unable to deal. Although we may not yet be justified in accepting Enderlein's 
view of actual sexual reproduction among the bacteria, we must accept the fact that 
the nuclear equipment and reproductive behavior of bacteria are highly complicated 
matters. We can no longer doubt that the hereditary mechanism in bacterial cells 
makes provision for amphimixis, so long denied to these forms; nor can we hesitate in 
accepting phenomena of gonidia formation, zygospore formation, and perhaps a kind 
of budding, as common methods of bacterial reproduction. In all of these matters 
bacteriologists as a class have combined in denying the existence of things that they 
have not been willing to take the trouble to search for. 

' Julianclle, L. A. : loc. cit. 

^ Sec Hadley, Philip: loc. cit. ^ Mellon, R. R.: /. Med. Rcscirch, 42, 6i. 1920. 



PHILIP HADLEY loi 

The acknowledgment of the existence of definite cyclostages in the development 
of the bacterial culture naturally concerns our appraisal of the thing that has passed 
as the "normal" bacterial type. The observations already presented dealing with this 
subject can lead us only to the view that the older notions of normality and immu- 
tability of culture type have determined a highly repressive and dangerous influence 
on the development of bacteriology — an influence which, even at the time of writing, 
is still menacing the progress of the science. The present conceptions of "normal type," 
"normal colony," and "normal" cytology we owe to the influence of monomorphism 
which, even in most recent textbooks, still clings like a barnacle to modern bacteri- 
ology. According to its dictates, whatever departs from the "normal" must be re- 
garded as an "involution form," a degeneration form, a mutant, or a contamination. 
On the other hand, whatever form of culture the bacteriologist succeeds in causing to 
develop most freely in his carefully standardized media, and under other standard- 
ized conditions of growth which he imposes, are "normal" cultures, while aberrant 
forms are of little consequence. In order that there may be no lack of means for mak- 
ing the species recognizable, we assiduously fill out data on neatly designed descriptive 
charts — all dealing with the "normal" type, usually the S. Are we, then, forced to the 
conclusion that cultures of the and R forms are not normal cultures? 

It is indeed time that we revised our notions on "normality" in bacterial species. 
The results of many studies dealing intentionafly or unintentionally with microbic 
dissociation force the conclusion that there is no such thing as "normal" type in the 
usual meaning of the term. The O type cultures are no less normal than the S; nor 
the R type cultures less normal than the O. They are all normal, and can be regarded 
in no other light than that of isolated states or stages of the bacterial species in its 
progress through the cyclode. And it may be said in passing that this view may no 
doubt be held equally for the filtrable forms of bacteria. These have often been re- 
ferred to as "fragments," or as minute "fractions," of the "normal" cell, and endowed 
with the power of "regeneration" into the original form. We shall perhaps do well to 
question whether these minute living elements may not represent one or more definite 
units (cyclostages) in the reproductive history of the species. From this viewpoint, 
whether we deal with filtrable or non-filtrable forms, it is essential that we should 
cease to regard "normality" in the old, absolute sense, but should come to regard the 
characters of bacteria as related to definite stages in their development. Thus we may 
have a normal growth of B. typhosus on plain agar, or on phenol agar, or at 42° C, or 
in immune serum, or under reduced oxygen tension. The growth is normal with ref- 
erence to a certain condition or environment, although it is likely to differ in each of 
the conditions mentioned above. That "pathological" growth forms may occur can- 
not be doubted; but at present we are not in a position to recognize them — any more 
than we are in a position to recognize bacterial mutations, so called — until we have 
gained a fundamental knowledge of the nature, limits, and sec^uence of cyclogenic 
variation. 



CHAPTER VIII 
BACTERIAL ASSOCIATIONS 

W. L. HOLMAN 
University of Toronto 

INTRODUCTION 

Bacterial association has taken in recent years a much more important place in 
bacteriological studies than formerly. This increased interest is largely due to the 
greater attention which is being given to the finer metabolism of bacteria and the in- 
teractions which occur between the bacteria and their environment. The idea long 
held by many that bacteria represent the lowest forms of life and are therefore com- 
paratively simple in their metabolic activity has been replaced by a realization that 
we are dealing with just as highly specialized and complicated functional activities 
as in any of the so-called "higher" plants or animals. It is true that we are dealing 
with unicellular forms of very small size and that these as suggested by Kendall' 
should be considered as similar to living colloids in which surface phenomena are so 
important. Many of the activities in the life of the bacteria can be appreciated better 
if this complicated metabolism is kept in mind, and the study of mixed or double cul- 
tures helps in an understanding of the actual life-processes. 

NOMENCLATURE 

It is generally recognized but often forgotten that under natural conditions mixed 
cultures are the rule, and the earliest work on bacterial associations is to be found 
largely in the studies which attempted to analyze such natural phenomena. The most 
variable results may be obtained in these mixed cultures, and there are many factors 
taking part which determine the final outcome. There may be simple mixtures with 
no demonstrable effect of one bacterium on another, but this is uncommon since one 
or the other usually dominates the picture. One microbe may favor the growth and 
activity of another or both may be benefited by the combination. The latter condi- 
tion is usually spoken of as "symbiosis," but true examples of this relationship are 
rare. The term "metabiosis" is sometimes used where one action follows another, and 
is well illustrated in innumerable examples in nature. Antagonism or antibiosis is 
often combined with the foregoing, but is mostly employed for the occurrences where 
there is a clearly demonstrable harmful effect of one micro-organism on another or 
when a characteristic product fails to be formed or disappears in the mixed culture. Be 
cause of the impossibility in many cases of determining the actual processes at work 
these terms must be used with reservations. It is better, I believe, to use the more 
general word "association" for all these phenomena and "synergism," introduced into 
bacteriological nomenclature by Kammerer,^ for those in which definite changes are 

' Kendall, A. I.: Colloid Symposium Monograph. 2, 195. 1925. 

^ Kammerer, H.: Klin. Wchnschr., 2, 1153. 1923; Deutschcs Arch.f. klin. Med., 141, 31S. 1923: 
ibid., 145, 257. 1924; Klin. Wchnschr., z, 723. 1924. 



W. L. HOLMAN 103 

demonstrable which indicate or suggest the combined work of two or more micro- 
organisms. Zoeller' has used "cumulative cultures" to express the results obtained by 
him in certain biological combinations. Synergism may be conveniently qualified, 
when one or the other result dominates, into an "antagonistic synergism" and a "be- 
neficent synergism." "Antagonism" alone should probably be retained for outstand- 
ing examples of one-sided harmful effect. 

GENERAL CONSIDERATIONS 

There are numerous examples of bacterial associations in every field of bacte- 
riology. In man and animals natural infection with more than one bacterium is rela- 
tively frequent, and the particular combinations which may occur often determine the 
course of the disease. There is an enormous literature on this phase of the subject but 
no very definite conclusions have been drawn. It would be futile to go into the prob- 
lems of tuberculosis and secondary infections, or those of typhoid fever, gonorrhea, 
influenza, and many other diseases. Certain phases of these I shall briefly discuss, but 
this article will deal largely with some of the outstanding phenomena studied experi- 
mentally, and I shall not confine myself to the pathogenic bacteria although the 
greater amount of work has been done on them. 

EARLY EXAMPLES OF ASSOCIATION 

Among the early observations we find the recognition by Pasteur'' of the harmful 
effect of "wild" yeast on the normal fermentation processes in the beer and wine in- 
dustries. He further noted the beneficial effect of aerobic forms which developing a 
scum on the surface, and using up the oxygen, favored anaerobic growth. Winogradsky^' 
isolated an aerobe which only fixed nitrogen from the air in the presence of other bac- 
teria. Burri and Stutzer^ demonstrated that horse feces split nitrate with the produc- 
tion of free nitrogen. He isolated from the feces B. coli communis and a strict aerobe, 
and these two in combination gave the same result. The B. coli could be replaced by 
B. typhosus, and therefore it was the strict aerobe which gave the actual gas produc- 
tion. Previous to this, Marshall Ward^ described a yeast and a bacterium which to- 
gether formed a ginger beer-like product in a saccharine fluid. 

ANAEROBES 

Nencki* reported, with a double culture of B. paralactici and B. chauvoei the for- 
mation from glucose of normal butyl alcohol, a substance not produced by either cul- 
ture alone. He believed his results might help to clarify certain difficulties in obtain- 
ing infections in animals with single pure cultures. Novy^ quoted Roger (1889) as 
the first to show that B. prodigiosus added to the bacillus of malignant edema ren- 

' Zoeller, C: Compt. rend. Soc. dc bio!., 92, 435, 497, 686. 1925. 

^ Pasteur, L.: Oeuvres de Pasteur (reunies par P. Vallery-Radot). Paris, 1922. 

3 Winogradsky, S.: Compt. rend. Acad, de sc., 118, 353. 1894. 

■* Burri, R. and Stutzer, A.: Centralbl. f. Bakteriol., I, Orig., i6, 814. 1894. 

sWard, Marshall: Phil. Tr. Roy. Soc, B, 187, 125. London, 1892. 

^Nencki, M.: Centralbl. f. Bakteriol., I, Orig., 11, 225. 1892. 

'Novy, F. G.: Ztschr.f. Hyg. u. Infektionskrankh., 17, 209. 1894. 



I04 BACTERIAL ASSOCIATIONS 

dered sublethal doses of this anaerobe fatal for rabbits, and he himself found that the 
injection into guinea pigs of B. proteus and his new anaerobe {B. oedcmatiens) resulted 
in rapid death and an enormous growth of the anaerobe in the animal body. The 
overgrowth was absent with pure cultures. He further was able, by adding B. proteus 
and other aerobes, to grow his anaerobe in the presence of air. Passing over numer- 
ous similar results we find Sturges' devising a method, based on bacterial association, 
for isolating spore-bearing anaerobes on open plates by growing them with B. coli 
or Staphylococcus aureus. Rhein^ used B. fecalis alcaligenes in bouillon for anaerobic 
growth because of the lack of saccharolytic and proteolytic activity in this aerobe. 
Inoculation of the mixed cultures into animals he considered practical because the 
aerobe is not toxic, but I believe from the work of many others that this procedure 
might well give faulty results. Barrieu^ noted that B. proteus and certain non-patho- 
genic spore-bearing aerobes found in wounds exalted, by their proteolytic activity, 
the virulence of pathogenic bacteria. Pringsheim^ grew Frankel's bacillus {B. welchii) 
with B. fecalis alcaligenes for ten transfers on agar slants and could see in the growth 
of the latter the opaque colonies of the anaerobe, A hquefying sarcina allowed B. 
welchii and B. hutyricus to grow in open tubes. After six days' growth the sarcina had 
disappeared from the B. butyricus culture, and he suggested this as an easy method 
to obtain a pure culture. Weinberg and Otelesco^ considered that many war-wound 
infections, looked upon as of pure anaerobic origin, may be due to an association with 
B. proteus since this latter organism increased the virulence of B. perfringens, V . 
septique, and others. Animals injected with B. sporogenes and B. proteus did not de- 
velop putrid lesions. This combined growth of aerobes and anaerobes on surface cul- 
tures I observed on a number of occasions in France while studying the bacterial flora 
of war wounds. Colonies picked from aerobic plates were not infrequently found to 
be mixed with anaerobes. 

Stillman and Bourn^ reported in 1920 the production of gas by 16 of 119 non- 
hemolytic strains of B. influenzae in i per cent dextrose agar with a little blood ex- 
tract. Four of 29 hemolytic strains also produced gas. Jordan and Reith^ also found 
gas production in certain of their strains. About four years ago, when working with 
cultures of a tiny anaerobe resembling B. pneumosintes^ (probably Staphylococcus 
parvulus of Veillon and Zuber), I mixed a culture of this anaerobe with a culture of 
B. influenzae and planted the mixture on blood-agar slants. A good growth of B. in- 
fluenzae occurred, and after five transfers I had no difficulty in recovering the gas- 
producing anaerobe in cooked-meat media. This anaerobe is practically always present 
in the oral cavity, and could easily contaminate cultures of B. influenzae. It can be 

' Sturges, Jr., W. S.: Ahslr. Bad., i, 63. 1917. 

^ Rhein, M.: Prcsse mcd., 27, 504. 1919. 

3 Barrieu, A. R.: ibid., 28, 40. 1920. 

"I Pringsheim, E. G.: Ccnlralbl. f. Bakleriol., II, 51, 72. 1920. 

5 Weinberg, M. and Otelesco, I.: Compl. rend. Soc. de biol., 84, 535. 1921. 

* Stillman, K. G., and Bourn, J. M.: /. Exper. Med., 32, 665. 1920. 

7 Jordan, E. O., and Reith, A. F.: 7. Infect. Dis., 34, 239. 1924. 

8 Holman, W. L.: .im. J. Ilyg., 3, 4S7. 1923. 



W. L. HOLMAN 105 

recovered from high dilutions of the saliva, as Hall and Wing' have shown, and be- 
cause of its morphological resemblance to forms of B. influenzae might very well be 
overlooked. I would suggest this as a possible explanation of the rather infrequent ob- 
servation of gas production by B, influenzae. 

The group of B. botiilinus and the effect of associated bacteria particularly on its 
toxin has received considerable attention. Hall and Peterson^ found that certain acid- 
producing aerobes inhibited toxin production in glucose but not in non-carbohydrate 
media, and some of these aerobes actually destroyed toxin in glucose broth. It would 
appear that the acid must be in the nascent state since acid itself was inefTective. 
Jordan and Dack'^ found that a mixture of a large amount of B. sporogcnes with B. 
botulinus interfered with the development of toxin and might cause its early disap- 
pearance. Francillon'' studied the same problem. He found that Staphylococcus au- 
reus, B. coli, B. proteus vulgaris, and other bacteria permitted the growth of B. botu- 
linus in open tubes of plain and glucose bouillon, but the growth was never as good as 
under other anaerobic conditions. A moist-meat medium gave somewhat better 
growth. Toxin was found in the mixed cultures in bouillon and meat, the amount vary- 
ing with the aerobe. The B. pyrcyaneus mixture gave no toxin in the bouillon but a 
strong one from the meat. There was but little effect on the toxin by two weeks' con- 
tact with B. proteus, B. coli, or B. pyocyaneus. Dack^ reported the gradual destruction 
of filtered toxin by the growth of B. sporogencs and other proteolytic and non-pro- 
teolytic anaerobes. 

Passini'' found that a putrefactive anaerobe B. putrificus verrucosus destroyed B. 
tuberculosis in nine days. The effect of similar anaerobes on the survival of anthrax 
spores in dead animals has been extensively studied. Among a great many other in- 
teresting anaerobic and aerobic synergistic phenomena I mention a few. Ome- 
liansky^ studied the fixation of atmospheric nitrogen as Winogradsky^ had done years 
before. He noted that in the surface layers of the soil numerous organisms used the 
oxygen and created anaerobic conditions for the B. Clostridium pasteurianum, but in 
addition some of these accompanying forms also supplied carbon compounds for the 
anaerobe. The Azotcbacter being alkaligenic used up such products from the anaerobe 
as butyric acid and thus favored the synergistic process. The other aerobes may at 
times do harm by depriving the Azotobacter of oxygen. These two nitrogen-fixing 
forms, one aerobic, the other anaerobic, worked very well together. The work of Kam- 
merer'and his associates gave interesting examples of synergistic action. They observed 
that emulsions of human feces reduced pure bilirubin and mesobilirubin to urobilin 
but had no action on biliverdin and that the feces of herbivora did not have this action 

' Hall, I. C, and Wing, H. U.: Am. J. Pub. Health, 15, 770. 1925. 

^ Hall, I. C, and Peterson, E.: /. Bact., 8, 319. 1923. 

3 Jordan, E. O., and Dack, G. M.: /. Infect. Dis., 35, 576. 1924. 

■* Francillon, M.: Arch.f. Hyg., 95, 121. 1925. 

5 Dack, G. M.: /. Infect. Dis., 38, id^. 1926. 

^ Passini, F.: CenlralU.f. Bakterlol., I, 81, 447. 1926. (Ref.) ^r'* '» \. J( /*\ 

7 Omeliansky, V. L.: Arch, de sc. biol., 18, i. 1915. S^ -y^\ 

' Winogradsky, S.: /oc. a7. ' Kammerer, H.: /oc. a7. ' ,.^ O,' 




io6 BACTERIAL ASSOCIATIONS 

because of its active fermentation. Filtrates had no effect. These changes they be- 
lieved were due to a synergism between B. putrificus and certain aerobes. B. coli 
either helped the bilirubin production or hindered it, depending on the presence or 
absence of fermentable material. They further demonstrated the development of hem- 
atoporphyrin from blood by a similar synergism and that sugar or bile inhibited it. 
A particularly important instance of bacterial association was reported by Speakman 
and Phillips.' During the war, acetone and butyl alcohol were produced on a large 
scale by fermentation of cereals and carbohydrates. Serious difficulties developed in 
the plants, owing to the contamination of the cultures of B. granulohacter-pectin- 
ovorum by the aerobic bacillus B. volutans. The acetone yield, as a result of the 
mixed culture, dropped or disappeared and the development of lactic acid increased. 
This increase was due to an altered metabolism of the acetone producer so that it 
formed more lactic acid and less acetone. The results varied with the relative numbers 
of each organism present. They considered it due to an inhibitory substance from the 
nitrogen metabolism. In actinomycotic granules there is found a bacterium named 
by Klinger^ B. actinomycetum coniitans, the presence of which was confirmed by 
Colebrook^ in 80 per cent of his twenty cases. The significance of this associate is 
not known, but Colebrook suggested a possible genetic relationship. There are many 
other examples of anaerobic and aerobic associations in the natural metabolism of 
the sulphur bacteria; in silage fermentation, the heat of which was definitely shown 
by Hunter'' to be due to bacterial action and not to cell respiration; in sewage decom- 
position and cellulose destruction in which Groenewege^ believed a symbiosis occurred 
but stressed the action of the aerobes and Khouvine*" gave chief importance to a 
strict anaerobe discovered by him, B. cellulosae dissolvens (n.sp.), but pointed out 
that five times as much cellulose was de5tro3'ed when in association as when alone. 
There are other examples in the natural breakdown of organic materials of all kind 3 
and in innumerable other bacterial activities in nature. 

THE ACroURIC GROUP (aND CERTAIN THERAPEUTIC USES) 

The effects of the aciduric group of bacteria on other bacteria has been closely 
Studied. They are usually facultative aerobes, and their action is considered as chief- 
ly antagonistic. Because of their use for therapeutic purposes there has collected an 
extensive literature brought together by Rettger and ChepHn^ and Kopeloff.* Certain 
points, however, may be briefly reviewed. Starting from the work of Metchnikoff 
with B. bulgariciis it was soon found that this organism could not be implanted in ih^ 
intestinal tract and B. acidophilus, a normal inhabitant, was substituted. B. bijidus 

' Speakman, H. B., and Phillips, J. F.: /. Bad., 9, 183. 1924. 
» Klinger, R.: Centralbl.f. BaktcrioL, I, Orig., 62, 191. 1912. 
3 Colebrook, L.: Brit. J. Expcr. Path., 1, 107. 1920. 
••Hunter, O. W.: /. Agric. Research, 10, 75. 1917. 
s Groenewege, J.: reference in J .A.M. A., 76, 279. 1921. 

* Khouvine, Y.: Ann. de Vlnst. Pasteur, 37, 711. 1923. 

7 Rettger, L. F., and Cheplin, H. A. : The Intestinal Flora with Special Reference to the Implantation 
of "Bacillus acidophilus." Yale University Press, 1921. 

* Kopeloff, N.: "Lactobacillus acidophilus." Williams & W'ilkins Co., 1926. 



W. L. HOLMAN 107 

found in the intestines of breast-fed infants, is responsible for inhibition of the growth 
of other bacteria, and its acid products are thought to be mild stimulants to the bowel 
walls. In the vagina, B. doederleini is believed to keep the reaction acid, thus inhibit- 
ing the growth of contaminating bacteria. Landau' was the first to use fresh beer 
yeast in the treatment of leucorrhea and considered the anticatarrhal action was due 
to mechanical overgrowth, the using up of food material and the action of metabolic 
products in injuring or destroying other bacteria, neutralizing the toxins and chang- 
ing the reaction to acid. He suggested injecting cultures of this yeast into the bladder 
in cases of cystitis with alkaline urine. Since this work there have been many sug- 
gestions and a variety of bacteria used to obtain such biological inhibitory action. 
The antagonistic action of B, acidophilus has been abundantly proved against putre- 
factive anaerobes, B. coli, and many other bacteria. Schiller,^ with a strain of B. 
acidophilus from a dog, found that it rapidly destroyed and dissolved many strains 
of streptococci in fluid media and suggested this as a useful way to obtain bacterioly- 
sis of cocci. He showed that this action was not due to lactic acid since it occurred in 
alkaline media and filtrates from a glucose broth culture living or killed (by heat or 
age) allowed a good growth of the streptococcus. The harmful substance was only 
formed by the B. acidophilus in the presence of the streptococcus or its products. 
Filtrates of the mixed culture, after thirty-six hours at 37° C. (when the streptococci 
are killed), were as toxic as when living cultures of the bacilli were used. Strepto- 
coccus cultures killed by heat or age had no toxic effect on living streptococci. He 
considered the phenomenon an example of induced antagonism and reported other 
examples in a series of four articles.-' In the first he used B. mesentericus and forced 
on it an antagonistic action against streptococci by growing it with the latter in a 
medium of poor-food value. It secreted a bacteriolytic substance which digested the 
living bacteria as it would any other insoluble albuminous material, and the amount 
depended on the number of sensitive streptococci present. It also acted when the B, 
mesentericus had been removed by centrifugation and after evaporation and drying. 
It was not completely specific. Schiller further showed that yeasts can be made an- 
tagonistic against bacteria including B. tuber ctilos is if the medium contained sugars 
but lacked nitrogenous materials. They acted in the same way as the foregoing, and 
a more active bacteriolytic substance was secreted in the presence of more resistant 
forms so that the enzyme induced by B. tuberculosis was even capable of attacking 
beeswax. The reverse was also found. Bacteria (staphylococcus, B. typhosus, B. para- 
typhosus, et al.) became antagonistic to yeasts in nitrogen-free media, and the secreted 
cytolytic substance was similar to the foregoing but had no effect on coagulated serum 
or egg albumin. This method of dissolving the yeast membrane he thought might be 
of interest in the study of zymase. 

Donaldson^ during the war used a strain of B. sporogenes in the treatment of slow- 
ly healing war wounds. The beneficial effects he ascribed, not to direct inhibition, but 
to the removal by the proteolytic anaerobe of the dead tissue (the pabulum for the 

'Landau, T.: Deutsche »icd. Wchnschr., 25, 171. 1899. 

^Schiller, I.: Centralbl.f. Baktcriol., I, Orig., 73, 123. 1914. 

3 Schiller, I.: ibid., 91, 68. 1924; 92, 124. 1924; 94, 64. 1925; 96, 54, 1925. 

4 Donaldson, R.: /. Path. &° Bact., 22, 129. 1918. 



io8 BACTERIAL ASSOCIATIONS 

pathogens in the wound), and, itself producing no harmful products, it further hy- 
drolyzed the toxic bacterial products present or being formed. Bumm' used a durable 
preparation brought out by Zeissler called "neocolysin." It was made up of living, 
albuminolytic bacteria and gave good results in chronic purulent conditions such as 
osteomyelitis. The bacteria were supposed to function as in Donaldson's method and 
continued growing as long as there was dead tissue available. Gratia and Dath^ dis- 
covered an aerobic streptothrix which had a powerful destructive action on a variety 
of bacteria. It did not act on B. tuberculosis and showed no lipolytic enzyme. Fil- 
trates were equally effective, could act without free oxygen, but were somewhat 
variable. The active substance was better developed in old cultures and was fairly 
stable. The dissolved bacteria caused specific response when used for vaccination. 
They referred to a similar organism reported by Lieske in 1921, but his studies were 
confined to solid media. Rosenthal worked with an organism apparently very much 
like the foregoing, and found it was antagonistic to many bacteria including B. diph- 
iheriae. He referred to the report of Gasperini of 1890 on a similar form acting against 
bacteria. Rosenthal and his associates'" found that it could be implanted in the in- 
testines of guinea pigs and that when injected parenterally it was enterotropic. 
Much and Sartorius^ used a strain of B. mycoides and showed similar effects, by cul- 
ture and filtrates, on many bacteria and that the dissolved bacteria had not lost their 
antigenic properties. A very interesting study by Gratia and Rhodes^ proved that 
living staphylococcus could live on killed suspensions of staphylococci in saline or in 
saline agar made cloudy with killed staphylococci. Thus we see that bacteria can and 
do remetabolize their own substances or that of other bacteria, and this helps in the 
understanding of the antagonistic action of many forms of bacteria. They are indeed 
cannibalistic. 

DIPHTHERIA GROUP 

Because of the pressing problem in carriers of the diphtheria bacillus, special at- 
tention has been given to researches for a possible biological method, through bac- 
terial antagonism, which would be effective in treating these cases. Streptococci and 
B. diphtheriae have long been considered mutually helpful in producing severe infec- 
tions in the throat. There is an extensive literature on this topic. Roux and Yersin,^ 
in studying the problem of the return of virulence in attenuated cultures of B. diph- 
theriae, were successful in accomplishing this by injecting the attenuated culture 
along with a non-fatal dose of an erysipelas strain of streptococcus. The virulence 
returned, and it was retained on successive cultures. They therefore warned against 
the use of Streptococcus erysipelatos to combat diphtheria as had been suggested by 

'Bumm: Arch.f. klin. Cliir., 138, iii. 1925. 

^Gratia, A., and Dath, S.: Compt. rend. Soc. de hiol., 91, 1442. 1924; 92, 461, 1125. 1925; 
93, 451. 1925; 94, 1267. 1926. 

3 Rosenthal, L.: ibid., 93, 77. 1925. 

'•Rosenthal, L.: Hid., 94, 309, 1059, 1926; 95, 10. 1926. 

sMuch, H., and Sartorius, F.: Med. Klin., 20, 347. 1924. 

^ Gratia, A., and Rhodes, B.: Compt. rend. Soc. de biol., 90, 640. 1924. 

' Roux, E., and Yersin, A: Ann. de I'Inst. Pasteur, 4, 385. iSgo. 



W. L. HOLMAN 109 

Babtchinski, Similar results were obtained by Barbier^ and Schreider.* Funck^ found 
the fact to be true but did not consider it as striking as had previous workers, and 
showed that the presence of the streptococci in no way affected the specific action of 
the diphtheria toxin. Klein^ also showed that streptococci enhanced the effect of B. 
diphtheriae. Arnold^ found httle or no evidence of any increased virulence in the he- 
molytic streptococci isolated from diphtheria throats, but that there was a decided in- 
crease in hemolytic streptococci during diphtheria. These strains showed limiting 
H-ion concentrations like pathogenic strains, but he believed this change was merely 
environmental. Gate, Papacostas, and Billa,'' although they found that filtrates of 
avirulent streptococci stimulated diphtheria-toxin production, reported that the in- 
creased virulence was not retained on further transfers. Zoeller^ showed it was possi- 
ble to produce in his cumulative cultures a diphtheria-streptococcus altero-toxin by 
growing a scarlet fever streptococcus in a diphtheria toxin to which had been added 
a little horse serum. Stovall, Scheid, and Nichols* reported that the presence of 
staphylococcus in mixed cultures changed the morphology of virulent B. diphtheriae 
so that they stained more solidly and that the non-virulent pseudo-diphtheria strains 
became more beaded. Streptococci had no such effect. 

The well-known overgrowth of B. diphtheriae by Staphylococcus aureus in cultures 
led many workers to try such cultures in patients following the report by Schiotz.' 
Among these, Lorenz and Ravenel'" had good results in nine carriers and eight clinical 
cases of diphtheria although nasal furuncles developed in some of them. Rolleston" 
found it helpful in ten carrier cases but ineffective in two cases of nasal infection. He 
considered it should only be used in chronic cases. There were also a number of un- 
favorable reports such as that of C, M. Davis'^ who reported the development of ton- 
sillitis following the use of the staphylococcus spray. Nicholson and Hogan'-' were 
encouraged by the results on nine acute cases, using sprays of B. bulgaricus and sour 
milk. Papacostas and Gate''' studied the question of the antagonism between the 
pneumobacillus of Friedlander and B. diphtheriae following the observation that clini- 
cal cases of such mixed infections were usually mild. Mixed cultures of these two bac- 
teria showed a progressive predominance of the former on serial transfers and the 

' Barbier, H.: Cenlralbl. f. Bakteriol., I, Orig., 11, 382. 1892. (Ref.) 

^ Schreider, M. von.: ibid., 12, 289. 1892. 

3 Funck, E.: Zlschr.f. Hyg. u. Infektionskrankh., 17, 465. 1894. 

1 Klein, E.: Thirty-third Ann. Rep. Loc. Gov. Bd., p. 431. 1903-4. 

5 Arnold, L.: /. Lab. df Clin. Med., 8, 387, 389. 1923. 

^ Gate, J., Papacostas, G., and Billa, M.: Compi. rend. Soc. de biol., 90, 500. 1924. 

' Zoeller, C.: loc. cit. 

» Stovall, W. D., Scheid, E., and Nichols, M. S.: Am. J. Pub. Health, 13, 748. 1923, 

' Schiotz, A.: see Diphtheria, p. 367. London: Medical Research Council, 1923. 

'"Lorenz, W. F., and Ravenel, M. P: J.A.M.A., sg, 690. 1912. 

" Rolleston, J. D.: Brit. J. Child. Dis., 10, 298. 1913. 

" Davis, C. M.: J. A. M.A., 61, 393. 1913. 

••i Nicholson, S. T., and Hogan, J. F.: ibid., 62, 510. 1914. 

'■» Papacostas, G., and Gate, J.: Compt. rend. Soc. de biol., 85, 859, 1038. 1921. 



no BACTERIAL ASSOCIATIONS 

morphology of the latter also changed toward a more homogeneous form on staining. 
By the use of filtrates of each culture they could not discover any evidence that the 
toxin of the former was able to neutralize diphtheria toxin in vivo or in vitro. If the 
two are grown together, however, no toxin is formed, nor is there any if the filtrate of 
the pneumobacillus growth is used to grow the B. diphtheriae. They suggested the 
therapeutic use of filtrates. In later studies on a larger number of clinical cases Gate 
et al.' and Chalier, Gate, and Grandmaison^ confirmed their impressions of the usual 
mild course of these mixed infections. 

Van der Reis\ having demonstrated an antagonistic action of B. coli to B. diph- 
theriae, showed that it was possible, by spraying B, coli into the mouth, to have it 
colonize there. In nine cases it was still present after fifty-four days. A careful study 
of the antagonistic activity of B. coli led him to conclude that there is formed a ther- 
molabile, volatile, non-dialyzable, non-filterable, inhibitory substance not adsorbed 
by charcoal, not identical with the normal metabolic products of the colon bacillus, 
but that it may be a special toxic product. It was tried in acute cases of diphtheria by 
means of sprays of B. coli and particles of B. coli agar with the result that the B. 
diphtheriae disappeared more quickly than in controls. Carriers could also be rapidly 
freed of their bacilli. On the other hand, Pesch and Zschocke,'' although confirming 
the crowding out of the B. diphtheriae by B. coli in cultures, were unsuccessful in treat- 
ing nasal carriers because the B. coli would not grow in the nose. Bloomfield^ failed 
in his attempts to implant Friedlander's bacillus from carriers to non-carriers, and 
even a foreign strain of the bacillus failed to establish itself in the throat of a carrier 
of another strain. Pringsheim* studied the inhibiting effect of a strain of B. mesenteri- 
cus vulgatus against a variety of bacteria but particularly against B. diphtheriae. He 
found that B. typhosus, B. paratyphosus A and B, B. fecalis alcaligenes, B. coli, and 
streptococcus were without effect on B. diphtheriae. B. pyocyaneus and an air staphy- 
lococcus were strongly inhibitive. Staphylococcus aureus was mildly stimulating as 
seen in larger colonies as was also a weakly sporing B. subtilis strain. On agar plates 
the effect of his B. mesentericus was to produce a circular zone of inhibition and just 
beyond this a ring of larger colonies. Filtered or heated cultures had no effect. Other 
proteolytic bacteria had no such action. It was tried on patients but the results were 
inconclusive. The findings of Zukerman and Minkewitsch' with B. mesentericus vul- 
gatus were somewhat different. The antagonism was inherent in the bacillus and was 
not increased by serial passage. It acted only on diphtheria and pseudo-diphtheria 
forms and not against a long list of other bacteria. Many other spore-bearers were 
either negative or but weakly active. Filtrates were very active, killing in four min- 
utes, and were fairly heat resistant. 

' Gate, J., el al.: ibid., 86, 929. 1922. 

' Chalier, J., Gate, J., and Grandmaison, L.: Paris med., 61, 205. 1926. 

3 Van der Reiss: Miinchen. mcd. Wchnschr., 68, 235, 1921; Zlschr. f. d. ges. expcr. Med., 30, 
1922. 

-• Pesch, K., and Zschocke, O.: Miinchen. med. Wchnschr., 69, 1276. 1922. 

sBloomfield, A. L.: Johns Hopkins Hasp. Bull., 32, 10. 1921. 

^ Pringsheim, E. G. : loc. cil. 

'Zukerman, I., and Minkewitsch, I.: Centralbl.f. Bakkriol., I, 80, 483. 1925-26. (Ref.) 



W. L. HOLM AN iii 

PNEUMOCOCCUS 

The pneumococcus is usually considered a rather delicate organism in culture 
media, but apparently it may have a striking antagonistic effect on the staphylo- 
coccus. Gromakowsky' discovered in eight sputum cultures a coccus which had a 
definite restraining action on the staphylococcus. This coccus resembled the pneumo- 
coccus in morphology, but he considered it different. Mixed with eight different 
strains of staphylococci and after twenty-four hours' incubation, transfers to agar 
gave no growth of the staphylococcus. It was irregular in action and also was an- 
tagonistic to streptococci from abscesses. Ahvisatos,^ working with twenty-eight 
strains of well-identified pneumococci and three strains of Staphylococcus albus, no- 
ticed, when the forms were mixed, interesting phenomena on ascites agar plates. 
Curious clear zones appeared about the colonies of the pneumococci, and the edges of 
the staphylococcus colonies were irregular and suggested the action of bacteriophage. 
These zones varied in size, and if enough pneumococci had been added no growth of 
staphylococcus occurred. He never found mixed colonies. Neither the virulence nor 
the agglutinating type of the pneumococcus was related to the extent of the phenom- 
ena. There was no demonstrable change in the cultures of either of the bacteria after 
these contacts. Living, growing pneumococci were necessary and filtrates were nega- 
tive. Eight strains of hemolytic, five of viridans, and one of mucosus streptococci 
gave negative results. In these cases mixed colonies were frequent, and he suggested 
that this characteristic might be used to differentiate pneumococci from closely sim- 
ilar streptococci. 

COLON-TYPHOm GROUP 

The antagonistic effect of soil bacteria against pathogenic forms has been exten- 
sively studied. The early work of Frost^ is important and includes the literature to 
that date. Limitation of space forbids a further discussion of this interesting subject. 
Fecal bacteriology, particularly of the colon-typhoid group, is replete with examples 
of supposed antagonism. It has long been held that the presence of slow lactose fer- 
menting B. coli, so frequently observed in stool examinations, is due to this phenom- 
enon (von Jeney"), and Henningson^ gave examples of inhibition of gas production 
and proposed the name B. coli anaerogenes for these. PrelP and many others have 
studied such defective strains. Nissle,^ having observed an inhibitory action in cer- 
tain stools seeded with B. typhosus, studied the antagonistic index of the B. coli to 
B. typhosus with various strains of the former. The difference seemed correlated with 
lactic acid production. The active coli strains also were inhibitory to other coli 
strains. He therefore gave these active cultures in capsules to persons carrying in- 
efficient B. coli strains and reported good results. R. P. Smith* found that B. coli 

' Gromakowsky, D.: ibid., Orig., 32, 272. 1902. 

^ Alivisatos, G. P.: ibid., 94, 66. 1925. 

3 Frost, W. D.: J. Infect. Dis., 1, 599. 1904. 

* von Jeney, A.: Ztschr.f. Hyg. u. Infektionskrankh., 100, 47. 1923. 
sHenningson, B.: ibid., 74, 253. 1913. 

^Prell, H.: CentralU.f. BakterioL, I, Orig., 80, 225. 1917. 
7 Nissle, R.: Deutsche mcd. Wchnschr., 42, 1181. 1916. 

* Smith, R. P.: J. Path. b° Boot., 26, 122. 1923. 



112 BACTERIAL ASSOCIATIONS 

strains from carrier cases were more active against stock cultures of B. typhosus than 
were stock cultures of B. coli. Unfortunately, he did not test the B. typhosus from the 
carrier with its own B. coli, but the evidence suggested an inhibition because of the 
difficulty he had in obtaining the B. typhosus in these cases. Vignati' described the 
reverse phenomenon in which fresh, actively growing cultures of B. typhosus inhibited 
the growth of B. coli, older cultures not being antagonistic. He explained the facts on 
Bail's theory of the spatial needs of each bacterium. Lisbonne and Carrere,^ by a 
method suggesting that of Schiller,^ forced a bacteriophage to develop by the antag- 
onistic action of B. coli against the Shiga bacillus. They found at the end of a series 
of passages that an active and transmissible lytic principle was developed by what 
they call a "vitiation" in the metabolism of the Shiga bacillus. B. proteus X19 gave 
identical results. They considered that this is what occurs in the intestines where an-' 
tagonistic conditions are always present. They later showed that this principle was 
not carried by the B. coli since the same strain was tested by Beckerich and Hauduroy 
who suggested such an explanation, and was not found to be lysogenic. It was def- 
initely the result of microbial interactions. Fabry also obtained a principle of the 
same kind through the antagonistic stimulus of a Staphylococcus albus on B. coli which 
also acted on the Shiga bacillus. Bordet^ reported a similar discovery with four pri- 
marily non-lytic strains of B. coli in which the lytic principle appeared spontaneously 
and was increased by passage. Gratia^ studied an example of antagonism between 
two races of B. coli as Nissle^ had shown from another point of view. Filtrates of B. 
coli V. inhibited B. coli and caused an agglutinative culture of the latter in fluid 
media. The same results were obtained with living cultures on agar, and in both 
cases the secondary colonies were resistant to the action of B. coli V, It resembled 
the Gratia principle but was not regenerated by the B. coli 0, being lost by the third 
passage, and did not act in high dilutions as bacteriophage does. On agar plates the 
area about the growth of B. coli V. was inhibitive to the growth of B. coli 4> but not to 
B. coli V, It was therefore not a vaccination of the medium. It was very resistant 
to storage, chloroform, and high temperatures (100° C, for thirty minutes). Bordet*^ 
has carefully analyzed the various interactions between the bacteria giving the re- 
sults that Lisbonne and Carrere' reported, but as this is encroaching on the problem 
of bacteriophage which is to be presented elsewhere'" in this book, I need go no further. 

THE THEOBALD AND D. E. SMITH PHENOMENON 

A most interesting example of the inhibitory effect of bacteria in association was 
reported in 1920 by Theobald Smith and D. E. Smith." They found that B. para- 
■ Vignati, J.: Compt. rend. Soc. de bioL, 94, 209. 1926. 

* Lisbonne, M., and Carrere, L.: ibid., 86, 569. 1922; 87, ion. 1922; 90, 265. 1924. 
3 Schiller, I.: loc. cit. 

■» Fabry, P.: ibid., 87, 369. 1922; 90, 109. 1924. 
sBordet, J.: ibid., go, g6. 1924. 

* Gratia, A.: ibid., 93, 1040. 1925. ' Nissle, R.: loc. cit. 

* Bordet, J.: Com pi. rend. Soc. dc bioL, 93, 1054. 1925. 
'Lisbonne, M., and Carrere, L.: loc. cit. 

'"Chapter xl. " Smith, T., and D. E.: /. General Physiol., 3, 21. 1920. 



W. L. HOLM AN 113 

typhosus B, after it had grown in lactose bouillon for four to six days, prevented the 
development of gas by B. coli when this was added. Members of the closely related 
hog-cholera group had no such action in the given time, but after eighteen days' 
growth they also inhibited the gas production for the B. coli added at this Time. A 
fuller analysis of this phenomenon is found in an article by Holman and Meekison.' 
Besson and De Lavergne^ confirmed the results of T. and D. E. Smith and found that 
B. aertrycki gave the reactions of the hog-cholera group. Brutsaert^ found the phenom- 
enon most inconstant and variable even in repeated tests of the same strain of bacillus. 
A hog-cholera type-agglutinating culture inhibited, but as a rule members of this 
group did not. Moreover, the phenomenon failed if, instead of lactose bouillon, a 
lactose peptone water were used. He found it too irregular for use in classification. 
Von Jeney" in studying this question used a bouillon previously freed from glucose by 
a twenty-four hour growth of beer yeast. (T. and D. E. Smiths presumably used B. 
coli for this purpose.) Both of these procedures may have an important bearing on 
the results since the effect of these preliminary cultures may be very great as is seen 
in many of the articles reviewed above and in the studies of Robertson^ on food ac- 
cessory factors in bacterial growth. Although such media may not interfere with gas 
production by the B. coli per se, it may have an effect on the combined metabolism. 
Von Jeney' investigated the subject very fully. He found five strains of B. para- 
typhosus B among twenty-six studied which increased the B. coli gas production. Also 
plates from the mixtures at times gave pure B. coli. The strains of the B. paratypho- 
siis, isolated from the mixture, did not always give the same results on retest. B, 
typhosus also inhibited. He searched for any evidence of bacteriophage action, but 
was only able to discover suggestions of such and no continuous passage was possible. 
Kauffmann* carried the work further and used besides human strains, as von Jeney 
had done, a large number of animal strains. He tested thirty different cultures of B. 
coli and found in pure culture that their gas production was most variable, ranging 
between o and 100 per cent in twenty-four hours, and for no known reason. He also 
used yeast-treated media. Some B. coli strains grown from the same stools as strains 
of B. paratyphosiis showed delayed gas production on glucose, but this was not the 
rule. On the other side he used thirty human B. paratyphosus B strains, Gaertner's 
bacillus, B. typhosus, and others and a further group of fifteen strains from animals. 
Besides the regular tests he used a number of heterologous and homologous combina- 
tions with the cultures from individual stools. A great irregularity was found through- 
out. After passage, certain strains of the B. paratyphosus increased in inhibitory 
powers, but the B. coli did not become more sensitive by such passage. There was, 
however, no regularity. The animal strains gave the same kind of results as the hu- 
man. Certain "pseudo-unstable" forms were sometimes seen on plates. In summing 

' Holman, W. L., and Meekison, D. M.: /. Infect. Dis., 39, 145. 1926. 

^ Besson and de Lavergne: Compl. rend. Soc. de biol., 86, 357. 1922. 

3 Brutsaert, P.: ibid., 88, 306. 1923. 

■* von Jeney, A.: loc. cit. s Smith, T., and D. E.: loc. oil. 

^Robertson, R. C: /. Infect. Dis., 34, 395. 1924; 35, 311; 1924. 

' von Jeney, A. : loc. cit. 

* Kauffmann, F.: Zlschr.f. Ilyg. u. Infeklionskrankh., 102, 68. 1924. 



114 BACTERIAL ASSOCIATIONS 

up these divergent and variable results, I feel that they tend to confirm the inter- 
pretation we have given. The results will depend on many factors such as the rela- 
tive ability of each bacillus to attack the salts of the organic acids, the effect of al- 
kaline reactions, and others. 

THE HEMOGLOBINOPHILIC GROUP 

The association of bacteria with the B. influenzae group has received unusual at- 
tention. The discussion of this part of the subject, even briefly, would take all my 
available space. I must merely observe that it has been studied from the earliest days 
of the discovery of the B. influenzae and gives striking illustrations of the importance 
of the subject. Many bacteria help the growth of B. influenzae on media, otherwise 
inhibitory, such as human blood agar, but they have little or no effect on properly 
heated blood agar. These beneficent bacteria are most variable in their characters, 
and include many listed as highly antagonistic such as B. pyocyaneus, an organism 
around whose antagonistic activity there has collected a comprehensive literature. 
B. influenzae will grow on hemoglobin-free media in association with many bacteria, 
although Putnam and Gay' and others were unsuccessful in doing this, and the im- 
portance of this and the possible bearing it may have in infection have been repeated- 
ly stressed. Eggerth^ was able to grow B. influenzae in plain broth within a collodion 
sac immersed in cultures of staphylococcus, streptococcus, or pneumococcus and sug- 
gested this method as useful for studying symbiosis of bacteria while keeping each 
in pure culture. It would be of great interest to learn if toxin production may be 
permanently increased by certain of these combinations. Certainly the virulence is 
readily raised by mixed injections, as Yanagisawa,-5 Hudson" and others have shown. 
I will not attempt to review the literature because this has been done by me^ and by 
Kristensen.^ I do not believe there has been anything new on the fundamental ideas 
of bacterial associations with B. influenzae since those reports. I cannot leave the 
subject of influenza without mentioning the importance OHtsky and Gates,^ in a long 
series of articles, have placed on the relationship between the B. pneumosintes and 
secondary infections. I may say that their controls were quite inadequate to justify 
the conclusion that this organism is more potent in encouraging secondary infections 
than a host of others. 

B. ANTHRACIS 

In anthrax it has been repeatedly shown that combined injections frequently 
prevent infection. I have found that guinea pigs did not die after large injections of 
washings from soil contaminated months before by B. anthracis in the slaughterin;^ of 

' Putnam, J. J., and Gay, D. M.: /. Med. Research, 42, i. 1920. 

' Eggerth, A. H.: /. Biol. Chem., 48, 203. 1921. 

3 Yanagisawa, S.: Kilasato Arch. E.vper. Med., 3, 85. 1919. 

■I Hudson, N. P.: /. Infect. Dis., 34, 54. 1924. 

sHolman, W. L.: Studies on Epidemic Influenza, p. 161. University of Pittsburgh, 1919. 

'' Kristensen, M.: IlaemoglohinopltUic Bacteria. Copenhagen, 1922. (In English.) 

'Olitsky, P. K, and Gates, F.L.: J. A. M. A., 74, 1497. 1920; 78, 1020. 1922; 81, 744, 21 19. 1923; 
/. Exper. Med., 33, 125, 361, 373, 713. 1921; 34, i. 1921; 35, i, 553, 813. 1922; 36, 501, 6S5. 1922; 
37. 303- 1923- 



W. L. HOLM AN 115 

a diseased cow, but gave typical results after injection of the cultures isolated from 
the soil. I have collected the literature on this subject, but it will not be included here. 
The explanation of the results rests largely on the fact that the anthrax spores are 
phagocyted by the leukocytes attracted to the site of injection by the other bacteria 
before they start developing and then are destroyed or eliminated. A most important 
discussion on the principles of such infections may be found in an article by Bail.' 

GAS SYNERGISM 

Holman and Meekison^ reported certain findings in gas production by bacterial 
synergism and have reviewed the literature on this phase of bacterial association and 
attempted to show that inhibition and stimulation are both based on the combined 
metabolism of the bacteria in the mixtures and that the same bacterium may use 
various methods in acting on the substances offered, depending on the environment. 
Sears and Putnam-' and Castellani^ have given many instances of its occurrence. 
Castellani has further discussed the whole subject of close association of different 
species and particularly its importance in the causation of certain diseases and their 
symptoms. He does not, however, review the literature. 

ALTERATION BY ASSOCIATION 

Dissociation of bacteria has been gradually attracting more and more attention, 
and since bacterial association is so important to appreciate the analysis of the re- 
sults obtained, a word or two may be added here. I have already given a number of 
examples of important alterations in the biochemical activities of certain bacteria 
living in association with others. Sometimes such changes were lasting on transfer, 
at other times fleeting. Rosenow^ claimed that he was able to change a hemolytic 
streptococcus to the viridans type by growth in symbiosis with B. subtilis. This P 
was unable to confirm, and I have reviewed the literature on the longevity of strepto- 
cocci in symbiosis and have shown the many chances of error from mixed cultures, 
particularly with closely similar forms, Pneumococci may live in intimate contact 
with non-hemolytic streptococci for long periods, and the demonstration by Alivi- 
satos' of the occurrence of mixed colonies of streptococci with staphylococci gives ad- 
ditional weight to the importance of such sources of error. The more sensitive organism 
will die out first, and retests may give quite different results from those found with 
the mixture. This danger was also emphasized by us^ for the gram-negative group 
of aerobic intestinal bacteria. Nevertheless, bacteria are living, reactive beings and 
as such are subject to alterations of many kinds. Bail' has shown how important this 
is in the infectiousness of bacteria (the change to the so-called "animal form"), and 

'Bail, O.: Ztschr. des. ges. Expcr. Med., 50, 11. 1926. 
^Holman, W. L., and Meekison, D. M.: loc. cit. 
3 Sears, H. J., and Putnam, J. J.: /. Infect. Dis., 32, 270. 1923. 

■• Castellani, A.: Brit. M. J., 2, 734. 1925; Proc. Soc. E.xper. Biol. & Med., 23, 481. 1926; 
J.A.M.A., 87, 15. 1926. 

5 Rosenow, E. C: /. Infect. Dis., 14, i. 1914. 

* Holman, W. L.: ibid., 15, 293. 1914. . * Holman, W. L., and Meekison, D. M.: loc. cit. 

' Alivisatos, G. P.: loc. cit. » Bail, O.: loc. cit. 



ii6 BACTERIAL ASSOCIATIONS 

similar adaptive alterations are to be expected in the growth in mixed cultures. How 
fundamental these changes may be is not as yet determined, and the study of dis- 
sociation should help us in our knowledge of the possible range which may occur in 
the bacteria as they are grouped by our present-day rather crude methods of classifi- 
cation. Bacteria may be forced to metabolize substances in a different way under 
certain environmental conditions than they would under other conditions, and this 
induced, but not necessarily new, function may become fairly well fixed by repetitions 
as a characteristic of the organism. In looking over the examples already given there 
are many instances to be found of such occurrences apparently brought out by bacterial 
association. LommeP has given an important instance of this. By growing a non- 
saccharose fermenting B. coli with B. typhosus, B. paratyphosiis, or the Shiga bacillus 
after some twenty passages it took on the function of actively attacking saccharose. 
She does not say how long this induced activity continued under non-associative con- 
ditions, as only a plate and agar slant intervened between the tests. She has, however, 
shown that certain B. coli lost their ability to ferment lactose by continued growth on 
malachite-green media and that this loss remained constant for some, but not for all 
strains after fifty-six transfers on plain agar. 

OTHER APPLICATIONS 

Passing by numerous other well-known examples of bacterial association such as 
that of B. fusijormis and Spirocheta vincenti, which Rukawischnikoff^ and others 
look upon as only stages in the growth cycle, and many mixed infections occurring in 
man and animals, I would give the interesting use made of this phenomenon by WoU- 
man.-' He used B. coli as an indicator to determine proteolysis by bacteria previously 
grown in horse serum, egg albumin, and similar substances through its ability, after 
such primary growths, to produce indol. He thus tested B. anthracis, B. subtilis, 
Staphylococcus aureus, and B. putrificus and later^ determined the proteolytic activity 
of streptococci by this method. Thompson^ was enabled by a symbiotic method with 
B. proteus to isolate an anaerobic B. acwe-like organism from cultures of B. tuber- 
culosis and suggested its relationship with the latter. I believe it might well have been 
present as a contaminant and brought to light by this technique. 

DISCUSSION 

Before closing I would call attention to the necessity of determining more care- 
fully than has been done the intimate metabolism of the bacteria we are studying and 
the environmental requirements necessary for the manifestation of their manifold 
characteristics, before any attempt is made to explain the phenomena I have re- 
viewed, those of bacterial association, or the active metabolism shown by the bac- 
teriophage. It is well known that d'Herelle^ considers the bacteriophage a living, sub- 

' Lommel, J.: Compt. rend. Soc. de bioL, 95, 711, 714. 1926. 
' Rukawischnikoff, E.: Ccntralbl.f. BaktcrioL, I, Orig., 100, 218. 1526. 
^ WoUman, E.: ConipL rend. Soc. de bioL, 82, 1263. 1919. 
^Wollman, E.: ibid., 87, 1138. 1922. 
5 Thompson, E. T.: Lancet, 2, 1S6. 1920. 

•^d'Herelle, F.: The Bacteriophage. 1922; Immunily in Natural Infectious Disease. 1924; The 
Bacteriophage and Its Behavior. 1926. Translated by G. H. Smith. WilHams & Wilkins. 



W. L. HOLMAN 117 

microscopic form, a strict parasite of bacteria, and therefore the most striking example 
of microbic association. This is discussed in other chapters of this book. 

There have been two main explanations offered in the interpretation of most of 
the phenomena of bacterial association which I have mentioned. The first is the effect 
of changes in reaction. Usually one of two organisms in a mixture may produce un- 
favorable H-ion concentration for the continued metabolism of the other organism, 
and indeed the latter bacterium may be killed. At other times these reaction changes 
may only alter the degree or kind of metabolism taking place, or the change may be of 
benefit to one or both. The other explanation is the production of enzymes of various 
kinds which directly affect the second organism in a mixture. This is undoubtedly a 
prime factor in many of the examples I have cited. 

A distinct advance has been made toward other explanations for the facts given 
above by M'Leod and Gordon' in grouping bacteria under their relative sensitivity 
to, and power to produce, hydrogen peroxide and a corresponding catalase. Certainly, 
if we take their table, with due consideration for variations in different strains we shall 
find a rather satisfactory explanation for many of the antagonistic and beneficial re- 
sults of bacterial associations. Burnet^ has analyzed many of these relationships, and 
his contributions have further made clear their application to our study. Bacteria 
sensitive to hydrogen peroxide will be inhibited by the presence of a strong hydrogen 
peroxide producer, and an organism with a well developed catalase production will 
assist another which forms much hydrogen peroxide to which it is itself sensitive. 
Anaerobes are very sensitive to this substance, and therefore a variety of aerobes 
producing catalase will benefit them in this respect as well as do those strongly aerobic 
forms which help the anaerobic conditions, 

A number of workers have stressed the role of carbon dioxide in the inhibition phe- 
nomena of bacterial cultures. Sierakowski and Zajdel' were able to show, by sealing cul- 
tures of various bacteria, that the H-ion concentration alone did not account for the 
growth inhibition, but they believed it due to the retention in the tubes of carbon diox- 
ide. Valley and Rettger-" showed that increasing amounts of CO2 raise the acidity and 
lessen the oxygen tension, and that the complete absence of CO2 in the atmosphere 
stops the growth of many bacteria. Other bacteria such as B. acidophilus were bene- 
fited by an increase in the atmospheric CO2. I^ have shown that anaerobes will form 
surface colonies on solid media in experiments in which their own gas production dis- 
places the fluid medium in inverted tubes. This may be a factor in wound infections. 

A further valuable contribution to our knowledge of these mutual relationships 
is to be found in the work of Gordon and M'Leod^ on the inhibition of growth by some 
amino acids. They found that the effect differed markedly as tested on different bac- 
teria, B. coli and staphylococcus were not at all affected while other more delicate 

' M'Leod, J. W., and Gordon, J.: /. Palh. cr Bad., 26, 326. 1923. 

^ Burnet, F. M.: Aiislnilian J. Exprr. Biol, iy M. Sc, 11, 65, 77. 1925; J. Path, b' Bad., 30, 21. 
1927. 

^ Sierakowski, S., and Zajdel, R.: Compl. rciid. Sac. dc hioL, 90, 1108. 1924. 

'•'Valley, G., and Rettger, L. F.: Ahstr. Bad., 9,344. 1925. 

sHolman, W. L.: Illinois M. J., 35, 289. 36, 10. 1919. 

^ Gordon, J., and M'Leod, J. W.: /. Path, of Bad., 29, 13. 1926. 



ii8 BACTERIAL ASSOCIATIONS 

bacteria were. Tryptophane, as an example, is most toxic and affects the widest variety 
of bacteria. Indol, a deaminization product of tryptophane, may account for the tox- 
icity of tryptophane since it is more toxic than carboUc acid. Serum prevents to a de- 
gree these inhibitory actions. Others are beneficial, such as taurine, aspartic acid, 
and alanine. It is readily seen that we have here an additional explanation for certain " 
of the phenomena being considered. Various bacteria will produce amino acids harm- 
ful or beneficial to others, and on these products will depend the effect on the asso- 
ciated bacteria in proportion to the relative sensitivity of the latter. The marked in- 
hibitory effect of certain proteolytic bacteria on agar plates against the nearby col- 
onies of other bacteria and the stimulating effect at some distance may well be due 
to differences in diffusibility of the products formed. There are also, of course, direct 
actions of proteolytic enzymes on the associated bacteria in certain cases as already 
suggested, and the lack of agreement in many of the examples quoted above may well 
be due to different factors having been at work. There is a pressing need to correlate 
the wealth of available material on the various products formed by many bacteria 
and the effect of these on associated forms. The work of Koser' and many others on 
the utilization of the salts of organic acids by bacteria makes clear other groups of 
phenomena and assists in the explanation of changes in H-ion concentrations as these 
occur in the reversed reaction of single forms and in bacterial associations. The rel- 
ative rapidity of growth of two forms, the continual alteration in their activity and 
sensitivity, the adaptability of bacteria to the form of food material offered at dif- 
ferent stages and under different conditions as aerobic or anaerobic, the changed 
metabolism under acid and alkaline influences, and a host of other factors determine 
the resultant metabolic products of mixed cultures. The analysis of these factors helps 
in understanding the metabolic processes involved. 

CONCLUSION 

Bacterial association occurs under natural conditions, and it plays an important 
part in many infections. At one time certain resistant but relatively harmless forms 
may ward off the body defenses and allow a more sensitive microbe to become es- 
tablished. At other times the reverse may occur and antagonistic bacteria may, and 
no doubt frequently do, prevent numerous infections. We have in bacterial associa- 
tion, then, a means of studying many natural phenomena and, as has been indicated, 
we touch on many fields of bacteriological study. In the routine bacteriological diag- 
nosis it must be always in mind that there are antagonisms in our media and methods 
for preventing them; that pure cultures are essential, and that curious results occur 
from mixed ones, often quite different from those with either culture alone; that the 
beneficent associations are to be found and that it must be realized that bacteria com- 
ing from varying environments may have been under inhibitory or stimulating in- 
fluences which alter the results obtained in our test tubes. In artificial animal in- 
jections and in natural human and animal infections and diseases bacterial associa- 
tions as seen in lowered or raised virulence or pathogenicity; the presence of secondary 
infections from the animal itself leading to faulty conclusions; the changes in bacterial 
flora as the conditions alter, as seen in war wounds and intestinal infections; and many 

' Koser, S. A.: /. Bad., 8, 493. 1923. 



W. L. HOLMAN 119 

similar considerations must be thought of to avoid error in interpretation of results. 
In the study of bacteriophage and bacterial dissociation the same considerations are 
needed. I would finally urge, after reading Claude Bernard's Introduction to the Study 
of Experimental Medicine^ and in attempting to get working hypotheses for the phe- 
nomena of bacterial association, that more and more attention be paid to the physi- 
ology of bacteria as reactive, living beings with as complicated metabolisms as our 
own, and that the study of their pathology, if such we may call it, requires this pre- 
liminary knowledge of the normal limits of their physiological activities, alone and 
together, in the test tube and in the animal body. Thus we may be able to understand 
better many phenomena which at present cause confusion, and may better appreci- 
ate the basic principles in many of the biological activities of bacteria. 

' Bernard, Claude: op. cit. Translated by H. C. Greene. Macmillan Co., 1927. 



CHAPTER IX 
CLASSIFICATION OF BACTERIA 

ROGER G. PERKINS 

Western Reserve University 

Bacteriological literature is crowded with classifications of bacteria of every sort, 
prepared by thoughtful workers, with painstaking labor. The literature is also crowd- 
ed with criticisms of these classifications for reasons which seem good to each author, 
though at times he is alone in his opinion. Therefore all I dare attempt is a summary of 
the principles under which these classifications and these honest criticisms have been 
made, and to present them as far as possible in their relations to one another and to the 
problem as a whole. 

Two main points of view are represented, quite distinct, although closely related. 
The ordinary student of bacteriology is chiefly concerned with the grouping of bac- 
terial forms to establish their general relations, involving the possibility of keys which 
shall enable him to place any new discovery in its approximate place. The taxono- 
mist, the specialist in bacterial classification, concerns himself further with the selection 
of the correct names for the divisions, and the evaluation of the proper sequence of 
order, family, genus, species, and variety, with an eye to the future. Stiles (1927), in 
a recent address, spoke of taxonomy as the grammar of the science and emphasized 
its essential value. He comments on the present status that "it has been the excep- 
tion — not the rule — that pupils who study zoology have been taught the grammar of 
the technical language they are called upon to hear, read, write and speak." This is 
no less true in bacteriology, and the student who does more than skim the subject of 
classification outside his own particular interests is rare. This is unfortunate, but 
perhaps if the grammar were less chaotic it would have more students. 

Taxonomists, not only in bacteriology but in general zoology and general botany, 
live in two camps : those who believe that for many reasons all names referring to in- 
dividuals, varieties, species, etc., should follow the Linnaean law of priority, laid down 
in 1 751; while the other camp holds that inasmuch as Linnaeus knew only what was 
known in 1751, and since so much totally new information regarding classification is 
available, we should no longer be restrained by the dead hand, but should be free to 
express ourselves. Stiles (1927) presents sharply his idea of these alternatives. 

First let every [zoologist] adopt any technical name he wishes, or second let us all a^ree to 
follow the Linnaean law of priority. The first alternative is subjective and leads to confusion, 
the second is objective and makes for uniformity in all objective cases .... In general I 
would evaluate a failure to apply the law of priority as the second most important formal 
factor in nomcnclatoriul confusion. 

And ])crhaps llic extreme in the other camp is Enderlein, with a com]i]ete new classifi- 
cation, and indeed a com|)lete new language. Neither side, of course, refuses lo admit 
new titles, new definitions, and new grou[)ings, nor does it liesitate to accept the iacl 
that our knowledge is progressive rather than fixed, l)ut the rules of the game differ. 

120 



ROGER G. PERKINS 121 

On the whole it seems as though, in spite of its defects, the work of the last twenty 
years has established sufficient recognized groups, especially of the higher ranks, to 
enable us, with the appreciation that bacteriology is a young subject, to develop these 
rationally, without any complete upset. At present there are certainly sufficient 
shades of difference between the extremes to accommodate anyone. 

Another important controversy, more marked perhaps among the adherents to 
Linnean priority, centers around the question whether a name should be descriptive, 
giving some indication as to character or the group or individual, or whether this is un- 
important, and the name, if historically correct, should be retained even if wrong or 
confusing. Enlows stated in 1920: 

It is to be hoped that the many inadequately defined genera here listed may serve as 
glowing examples of errors to be avoided by future contributors. A plea is made, too, for 
the introduction of generic names which are descriptive, since many names of this sort define, 
and in a way, classify. Proper names converted by the addition of -iiica, -cUa, etc., are very 
alluring because of the acknowledgment of the debt we owe our leaders, but they are not de- 
scriptive terms, and offer no aid whatever to any system of classification. 

On the other hand, the Committee of the Society of American Bacteriologists stated 
in 1917 that "the name need not be appropriate, it need only be stable. It is an ar- 
bitrary description." Stiles stated in 1905: "It is essential to recall that names are 
not definitions; they are merely handles by which objects are known." These last 
indicate the majority opinion, save in the more independent classifications, such as 
Orla-Jensen (1921), Enderlein (1925), and others, where the attempt at descriptive 
titles is prominent. In connection with Enlows' {loc. cit.) remarks about the eponymic 
groupings, it is interesting to note that in medicine there is a growing tendency to- 
ward correctly descriptive names for diseases, with corresponding abandonment of 
proper names. 

It seems as though neither the apologists nor the higher critics are satisfactory or 
satisfied, nor is it surprising in a science in formation. The essential contention ap- 
pears to center about the proper, relative proportion of a conservatism which objects 
to the removal of well-known signposts, partly on the basis that their familiarity more 
than overbalances their misdirections, and a liberalism which wishes to accept only 
such names and descriptions as fit our present knowledge. What shall be the mixture 
and who shall mix it? 

It is stated with much force of logic that constant change in names and in qualifi- 
cations is confusing, and that even now the bacteriologist has to have at least one key, 
if not more, at his elbow when he reads his literature, and that historical articles will 
soon be quite unusable, a mere hieroglyphic literature, understandable only by a few. 
It is true also that if in the last thirty years or less the discoveries have been such as 
to invalidate old classifications, what warrant have we that the next thirty years may 
not repeat the process? Further advance in technicjue may reveal details of form yet 
unknown to us, may crystallize our information, and may yet uncover unsuspected 
biological relations. On the other hand, it is clear that at present our known details 
of morphology of any degree of constancy are numbered, and that chemical and phys- 
ical differentiations have become more and more stabilized within certain group lim- 
its. Although it may be true that new information may and probably will necessitate 



122 CLASSIFICATION OF BACTERIA 

changes in another quarter-century and even in a decade, it is likely that the new in- 
formation will not be morphological. 

When acknowledged experts disagree widely on so complex a subject, an ordinary 
bacteriologist like myself must avoid as far as possible technical taxonomies end ex- 
tensive references to authority, of which there is a sufficiency so arranged that any 
diligent reader can make his own evaluations. To the general reader the main lack is 
in summaries of opinions which make it possible to omit the individual technical 
articles. 

The recent classifications and keys due to the industry of Bergey (1923), Buchan- 
an (1916-25), and the Committee of the Society of American Bacteriologists (191 7- 
20) with the critical summaries of taxonomic authority by Buchanan (loc, cU,), En- 
lows (loc. cit.), and others make the entire history of the development of classification 
readily available. One should note also that not the least service of such attempts at 
complete classification of a series of organisms, concerning which the authors them- 
selves are the first to admit our information deficient, is their courage in presenting 
their plans and ideas for criticism. Unless broad plans of this sort are accessible, re- 
vision is impossible, since most of us are more particularly concerned with some re- 
stricted portion of the whole. It is far easier to criticize than to construct, but only 
out of a combination of construction and constructive criticism can we hope to reach 
stability. 

At first the whole affair looks extraordinarily discouraging. Each attempt at im- 
provement meets with a volley of objections (Hall, 1927), and such attempts as seek 
to break away from conventions of taxonomy have few adherents. Closer study, how- 
ever, offers more hope. One finds that there is a great deal of acceptance of main 
groups, and that most of the trouble comes from disagreement as to the rank of these, 
and from persons studying a comparatively small group and believing that their ar- 
rangement is the only proper one. 

To my mind the first essential, as brought forward by many, and ignored by as 
many more, is that bacteria in comparison with higher plants and with animals are 
notably unstable, and that such conformity to type as we see in pure cultures of Plym- 
outh Rock chickens, or white rats, or Lima beans is not to be expected. 

The more we study them, the less stable appear the characters on which we base 
much of our classification. If zoologists find, as they do, that complex multicellular 
organisms are altered by environment to a point where, had they not been followed 
through, they would be thought different genera, and the botanists present similar 
evidence, how much more may we expect environmental modifications of a temporary 
or permanent character among bacteria? One must admit, of course, that unicellular 
organisms which divide by simple fission carry heredity in a manner very different 
from that of more complex forms, each half being supposedly identical with the other; 
but recent work in variation and in mutations in single-cell cultures has forced a modi- 
fication of those ideas. There is no a priori reason why a colony of anthrax might not 
undergo in nature conditions similar to those by which we modify it artificially. Evi- 
dence that the citrate-using B. coli preceded or followed the non-citrate-using is not 
yet conclusive. We are told that the development of terminal flagella on a coccus 
tends to change it to a rod form, and we find that an organism may be changed from 



ROGER G. PERKINS 123 

a gram+to gram — by the use of suitable chemicals. Winslow quotes Wolf as re- 
porting "a considerable number of temporary modifications and some permanent in- 
heritable ones stimulated by exposing bacteria to the action of chemicals. White and 
dark-red strains were thus produced from a normal B. prodigiosus, the resulting modi- 
fications breeding in each case true to their new type." Winslow calls these changes 
"impressed variations," and further on says: 

The fact that all cells are potentially reproductive removes any bar against the inherit- 
ance of acquired characteristics. Again, the absence of sexual reproduction must operate to 
preserve variations which arise from within or without .... with fission as the normal mode 
of reproduction every variation which can arise can be handed on unchanged; .... there are 
sharp limits to the variability even of the bacteria, and for practical purposes we find the 
larger groups quite constant in their general properties .... in part at least I am inclined 
to believe that this is due to the direct or selective action of similar environmental conditions. 

Since we are dealing not only with variables, but with variables in a group of or- 
ganisms susceptible to permanent and hereditary change, we must select as most im- 
portant those characters which as nearly as possible approach constants, and which 
seem least susceptible to these permanent changes. One method of approach is well 
exemplified by the work of Winslow (1906) in his study of the Coccaceae, and the 
work of Hucker (1924) in a similar study. Believing that improvement might result 
from a statistical analysis of a variety of characters in the hope of determining cor- 
related groups, these extensive and painstaking studies were undertaken. The fact 
that the authors are often in marked disagreement shows that the method of pro- 
cedure, while suggestive, is as yet unsatisfactory. 

We must not forget that the actual number of bacteria described is but a small 
fraction of those which exist. This is of course true in botany and zoology as well. 
Stiles (1927) states that there are hundreds of thousands, possibly millions, of genera 
and species still to be given technical baptismal certificates. Aldrich (1927) quotes 
Horn as saying; "Whoever as an entomologist looks into the future knows full well 
that we are steering into a shoreless sea, no matter whether he estimates the total num- 
ber of insect species at three, ten or fifteen millions. In the near future any beginner 
will be greyheaded before he has caught up with what is already known." Although we 
have not quite reached this status with bacteria, partly because the details of struc- 
ture and other characteristics are so much less complex, it is already practically im- 
possible for anyone to be an expert in all the lines of bacteriology, and especially in 
their finer taxonomic relations. The student in medicine interests himself in the non- 
pathogenic forms only in their relation to the pathogenic (many early classifications, 
such as Fliigge, were of pathogens only), and even neglects the organisms relating to 
those diseases of animals that are not transferable to man, to say nothing of those 
which cause disease in plants, while the veterinary and the plant bacteriologists are 
similarly exclusive. It is only very recently that the workers on filterable viruses have 
appreciated that the botanist, the zoologist, and the medical biologist are working on 
an identical problem and that in their combined effort may lie the key to success. 

Students of the non-pathogenic organisms naturally tend to the study and iso- 
lation of those groups which have economic relations with soil values, with fermen- 
tations, with decompositions, and consider all others as merely annoying contami- 



124 CLASSIFICATION OF BACTERIA 

nations. Yet new perfections in technique constantly cast new organisms on the shores 
of the bacterial sea, where they become accessible. Workers with the anaerobic group 
tell us that it is c^uite possible to describe totally new organisms at the rate of several 
a week, and it is only when someone gets interested in a special problem that we get 
a list of the organisms concerned. It is clearly impossible to foresee new discoveries 
beyond what may be safely prophesied from analogy, and the best we can do is to 
make and preserve a scheme which we believe will act as a framework on which to 
hang not only the groups we have, but those which may turn up from time to time. 

It seems clear, then, that in common with students of other biological groups we 
have reached only a small fraction of the varieties which make up the mass, and that 
it is logical to believe that the changes in our knowledge and beliefs which have fol- 
lowed the lights of informative changes in technique are only an earnest of what is to 
follow. Bacteriology, as we now understand it, is less than fifty years old, and inas- 
much as bacteria are either plants or animals, it fell heir to all the taxonomic litera- 
ture relating to both. The discovery of the principles of pure-culture study resulted 
in such a sudden burst of investigation that it was a lost month in which a new organ- 
ism was not described, catalogued, and laid away, very frequently in the wrong grave. 

With this introduction we can enter upon a discussion of the criteria used in past 
and present times to establish the various groups from order to variety with an at- 
tempt at a critical summary of modern opinion, 

BASES OF CLASSIFICATION 

In the minds of many, perhaps most persons, the actual definition of the words 
used in taxonomy are vague, and some definitions may be helpful. Inasmuch as the 
"genus" is the center, so to speak, the grouping which brings together a collection of 
species, and itself forms the base for larger groupings, let us begin with it. Agassiz de- 
fined genera as "most closely allied groups of [animals] differing .... simply in the 
ultimate structural peculiarities of some of their parts." The Century Dictionary 
(iQoi) speaks of it as a 

classificatory group ranking next above the species, containing a group of species (sometimes 
a single species), possessing certain structural characters differing from those of any others. 
The value assigned to a genus is wholly arbitrary, that is, it is entirely a matter of opinion 
or current usage what characters shall be considered generic .... a genus has no natural, 
much less necessary, definition, its meaning being at best a matter of expert opinion, and the 
same is true of the species, family, order, class, etc. A genus of the animal kingdom in the 
time of Linnaeus was a group of species approximately equivalent to a modern family, some- 
times even to an order. 

Stiles {loc. cit.) defines it as "a taxonomic complex of specimens grouped for the mo- 
ment (according to our subjective and never absolutely perfect knowledge) around a 
genotype." 

These definitions emphasize that it is impossible to lay down rules which will hold 
future experts, at least beyond a certain general plan, and also show why it is that in 
so many cases the desire for revision starts with the study of a genus, and later in- 
volves its position in connection with other genera. 

Perhaps one of the most important points in the description of genera is the selec- 



ROGER G. PERKINS 125 

tion of the genot>'pe. If this is properly done, the personal equation involved in the 
selection of various group characters for inclusion is checked and crystallized by the 
selected type example. This has long been theoretically necessary, but has been much 
neglected. Buchanan {loc. cit.) and Bergey {loc. cit.) have been the most consistent, 
not only in their demands for this, but in actually supplying the types. 

There are five main headings which influence classification. Beginning with the 
earliest, "Morphology," we have added "Chemistry and Physiology," "Evolution," 
"Habitat," and "Immunology." The most successful work has been accomplished 
with the first two. 

MORPHOLOGY 

In the beginnings of biological classification, this was the only basis, the alpha and 
omega, and until discoveries multiplied was quite satisfactory. But as more and more 
individuals were discovered, groups became very large and needed further or differ- 
ent subdivisons. Under the head "Morphology" we generally admit not only form 
and arrangement, including organelles, capsules, and spores, but also tinctorial dis- 
tinctions such as gram stain, acid fastness, and granule formation, though of course 
these might also be considered chemical. But even with these admissions the number 
of distinctive characters is limited. Moreover, as noted earlier, even form is unstable, 
and subject to temporary or permanent modification which may readily become in- 
herited. 

But whatever may be the changes in morphology under extraordinary conditions, 
we must still admit that under ordinary conditions, or under readily obtainable stand- 
ard laboratory conditions, a rod form remains a rod form, a sphere continues as a sphere 
a spiral form clings to its spirals. Moreover, a coccus with the habit of dividing in 
certain planes tends to retain this habit with some obstinacy, and a rod form which 
after division tends to retain two or more individuals in a chain may be reasonably 
expected to continue this activity. What constants may we add to form and planes of 
fission? Obviously, the most conspicuous are motility and spore formation. 

Migula (1897-1900) went so far as to make a major division on the basis of mo- 
tility, speaking of the motile form as "bacillus," the non-motile as "bacterium." Al- 
though this was rather widely accepted for a time, it finds little support at present. 
Leaving aside the question as to whether motility is a higher or lower divisional point, 
there is marked variation in this character, to such a degree that insistence upon it 
will make wide separations of closely allied strains. In this group, moreover, not only 
is the actual motility a differentiating feature, but inasmuch as in the Eubaderiales 
(Buchanan) motility is dependent on flagella, the arrangements of these may also be 
important. Orla- Jensen's two orders are based on such arrangement, separating those 
with flagella at the end from those with flagella all around. Breed and others (191S) 
state: 

In passing, it is of interest to notice that there is a close analogy between the generally 
recognized groups of bacteria and those of protozoa. Thus the cephalotrichic and peritrichic 
bacteria find their analogies respectively in the flagellates and ciliates. This analogy goes 
further than a mere resemblance in the arrangements of organs of locomotion ; for the ciliates 
and peritrichic bacteria are both highly specialized grc ups, while both flagellates and cephalo- 
trichic bacteria contain all gradations between primitive forms and highly specialized human 
parasites. 



126 CLASSIFICATION OF BACTERIA 

I must confess, however, that on tabulating the one group against the other, save for 
the fact that more of the peritricheae are pathogenic, the evidence of superiority ap- 
pears inconclusive. It does not seem to me that our knowledge of bio-chemistry is 
sufficient to show that the power to utilize or break down certain chemical compounds 
proves more or less "advancement," and the analogies with the protozoa, while un- 
questionably fascinating, seem to me to stop at that point. 

Without plunging too deeply into controversy, one may say that the division on 
the basis of spore formation is logical, as showing a major activity, and is widely ac- 
cepted, though the resultant group is variously placed. Some (Table I) accept this 
character as a basis for families under the Euhacteriales , others prefer tribal divisions 
under the family of rod forms, others as divisions of even lower rank. Classifications 
such as Orla-Jensen's (1921), fundamentally based on physiology and chemistry, are 
much less interested in spore formation. 

How far should the differentiation by morphology carry? It seems to me that 
this basis can certainly carry us as far as genus, and Hall (1927) goes further, saying: 
'T submit that morphologic criteria should enable us to identify the genus, and I be- 
lieve that the definition of orders, families and genera should be based solely upon 
morphologic data." With this sentiment many but by no means all agree. 

CHEMISTRY AND PHYSIOLOGY 

The most extensive changes have been due to attempts to use the activities of the 
bacteria rather than their form and arrangement as differentiating factors. The 
changes have ranged from a practically complete substitution, such as Orla-Jensen's 
(after the original flagellar division), to studies of small fractions of the problem. The 
general tendency of taxonomists is to emphasize morphology in the larger groupings 
and to reserve biological activities for smaller collections. Pigment formation, prob- 
ably the earliest observed of the definite chemical activities, has been graded all the 
way from family to variety, sometimes in connection with other group characters, 
sometimes practically alone. No very adequate reason has been adduced why groups 
of chromogenic organisms with different metabolic activities should be classed by 
their pigments rather than distributed according to their other characteristics. It is 
true that we have a very moderately studied collection of pigment-formers with little 
else by which to classify them and that this procedure may be temporarily convenient. 
But why the power to use fixed oxygen rather than free oxygen, or to break down sug- 
ars, or to form complex toxins or proteolytic ferments is not as remarkable as the 
power to form pigments, has never been demonstrated to my satisfaction. Hucker {loc. 
cit.) states in regard to the cocci: 

It seems evident that pigment production by the mass-forming cocci is a very important 
character for use in classification. However, due to its general lack of correlation with other 
characters, it does not appear that the group should be divided into genera with differences 
in chromogenesis as the chief diagnostic feature. On the other hand it seems that pigment 
production is sufficiently constant to warrant its use as a character of importance in differ- 
entiating species, and when so used should be interpreted along broad lines rather than to 
attempt to make fine distinctions in shades of color. 

In the same way the "nitro-" bacteria, placed as high as a family by some, a sub- 
family or tribe by others, seem to be getting more attention than they deserve. As 



ROGER G. PERKINS 127 

will be seen later, even the arguments for early historical precedence are not generally 
accepted. In other words, are not most of these chemical and physiological characters 
even less definite and stable than morphology, and should not their use begin where 
the criteria of morphology alone are inadequate? It has, of course, been frequently 
brought out that characters important in one group are unimportant in another, as, 
for instance, fermentations in the cocci and in the bacilli, or character of division in 
the same groups. It is probably impossible to formulate as a rule whether a given 
character should always define species or confine itself to varieties. 

HABITAT AS A DIFFERENTIAL POINT: PATHOGENICITY 

"Pathogenicity may be taken as a type of those powers of the organism which are 
easily and profoundly modified by external conditions." This quotation from Wins- 
low in 1906 seems incontestable, yet later he uses "habitat" in the separation of his 
genera in the Coccaceae. This seems rather inconsistent even though he insists upon 
groups of characters rather than individual ones. Here, again, there is marked dis- 
agreement, as Hucker differs from Winslow in a similar paper, as follows: 

Due to the fact that the micrococci are found in such a wide variety of sources, and due 
to their ability of adaptation to various environments, the use of habitat alone as a char- 
acter in separating the general group is precluded. It is true also that no other character will 
definitely correlate with the source of the different types and bear out any conclusions that 
different natural groups of micrococci can be secured from different habitats. 

It would seem that if this applies to this group, other groups in which the same ob- 
jections hold true would be similarly affected. 

It has always seemed to me that the selection of pathogenicity as a prime factor 
of division is only part of the pride of the human race in its superiority, a pride which 
has from time to time admitted some of the lower animals under the wider cloak of 
animate, moving life. In the consideration of bacteria, however, why should one 
metabolic activity be superior to another? We believe that pathogenicity is more or 
less of an accident, that bacteria are not toxic in order to be pathogenic, but pathogenic 
because they happen to be toxic. Because some by-product of digestion when applied 
to a mucous membrane causes death and breaking down of the local cells, whereas 
other products of digestion can break them down only after cell death, may be a 
matter of degree rather than character. Non-bacterial poisons may have the same 
effect, but are not classified on this basis, but rather on the general character of their 
possible chemical combinations. Should we not therefore consider the chemical activ- 
ities of bacteria, due to metabolic products with various chemical characters, rather 
on the basis of these characters alone than on some special reaction, which is, as Wins- 
low (1905) calls to our attention, readily modifiable by environment? Breed, Conn, 
and Baker {loc. cit.) criticize the Society of American Bacteriologists' Committee 
(1917) in that "too great weight has been placed on pathogenicity," attacking special- 
ly Hemophilus, Pasteurella, and Erwinia. It seems to me dangerous in a general clas- 
sification to say that a group is generally, or usually, or essentially parasitic, in view 
of the well-known fact, already recalled, that our knowledge of bacteria is confined 
to a very small fraction of the total in existence, and since scarcely any groups which 
contain pathogens do not include forms closely related but without pathogenicity. 



128 CLASSIFICATION OF BACTERIA 

Can we determine habitat by a limited series of findings? Any organism that requires 
oxygen, moisture, warmth, and broken-down organic matter may develop on the 
surface of the body and remain there for various periods of time. It will also survive 
or develop anywhere else under similar conditions, but would not be called parasitic 
if found on a filthy blanket, 

HABITAT OTHER THAN PATHOGENIC 

The same general criticisms are applicable. It is unquestionably valuable to know 
that most of a group have been isolated from water or soil or mucous membranes, 
but the connotation to the average reader is that there is some inherent relation be- 
tween the organisms and the particular environment. And this may or may not be 
the case. This is a dififerent question from that of taxonomic names, and whether such 
a definition is misleading or not is a matter of sharp controversy. 

CHRONOLOGICAL CLASSIFICATIONS 

Orla-Jensen (Joe. cit.) and Kligler (1913) have presented more or less elaborate 
classifications on this basis, and a large part of the former's grouping, especially that 
relating to the nitro-bacteria, has been taken over by the Committee of the Society 
of American Bacteriologists. Breed, Conn, and Baker {loc. cit.) make a well-consid- 
ered attack on both these systems, emphasizing that we are now dealing with end- 
processes of evolution and not the primary forms except in so far as environmental 
conditions have remained more or less permanent. (It is likely that conditions even 
in water are not now identical with those of the period of origin of bacteria.) They quote 
the Committee in their definition of the Nitro-bacteriaceae, and state that if we ac- 
cept this family, we indorse the theory that its members are modern representatives 
of the primordial bacteria. They object to this, and indicate that the theory, while 
interesting, is without adequate proof. (Buchanan [1918] attacks Kligler's paper even 
more sharply.) The most logical reason adduced for family rank is the close relation 
of rods and spheres in this group and the inconvenience of separation. 

It seems to me also that in this question of primordial relations as a basis for di- 
vision, too little notice has been taken of the well-recognized fact that characters may 
be lost as well as acquired. An organism once able to utilize and synthesize simple ma- 
terials may, under conditions of parasitism or even symbiosis, lose those powers, and 
in the vast numbers of generations of bacteria it is not far fetched to consider the 
possibility that strains with certain powers may lose part of them under changed en- 
vironment, and under subsecjuent changes may either regain what was lost, acquire 
new powers, or any combination of these two changes. Orla-Jensen shows that it is 
quite probable that actual changes in form from sphere to rod may have taken place, 
and botanical and zoological alterations due to environment under short-time ex- 
perimental conditions are well known. Conclusions drawn from the conditions of 
light, heat, available food, etc., in those geological periods during which bacteria de- 
veloped are based on disputable evidence, and must be considered cautiously. 

IMMUNOLOGY 

This division of scientific work is still in its infancy, and its application to classifi- 
cation of bacteria has been unsuccessful, save in the differentiation of varieties, and 



ROGER G. PERKINS 129 

rarely in confirmation of species which are already shown to be in close relation. As an 
aid to differentiation, for such groups as the pneumococci, its value is high. 

THE PROBLEM OF A NEW CLASSIFICATION 

With all these difBculties of a classification based on a variable combination of 
morphology and chemistry, what are the possibilities of a complete revision? I con- 
fess it seems to me that unless someone is sufificiently omniscient to foresee the new 
developments, and to select characteristics which will remain stable for the next hun- 
dred years there is little hope for such a classification. Man is conservative and 
would demand proofs of omniscience and prophecy which would be hard to offer. The 
boldest now in view is that of Enderlein (1925), who believes that morphology is ab- 
solutely the only important criterion, especially in its development. He decries mono- 
morphism, mutations, and monocytism, and goes into extensive details as to the 
minute anatomy of the bacterial cell, with nucleus, sexual and asexual division, re- 
consideration of the spore, etc. The entire vocabulary is different, so that no com- 
parison can ])e made. 

PRESENT STATUS OF BACTERIAL CLASSIFICATION 

Reviewing the present status of bacterial classification, what hope does it offer 
for the future? There is practical unanimity in accepting the class Schizomycetes as 
first brought forward by Naegeli in 1857. Vuillemin gave a good description and 
Buchanan (1925) modified it somewhat. Vuil'.emin stated: 

They are simple organisms, formed of a single element without septa and unbranched. 
The element is circumscribed by a rigid vegetable-like membrane, elastic, but not contractile, 
sometimes also with a capsule. It may undergo plasmolysis or plasmoptysis. The protoplasm 
is less differentiated than that of most cells; the chromatin particles do not form an individual 
nucleus of a permanent type. Division is amitotic. Some forms are motile with flagella which 
traverse the membrane at points characteristic of the species (polar or diffuse). They are 
not broader than 5 ix when not in bunches or unimpregnated with metal or colloidal coloring 
material. The resting stage may be either an arthrospore resulting from a simple modifica- 
tion of the membrane or an endospore. In some cases the spore-bearing element retains its 
form or is modified passively by the enlargement of the spore, in other cases it is a specialized 
element for spore production. Only the latter type of sporulation is satisfactory for generic 
characters. The amitotic division usually occurs by a pinching transversely with rapid sepa- 
ration of two individuals. These may remain united into families, in chains, la3'ers or packets 
as determined by the successive planes of deviation. 

He emphasizes that all forms showing contractility should be placed in the protozoa, 
that the myxobacteria constitute a distinct group, and that forms like the tubercle 
bacillus which show branching should be placed in the mold group Microsiphones. 

Buchanan used the following characterization: 

Typically unicellular plants, cells usually small and relatively primitive in organization. 
The cells are of many shapes, spherical, cylindrical, spiral or filamentous; cells often united 
into groups, families or filaments; occasionally in the latter showing some differentiation 
among the cells, simulating the organization seen in some of the blue-green, filamentous 
algae. No sexual reproduction known. Multiplication typically by cell fission. Endospores 
are formed by some species of the Eubacteriales [see below], gonidia (conidia, arthrospores) 



I30 CLASSIFICATION OF BACTERIA 

by some of the filamentous forms. Chlorophyll is produced by none of the bacteria (with the 
possible exception of a single genus). Many forms produce pigments of other types. The 
cells may be motile by means of fiagella; some of the forms intergrading with the protozoa 
are flexous, a few filamentous forms (as Beggiatoa) show an oscillating movement similar to 
that of certain of the blue-green algae (as Oscillatoria) . 

The Committee used the following description: 

Minute, one-celled, chlorophyll-free, colorless, rarely violet-red or green-colored plants, 
which typically multiply by dividing in one, two or three directions of space, the cells thus 
formed sometimes remaining united into filamentous, flat, or cubical aggregates. Capsule 
or sheath composed in the main of protein matter. The cell plasma generally homogeneous 
without a nucleus. Sexual reproduction absent. In many species resting bodies are pro- 
duced, either endospores or gonidia. Cells may be motile by means of flagella. 

Under the Schizomycetes a number of orders have been proposed, in some of which 
there is general agreement and in some less. In the discussion following, except where 
a definite published group is referred to, I shall attempt to avoid the technical terms, 
using groups, forms, kinds, etc., as well as the common term "bacteria" or "cocci" to 
indicate rods or spheres. 

Among the groups generally known as "higher bacteria" and less important to 
the medical bacteriologist there is a great deal of agreement. 

Under the Schizomycetes the groups of the sulphur {thio-) bacteria, the sheathed 
(chlamydo-) bacteria, and the pseudoplasmodial (myxo-) bacteria are fairly well ac- 
knowledged and contain much the same material the world over, though the rank is 
not always the same. This leaves us the "true bacteria" {Eubacterlales [Buchanan]) 
with the border-line groups of "actino-" and spiral forms. 

Taking up first the border-line groups of thread forms and spiral forms, the sum- 
mary of their history leading to the present status is about as follows: 

Actinomyces — In 1877 Harz described the thread fungi in "lumpy jaw" of cattle 
as "actinomyces" and started the group on its quarrelsome path. Rivolta in i8/3 
preferred "discomyces," and various observers (Blanchard [1895], Brumpt [1910], and 
Merrill and Wade [1919]) agree with him. Streptothrix (Cohn) is accepted by others, 
but there is apparently reason to believe this term invalid (Buchanan, p. 497), though 
it still has plenty of adherents. Study of the group brought out the fact that there are 
two distinct methods of reproduction within the group, and Trevisan in 1899 sug- 
gested the name Nocardia for those forming spores, as distinguished from the non- 
spore-forming ''actinomyces,'" a term accepted by Wright in 1904. Buchanan (p. 405) 
regards it as a synonym. Thus we have four names, each more or less ably supported 
as the ten pages devoted to the subject by Buchanan indicate. The Committee adopts 
Actinomycetales as an order, as do also Buchanan and Bergey, while Breed, Conn, and 
Baker consider them as a family — Actinomycelaceae. 

Whatever name we prefer and select, new arguments arise as to the contents of 
the group (see Table I). Some authors under the prefix "actino-" include the thread 
fungi and also the tubercle-bacillus group and the diphtheria group, others include the 
same series under the prefix "myco-," while others separate the thread fungi from the 
"myco-bacteria," placing these last directly among the non-spore-forming rods. The 
modern tendency, since Lehmann and Neumann, seems to be to place tuberculosis 



I 



ROGER G. PERKINS 131 

and its associates with diphtheria and its associates and to consider them both as re- 
lated to the thread fungi which have true branching. It is also the prevailing practice 
where Nocardia is used to confine this to the special group noted above. It is to be 
hoped that a better agreement may develop in this group. 

Spiral fonns. — There has naturally been much contest as to the placing of these. 
One school places all spiral forms, from the cholera organism to the treponema and 
its associates, in one group, subdivided into: (c) simpler forms long known to us 
as the cholera group; and {b) the more "protozoan-like" (if that means anything), 
including treponema, leptospira, spirochaeta, etc. Another school places this second 
group in a completely separate division, leaving the others in Spirillaceae {q.v. infra). 
Some believe the "protozoan-like" group to be intermediate between bacteria and 
protozoa but it is interesting to find recent textbooks on protozoa omitting it entirely 
(Hegner and Taliaferro, Craig). (See Table I for details). In general, the first of the 
two groups corresponds to Spirillaceae Migula (1894) emended Committee, So- 
ciety American Bacteriologists (1917), with the following description: "Cells elon- 
gate, more or less spirally curved. Cell division always transverse, never longitudinal. 
Cells non-flexuous. Usually without endospores. As a rule motile by means of polar 
flagella, sometimes non-motile. Typically water forms, though some species are in- 
testinal parasites." The second corresponds in the same way with Spirochaetaceae 
(Swellengrebel) described by the Committee as follows: "Free living or parasitic 
spirilliform organisms with or without flagella, with undulating or rigid spiral twists. 
Reproduction by transverse division and by 'coccoid bodies,' the equivalent of 
spores." 

This brings us to the group of true bacteria (Eubaderiales , Buchanan) (see 
Table I). 

This vast group of simplified forms, aerobic and anaerobic, spherical and rod 
shaped, pigment formers or non-pigment formers, fermenters or non-fermenters, mo- 
tile or non-motile, gram positive or negative, spore-formers or non-spore-formers, and 
with many other positive and negative characters, has been divided and subdivided 
on the basis of combinations of these until there is practically no theory which may 
not find substantiation in published work. The minimum number of divisions under 
this head is three: the spherical forms, the rod forms, and the spiral forms. 

Spherical forms. — This group was recorded as Coccaceae by Zopf in 1884 and is 
the most widely accepted of all, including all spherical forms of whatever arrange- 
ment, with certain minor and not undisputed exceptions. 

In spite of possibilities that in the development of bacteria there may be a ten- 
dency for cocci, especially when there are one or more flagella, to elongate, it seems 
agreed by most observers that the spherical forms are worthy of a grouping of their 
own, and the rod forms likewise. If one selects habitat, or fermentation, or color, or 
motility, or pathogenicity as prime factors, this grouping will at once disappear. Some 
observers (Orla- Jensen) believe that the chain-cocci and the chain-rods are to be con- 
sidered together, but their arguments, though interesting, are necessarily based on 
theoretical grounds, and, as noted elsewhere, our actual, objective knowledge of the 
evolution of bacteria from the original, whatever it was, is mostly speculative. 
Whether the modern work on variation will reach a point from which we may argue 



1.^2 



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ROGER G. PERKINS 133 

backward is an open question, and in the meantime we find in general that, at least 
under ordinary conditions, in the laboratory of nature rather than in that of the 
specialized scientist, the coccus remains coccus. There are two qualifications to the 
acceptance of a single group for all spherical forms. Some desire to place the so-called 
''primitive" nitro group together, both the spheres and the rods, on the basis of sim- 
ilar physiological characters regardless of form. In a similar manner there are those 
who believe that organisms of the lactococcus type should be placed with those of the 
lactobacillus type. In the first report of the Society of American Bacteriologists Com- 
mittee in 191 7, Lactobacillaceae was recorded as a family, and in the final report it 
was placed as a tribe under Baderiaceae. 

Further subdivisions of the family have been made in various ways. Winslow, 
Bergey, Castellani and Chalmers among recent workers made a primary division on 
the basis of parasitism, following this by morphology. The Society of American Bac- 
teriologists follows Winslow closely, but many writers feel that the establishment of 
habitat, or its subdivision parasitism, is likely to be misleading and does not produce 
natural groups. 

Hucker {loc. cit.) considers pigment as a species rather than a generic distinction, 
and sees no reason for the retention of the red pigment formers as a genus among thp 
cocci, the rest having already been generally abandoned in this group. In general, all 
the chemical and physiological characters are best used in the differentiation of 
species and varieties. 

Rod forms. — There remains the large group of rod forms about whose classifica- 
tion there is a more or less consistent disagreement. As will be noted by the chart, 
the tendency of foreign writers is to group all of these under one head, sometimes with 
one name, sometimes with another, though Orla- Jensen has made much wider sepa- 
rations, from which one of the American expansions has come. This is the group of 
supposedly "primitive" organisms, mentioned in this paper as the "nitro"- forms, 
accepted with more or less modification by Buchanan, the Committee, and Bergey and 
criticized by others, as noted earlier in the paper. An additional family, the Pseudo- 
monadaceae, including pyocyaneus and allied forms, is found as such only in the 
Society of American Bacteriologists and in Breed's comments (1917), elsewhere hav- 
ing a maximum rank of genus. Another American expansion is the elevation to fami- 
ly rank of both the spore-formers and the non-spore-formers, as Baderiaceae and Ba- 
cillaccae, a proper division, though there is argument as to the rank which should be 
given these groups. Buchanan and Castellani, in agreement with most of the Euro- 
pean authors, prefer subfamily rank. The variety of further divisions is unending, 
and, as noted earlier, far more work has been done on species and variety than on 
genus and higher ranks. It is almost impossible to summarize within any reasonable 
space the variety of opinions. Primary difi"erentiations, after or before relation to 
spore formation, are made on the basis of pigment formation, habitat, morphology, 
and physiology. Bergey, in his eleven tribes of non-spore-formers, names two on the 
basis of pigment formation, three on parasitism, five on cultural characteristics, and 
one on morphology. Buchanan makes two divisions of the same group by morphology, 
with a second division by physiological needs and further subdivisions on different 
bases for different groups. Castellani and Chalmers divide their family of Bacillaceae 



134 CLASSIFICATION OF BACTERIA 

into ten tribes, primarily on the cultural characters, or physiological activities. This 
is sufficient to show clearly how little bacteriologists have been able to agree on evalua- 
tion of the actual relations of these various characters. 

I SUMMARY OF "eUBACTERIALES" 

1 

Within the Eubacteriales the final report of the Committee forms the largest rium- 
ber of families, and may consequently be used as a basis. The accompanying chart 
shows the general relations and the variations in content of most of the European 
and American classifications, except those which depart so far from the prevailing 
methods as not to be readily comparable. It will be at once noted that the American 
group is more detailed. One may summarize the chart somewhat as follows: 

The "spherical" group is generally accepted. 

The "spiral" group is generally accepted, but the content varies, some writers 
including both the spirals typified by the cholera organism, and those of the "spiro- 
chaete" type, ochers placing the latter group separately, usually in an order by itself. 

The "cylindrical" or rod group is the most varied. Under it, however, certain 
groups are usually put together, such as the tuberculosis-diphtheria combination, 
unless this is placed with actinomyces. 

The "spore-formers" and "non-spore-formers" are usually differentiated, but the 
rank varies. 

The "nitro" -bacteria are generally kept together though again with different 
ranks and occasionally placed with the pyocyaneus-fluorescens group. 

The "pigment formers" are also kept more or less together. 

In the various classifications such characters as motility, flagella arrangement, 
gram stain, fermentation, proteolysis, etc., are mostly well down the line. 

REFERENCES 

No attempt has been made to develop or extend a list. Any student of classifica- 
tion must necessarily consult Buchanan, who gives a full bibliography. This is supple- 
mented by Enlows as concerns genera, and the various articles by Winslow and his 
associates add enough references to occupy the student for some time. Save when 
otherwise specified, classifications recorded by these writers have been accepted and 
are referred to in the published articles. 

Aldrich, J. M.: "The Limitations of Taxonomy," Science, 65, 381-8.15. 1927. 

Bergey, D. H., et al.: Manual of Determinative Bacteriology, 1923. 

Breed, R. S., Conn, H. J., and Baker, J. C: "Comments on the Evolution and Classification 
of Bacteria," /. Bad., 3, 445-59. 1918. 

Breed, R. S., and Conn, H. J. : "Nomenclature of Actinomycetaceae," ibid., 4, 585-602. 1919; 

I ihid (Addenda), 5, 489-90. 1920. 

Buchanan, R. E.: General Systematic Bacteriology. 1925; "Nomenclature of the Coccaceae/' 
/. Infect. Dis., 17, 528 41. 1915; "Studies in Nomenclature and Classification of Bac- 
teria," ibid., 1-3. 1916-18; "The Evolution of Bacteria," Science, 47, 320-24. 1918. 

Castellani, Aldo, and Chalmers, A. J.: Manual of Tropical Medicine. 1919; Ann. dc I'lnst. 
Pasteur, 34, 600-621. 1920. 

Chester, F. O: Manual of Determinative Bacteriology. 1901. 

Enderlein, G.: Bakterien-Cyclogenie. Berlin and Leipzig, 1924. 



I 



ROGER G. PERKINS 135 

Enlows, E. M. A.: "Generic Names of Bacteria." Hyg. Lab. Bull. 121. 

Felt, E. P.: "Nomenclatural Efficiency," Science, 65, 489-Qi. 1927. 

Hall, I. C: "Some Fallacious Tendencies in Bacteriologic Taxonomy," /. Bad., 13, 245-53. 
1927. 

Hitchcock, A. S.: "How the Taxonomists May Utilize the Instructional Committee on 
Nomenclature." Science, 65, 412-15. 1927. 

Hucker, G. J.: "Studies on the Coccaceae," Tech. Bull. 99-103. Geneva, N.Y.: New York 
State Agricultural Experiment Station, 1924. 

Kligler, I. J.: "A Systematic Study of the Coccaceae in the Collection of the Museum of 
Natural History," J. Infect. Dis., 12, 432. 1913. 

Lehmann, K. B., and Neumann, R.: English translation of second German edition by G. H. 
Weaver. 

Merrill, E. D., and Wade, H. W.: "Validity of the Name Discomyces, etc.," Philippine 
J. Sc, 14, 55-69. 1919- 

Migula, W.: System dcr Baktcrien. 1887-1900. 

Orla- Jensen, S.: "Hauptlinien des natiirlichen bakterien Systems," Centralbl. f. Bakterio . 
Abt. II, 22, 305-46. 1909; "The Main Lines of the Natural Bacterial System," /. Bad., 
6, 263-73. 1921. 

Stiles. C. W.: "International Code of Zoological Nomenclatures as Applied to Medicine." 
Hyg. Lab. Bull. 24; "Underlying Factors in the Confusion in Zoological Nomenclature," 
Science, 65, 195-99. 1927. 

Winslow, C.-E. A., d al. (Committee of the Society of American Bacteriologists on Character- 
ization and Classification of Bacterial Types.) "The Families and Genera of Bacteria" 
(Preliminary Report), /. Bad., 5, 191-229. Baltimore, 1917; "The Families and 
Genera of Bacteria" (Final Report of the Committee of the Society of American Bac- 
teriologists on Characterization and Classification of Bacterial Types), ibid. Baltimore, 
1920. 

Winslow, C.-E. A., and Rogers, Anne F.: "A Revision of the Coccaceae," Science (N.S.) 
21, 669-72. 1905; "A Statistical Study of Generic Characters in the Coccaceae," 
J. Inject. Dis., 3, 485-46. 1906. 

Winslow, C.-E. A., and Winslow, Anne Rogers: The Systematic Relationships of the Coccaceae. 



CHAPTER X 



"T 




> 

f^ 


f 




[} 


Jd 


P 


r 




c 


N 


> 



(<^) 



ATOMS, IONS, SALTS, AND SURFACES 

WILLIAM D. HARKINS 
University of Chicago 

ATOMS, IONS, AND SALTS 

THE ARRANGEMENT OE ATOMS IN SOLIDS 

Several different types of evidence indicate that solid substances are in general 
built up from minute building blocks called "atoms," whose diameters vary slightly 
with the element involved, but are of the order of a hundred-miUionth of an inch. The 
atoms attract one another in a way which is suggestive of the action of small magnets; 

it is believed that the forces involved 
are largely electrical but partly magnet- 
ic. In any crystal the atoms or mole- 
cules are arranged in a definite pattern 
or lattice, and this pattern largely de- 
termines the outer form of the crystal. 
Thus in rock salt (Fig. la) sodium and 
chlorine occupy alternate corners of 
the cubes of the lattice, and each atom 
of chlorine is surrounded by six atoms 
of sodium and each atom of sodium by 
six atoms of chlorine. Calculations 
based upon the density of rock salt, 
its molecular weight and the number 
of molecules in the molecular weight 
(6.06 X lo-^), indicate that the distance 
between the center of any sodium and 
any adjacent chlorine atom is 2.81 
hundred-millionths of a centimeter, or 
2.81 A units.' 

Crystals have 32 types of outer form and 14 types of atomic pattern which 
may be arranged in 230 different ways. 

THE COMPOSITION AND STRUCTURE OF ATOMS 

All known material substances are considered as built up from 92 simple forms of 
matter known as the "chemical elements." Of these 90 have been discovered and 2, 
with element numbers 85 and 87, have not been found. Each of these elements was 
formerly supposed to consist of only one kind of atom of a definite atomic weight, but 
in recent years it has been found that an element may consist of from one to eleven 
(probably even more) atomic species which are almost identical in their chemical 

' A glossary of symbols and terms is given on pages 176 and 177. 

136 



;^ 





y 


y 




■7> 


^ 




y 




^ 


^ 




D 


y 


c 






E 






^ 






y 






/" 


% 




y 


B 


^ 









A 






^ 




^ 


<^ 




A , 


K^ 



(6) 



(O 



Fig. I. — {a) Space lattice of sodium chloride, 
(6) face-centered, and (c) body-centered cubic lat- 
tice. 




Fig. 2. — Tni 



electrons (C. T. R. Wilson). Note that the tracks are dotted and irregular 




P'iG. 3a. — ( Fig. 2,l> shows how to find the track of a positive electron in this figure.) Tracks of 
positive helium atoms, and a track of the positive electron (proton or nucleus [H+] of the hydrogen 
atom). This photograph illustrates the synthesis of an atom, since a helium atom strikes a nitrogen 
atom and forms a (heavier) o.xygen atom and a hydrogen atom. (Photograph by Harkins and 
Shadduck.) 



WILLIAM D. HARKINS 



137 



behavior. Since all of the species which constitute an element are given only one 
place in the periodic system of the chemists, they are called "isotopes" (iso = "sa.me," 
to pe = "place"). 

The lightest-known atoms are those of hydrogen with a mass of i.66Xio~^-'gm., 
but the relative or atomic weight is taken as 1.0078 in order that the atomic weight 
of oxygen may be exactly the whole number 16. 

The mass of a projectile may be determined by the diilculty of deflecting it from 
its path when moving at any definite speed, since the momentum (mv) is proportional 
to the mass at a given velocity. Certain negatively charged particles are thus de- 
flected about two thousand times more easily than hydrogen atoms. These particles 
are designated as "negative electrons," and their charge 
is exactly ecjual in magnitude, within the limits of error of 
the most exact work, to that carried by positively charged 
hydrogen in the electrolysis of an acid in water. The 
tracks of negative electrons in air are shown in Figure 2. 
It may be noted that these tracks are very irregular, while 
the tracks given by hydrogen or other atoms are straight. 

Thus, this is true of the atom tracks shown in Figure 
3. The dim track in the right-hand view is that of posi- 
tive hydrogen, which is the lightest positively charged 
particle known, so it is considered as the positive elec- 
tron, often called the "proton." 

All uncharged atoms are built up from equal num- 
bers of protons and electrons. In the hydrogen atom 
there is one proton, which contains almost all of the mass 
of the atom and acts as its central sun or nucleus, and one 
electron which is supposed, in the Bohr theory, to move 
in an orbit around the proton somewhat as the moon 
moves around the earth. 

It is supposed that the atomic weight of an atomic 
species gives the number of protons in its nucleus and also the total number of elec- 
trons in the atom. The nucleus of the helium atom consists of four protons and two 
electrons so that there is a net positive charge of two on the nucleus and there are, 
therefore, two negative planetary electrons in the outer part of the atom. Helium is 
the second element in the chemical periodic table, so its atomic number is 2. It may 
be noted that the atomic number expresses also the positive charge on the nucleus 
and the number of outer planetary electrons. 




Fig. 3?;. — Shows two views 
of the synthesis and disinte- 
gration of atoms given in Fig. 
3(1. The track marked H rep- 
resents the track of a positive 
electron or proton, shown at 
the left-hand side of the right- 
hand view of Fig. s(^. The 
angles are the same as those 
in the photograph. 



MASS, ENERGY, AND FREQUENCY OF VIBRATION 

Mass and energy may be considered as the same but measured in different units. 
Thus I gm. of mass is equivalent to i times the square of the velocity of light (c = 3X 
10" cm. a second, soc^ = 9Xio"'), or 9X10'" ergs. Thus a i-gm. weight (mass = i gm.) 
is 9X 10^" ergs of energy. 

In 191 5 Harkins and Wilson showed that, according to the special relativity the- 



138 ATOMS, IONS, SALTS, AND SURFACES 

ory of Einstein, the conversion of hydrogen into helium should liberate an enormous 
amount of energy. The reaction may be written: 

4H^He 

4.0312-gm. mass-^4.ooo-gm. mass+o.o3i2-gm. mass in the form of radiation 
0.0312-gm. mass is equivalent to 0.0312X9X10^° ergs = 
2.81X1C ergs = 6.71 X 10" calories 

In more ordinary terms, the conversion of i lb. of hydrogen into helium should give 
off as much energy as radiation — which could be transformed into heat — as would be 
given by the burning of 10,000 tons of coal. 

The frequency of light- vibrations is very great. According to the quantum theory, 

if this frequency is multiplied by a con- 
stant //, the quantum constant, the 
energy of one quantum of the radiation 
Q is obtained, E = hv. Conversely, the 

energy quantity divided by h gives the 
frequency of the vibration, which is the 
Q velocity of light (c) divided by the 

wave-length (X). From these relations 
x-v we find that if four hydrogen atoms 

^^ were to be converted into one helium, 

Q all of the particles having small veloci- 

ties, the wave-length emitted would be 
0.00046 A, or this would be of the type 
of the cosmic radiation which falls upon 
the earth from outside. The reaction 
Fig. 4 -Neutral sodium atom (diagrammatic probably occurs in several steps, but a 



O 



representation in a plane: the structure is un- ■ r ^u 1 ^\, v.^ 1 i. u 

^.^^^^^^,i part of the wave-lengths emitted should 

be of this general order of magnitude. 



known). 



IONS 

Atoms, or molecules which are not electrically neutral, are called "ions," so ions 
contain either a larger or a smaller number of electrons than of protons. The non- 
nuclear, or planetary, electrons of an atom are classified in sets. Thus in the sodium 
atom there are supposed to be three of these sets : the innermost with two, the next 
with eight, and the third or outermost with one electron. These numerical relations 
are represented by Figure 4. The chemical properties of an element are supposed to 
depend to a large extent, but not wholly, upon the number of electrons in the outer set. 
Thus the atoms of the alkalies, lithium, sodium, potassium, rubidium, and caesium, 
are all supposed to have one electron in the outer set. They ionize, i.e., lose this outer 
electron, more easily than any other elements. All of them react violently with water, 
and in all of their other chemical, and in most of their physical, properties act almost 
alike. 

With helium the outer set of electrons is complete when two electrons are present, 
but with heavier elements completeness in this set seems to be reached only when 
eight electrons are present. 



WILLIAM D. HARKINS 



139 



Sodium, magnesium, and aluminium have, respectively, one, two, and three outer 
electrons. Such atoms lose electrons to form positive ions. If, however, the outer set 
of electrons is more than half complete in the neutral atom, there is a much greater 
tendency to pick up electrons than to lose them, so such atoms usually form negative 
ions. If the three elements mentioned above are in the gaseous form and the sodium 
atom loses one (Na+), the magnesium two (Mg++), and aluminium three electrons 
(A1+++), then ten planetary electrons are present in each of these ions (Fig. 5). This 
is the number of planetary electrons present in the neutral neon (Ne) atom. Since it 
is supposed that the arrangement of the electrons is largely conditioned by their 
number, it is supposed that Ne, Na+, Mg++, and A1+++ have the same structure ex- 
cept that the electrons are bound somewhat more tightly as the charge on the nucleus 
changes from 10+ for neon to 13+ for aluminium. That the idea of a common struc- 
ture is justified is indicated by the fact that in so far as they have been investigated 
the spectra of these ions upon further dissociation is like that of neon. 



O 



o 



o 



o 



o 



o P^ o 



o 



00 GO 

Neutral neon atom Triply positive aluminium ion (A1+ + +) 

Fig. 5. — The composition of Ne, Na+, Mg++, or A1+++. The nuclear charges are 10+, 11+, 
12+, and 13+, respectively. 

IONS IN SOLID SALTS 

In rock salt the mean distance from the center of a sodium atom to that of any of 
the six adjacent chlorine atoms is, as has been stated, 2.81 A (2.81X10"* cm.). The 
fact that the analysis of the structure of such a crystal by X-ray methods has given no 
evidence that the sodium atom is more firmly attached to any one of these atoms than 
to the five others has led to the hypothesis that no molecules exist in such a crystal, 
and that the chlorine atoms are attracted to the sodium atom because the former are 
charged with a negative (Cl~) and the latter with a positive (Na+) electrical charge. 
Thus the assumption is that the crystal is built up from individual positive and nega- 
tive ions held together by the attraction between charges of like sign. The attraction 
between the ions of unlike sign of charge is assumed to follow Coulomb's law that at- 
traction or repulsion between electrified particles varies as the inverse of the square of 
the distance between them: 



F= 



6162 



(i) 



I40 ATOMS, IONS, SALTS, AND SURFACES 

Madelung and Born find that if Coulomb's law is assumed to hold for the attractive 
forces, it is necessary, in order to accord with the known values of the compressibility 
of solid salts, to assume that there is in addition a repulsive force between the ions 
which varies as about the inverse tenth power: 

ylO 

According to this type of theory, solid salts consist entirely of ions, and in this 
sense the salt is already completely ionized. 

THE IONIZATION OF SALTS IN AQUEOUS SOLUTIONS 

The dielectric constant {D) of ordinary solid salts is about 5, while that of water at 
ordinary temperatures is about 80. Since the attraction between charged particles 
(equation |i]) varies inversely as the dielectric constant, the attractive forces between 
ions of op])osite sign of charge should be very much less in water than in solid salt. If, 
therefore, it is assumed that the solid salt is completely ionized, it would be unreason- 
able to assume that the same salts are less than completely ionized when dissolved in 
water. 

The idea that salts in aqueous solution are completely ionized was suggested by 
Sutherland in 1907. The theory has been put in more definite form by Bjerrum, by 
Milner, and by Debye and Hiickel. Their fundamental idea is that the electrical at- 
tractions between ions of unlike sign, and repulsion between those of like sign, give 
rise to the following effect: on the average any positive ion will be immediately sur- 
rounded by more negative than positive ions, while any negative ion will be surrounded 
by more positive than negative ions. The most important result is that when the solu- 
tion is diluted the separation of the ions involves the expenditure of energy.^ 

' Debye and Hiickel have calculated this electrical internal energy by assuming that the charges 
on each ion are concentrated at a point, and that the distribution of these points is determined by 
probability. They use the probability relation of Boltzmann, developed in connection with the kinetic 
theory of gases, together with the equation of Poisson derived from the laws of electrostatics, includ- 
ing Coulomb's law. 

Suppose that we have an ion of valence + n and charge + nE. In any concentric shell of thick- 
ness dr, the potential is P and the density of the electric charge is p. 

The average kinetic energy of the molecules at a temperature Tis 3/2 kT. The Boltzmann constant 

k is equal to the gas constant 7? divided by the number of molecules in a gram molecule {6.o6Xio^'5). 

According to Boltzmann's principle, if the molecules are distributed in a field of force, such as in 

an electrical field, the distribution will be such that the number of molecules, instead of being equal 

-E 

to N, the number present in the absence of the field of force, will be equal to Ne *^ . Thus the number 

present is modified by a factor in which the base of Naperian logarithms is raised by a power equal 

to the potential energy of the molecule divided by two-thirds of its mean kinetic energ>'. 

The equation of Poisson applies to the variation of the potential P around a point when it is 

distributed with spherical symmetry. It may be expressed as follows: 



r^ dr \ dr ) ~ dr' 
The electrical density is represented by p. 



2 dP _ 47rp 
'^~r'dr'~ D' 



WILLIAM D. HARKINS 141 

LAW OF MASS ACTION 

Suppose that two substances, A and B, react to form the substances E and F, and 
that the reaction has proceeded until equilibrium has been attained. The chemical re- 
action may be expressed: 

aA+bB = cE+fF. (i) 

It was found by Guldberg and Waage that in such a case a definite law, known as the 
"mass law," determines the condition of the system at equilibrium. If the substances 
are gases this may be expressed 

Pe-Pf j^ / X 

Pa-Pb 
in which Kp is a constant. 

In the case of the simple dissociation 

A^B^C, (3) 

the mass law becomes 

-^ '^P' (4) 

Pa ^ 

In the ordinary development of the mass law it is assumed that the gas law {pv = 
NRT) is true, so the mass law fails to hoM in any case in which the gas law is invalid. 
The gas law may be written 

p^K^ = CRT. (S) 

V 

If this is substituted in (4) the following expression is obtained: 

^^ = ^ (6) 

C^ RT ' ^ ' 

but since the temperature is constant and R is the gas constant, Kp/RT is a constant, 
or 

^^ = K,. (J) 

^A 

DEVELOPMENT OF THE LAW OF MASS ACTION 

The law of mass action may be developed very simply by a consideration of the 
amount of work necessary to compress a gas reversibly, i.e., in such a way that the 
same amount of work may be regained when the gas expands. The amount of work 
done when a force (F) acts through a distance (S) is 

W = F.S. (i) 

If a gas expands in a cylinder provided with a piston, the force (F) is equal to the 
pressure (p) times the area (A) of the piston, so 

W=pAS=p-Av. (2) 



142 ATOMS, IONS, SALTS, AND SURFACES 

If the increase of volume (A v) is infinitesimal, it is designated by dv, so 

dW=p-dv. (3) 

But by the gas law 

P —, (4) 

so 

dW=NRT - . (S) 

The work (W) done by the reversible expansion of the gas from a volume to a 
volume V2 is 

W= I 'dW = NRT\ '^ = NRTln^. (6) 

Since, however, the volume of a perfect gas varies inversely as its pressure (/>), 

W = NRTln^. (7) 

P2 

Consider a chemical reaction 

aA+bB%eE+fF , 

in which all of the substances are in the gaseous state. Let the initial pressures of the 
gases A and B be p^ and p^ and the final pressures of E and F be p^ and /?/. Assume 
that a large box contains all of these four gases in equilibrium with one another at 
pressures pA, pB, pE, and pp- Let the gases A and B at pressures p^ and p'g be con- 
tained in two cylinders provided with pistons. 

The first step in the process is to change the pressure /?j to pA- The work done by 
a gas in such a process equals the number of mols (c) of gas times RT times the loga- 
rithm of the initial pressure divided by the final pressure, or 

P'a 

W, = aRTln^. (8) 

Pa 

By the use of a well-known characteristic of logarithms, this becomes 

W. = RTln^. (9) 

Pa 

The similar equation for the gas B is 

W2=RTln^. (10) 

Pb 

The gases A and B are now at pressures equal to their partial pressures in the 
large equilibrium box. Suppose that the cylinders which contain these gases are set on 



I 



WILLIAM D. HARKINS 



143 



the equilibrium box and that at the bottom of each cylinder there is a membrane 
permeable only to the gas in the cylinder (Fig. 6). 

Force the a mols of the gas A and the b mols of the gas B slowly into the box, and 
as rapidly as these react take out the e mols of the gas E and the/ mols of the gas F 
which are formed. 

Since work must be done upon the gases .1 and B to force them into the box, the 
work they do is negative, or 

Wi = p■^v==-aRt , (11) 

which is obtained from equation (2) by substituting in it the gas law 

p.Av = NRT iN=-a) . 





1 




I 




1 




1 






Gas 
A 






Gas 
E 


Gas 
F 






Gas 
B 




Equilibrium Mixture of A, B, E, and F 



Fig. 6 



For the gas B 

W,= -hRt. 

Since the gases E and F come out of the box, they do work, and 

Ws=^eRT, 
Wt=JRT . 



(12) 

(13) 
(14) 



The gases E and F are now at pressures pE and pp. Let them now be changed to 
their final state in which the pressures are p^ and pp: 



W^ = RTln-^^, 
Pe 



(is) 



W%=RTln-^ 



P± 
P'/ 



(16) 



The total work (W) is the sum of these eight quantities of work, or 

p^ pf p'ep'f 

W^RT In ^-RT In Y^M^+f -^-b) RT . 

^A^B ^A ^E 



(17) 



144 



ATOMS, IONS, SALTS, AND SURFACES 



Now, e-\-J—a—b is simply the increase (A n) in the number of molecules when the 
given reaction takes place, so 



P%P-i 



Et^F 



W = RTln'^^-RTln 
(Term i) (Term 2) 



P'^P'J 






(18) 



(Term 3) 



If the reaction takes place at a constant temperature, term 2 is a constant, since 
the pressures designated by primes are fixed values. Term 3 is also constant, sine • 
An, the increase of the number of mols of gas, is fixed by the reaction. 

The work done in a series of reversible changes depends only on the initial anrl 
final states, and is therefore a fixed quantity for the given reaction. Equation (18) 
may now be written: 



K, = RTln 



KPi 



or 



RT 



P%pfn 



4^5 



pU'^"'^"' 



■ Ki — K2 — K^ = K^ , 



Therefore 



pipV 



K, 



(19) 



(20) 



(21) 



which is the law of mass action. This equation gives the mass law in a general form. 
For a reaction in which only i mol of each substance is involved, 



it takes the form 



A+BZE+F , 
PeXPf 



Pa^Pi 



K 



Thus, according to the mass law, the product of the partial pressures in the equi- 
librium mixture of the substances formed in a chemical reaction, divided by the prod- 
uct of the partial pressures of the substances from which they are formed, is equal to 
a constant at any given temperature. If more than i mol of any of the substances is 
involved in the reaction, the corresponding partial pressure must be raised to a power 
equal to the number of mols. 

ESCAPING TENDENCY OR ACTIVITY 

According to the mass law, the effect of any substance present upon the chemical 
equilibrium is proportional to its concentration. Thus, the activity (a) of a substance 
may be expressed by its concentration, or 



CbCc 



= K 



(8) 



WILLIAM D. HARKINS 145 

While the activity of a perfect gas, or a substance which in solution obeys the per- 
fect-gas law, is expressed by its concentration, this is not the case if the gas law does 
not hold. The activity may then be defined as that quantity which, when substituted 
for the concentration of a substance in the mass-law equation, expresses its effect in 
determining the equilibrium. 

If a small quantity of iodine is shaken with water and carbon disulphide at 25° C. 
until equilibrium is attained, it is found that a unit volume of carbon disulphide con- 
tains six hundred times more iodine than the same volume of water. Since there is 
equilibrium, the tendency of iodine to escape from the water is the same as that from 
carbon disulphide, i.e., the vapor pressure of iodine should be the same over the two 
solutions, and this is found to be true. The identity of vapor pressures of the iodine 
indicates that the activities are equal. 

If it is considered that the activity of the iodine in the carbon disulphide is equal 
to its concentration, then the activity in water, which is the same, must be six hundred 
times the concentration of the iodine in the water. Thus carbon disulphide is a better 
solvent than water for iodine since it can hold six hundred times as much of the latter 
and still give no more activity to the iodine.' 

1. When the activity of a constituent is the same in two difTerent phases the con- 
stituent will not increase its concentration in one phase at the expense of the other un- 
less energy is supplied to effect the transfer. 

2. If the activity of a constituent is greater in one phase than another the con- 
stituent (if transferred at all) will pass from the phase in which it has the greater into 
the one in which it has the lesser activity. 

THE ACTIVITY COEFFICIENT OF ELECTROLYTES 

A solution of common salt which contains 58.5 gm. of sodium chloride to 1,000 gm, 
of water is considered to be i molal in concentration. If m represents the molality of 
a solution, its activity coefficient (a) is defined as the ratio of its activity to its mo- 
lality, or 

a 
a = — ; so a = ma . 

m 

The activity coefficient for a salt in its extremely dilute solution is i.oooo by defi- 
nition. At a molality of o.oi the activity coefficient for sodium chloride in its aqueous 
solution is 0.922, while at o.im it is 0.798. Now the activity coefficient plays the same 

' The escaping tendency may be expressed also in terms of the "fugacity" (/), a term introduced 
by G. N. Lewis. The fugacity of an ideal gas is equal to its pressure. Any gas is practically ideal at 
low pressures. At higher pressures its fugacity is the geometric mean of the actual pressure {P) of the 
gas and the ideal pressure {Pi) calculated from the gas law, so that 

^ Pi 

The activity of a constituent may now be defined as its relative fugacity (/) as compared with its 
fugacity in some standard state (/J, or 



146 



ATOMS, IONS, SALTS, AND SURFACES 



part in the newer work on the ionization of salts in solution as that of the degree of 
ionization in the older theory. Thus the degree of ionization of sodium chloride in its 
0.1m aqueous solution was given as 0.86, while the activity coefhcient as given above 
is 0.798. The activity coefficient is sometimes designated as the "thermodynamic de- 
gree of dissociation." 

Table I gives the activity coefficients for sodium and potassium chlorides and a 
few salts of higher types. It may be noted that as the product of the valence of the 
ions of the salt increases the activity coefficient decreases. 

TABLE I 

Activity Coefficients of Salts in Aqueous Solution .4t 25° C, as 
Calculated from the Lowering of the Freezing-Point 



Molality 
m 


Activity 
«NaCl 


Coefficient 
"KCl 


"Bad. 


<iLa(N0j)3 


"MttSO, 


001 


0.966 

•953 
.928 

■903 

.871 
.821 
.778 
•734 
.671 
■634 
•645 
.686 
.746 
.831 
0.852 


0. 965 

•951 
.926 
.899 
.865 
.809 
.762 
•715 
■654 
0.605 








002 








• 005 

.01 








0. 716 

•655 

•568 

50* 


0.571 
.491 

•391 

.326 

0.271 


0.404 


.02 

•05 

. I 

2 


•321 

.225 

.166 

0. 119 


0.5 

I 










20 ... 








3-0 

40 

S-o 

5-2 











































THE IONIC STRENGTH 



The activity of ions in a mixture is determined by the concentrations and elec- 
trical charges of all of the ions present. Lewis and Randall found that if the molality 
of each ion is multiplied by the square of its valence, the sum of these quantities (di- 
vided by 2, since both positive and negative ions are included) is an important quan- 
tity, designated as the ionic strength (fx). 

It is found that in dilute solutions the activity coefficient of a given electrolyte is the 
same in all solutions of the same ionic strength. 

The activity coefficient (a) in water of a salt composed of two ions is given by an 
extremely simple equation: 



— logia a = o.5oZi ZjV /i , 
in which Zi and Z, are the valences of the ions of the salt, and 



(i) 



(2) 



calculation of the activity coefficient from solubility 
If a solid .1 is in contact witli its saturated solution, the relation may be expressed: 



.1 (solid) l,-'l(ilissolve<l) , 



(3) 



WILLIAM D. HARKINS 147 

so the activity of the dissolved substance is equal to the activity of the solid: 

a(solid) = ^(dissolved A) = Const. ; (4) 

but since 

a(dissolved) = ;»a , (5) 

moao = m,ai = m2a2 , (6) 

where Wo designates the solubility in water and nij and W2 refer to the solubility of the 
substance A in aqueous solutions to which different salts have been added. From (6), 

m (7) 

Wo 

If the activity coefficient of the pure substance in its aqueous solution is arbitrarily 

fixed as unity, then 

Wo (8) 

a = — . ^ 

m 

If the solute (dissolved substance) is a salt, its mean molality (w ± ) should be used. 

a = Const.-i-. ^9) 

The mean molality is a geometric mean as defined by the equation 

/„Z+„Z-\i/Z 

tn^=myZ Z_ j , 

and Z = Z+-\-Z-. 

The extremely simple relation which emerges is that iJie activity coefficient for a 
saturating salt in solutions of other salts is inversely proportional to the (geometric) mean 
molality of its own ions.^ 

IONIZATION OF SUBSTANCES NOT COMPLETELY IONIZED 

Many acids and bases, and some salts, are not completely ionized. In general, the 
activities of such substances in aqueous solution have not been determined; but the 
percentage ionization (aj) has been calculated from the ratio of the electrical con- 
ductance (A) of the solution at the given concentration to its conductance at zero 
concentration or from this conductance ratio corrected by the ratio of viscosities (77), 

An 
Ac?/, • 

Table II gives the percentage ionizations of a few acids and bases. At the same 

' This system gives entirely correct values of the activity coefficient, but the values become 
greater than unity in extremely dilute solutions. It is more common to extrapolate some function 
oimjni to zero concentration. Thus in dilute solutions if log {m/in^) is plotted against ^2, a straight 
line is commonly obtained. From (8) — log a = log {m/ni^. If for m = o, log {m/m^) is put equal to 
zero, then a becomes unity, and all of the other activity coefficients are on this basis: thus a is unity 
at zero concentration of the salt. This is the svstem used in Table I. 



148 ATOMS, IONS, SALTS, AND SURFACES 

concentration the values indicated are 45 per cent for cadmium chloride and less than 
0.1 per cent for the mercuric halides. 

TABLE II 

Percentage Ionization of Acids and Bases in Water at o.i 
Normal and 25 °C. 

Per Cent 

Sulphurous acid (into H+ and HSO3) 34 

Phosphoric acid (into H+ and H2PO4) 28 

Nitrous or hydrofluoric acid 8 

Hydrogen sulphide, carbonic acid, and hypochlorous acid. . . o. i 

Hydrocyanic acid and HBO2 o .002 

WEAK ELECTROLYTES OR SLIGHTLY IONIZED SUBSTANCES 

Slightly ionized substances follow the mass-law relation with respect to ionization 
at small concentrations, provided strong electrolytes are not present. Thus, with 
acetic acid (HAC), 

— — =Ai = o. 000018 . 

Chac 

The ionization constants (/v,) for a few weak electrolytes are given in Table III. 

TABLE III 

Ionization Constants at 25° C. 

Acids lo'iC 

Hydrocyanic o . 0005 

Boric (HBO,) .0017 

Hypochlorous o . 044 

Nitrous 400 

Hydrofluoric 790 

Formic 210 

Acetic 18 

Propionic 13 

n-Butyric 15 

Monochlor acetic i >SSo 

Benzoic 60 

Ortho 

Hydroxy benzoic i ,020 

Chlor benzoic 1,320 

Nitro benzoic 6, 160 

Bases 

Ammonium hydroxide 18. 

Methyl ammonium hydroxide . . 500 . 
Phenyl ammonium hydroxide. . 0.0004 

HYDROGEN-ION CONCENTRATION AND pH 

Suppose that a primary cell consists of an electrolyte with two hydrogen elec- 
trodes. At the electrode on the left-hand side the pressure of hydrogen is 10 atm. 
(atmospheres), and on the right-hand side i atm. The cell is 

M+IIz (10 atm.) , Electrolyte with //+ ions , M + II2 (i atm.) . 



Meta 


Para 


87 


29 


15s 


90 


345 


396 



WILLIAM D. HARKINS 149 



The change of state is 

Left 



— (10 atm.)-^ n+ (at concn. x) , 
2 



Therefore, the total change is 



Right 

H+ (at concn. x)-^ — ^ (i atm.) 
2 



^(10 atm.) -^ — ^(latm.) 

2 2 



The electromotive force ((5) of the cell is an energy quantity and may therefore 
be calculated as the equivalent of the maximum work which would be obtained if § 
mol of hydrogen gas expands from a pressure of 10 atm. to that of i atm.' 

Now the amount of work done by a force F in acting through a space 5 is 

W^FS . 

But since force equals pressure times area (A), 

W^pAS^pAv, 

a A V represents the increase of volume. Thus for a small amount of work 

dW^pdv , 
but since 

dW^NRT'^, 

V 

or by integration between the limits V2 and Vi , 

W = NRT In ^-- = NRT In ^ . 

This gives the amount of mechanical work which is equal to the change of electrical 
energy (S'DfJ, if '^l is the number of equivalents of electricity transferred. '^, the 
value of one equivalent of electricity, is equal to 96,500 coulombs. So 

^m=NRTln^, 

p2 

or 

^NRlli^^ log t, 

^i 96,500 p2 ' 

' The small amount of work involved in the change of volume of the liquid is neglected for the 
sake of simplicity. 



I50 ATOMS, IONS, SALTS, AND SURFACES 

Pi 
In the special case given above, pi = io and p2 = i, and log t^=i, the number of 

P^ 

mols of hydrogen (N) transferred is \ and the number of charges transferred (91) is 
I, so 



96,500 



If the temperature is 25° C, then r= 270+ 25 = 298° K., and (g = °-°59i5 volts = 

0.02957 volts. 

A cell may contain two aqueous solutions with diliferent hydrogen-ion concen- 
trations, such as 

M+H2 {p atm.), H+Cl- {nh molal), H+Cl- (w. molal), M+fl^, {p atm.) . 

Now if I Faraday of positive electricity is passed from left to right, the following 
changes occur: 

Left Electrode 

IT 

— ip atm.) -^ H+ (;«, molal) , 
2 

Liquid Junction 

T^jj + )H+ (nil molal) -^ ^(/7 + ) ^'^ (^2 molal) , 
T(^Qi^^Cl~ [niz molal) -^ T^ (Ci-) CZ+Cmi molal) , 

Right Electrode 

U+ (w. molal) — [p atm.) . 
2 

If the changes at the electrode are alone considered it is found that as much hy- 
drogen {H2/2) appears at p atm. as disappears at that pressure, so this change involves 
no work, and therefore no electromotive force. 

If the liquid junction is neglected for the moment, the total remaining change is 

H+{tn2 molal) -> H+ (nii molal) 
and 

@£ = -sT?r- ^« — (Approximate) , 
or 

^E = -^^r^ In - (Exact) . 

Here iV/9^ = i. For the liquid junction potential, 

K r,. , RT , Wi I ^ RT , Ml 

g nii iS nh 

rj. , RT m2 . rj. RT , W2 

= -T{H+) -^ In \-T(Ci-) -^ In ~ , 



or since 



WILLIAM D. HARKINS 151 

Tci-+Tn + = i, 
T(ci-)-{i — Tci-) = {2Tci- — i), 






.*. © = @£+@L = 2r(a-) -^ In —^ ; 



5 Wi 



or more exactly: 



S = 2r(c/-) -^ In— . 

15 fli 



By the use of such concentration cells it is possible to find the hydrogen-ion ac- 
tivity in one solution, provided its activity in some other solution with the same anion 
is known. If the anion is not the same, difficulties arise in the calculation of the liquid- 
Hquid junction potential. 

The results of part of the research by biologists upon hydrogen-ion concentration 
have been based on the assumption that two different liquid-liquid junctions give the 
same potential, and can therefore be canceled, which is in general not true. 

Since the electromotive force, and therefore the maximum work or free energy, 
varies as the logarithm of the activity (or inexactly as the concentration) ratio, the 
hydrogen-ion activity or concentration is often expressed as the logarithm. Thus in 
pure water at 25° C. the hydrogen-ion concentration is considered to be io~' molal, or 
the logarithm is— 7. This is often expressed according to the system of Sorensen as pH 
= 7. If, for example, pH = 5.2, the hydrogen-ion concentration is lo""^"^ molal, and the 
solution is on the acidic side of the neutral point (Cfl + = o.63 lo"^ mols per liter). 

THE SOLUBILITY PRODUCT 

Suppose that we have two saturated solutions (i and 2) of thallium chloride sep- 
arated by a partition which consists of a single crystal of the salt. Let solution i be on 
the left and solution 2 on the right. 

Tl++Cl-^{TICI (solid)5r/++a- . (i) 

The mass-law relation is 

ari +Xaa-= flsoiid Tici =aTi+Xaa- = K . , (2) 

This is known as the "solubility product." In the case cited, the solid salt of the mid- 
dle phase has a constant activity; but even if other salts, such as TI+R~ or NaNOj, 
are present, the relations between the activities in the right- and left-hand phases may 
be expressed: 

-77— =-7^' \3) 

aTi+ aci- 



152 ATOMS, IONS, SALTS, AND SURFACES 

THE MEMBRANE EQUILIBRIUM 

Let the solid salt be replaced by a membrane, and let one of the salts in the left- 
hand solution consist of one ion {Na'^), which is, and one (i?~), which is not, diffusible 
through the membrane. The equilibrium relations are represented below: 



Equilibrium 

Na+ Cl+ 

a" a" 



Na+ Cl- 

R- 

QR- 

If both solutions are considered infinite in volume, then the transfer of i mol of 
sodium chloride from left to right will not disturb the equihbrium. If i mol is thus 
transferred, the decrease of free energy ( — A/^) is 

aifa+ aci~ 

But the characteristic of an equilibrium process is that the change of free energy 
(— AF) is zero, so 

aNa+ aci~ 
or 

aNa+ aci- 

Ijut this is identical with (3), the relation which expresses ratios obtained from the 
solubility product. Thus, to this extent the membrane equilibrium is identical with 
the equilibrium with a solid. Equation (6) may be written 

iiNa + X aci - = axa + X aci , (7) 

which is the same as the solubility-product relation between the two solutions (2) but 
differs in that no solid of constant activity is concerned in the equilibrium. In the 
solubility product the product of the activities of the ions of the saturating salt, in 
either of the solutions on the two sides of the solid salt, equals the activity of the solid 
salt which is constant. In the membrane equilibrium the same product is equal to the 
activity of the salt in the membrane between the two solutions, but this activity is 
variable. 

In order to calculate the cciuilibrium in any special case, the activities under the 
conditions of the membrane equilibrium must be known. An attempt was made by 
Donnan to solve this problem without a knowledge of these activities, but there is no 
conclusive evidence to indicate that such a solution corresponds with the actual equi- 



WILLIAM D. HARKINS 



153 



librium. However, the method of treatment for a simple case is presented below, 
where the initial and equilibrium conditions are represented. 



Initial 



Na+ 


ci- 


Na+ 


Cl~ 


2C 


c 

R- 
C 


C 


c 




Equilibrium 




Na+ 


ci- 


Na+ 


ci- 


2C-X 


c-x 

R- 
C 


OX 


c+x 



Here A' represents the number of mols of NaCI transferred from left to right. If X 
happens to be negative, the equations will reveal that this is the case. 

The membrane product relation, written in the inexact form in which the con- 
centration (C) is substituted for the activity, is 

{2C-X){C-X) = {C+Xy-, 
or 

20-2,CX+X' = 0^-2CX+X' , 
so 

X = iC . 





Equilibrium 


Na+ 


ci- 




Na+ Cl- 


liC 


ic 

R- 
C 




liC • liC 


se is outlined below: 






Initial 




Na+ 


a- R- 


Na+ Cl- 


iiC 


C loC 




C C 



(xiC-x) (C-x) = {C+xy, 
iiO-i2Cx+x^ = 0-\-2Cx-irx\ 



Na+ 



ci- 

}C 



Equilibrium 

R- 

loC 



Na+ 



Cl- 

ifC 



MEMBRANE "POTENTIAL" 

The equations for calculating the electromotive force for a liquid-liquid junction 
are well known in the case in which different concentrations of the same salt are pres- 



154 ATOMS, IONS, SALTS, AND SURFACES 

ent on the two sides of the junction. The same equations have been applied without 
change when a membrane has been supposed to be present. Thus, with the cell given 
below 

Na+ Cl- 

aNa+ aci- 



Na+ CI- R- 

III 

aNa oci aR~ 



let I Faraday of positive electricity be passed from left to right. The decrease of free 
energy is 

-AF=TNa+ RT In ^^+Tcr RT In ^ = {2TNa+-i) RT In ^ . (8) 

aNa+ act- aNa+ 

Since the number of equivalents of sodium and chlorine ions which cross the boundary 
are given by their transference numbers {T!fa+ and Tci-) the decrease of free energy 
is also equal to the electrical work done, or 

-AF=(S9^5 (9) 

or, since in the special case given above the number of equivalents of electricity (^l) 
transferred is one, 

^==(2TNa+-i)^ln^, (10) 

^ aNa + 

which is the equation commonly given for the membrane potential. In general, 

(^ = iTc-TA)^ln^. 
F a+ 

Up to the present time no trustworthy experimental verification of these equa- 
tions has been obtained from any membrane equilibrium, since in the tests of these 
relations thus far the assumption has been made that other liquid-liquid junction 
potentials involved are negligible, without proving that this is true. 

SURFACES AND SURFACE ENERGY 

The importance of the energy stored up in the surfaces of bodies and in the inter- 
faces between the particles of the bodies is due in part to the influence which the sur- 
face tension exerts upon the form of the bodies and the particles. However, the effect 
of the surfaces upon the chemical composition and action, and upon the electrical 
phenomena, are of even greater moment. 

Interfaces are of particular significance in living organisms, since the motion of an 
organism as a whole is evidently brought about by transformation of one kind or 
another of the interfacial energy resident in it. The term "surface" unfortunately im- 
plies the entire absence of a third dimension in space, that of thickness, but physical 
surfaces and interfaces, sometimes designated as "phase boundaries," although they 
are exceedingly thin, commonly have a thickness as great as the sum of the diameters 
of several atoms, or a distance of the order of a millionth of a millimeter (10 A), which 
is by no means negligible. At many interfaces films or membranes collect, and these 



WILLIAM D. HARKINS 



155 



are of particular importance in biological systems, particularly in the human body 
itself. 

If a cubic centimeter of water is sprayed into spherical droplets o.oi ix (100 A) in 
diameter, the area of the surfaces thus formed is 600 sq.m. or approximately one- 
eighth of an acre. The free surface energy {yy^A) for this area at ordinary tempera- 
tures is about 2.2X10^ ergs or 10.5 calories, while the total surface energy {H) is 
larger and equal to 16.6 calories. This is one-third as large as the total energy of heat 
vibration of all of the molecules in the water. 

Any system in which the area of the surfaces (interfaces) becomes large enough so 
that the surface energy is appreciable in comparison with the energy of (heat) vibra- 
tion of the molecules of the disperse phase is considered as a "colloid." 

It is commonly observed that water drops on a hot 
stove or on very dry dust assume a spherical shape, which 
is the form assumed by a balloon surrounded by a uni- 
form elastic membrane. This suggests that every liquid 
is surrounded by an elastic film, the tension of which 
causes the surface to contract to the smallest possible 
area for the volume of the liquid, provided other forces 
(such as gravitation) do not act to change the form. 

If a faucet with a narrow orifice is turned on very 
slightly, a drop may be seen to form, and hang for some 
time, after which it drops very suddenly. The drop is 
supported before it falls by the vertical component of 
the surface tension. If a capillary tube is dipped into 
water, the liquid inside the tube rises higher than that 
outside. Here, also, the film of liquid on the inside wall 
of the tube exerts an upward pull. If a camel's-hair 
brush is dipped into water, the hairs remain spread 
apart as if they were dry and in the air, but when the 
wet brush is pulled out of the water, the pull of the surface tension of the water 
binds all of the hairs compactly together. 

A soap film stretched on a wire frame such as that shown in Figure 7 has two sur- 
faces. If the distance AB is | cm., then this lower wire is in contact with ^ cm. of the 
film or I cm. of the surface. The pull on the film as measured by the weight of the 
wire and the weights suspended from it at W gives the surface tension of the surface 
per unit length. This may be expressed in dynes. If the wire is pulled downward i cm., 
then the surface increases in area by i sq.cm., so the work done is force times area 
equals 7X1=7 ergs. This energy may again appear as work when the film contracts 
to its original position, so it possesses the characteristics of free energy. Thus 72.8 
dynes per centimeter is the surface tension of water at 20°, and 72.8 ergs per square 
centimeter is the free surface energy of water at this temperature. 




Fig. 7. — Maxwell frame for 
the determination of the surface 
tension of a soap film. 



TENSILE STRENGTH AND TENSILE ENERGY 

There are a number of phenomena which indicate that the forces between ad- 
jacent molecules in soUds and liquids are very high. Tensile-strength tests on bars of 



156 ATOMS, IONS, SALTS, AND SURFACES 

steel show that it is necessary to apply a force of 100,000 lb. to rupture a bar i sq.in. in 
cross-section. If it were possible to carry out a tensile-strength test in an ideal way 
such that the bar (of i sq.cm. cross-section; see Fig. 12) would not be deformed before 
the break occurs, and so that the rupture would give two plane surfaces at right angles 
to the longitudinal axis of the bar, then the energy used would be equal to twice the 
free surface energy (27) per square centimeter at the temperature of the test. This is 
true because all that occurs in such an ideal rupture is the formation of a new surface on 
the steel of 2-sq.cm. area. This is equal numerically to twice the surface tension of 
steel per centimeter. The work necessary thus to pull apart a bar of unit cross-section 
may be designated as the work of cohesion {Wc). 

Wc = 2y. (i) 

If an endeavor is made to apply such a tensile-strength test to a bar of liquid, it is 
found that certain experimental difficulties arise. Nevertheless, the numerical value 
of the work of cohesion is known with considerable accuracy in such a case, since it 
may be obtained from the surface tension of the liquid. 

The surface tension of water at 20° is 72.8 dynes per centimeter, so its work of co- 
hesion is 145.6 ergs per square centimeter. This small value may seem to indicate a 
small tensile strength (force of cohesion) in water, but just the opposite is true since 
the distance to which molecular attraction remains appreciable is very small, and is 
only of the order of molecular dimensions. Furthermore, it decreases as a moderately 
high power of the distance. Suppose that the summation of this rapidly decreasing 
force is equivalent to the action of a constant force through io~^ cm. Then the force 
of cohesion would be 

■ _ or 1 .4s6X 10'° dynes = 1 .48X lo^ gm. per square centimeter , 
10 * 

or about 14,000 atm. The theory of van der Waals indicates a value of about 11,000 
atm., while other methods of calculation usually give between 10,000 and 15,000. 

LATENT HEAT OF A SURFACE 

According to the rule of Le Chatelier, if the state of a system is changed, the sys- 
tem alters in such a way as to oppose a resistance to that change. Thus if the solubil- 
ity of a salt increases with the temperature, the last amount of salt which dissolves to 
saturate the solution produces a cooling, since this cooling lowers the solubility, and 
thus opposes the solution of the salt. Now, since the surface tension decreases with rise 
of temperature (Fig. 8), a surface must cool if it is expanded, since by cooling the surface 
tension is increased, and this opposes an extra resistance to the further extension. 

That heat should be used up in the formation of a surface is to be expected on 
other grounds. In the vaporization of a liquid the kinetic energy of molecular vibra- 
tion of the molecules of the liquid, which determines the temperature, is partly con- 
verted into molecular potential energy, i.e., the molecular energy of motion is utilized 
in the separation of each molecule from its neighbors and against the attraction which 
they exert. Now since a molecule which is in the interior of a liquid must move into 



WILLIAM D. HARKINS 



157 



the surface against the attraction of the surrounding molecules, as a part of its migra- 
tion into the vapor phase, it seems probable that in surface formation as well as in 
vaporization molecular kinetic energy would be utilized and transformed into poten- 
tial energy of the surface. That heat is actually used in the formation of the surface is 
shown by the thermodynamic equation of Clapeyron, which gives the latent heat (/) 
of the surface as 

87 



l=-T 



hT 




Fig. S. — The free surface energy (or surface tension) of organic liquids. This equals one-half the 
tensile work (or work of cohesion Wc) per square centimeter. 

Here I gives the amount of molecular kinetic energy which is transformed into molecu- 
lar energy of position when i sq.cm. of surface is formed. 



TOTAL SURFACE ENERGY 

The total energy Qi) of a surface is equal to the sum of the free energy (7) and the 



latent heat (/), 



h=y-\-l=y-T 



dy 
dT' 



In the formation of a surface a part of the energy must be supplied in the form of 
work in order to give rise to the free surface energy, and a part comes from the kinetic 



158 



ATOMS, IONS, SALTS, AND SURFACES 



Air 



energy which the molecules themselves possess. Thus, if a person extends a surface by 
doing work upon it, the liquid will also contribute its share to the total energy. The 
Clapeyron equation tells us that the temperature, i.e., the wealth of the molecules in 
kinetic energy, is an important factor in determining the extent of this contribution. 

If a surface is to be formed on a definite liquid at a definite temperature, a definite 
amount of energy must be contributed and converted into potential energy. 

THE ORIENTATION OF MOLECULES IN SURFACES 

The ordinary observation of large-scale objects, such as logs or ships, as they lie 
upon the surface of a body of water, indicates that these objects exhibit a character- 
istic orientation with respect to the surface. Thus logs, when not too closely crowded 

together, lie flat upon the water, i.e., the longitudinal 
axis is parallel to the surface. However, if one end of 
each log is loaded with a mass of iron or brass of the 
proper weight, it floats upon the surface and the longi- 
tudinal axis becomes vertical. If there is just a suffi- 
cient number of logs, the surface becomes covered with 
a single layer of vertical logs with their sides more or 
less in contact, while with any greater number, bunches 
of logs are found raised above the common level in 
certain places. If the number is smaller, a part of the 
surface remains uncovered. These phenomena may be 
illustrated by the use of a large number of cylindrical 
sticks of wood 3 mm. in diameter and 14 cm. long, 
weighted by a small cylinder of brass placed at one end. 
These are thrown upon the surface of the water in a 
large glass cylinder. This is represented in a diagram- 
matic way in Figure 9. If one of the vertical sticks is 
taken from the water, the brass weight removed, the 
stick dropped upon a vacant space upon a water sur- 
face, it at once assumes a horizontal position, thus 
exhibiting another type of orientation. 

It is well known that the molecules or ions which make up a crystalline solid are 
arranged in an orderly way. A certain type of orderly array of very long and highly 
symmetrical molecules is found also in certain liquids, which are said to contain 
liquid crystals. Ordinary liquids are often supposed to be characterized by a complete 
disorder in the arrangement of their molecules, but it is probable that this disorder has 
been overemphasized. For example, in organic liquids of the type of acetic acid, the 
molecule of which consists of the polar carboxyl group, and the "non-polar" methyl 
group, there is some evidence of molecular association, presumably a type of orienta- 
tion in which two or more polar groups come close together in the pure liquid as they 
do in benzene' (Fig. 11, upper part). The theory that the molecules in the surface of a 

' The number of molecules which unite in this manner must be large in some groups, as they give 
definite X-ray patterns. The grouping may be more analogous to that given by the bristles of two 
brushes which are set with bristles together; this is called the "cybotactic state." 



-- j^ Water r^ 



Fig. 9. — Orientation of mole- 
cules of an alcohol (or an organic 
acid) at the surface of its aqueous 
solution. 




WILLIAM D. HARKINS 159 

liquid are oriented in a characteristic fashion is of comparatively recent origin. It 
seems peculiar that the birth of so obvious a conception should have been so long de- 
layed, but it is probable that this is due to the general habit of 
considering molecules as spherical, even when their formulas are 
highly elongated. In such cases the former conception was that 
it would roll itself up into a sphere. It is obvious that even in a 
dissymmetrical field of force, such as may be assumed to exist at 
the surface of a liquid, a molecule which is a perfectly sym- 
metrical sphere could exhibit no orientation. However, even in a 
uniform gravitational field a perfect sphere may orient itself Fig. 10.— Illustrates 
provided its mass is not uniformly distributed, as will be seen if the orientation of a 
a sphere is weighted on one side, so that even if all molecules weighted sphere under 
were spherical, molecular orientation would not be at all impos- theintiuence o 
sible (Fig. 10). ^""'^- 

SOLUBILITY AND MOLECULAR ORIENTATION IN SURFACES 

The cohesion in liquid ethane, 

H H 

1 I 
H— C— C— H , 

I I 
H H 

is extremely low, so low that liquid ethane cannot exist at ordinary temperatures. 
The introduction of one oxygen atom gives rise to ethyl alcohol in which the cohesion 
is equivalent to a pressure of 3,000 atm. The hydrocarbon molecule (ethane) is non- 
polar, but the hydroxyl group ( — OH) of the alcohol is polar. The attraction between 
two such polar groups is very much greater than that between two non-polar groups 
or that between a polar and a non-polar group. 

The solubility of an organic acid, an alcohol, or an amine in water is due to its 
polar group which is greatly attracted by the water. The solubility of such a substance 
in hexane is, on the other hand, due to the presence of the hydrocarbon groups. Thus, 
all saturated hydrocarbons are practically insoluble in water. The old rule is: Similia 
similibus solvimtur ("Like dissolves like"). 

If, now, we have a two-phase system consisting of water below and hexane above, 

and add butyric acid, 

H H H 

I I I 
H— C— C— C— C— 0— H , 

I I I II 
H H H O 

the hydrocarbon end, which we may designate by CZZl , is soluble in the hexane, but 
not in the water, and the carboxyl group O is soluble in water but not in the hexane. 
However, the carboxyl group will drag some molecules of the butyric acid into the 
water, while the hydrocarbon group will drag others into the oil. 

At the interface between the water and the oil, however, the hydrocarbon of the 



i6o 



ATOMS, IONS, SALTS, AND SURFACES 



molecule may dissolve in the oil and the polar group in the water. Thus, each end of the 
molecule is highly soluble in the liquid toward which it turns. If this is true: (a) butyric 
acid should be very much more soluble in the interface than in either the water or the 
benzene; {b) the butyric acid molecules should be oriented with their polar ends toward 
the water and their non-polar ends toward the hexane. (Fig. ii). 




_ — Water Phase —-^ 



__^. 



GENERAL STATEMENT OF THE THEORY OF ORIENTATION OF MOLECULES IN SURFACES 

The following statement, written in 1916, gives in concise form the general funda- 
mental principles of the orientation theory: 

I. The molecules in the surfaces of liquids 
seem to be oriented, and in such a way that the 
least active or least polar groups are oriented 
toward the vapor phase. The general law for 
surfaces seems to be as follows : // we suppose 
the structure of the stirface of a liquid to be at first 
the same as that of the interior of the liquid, then 
the actual surface is always formed by the orienta- 
tion of the least active portion of the molecule 
toward the vapor phase, and at any surface or 

INTERFACE THE CHANGE WHICH OCCURS IS SUCH 
AS TO MAKE THE TRANSITION TO THE ADJACENT 

PHASE LESS ABRUPT. This last Statement ex- 
presses a general law, of which the adsorption 
law is only a special case. If the molecules are 
monatomic, and s^onmetrical, then the orienta- 
tion will consist in a displacement of the 
electromagnetic fields of the atom. This molec- 
ular orientation sets up what is commonly 
called a ''double electrical layer" at the sur- 
faces of liquids and also of solids. 

This law, if applied to special cases, indi- 
cates for a few pure liquids the following orientation: In water the hydrogen atoms 
turn toward the vapor phase and the oxygen atoms toward the liquid. With organic 
paraffin derivatives the CH3 groups turn outward, and the more active groups, such 
as NO. CN, COOH, COOM, COOR, NH„ NHCH3, NCS, COR, CHO, I, OH, or 
groups which contain N, S, O, I, or double bonds, turn toward the interior of the 
liquid. 

If any of these organic compounds are dissolved in water, their orientation in the 
water surface is the same as that just given, with the active groups inward. 

At interfaces between two pure liquids the molecules turn so that their like parts 
come together in conformity with the general law. With solutions, the solute mole- 
cules orient so that the ends of the molecules toward the liquid A are as much like A 
as possible, and the ends toward B are as much like B as possible. So at interfaces be- 
tween organic liquids and water, for example, the organic radical sets toward the or- 
ganic liquid, and the polar group toward the water. 



Fig. II. — A two-phase system of water 
and benzol which contains butyric acid. 
The greatest concentration of the acid is at 
the interface. The acid is more or less as- 
sociated in the benzol. 



WILLIAM D. HARKINS 



i6i 




2, If at an interface the transition from a liquid A to the liquid B is made by a 
saturated film of solute molecules which we may call A-B, i.e., they have one end like 
A and the other like B, then the free surface energy is greatly reduced. 
For example, with water and benzene with sodium oleate as the solute, 
the free energy falls as low as 2 ergs per square centimeter. 

3. If the solvent is polar, such as water, then solutes will in general 
be positively adsorbed in the surface if they are less polar than water (or 
if a part of the molecule of the substance is less polar than water), and 
the least polar end of the molecule will be turned outward. Solutes more 
polar than water are negatively adsorbed. 



EVIDENCE FOR ORIENTATION OF MOLECULES IN SURFACES 
AND INTERFACES 

I. Evidence from the energy of rupture. — The orientation theory indi- 
cates that if a bar of liquid octyl alcohol (Fig. 12) were to be pulled 
apart, one of the steps in the process would be for the molecules to 
orient where the break is to occur (Fig. 13) in such a way that this will 
become the weakest part of the bar. Evidently this means that the final 
break will occur between the non-polar ends of the molecules. If octane 
(CsHis) is ruptured, the work done for a bar of i sq.cm. cross-section (Wc) 

is 43.5 ergs. When octyl alcohol 



— - - :Alcphol-_ - z-i 



Fig. 12. — 
Bar of liquid 
of unit cross- 
section. 



is pulled apart, additional energy 
must be utilized in orienting the 
molecules, so it is not surprising that the work 
of rupture (Wc) is slightly higher (55.0). 

If, however, the octyl alcohol is to be 
pulled away from water, and the molecules of 
the alcohol are oriented in the interface, then 
t/ie final break must come between the polar 
hydroxyl ( — OH) groups of the alcohol and the 
polar molecides of water (Fig. 14): therefore 
(the orientation theory predicts that in this 
case) the work of rupture (work of adhesion, 
Wa) should be high. The experimental results 
show that this prediction of the theory is 
justified, since the work of adhesion is found 
to be 92 ergs, or 60 per cent higher than the 
work required to rupture the alcohol. Even 
more remarkable is the fact that this is 1 11 per 
cent higher than the work required to rupture 
octane, and 1 10 per cent higher than the amount of work necessary to separate octane 
from water. The extremely remarkable nature of these results is evident when it is 
considered that the molecule of octane contains 26 atoms, while that of octyl alcohol 
contains these same 26 atoms and only one more, an atom of oxygen which gives the 
polar nature to the molecule. Thus an increase of less than 4 per cent in the number 



D 



Alcohol 



Fig. 13. — Represents the orientation 
of the molecules which occurs if a bar of 
alcohol is pulled apart. 



l62 



ATOMS, IONS, SALTS, AND SURFACES 



of atoms increases the work of attraction for water by i lo per cent, which is suffi- 
cient evidence that the oxygen of the alcohol must be oriented toward the surface of the 
water. 

If the alcohol surface is pulled from the water surface at the interface between the 
two the interface disappears and a water {A ) surface and an alcohol {B) surface ap- 
pear. The work done is aided by the free 
energy of the surface which disappears, and 
hindered by those which appear, so 

WA=yA-]-yB—yAB . 

Values of the interfacial tension (t^b) for 
a number of liquids are plotted in Figure 15, 
and for the work of adhesion in Figure 16. 

THE SPREADING OF ONE LIQUm ON THE 
SURFACE OF ANOTHER 

Most organic liquids will spread on water, 
but water spreads on almost no organic 
liquids. It is easy to show that spreading or 
non-spreading is determined by the work of 
adhesion {Wa) between the liquids and the 
work of cohesion (Wc) for the upper liquid 

S=Wa-Wc(B). 

If S is positive, the liquid B will spread on the 
surface of A;ilS is negative, it will not spread. 
The presence of polar groups in the organic 
liquid is not essential for spreading, since 
hexane and octane, as well as benzene, spread 
on water. Not only organic liquids, but water 
as well, spread on a clean surface of mercury: 




Fig. 14. — Octyl alcohol over water. Illus- 
trates the oreintation of the alcohol mole- 
cules at the interface. 



S = yA-(y -{-y ) . 



2. Evidence for orientation of molecules in surfaces of pure liquids; comparison of 
energy of surf ace formation with heat of vaporization. — If a liquid consists of molecules 
with one end polar and the other end non-polar, the energy required to lift the non- 
polar end (the "light" end from the standpoint of electrical forces) into the surface is 
much less than for the polar end, so the orientation theory predicts that in the outer 
layer of molecules the non-polar groups will be at the surface. However, if such a 
molecule passes into the vapor state, the polar end of the molecule must be separated 
from the liquid, and thistwill require a relatively large amount of energy. Therefore, 
according to the theory, the energy per molecule of surface formation (/?) should be 
small as compared with the energy of vaporization (X). If the molecule is symmetri- 



WILLIAM D. HARKINS 



163 



cal, then h/\ should be much larger, since there is no "light" end which can be lifted 
into the surface. 

In entire confirmation of these ideas, h/\ is very small (0.18) for the lower al- 
cohols which have highly unsymmetrical molecules, and much larger (0.50-0.60) for 
highly symmetrical molecules, such as those of oxygen, nitrogen, or mercury. These 
values are those found at a corresponding temperature of 0.7 (T = o.'jTc), but the same 
general relations are followed at other temperatures. 




/o £0 JO 40 so 

Fig. 15. — The free interfacial energy (or interfacial tension) between organic liquids and water 

This is the only evidence thus far found which indicates strongly that the mole- 
cules in the surfaces of pure liquids are oriented. 

3 . Evidence for orientation in relations of monomolecular films. — If a long-chain fatty 
acid, such as stearic acid, is dissolved in hexane it spreads on water to form a dilute 
film I molecule thick. This may be compressed between the movable barrier (back 
of Fig. 17) and the floating barrier attached to a film balance (front of Fig. 17). When 
the film is compressed until it is tightly packed, but still monomolecular, the area per 
molecule is 19.3 A units of area (19.3 A^ = 19.3X10"'^ sq.cm.) (Fig. 18). The square 
root of this area, 4.4 A, gives an idea of the diameter of the area occupied by the 



164 



ATOMS, IONS, SALTS, AND SURFACES 



molecule. This area is found to be nearly the same for long-chain compounds of from 
16 to 30 carbon atoms long, which indicates that the molecules in the film are oriented. 
The thickness of such a film, calculated on the basis of the idea that its density is that 




s& 



'f2- 



3^ 



Fig. 16.— Adhesional work, ergs per square centimeter, between organic liquids and water. (The 
names of the substances represented by the curves are given at the right while the names given in the 
middle of the diagram represent substances for which the values are given at 20° only.) 

of the pure organic substance, indicates that each CH2 group adds about 1.4 A to the 
thickness of the film, while X-ray measurements on the solid substance give a mean 
value of about 1.15 A. 

FILMS ABSORBED FROM LIQUIDS 

The best evidence at present available indicates that the films on water of butyric 
acid, amyl amine, octyl alcohol, phenol, resorcinol, and all analogous substances are 



WILLIAM D. HARKINS 



165 



essentially monomolecular at sufficient concentrations of the solutions. With long- 
chain (10 carbon atoms) compounds, only dilute aqueous solutions are obtained, so 
the time necessary for the diffusion of sufficient organic substance into the surface to 




Fig. 17. — Perspective drawing and photograph of filmometer (design of B. B. Freud, a modi- 
fication of the filmometer of N. K. Adam). 



i66 



ATOMS, IONS, SALTS, AND SURFACES 



give a monomolecular film which is in equilibrium with the solution is considerable — 
more than thirty minutes in the case of decylic acid (lo carbon atoms) (see Fig. 19). 




Fig. 18. — Areas per molecule (X-axis) for monomolecular films on water. The F-axis represents 
the "force of compression" which is measured by the film balance, and is equal to the surface tension 
of water minus the surface tension of a water surface covered by a film of the organic substance. The 
curves for palmitic acid, and for stearic acid alone, are of the more usual t>'pe. These films are under 
high pressures. Under low pressures the curves are similar to the p, v curves for gases. For com- 
parison, two curves for polymolecular films of phenanthrene and triphenjdmethylcyanide are given. 
The areas per molecule for these two substances are from 2 to 6 A, which is too small an area for a 
monomolecular film. The line for palmitic acid extrapolated to zero pressure gives 20.2 A, and for 
stearic acid on water alone the two lines give 19.1 and 19.2 A of area. Laurie acid forms a dilute 
monomolecular film. 



WILLIAM D. HARKINS 



167 



The equation of Gibbs, which gives the number of gram molecules (u) absorbed 
per square centimeter of surface formed, may be expressed: 

I 87 



RT d In a 



.08 



< 



Fig. 19. — Effect of time on the drop weight (surface tension) of decylic acid, 0.0015 N 




Form. ; 
Acetic 
Prop/ on I c 



'ric 



Va/en 
CoprdfC 



Hepti/(c 

NONl/i 

Decy/ 



c 



-a 



-1 



■ WatS/- 



LoK of Concentration 



Fig. 20. — Adsorption curves for fatty acids 



i68 



ATOMS, IONS, SALTS, AND SURFACES 



a In Cj 
, but the correction is less than lo per 



4 
3 
Z 




^^ 


^ 








lie, 






^-^.^^ 


KCI 














c^ci^ 


















The difficulty in the application of this equation is that activities in solution are 
known for almost no organic substances. Figure 20 shows how the surface tension of 

solutions of organic acids varies with the logarithm of the concentration 

The slope of k-t — is slightly greater than ^r-. — - 
o In a o-'o d In C 

cent in the case of the condensed monomolecular film of butyric acid. The nearly iden- 
tical slopes of the 7, log C, curves indicate that the adsorption is nearly independent 

of the number of carbon 
atoms in the molecule for 
the tightly packed mono- 
molecular films. 

The calculations made 
thus far indicate that the 
area per molecule in the 
film for these soluble sub- 
stances is of the same order 
as that for the longer mole- 
cules which are insoluble. 
Data on the surface and 
interfacial tensions indi- 
cate that between water 
and benzene, or between 
water and hexane, the num- 
ber of butyric acid mole- 
cules in unit area of the film is exactly the same, within the limits of experimental 
error, as between water and vapor. This is additional evidence that the number of 
molecules in a condensed film depends mainly on the size of the molecules and their 
orientation. 

MEAN THICKNESS OF THE WATER FILM ON SALT SOLUTIONS 

Water is positively absorbed on aqueous salt solutions, i.e., the surface contains 
much less salt than the solution. It is possible to calculate a mean thickness for the 
water film if it is assumed that the film consists of pure water, and that just below the 
film the concentration of the solution is the same as farther inside. 

The remarkable relation which emerges from such calculations is that in very dilute 
solutions the calculated film thickness is almost exactly the cube root of the volume occupied 
in water by a water molecule (3.1 A). Figure 21 plots the thickness of the water film as 
a function of the concentration of the salt. 

The water film between water and benzene is found also to have practically the 
same thickness at the same concentration as between water and air. 

Thus, either between aqueous solution and vapors, or between the aqueous phase 
and a non-polar liquid, the water film is monomolecular in dilute solutions, and even 
thinner in concentrated solutions. Both between salt solution and vapor and between 
salt solution and benzene the surface tension increases as a linear function of the mo- 
lality of the salt in the solution. 



Fig. 21.— 
Strom units). 



Mols of salt per i,ooo-gm. water 
■Thickness of the water film on salt solutions (Ang- 



WILLIAM D. HARKINS 169 

ELECTRICAL CHARACTERISTICS OF EMULSIONS AND SUSPENSIONS 

If an emulsion or a suspension is placed between a positive and a negative elec- 
trode, it is fcund that the particles move (with respect to the water) toward one of the 
two electrodes, and that in general all of the particles of the same material in the same 
medium move at the same speed, whether they are large or small. This phenomenon 
of the movement of small particles in the electric field is known as "cataphoresis." It 
may be considered that this is analogous to the conductance of electricity by the ions 
of a salt in electrolysis. 

If equal parts of hexane and an aqueous solution of a sodium oleate soap are mixed 
together by shaking or by stirring with an egg-beater, it is commonly found that the 
droplets vary from less than 0.2 /x to about 10 tx in diameter, with the largest number 
of drops at a diameter of 1-1.5 m. 

Determinations of the amount of soap absorbed indicate that each droplet of oil in 
a stable emulsion is surrounded by a monomolecular film of soap. Since soap is part- 
ly hydrolyzed, the film should consist of molecules of sodium oleate and of oleic acid. 
The total number of oleate molecules in the film around a droplet of a diameter of i /u 
is of the order of 10,000,000 or 15,000,000. 

According to the orientation theory, the hydrocarbon groups of the soap are 
turned toward the oil and the polar groups with their positive sodium ions toward the 
water. It is to be expected that such a droplet ivill act like a highly polyvalent salt mole- 
cule, and that sodium ions will diffuse off into the solution, leaving a negative charge 
on the droplet. The question now arises, To what extent is the droplet ionized? Since, 
however, the different sodium ions diffuse to different distances, the ionization should 
be expressed as a relation concerning the distribution. However, it is easy to calculate 
a mean or effective ionization by the use of the law of Stokes for the motion of a 
spherical droplet in a viscous medium. 

In this way it is found that the velocity of such a particle (4 /j, per second per volt 
per centimeter) corresponds to a negative charge of 2,430 electronic charges on a par- 
ticle I (X in diameter, i.e., if all of the 10,000,000 or 15,000,000 molecules of the soap 
except the 2,430 were completely un-ionized, and if the 2,430 molecules of sodium 
oleate were so completely ionized that the 2,430 Na+ ions are at an infinite distance, 
then the oil droplet should have a negative charge equal to that of 2,430 univalent 

negative ions. The equation is 

j^T_ 6Trrr] v 
e A 

in which e is the charge on the electron, N is the number of charges, 77 is the viscosity 

V 

of the solution, and ^ is the mobility or the velocity for unit potential gradient. 
The potential of a charged sphere is 

, Ne , dirri V 

<p = -— ; so (i) = — . 

^ Dr' ^ D X 

The use of this potential instead of the fictitious zeta potential was suggested to 
the writer by Professor A. C. Lunn. According to this equation, the potential is 84 



I70 ATOMS, IONS, SALTS, AND SURFACES 

millivolts for the droplet in question. The potential </> obtained by the use of the 
equation of Stokes is always 3/2 the fictitious zeta (f) potential usually given in 
books on colloid chemistry. 

According to the relations given above: (i) the effective ionization (A^) of a col- 
loidal particle varies directly as its radius. (2) The effective ionization per unit area 

A^\ 

— ) varies inversely as the radius of the particle, and therefore directly as the curva- 
ture of the surface. (3) The potential for the particle is independent of the radius. 
Therefore the potential is the same for all spherical particles of the same material in 
any certain solution. (4) The effective ionization (N) and the potential (</>) depend 
upon the nature of the particle and upon the nature of the medium in which it is 
suspended. 

The Helmholtz-Lamb equation for the velocity in cataphoresis is 

<f)rXD I 

v= -.. 

4 TTT? a 

Here D, -q, and d refer respectively to the dielective constant, viscosity, and thickness 
of the electrical double layer, / is the coefficient of slip, and X is the impressed 
potential gradient. Smoluchowski simplified this equation to 

4x7? ■ 
If 77 is taken to be the viscosity of the solution and not of the double layer, 

v=- -, 

or 

^~ Wx 

gives the value of the fictitious zeta potential, so 

The equations give the potentials in electrostatic units. The value of in volts is 
given by 

= 67r -^ y,X (300)2 volts . 

According to Debye and Hiickel, the constant of the Helmholtz-Lamb-Smolu- 
chowski equation should be bir for spherical particles. On this basis the value of 
f should be equal to that of 0. 

POTENTIAL AND STABILITY' 

According to the theory of Hardy, the stability of a colloidal suspension is de- 
pendent upon the electrical repulsion of the charges of like sign upon the particles. 

' Cf. also chaps, xlii and Iviii in this volume. 



WILLIAM D. HARKINS 



171 



On account of their Brownian movement the particles would collide, and in some 
cases unite, if they were not kept apart by this repulsion. However, some of the par- 
ticles have very high velocities, so even with high potentials the solution may not be per- 
manently stable. If the particles collide there may be an actual union. This is ac- 
companied by a decrease in surface energy. The union of two such particles involves 
the deorientation of the films between them, and this may require the expenditure of 
an appreciable quantity of energy. The particles which collide may merely adhere 
(agglutination), or they may merge and lose their identity completely. 

In 1906 Burton made some interesting experiments on the cataphoretic velocity 
of colloidal gold particles in a gold sol to which different amounts of an aluminum salt 
were added (see Table IV). Such experiments indicate: (a) that the positive alumi- 
num ions are adsorbed at the interfaces between the gold and the water; (b) that this 
adsorption may be great enough to change the original negative charge on the par- 
ticles of gold into a positive charge, the number of ions adsorbed increasing rapidly 
with the concentration of the aluminum soap; (c) that instability of the colloidal solu- 

TABLE IV 

Effect of Aluminum Ions (Al+ + +) upon the Charge of Particles of 
Colloidal Gold 



Milligrams Aluminum 
per Liter 


Cataphoretic Velocity in m per 
Volt per Cm. per Sec. 


Stability 




0. 19 


330 (toward anode) 
171 (toward anode) 


17 (toward cathode) 
135 (toward cathode) 


Indefinitely stable 
Flocculated after four hours 
Flocculated immediately 
Flocculated after four hours 
Not completely flocculated 
after four days 


0.38 

0.63 



tion increases as the charge on the particles (cataphoretic velocity) approaches zero 
from either the positive or the negative side. The point at which the cataphoretic ve- 
locity, and therefore the charge on the particle, becomes zero is called the "iso-electric 
point." 

It is believed that a small concentration of salt is essential for the stability of a 
colloidal sol (such as mastic). An excess of salt destroys the stability and causes floc- 
culation. If the salt is of the type of AlCl, and the sol is negative, further addition of 
the salt produces a positive stable sol, but still further additions of the salt bring about 
another flocculation. These peculiar phenomena are designated by the technical term 
''irregular series." 

EFFECT OF THE VALENCE OF THE ADSORBED ION 

It is impossible to add negative salt ions to a solution without the addition of pos- 
itive ions, and vice versa. However, it has been found that with negatively charged 
suspensoids, such as the arsenic trisulphide or the gold sol, the flocculation produced 
is a function of the valence of the positive ions of the salt, and with positively charged 
suspensoids it is a function of the valence of the negative ions of the salt. 

Thus with an emulsion of a paraffin oil the value of phi (<^) is reduced from 69 
millivolts, the value for water, to 55.5 millivolts by the addition of the specified 



172 ATOMS, IONS, SALTS, AND SURFACES 

amount of salt (see Table V). Thus it requires twelve hundred times more equiva- 
lents of potassium chloride than of thorium chloride to reduce the cataphoretic ve- 
locity from 3.1 to 2.5 fjL (which corresponds with the lowering of (</>) from 69 to 55.5 

millivolts). 

TABLE V 

Lowering Effect of Salts upon the Cataphoretic Mobility (y), the Zeta 

Potential (f), or the Phi Potential (0) for Droplets of Oil in Water 

No. of Milliequivalents of Salt 
Electrolyte Required to Lower f from 46 to 37 

Millivolts (or ^ from 6g to 55.5 Millivolts) 

KCl 24 

BaCU o. 90 

AICI3 03 

ThCl4 0.02 

The same relationship concerning the valence of the ion of opposite charge is ap- 
parent in the flocculation values. Thus, for the negatively charged arsenic trisulphide 

TABLE VI 

Flocculation Values; Number of Milliequivalents of Salt per Liter 
Required to Flocculate the Arsenic Trisulphide Sol 

c 1. Ajj J Flocculation Value Milliequivalents 

Salt Added ^f g^H pg^ Liter 

Monovalent Cations 

LiCl 58 

NaCl 51 

KCl 50 

KNO3 SO 

^K.S04 63 

HCI 31 

Divalent Cations 

MgS04/2 1.62 

MgC]2/2 1.44 

CaCl2/2 1.30 

ZnCl2/2 1.36 

Trivalent Cations 

AICI3/3 0.279 

Al(N03)3/3 285 

Ce(N03)3/3 0-24 

Tetravalent Cation 
Th(N03)4 0.36 

sol the number of millimols per liter of salt required to cause flocculation decreases 
very rapidly with the valence of the positive ion (Table VI). 

The work thus far done upon the carrying down of ions by precipitates shows 
that with a negatively charged sol flocculation is produced by about the same number 
of equivalents of one positive ion as another (Table VII). This indicates that inor- 
ganic ions of high charge (valence) are much more highly absorbed than those of low 
charge (valence). 



WILLIAM D. HARKINS 

TABLE VII 

Flocculation Values 



173 



Ion 


In Milliequivalents 
per Liter 


Milliequivalents of 

Cation Absorbed by 

25 Gm. AsjSj 


K..... 

(Aoneline) 


SO 
2-5 

0. I 

1. 2 
I. 2 
I. 2 
I. 2 
0.27 


2.05 

1.8^ 


(New fuchsin) 

Ca 

Sr 

Ba 

uo. 


1.90 
2.50 
2. 05 

215 

2. 20 


Ce 


1.72 



STABILITY OF AN EMULSION AND THE MONOMOLECULAR FILM 

An emulsion produced from equal volumes of water and oil by the use of sodium 
oleate (a soap) as an emulsifying agent is found to be stable for a period of years if a 
o.i-molal solution of soap is used, but changes greatly in a few days if produced by 




Fig. 22. — Shows how the change from the soap of a monovalent metal, such as sodium, to that 
of a bivalent metal, such as calcium, may change the emulsion from one of oil droplets in water to an 
emulsion of water droplets in oil. 

0.005-molal soap. In the latter case not enough soap is present to form a condensed mono- 
niolecidar film around the particles of oil, so the droplets of oil unite in order to de- 
crease the interfacial area and thus increase the concentration of the soap in the film. 
With o.i-molal soap a condensed fikn of approximately monomolecular thickness is 
present, and this gives a considerable degree of stability to the emulsion. If the soap 
is the salt of a univalent metal, the droplets will be of oil, with water outside; but 
with bi- or trivalent metals several hydrocarbon chains are present for each single 
positive ion, and (Fig. 22) the droplets are of water, with oil outside. 



174 ATOMS, IONS, SALTS, AND SURFACES 

THE DETERMINATION OF SURFACE AND INTERFACIAL TENSION 

The use of what is often called the "drop-number method" of determining the 
surface or interfacial tension is not only a waste of time but fills the literature of bi- 
ology with worse than useless data, since they are so extremely deceptive. 

Drop-weight method. — The capillary-height and the drop-weight methods are the 
most accurate now known. The drop- weight method is in general much the more 
suitable of the two for biological investigations. It depends upon the fact that if a 
drop of liquid is allowed to fall from a horizontal circular disk (end of a glass capillary 
tube) with sufficient slowness, the weight of the drop is a definite function of the surface 
tension, the radius of the tip, and the volume of the drop which falls, so 

W = Mg = 2 -nryf , 
where/ stands for the value of the function. Thus 

2-Kr] 



or if 

V— 

2 7r/' 



F=-' 



7 = M^F. 
r 

To calculate the surface tension: (i) The volume (F) of the drop is its mass in 
grams divided by its density ( ^ = ^ ) • (2) Multiply F by - , in which r is the radius 

V . 

of the circular tip. (3) Find — in Table VIII, and note the corresponding value of F. 

(4) Divide g (in dynes per sec.^ = 980.3 at Chicago) by r^ {r in centimeters), and multiply 
by M in grams, and by F. The result gives the surface tension in dynes per centimeter. 

In the determination of interfacial tension the volume of the drop is measured di- 
rectly. The weight of the drop as it hangs in a second liquid is V {d^—d^, in which d^ 
is the density of the heavier and d^ that of the lighter liquid. The liquid which is 
dropped should always be the aqueous phase, so if the oil is the heavier the tip should 
face upward. 

Ring method for determination 0] surface tension. — The pull on a ring just being de- 
tached from the surface of a liquid does not give the surface tension, but the pull must 
be multiplied by a factor F, as given by Young and Cheng and the writer in a recent 
paper in Science. The outline of the method is as follows: 

1. Determine the total pull in grams (M) necessary just to detach a circular ring 
of diameter R, of circular wire of diameter r, from the surface of the liquid by the use 
of a chainomatic balance or a torsion balance, such as that of Du Nouy. 

2. The surface tension is given by the equation 

3. Obtain the value of F, the correction factor, from Figure 23. F is plotted on the 

Ri 
F-axis. To do this find the value of vf > the cube of the radius of the ring divided by 



WILLIAM D. HARKINS 175 

M 
the volume of liquid (F) upheld by the ring. V= — , or the volume of liquid is equal 

to the weight given by the balance, divided by the density of the liquid. 

4. After finding the value of ..j on the A'-axis, find a point on one of the curves 

TABLE VIII 

Drop-Weight Surf ace-Tension Corrections (Factor for Multiplication = F), 
Based on the Value 72. 75 as the Surface Tension of Water at 20° C. 

I F 

(°o) 0.159 

5000 .0 172 

250.0 198 

58.1 215 

24.6 22561 

17 -1 23051 

13-28 23522 

10 . 29 23976 

8 . 190 24398 

6.662 24786 

5S22 2513s 

4653 25419 

3-975 25661 

3-433 25874 

2 . 995 26065 

2.637 26224 

2.3414 26350 

2.0929 26452 

1 . 8839 26522 

1 . 7062 26562 

I -5545 26566 

1-4235 26544 

1-3096 26495 

1 .2109 26407 

1. 1 24 ' 26324 

1.048 2617 

o . 980 2602 

.912 2585 

0.865 0.2570 

•n 

which has the same value of x. To do this it is necessary to know — . Curves are 

given for values of — equal to 29.5, 40.2, 59.1, and 78.5. If the ring corresponds to 

some other value of this ratio, it is necessary to interpolate. 

If the correction factor F is not used, the results will in general be worthless, since 
they are not even relatively correct, as may be seen from the high curvature of the 
correction curves. 



176 



ATOMS, IONS, SALTS, AND SURFACES 




Fig. 23. — The values of — for the curves in this figure, beginning with the bottom curve, are: 
29.5, 40.2, SQ.i, and 78.5. 

A = Area 

A= I Angstrom unit of length = io~^ cm. 
A^= I Angstrom unit of area = 10-'*' cm.^ 
0= Activity 
a = Activity coefficient 

c = Concentration (respectively, the velocity of light = 2.9986X10'° cm./sec. 
8 = Partial differential 
D = D ielectric constant 
E = Energy 

@ = Electromotive force 

e = Charge on the electron=4.774Xio~"' electrostatic units 
e = Epsilon, base of Naperian or natural logarithms 
f = Zeta, fictitious electrical potential at a phase boundary 
/^ = Force (respectively, a correction factor of multiplication) 
S = I Faraday — 96,500 coulombs 
AF = Increase of free energy (zeta function of Gibbs) 
g= Acceleration of gravity ( = 980.278 cm./sec.^ at the University of Chicago) 
/? = Heat function for unit area of surface (total surface energy, respectively, for the 

area occupied by i mol); quantum constant ^ = 6.554 erg. sec. 
K = A constant 

^ = Gas constant per molecule (Boltzmann) =1.372X10-'* ergs per degree per molecule 
1 = Latent heat of the surface per unit area 
X = Lambda, latent heat of vaporization per molecule 



WILLIAM D. HARKINS 177 

A = Lambda, equivalent conductance 
M = Mass 

w = Molality, or concentration in mols per 1,000 gm. of solvent 
/x = Mu, ionic strength (respectively, a unit of length = io-^ cm.) 
iV = Number of mols (respectively, number of electronic charges on a particle) 
1^ = Frequency of vibration = 6"/X 
P = Potential 
p = Pressure 
i?= Gas constant 

= 8.317X10' ergs per degree per molecule 
= 1.9885 calories per degree per molecule 
/' = Radius 

p = Rho, density (respectively, electrical density) 
5 = Spreading coefficient 

r = Temperature on the Kelvin (absolute) scale 
Tc — Critical temperature 
T^A^a + = Transference number of the sodium ion 
I) = Volume 

= Phi, electrical potential at a phase boundary 
IF = Work (respectively, weight) 
Wa = Work of adhesion 
Wc = Work of cohesion 
X = Field strength in electrostatic units 
Z = Valence of an ion 

BIBLIOGRAPHY 

CRYSTAL STRUCTURE 

1. Bragg, Sir W.: Concerning the Nature of Things. New York: Harper & Bros., 1925. 

2. Bragg, W. H. and W. L.: X-Rays and Crystal Structure. New York: Harcourt, Bruce & 
Co., 1924. 

3. Clark, G. L.: Applied XRays. New York: McGraw-Hill, 1927. 

ATOMIC STRUCTURE 

1. Andrade, E. N. da C: The Structure of the Atom. London: G. Bell & Sons, 1927. 

2. Lewis, G. N.: Valence and the Structure of Atoms and Molecidcs. New York: Chemical 
Catalog Co., 1923. 

3. Harkins, W. D.: "The Stability of Atom Nuclei, the Separation of lostopes, and the 
Whole Number Rule," /. Frankl. Inst., August, 1922, to April, 1923. 

4. Born, M.: Probleme der Atommechanic. Berlin: Springer, 1926. 

5. Schroedinger, E.: "Quantisierung als Eigenwert Problem," Ann. d. Phys., 79, 361, 489. 
1926; 80, 437. 1926; 81, 109. 1926. 

IONIZATION OF SALTS IN SOLUTION AND THE ACTIVITY COEFFICIENT 

1. Lewis, G. N., and Randall, M.: Thermodynamics. New York: McGraw-Hill, 1923. 

2. Eucken, A., Jette, E. R., and la Mer, V. K.: Physical Chemistry. New York: McGraw- 
Hill, 1925. 

3. Debye, P., and Hiickel, E.: Physikal. Ztschr., 24, 185, 305. 1923. 



178 ATOMS, IONS, SALTS, AND SURFACES 

SURFACES AND SURFACE ENERGY 

1. Harkins, W. D.: "Surface Energy and Surface Tension," Colloid Chemistry (ed. J. 
Alexander), chap. viii. New York: Chemical Catalog Co., 1926. 

2. Harkins, W. D.: "Surface Energy in Colloid Systems," ibid. (ed. R. H. Bogue), chap. vi. 
New York: McGraw-Hill, 1924. 

3. Harkins, W. D.: "The Orientation of Molecules in the Surfaces of Liquids," Colloid 
Symp. Mono., Vol. 2, chap. xv. New York: Chemical Catalog Co., 1924; see also Vol. 

5, 1927- 

4. Debye, P., and Hiickel, E.: "Cataphoretic Velocity of Suspended Particles," Physikal. 
Zischr., 25, 49. 1924. 

5. Willows, R. S., and Hatschek, E.: Surface Energy and Surface Tension. Philadelphia: P. 
Blakiston's Son & Co., 1923. 

coLLoros 

1. Kruyt, H. R.: Colloids. New York: John Wiley & Sons, 1927. 

2. Freundlich, H.: Colloid and Capillary Chemistry. New York: E. P. Button & Co., 1926. 

3. Books edited by Alexander and by Bogue listed in preceding section. 

4. Burton, E. F.: The Physical Properties of Colloidal Solutions. New York: Longmans, 
Green & Co., 1921. 

5. Clayton, W., and Churchill, J. and A.: The Theory of Emidsification. London, 1923. 

DETERMINATION OF SURFACE TENSION 

(Capillary-Height Method) 

1. Richards, T W., and Carver, E. K.: /. Am. Chem. Soc, 43, 827. 1921. 

2. Harkins, W. D., and Brown, F. E.: ibid., 41, 499. 1919- 

(Drop-Weight Method) 

3. Harkins, W. D., and Brown, F. E.: ibid., p. 499. 1919. 

(Drop-Weight Method for Interfacial Tension) 

4. Harkins, W. D., and Humphrey, E. C: ibid., 38, 228, 236. 1916. 

(Ring Method) 
S- Harkins, W. D., Young, T. F., and Cheng, Y. C: Science, 64, 333. 1926. 

Note. — Acknowledgment is made to Dr. Harry N. Holmes and the Chemical Catalog Com- 
pany, editor and publishers respectively of Colloid Symposium Monograph, Vol. H, for permission 
to reproduce Figs. 7, 9, 10, 11, 12. 13, 14, and 22 from the above-mentioned volume. 



CHAPTER XI 

THE EFFECT OF THE SURFACE TENSION OF THE MEN- 
STRUUM UPON BACTERIA AND TOXINS 

W. P. LARSON 

University of Minnesota 

INTRODUCTORY 

Bacteriologists have been slow to recognize the influence of the surface tension of 
the menstruum upon bacteria and toxins, although surface energy and the phe- 
nomenon of adsorption have been studied by physical chemists, and even biologists, 
for many years. It is not our purpose here to discuss the fundamental problem of 
surface energy, since a complete treatment may be found in works devoted specifically 
to that subject.' 

It may be recalled that the surfaces of all liquids (and solids) are in a state of 
stress or tension due to the play of intramolecular forces, the degree of tension being 
determined by the nature of the fluid and the solutes which it contains. 

Surface energy may be defined as the product of the surface area and the surface 
tension. Since free energy constantly strives toward a minimum, a liquid will assume 
the form giving it the smallest possible surface area, as the sphere of a raindrop, and 
many solutes which it contains will be forced — adsorbed — into the surface, provided 
the attraction of the molecules of the liquid is greater for each other than for the 
molecules of the solute. 

Surface tension may be defined as the force per centimeter, in the plane of the 
surface, required to overcome the tendency of a liquid to maintain a minimum surface 
area. The unit of measurement is the dyne. Several methods for measuring the sur- 
face tension of a liquid are in use, the best known of which are: 

The drop-weight method, the capillary-rise method, the jet method, and the method of 
measuring the force required to pull a disk or ring from the surface of a liquid. 

The physicist, who usually works with pure liquids, seems to prefer the capillar3'-rise 
method in preference to others. After having tried out all the foregoing methods I have found 
the drop-weight method best suited to the work of the bacteriologist who never, or seldom, 
works with pure liquids but rather with a solution of several substances in water. 

Morgan^ and Harkins and Brown' have perfected the drop-weight method to a point 
where most of the errors have been eliminated. In fact, their method is more delicate than 
the routine bacteriologist requires. Green' has recently developed a torsion balance which is 
rapid and yet sufficiently accurate for bacteriological work, where a high degree of accuracy 
is not important. When hundreds of measurements are required, I have found Green's 

• Harkins, W. D., in Alexander, J.: Colloid Chemistry, New York, i, 192. 1926. See chap, x by 
Dr. Harkins in this volume. 

' Morgan, J. L. R.: Jour. Amer. Chem. Soc, 33, 349. 191 1. 

J Harkins, W. D., and Brown, F. E.: ibid., 41, 499. 1919. 

* Green, R. G.: Indus, and Eng. Chem., 15, 1024. 1923. 

179 



i8o SURFACE TENSION OF THE MENSTRUUM 

torsion balance a great convenience. The average of fifty or a hundred readings may be 
checked with the method of Morgan, or Harkins and Brown. In this way much time may be 
saved without the sacrifice of accuracy. 

Clowes' has shown that it is often desirable to measure the interfacial tension between 
liquid and oil. Clowes's method consists in immersing the tip of an ordinary stalagmometer 
beneath the surface of parafiin oil and counting the number of drops in the usual way. This 
method gives very uniform results. 

The surface tension of ordinary bacterial culture media varies from 57 to 63 dynes per 
centimeter. The surface tension of standard veal infusion broth is approximately 58 dynes. 
That of I per cent peptone solution approximately 63 dynes. Since the surface tension of 
water is 73 dynes, it may be seen that peptone depresses the air-liquid tension about 10 dynes. 

The surface tension of culture media may be raised by treating them with charcoal. 
This raises the surface tension by removing some of the substances from solution. This 
treatment removes nutritive elements from the medium. The growth of bacteria^ may like- 
wise, in some cases, raise the surface tension of the medium in which they are "grown. 

On the other hand, many things may be used to lower the surface tensions of a liquid. 
Alcohol which has a surface tension of 22 dynes will lower the tension of a fluid in proportion 
to the percentage added. However, it does not lower the surface tension by being adsorbed 
into the surface of the liquid, and, therefore, it should not be regarded as a true surface- 
tension depressant. Soaps, saponin, and many organic compounds are true surface-tension 
depressants since they are concentrated in the surface of a liquid, and in this way small 
quantities exert a marked effect in lowering the surface tension. 

A small amount of soluble soap or saponin lowers the surface tension relatively more than 
larger amounts. The curve expressing such a relationship is that of a parabola. 

For bacteriological work I have found the sodium soaps of the unsaturated fatty acids 
most satisfactory as surface-tension depressants. Potassium soaps are also very effective, 
but there is no point in using a potassium soap in a medium containing the sodium ion, since 
in this case the sodium ion would invariably replace many of the potassium ions, and the final 
product would be essentially a sodium soap. 

Sodium ricinoleate presents certain advantages over the soaps of other fatty acids from 
the standpoint of the bacteriologist in that it is very soluble, is readily purified, remains in 
solution at low temperatures, exerts its maximum action at the pH optimal for the growth of 
bacteria and production of toxins, and forms perfectly clear solutions. 

It is important to use media which contain no salts of either calcium or magnesium, 
since these elements form insoluble soaps and would, therefore, remove the latter from solu- 
tion. 

An important step in preparing glassware — only hard glass should be employed for this 
work — is to wash it with a hot soap solution in order to remove the surface film usually pres- 
ent. It is desirable to sterilize the medium and soap solution separately and mix when cool. 

The air-liquid surface tension of water or broth may be lowered to approximately ^2 
dynes per centimeter by adding a suitable amount of soap of an unsaturated fatty acid. 

In studying the effect of the surface tension of the culture media on bacterial growth, 
one should not lose sight of the fact that the air-liquid or even the oil-liquid interfacial 
tension gives no accurate information as to the tension at the interface of the bacteria and 
water (medium). (Mudd and Mudd-* have studied the effect of interfacial tensions of two 
phase liquids on bacteria.) 

' Clowes, G. H. A.: Jour. Phys. Cliem., 20, 407. 1916. 

" Larson, W. P., and Evans, R. D.: Proc. Soc. Exp. Biol, and Med., 21, 133. 1923. 

i Mudd, S., and Mudd, B. H.: Jour. Exp. Med., 40, 647. 1924. 



W. p. LARSON 



iSi 



At the present time interfacial tensions cannot be measured or even calculated 
with any degree of accuracy. The studies of Harkins' and Halvorson and Green^ indi- 
cate there are all gradations of zones at the particle-water interface. Such zones may 
vary from one extreme, where they must be well defined, to the other, where they 
flow together, with all possible intermediate gradations. A pellicle-forming micro- 
organism rich in "fat," like the tubercle bacillus, probably is a good example of the 
first case, while the pneumococcus in a solution of bile salts represents the latter 
(not "wet" in one case and dissolved in the other). 

SURPACE TENSION AND BACTERIAL BEHAVIOR 

In discussing the effect of the surface tension of the culture medium on bacterial 
growth it may be well to begin with the pellicle-forming organism, since its effect is 
more apparent with this group. 

The earlier bacteriologists regarded pellicle formation as an expression of obligate 
aerobiosis. It is true that pellicle-forming bacteria are aerobic, but they are not all 
obligate aerobes. Bacteria which are facultative anaerobes may be developed to 
grow in pellicle. Upon reflection it is apparent that the quality of aerobiosis is not 
the mechanism which causes bacteria to grow upon the surface of a licjuid medium 
since pellicles are invariably of greater density than the fluid upon which they are 
growing. Benton^ has shown that the pellicle of B. subtilis may be sedimented at 
every stage of its development by centrifugation. It is obvious, therefore, that 
bacteria growing on the surface of a liquid are supported in this position by some 
force. Since aerobiosis does not constitute a force, we must look elsewhere for the 
explanation. It is undoubtedly the tension in the surface of the medium which sup- 
ports the bacteria in this position. An analogy may be found in the floating steel 
needle. If a steel needle is coated with a film of oil and carefully placed upon the 
surface of water it also will remain supported upon the surface. Here there can be no 
question of aerobiosis. In the language of the physicist, the needle is not "wet" by 
the water; its weight is not sufficient to break the water surface. It is thus supported 
by the surface tension of the liquid. Reducing the surface tension by the addition of 
a little soap or other surface-tension depressant causes the needle to sink promptly. 
There is little doubt but that pellicle formation is a property of the surface tension 
of the medium, although the character of the bacterial surface is also an important 
factor, as we shaU see presently. 

If the surface tension of broth is depressed to some point below 40 dynes by the 
addition of sodium ricinoleate and inoculated with the hay bacillus,'' it will be ob- 
served that the growth is either diffuse throughout the medium, or, as often occurs, 
it grows at the bottom of the flask. In this connection the question arises as to the 
relation of the surface tension of a liquid to its ability to dissolve oxygen. If oxygen 
were more soluble in water of reduced surface tension, this might account for the 
different type of growth. 

' Harkins, W. D., in Alexander: op. cit., p. 192. 1926. 

^Halvorson, H. O., and Green, R. G.: Colloid. Symp. Monograph, 2, 185. 

3 Benton, A. G.: Proc. Soc. Exp. Biol, and Med., 20, 513. 1923. 

^ Larson, W. P., Cantwell, W. F., and Hartzell, T. B.: Jour. Infect. Dis., 25, 41. 1919. 



i82 SURFACE TENSION OF THE MENSTRUUM 

Green' studied this problem, using standard methods of gas analysis. She found 
that the dissolved oxygen did not vary with the surface tension. The conclusion, 
therefore, that pellicle formation is a property of the surface tension of the medium 
rather than the property of aerobiosis seems to be justified. 

Since wetting is a function of the surface tension, it may be stated that bacteria 
will grow on the surface of a liquid medium if the latter is unable to wet the surface 
of the organism. Since not all bacteria grow upon the surface of broth of a given sur- 
face tension, it follows that some of the factors determining the type of growth are in- 
herent in the bacterial cells. 

In discussing this phase of the question it may be assumed that the surfaces of 
bacteria attract water in varying degrees. In case the water molecules have a greater 
attraction for each other than for the bacteria, the latter are caught and held in the 
surface of the fluid, and, finding conditions favorable to growth, develop in pellicle. 
On the other hand, where there is greater attraction between water and bacteria — 
as is the case between water and a clean, fat-free, glass surface, the bacteria are drawn 
into the medium, and, finding conditions favorable to growth, grow diffusely. Thus 
it is apparent, if this conception be true, that the type of growth in a liquid medium 
will depend upon two factors, viz.: the liquid-air tension of the medium and the 
nature of the bacterial surfaces. 

Since wetting is a function of the surface tension, it follows that an organism may 
behave differently in two media of different surface tension, as is the case with the 
hay bacillus in media with surface tensions of 60 and 35 dynes. 

If pellicle-forming bacteria are extracted with acetone and ether and the extract 
compared with that of non-pellicle formers, it will be found that the former are rela- 
tively rich in acetone-ether soluble substances. The tubercle bacillus is a striking 
example of this group of bacteria, possessing as high as 40 per cent of fatlike sub- 
stances, compared with about 7 per cent in non-pellicle-growing organisms. 

Bacteria, which ordinarily grow diffusely throughout liquid media, will develop in 
pellicle' if grown on a medium containing a carbohydrate or glycerol, which they will 
not ferment. The staphylococcus, for example, when grown for a few generations 
upon 3 per cent glycerol broth, produces a pellicle resembling very much the growth 
of the tubercle bacillus; the medium beneath the pellicle remains perfectly clear, and, 
macroscopically, it resembles the growth of the tubercle bacillus. The ether-acetone 
extract of such a culture represents 39.9 per cent of the weight of the dry matter of 
the organisms, as compared with approximately 7 per cent of the same strain of the 
staphylococcus grown upon broth without glycerol. 

It has been shown by numerous investigators that surface-tension depressants 
have a marked effect upon bacterial growth. Indeed, it is the operation of the laws 
of surface energy which makes it possible for bacteria to obtain food. It has been 
pointed out earlier in this chapter that many organic compounds are surface-tension 
depressants; that is to say, that they concentrate in the surfaces and interfaces of the 
liquid. The nutritive material in culture media must, therefore, be concentrated at 
the bacteria-water interfaces, which make them immediately available to the bacterial 

' Green, B. S.: Papers from Mayo Found, and Med. School, Univ. of Minn., p. 578. 1921-22. 

^ Larson, W. P., and Larson, L. W : Joiir. Infect. Dis., 31, 407. 1922. 



W. p. LARSON 183 

cells. It will perhaps not be considered too theoretical to assume that the rate of 
growth is influenced by the effect the most immediate surface tension depressants 
have upon the organism. If it happens to be an idealfood for the particular bacterium, 
it constitutes an ideal culture medium. If, on the other hand, the surface-tension 
depressants are toxic, there will be little or no growth. The use of bile in culture 
media may illustrate the point in question. It has been known for many years that 
bile salts, when added to culture media, stimulate the growth of the colon-typhoid 
bacteria, whose normal habitat is the intestinal tract. The pneumococci and strepto- 
cocci, on the other hand, are very sensitive to bile salts and soluble soaps. 

Studies published from this laboratory have shown that relatively low concentra- 
tions of sodium ricinoleate deprive pneumococci,' streptococci, and tubercle bacilli^ of 
their power to infect. Netter and his collaborators^ have recently confirmed and some- 
what extended this observation. Ayers et al.,^ studying the effect of the surface ten- 
sion of the culture medium on the growth of streptococci, suggested that the surface 
tension of the medium may be used as a basis for classification. Frobisher^ has empha- 
sized the importance of using high-tension medium in growing pneumococci. Albus 
and Holm* have further shown that B. biilgaricus is more sensitive to surface-tension 
depressants than B. acidophilus. They believe the difficulty of successful implantation 
of B. biilgaricus in the intestinal tract i? due to its inability to grow at the low-surface 
tension created by the bile salts. Hansen,^ Frobisher,* and others have shown that the 
action of some disinfectants is enhanced by surface-tension depressants. 

It is an interesting observation that the micro-organisms which thrive well upon — 
even prefer — a low-tension medium are splendid antigens, and as a rule produce 
infections which are followed by an immunity which is reasonably permanent, as is 
the case with typhoid, paratyphoid, and cholera vibrio infections. 

Infections by the pneumococci, streptococci, and tubercle bacilli, on the other 
hand, confer no such degree of immunity. It is not possible at the present time to 
show a definite relationship between the optimal surface tension for an organism and 
its antigenic properties. The apparent relationship may be purely incidental, but 
nevertheless interesting. 

The nature of the interfacial zones mentioned above undoubtedly plays an im- 
portant role in antibody reactions. Where bacterial surfaces are of such a nature as to 
resist being wet by water it might be expected it would have an influence upon the 
union between antibody and bacterium. Studies in this connection were made by 
Larson and Greenfield.' A staphylococcus was grown for several generations on 
glycerol broth until it grew in a pellicle which resembled the growth of the tubercle 

' Larson, W. P., and Nelson, E. N.: Proc. Soc. Exp. Biol, and Med., 22, 357. 1925. 

^Larson, W. P., and Montank, I. A.: ibid., 20, 229. 1923. 

•5 Netter, A., Andre, E., Cesari and Contoni: Compl. rend. Soc. de bioL, 96, 184. 1927. 

* Ayers, S. W., Rupp, P., and Johnson, W. T., Jr.: Jour. Infect. Dis., 33, 202. 1923. 
5 Frobisher, M.: ibid., 38, 66. 1926. 

'' Albus, W. R., and Holm, M. L.: Proc. Soc. E.xp. Biol, and Med., 22, 337. 1925. 
7 Hansen, T.: Comp. rendu, soc. biol., 86, 215. 1922. 

* Frobisher, M.: Jour. Bad., 13, 163. 1927. 

' Larson, W. P., and Greenfield, R.: Proc. Soc. Exp. Biol, and Med., 20, 348. 1923. 



1 84 SURFACE TENSION OF THE MENSTRUUM 

bacillus. The ether-acetone extract of such a culture represented 39.9 per cent of the 
weight of the dry matter of the organisms, as compared with 7 per cent for the control 
culture, which was grown on broth without glycerol. Sera prepared against these 
two strains agglutinated the normal strain of staphylococcus in about two hours, while 
only 30 per cent, by actual count, of the "fat" strain was agglutinated after thirty 
hours' incubation. 

The data available are not yet sufficient to permit final conclusions as to the 
mechanism involved, yet in view of our conception of the mechanism of pellicle for- 
mation the thought suggests itself that the attraction between the antibody and the 
water molecule may be greater than between antibody and micro-organism. In other 
words, if water will not wet the surface of the micro-organism rich in fat or related 
substances, the antibodies which are dissolved or suspended in water, for which they 
have greater attraction, cannot come in sufficiently intimate contact with the bacteria 
to be adsorbed. 

The writer is of the belief that by the choice of suitable surface-tension depres- 
sants the wetting of bacteria by specific antibodies may be enhanced. 

EFFECT OF SOAPS ON TOXINS 

Studies have been conducted in our laboratory on the effect of surface-tension 
depressants on various toxins.' Many bacterial toxins, snake venoms,^ and indeed 
some of the vegetable toxins are instantly detoxified by soluble soaps, particularly 
the soaps of the unsaturated fatty acids. Although this work was begun by studying 
the results from the standpoint of a surface-tension phenomenon, it soon became 
evident that factors other than the surface tension of the menstrua must be con- 
sidered. Indeed, some soaps, especially those of the fatty acids of the odd-numbered 
carbon chain, which have a marked effect in lowering the surface tension, have very 
little detoxifying action on toxins. The soaps of the saturated fatty acids are far 
inferior to those of the unsaturated series as detoxifying agents. The double bond of 
the carbon atom apparently plays a role in this reaction, although, as will be pointed 
out later, the reaction cannot be regarded as a true chemical union. 

Diphtheritic, tetanic, streptococcic, and other toxins are instantly rendered non- 
toxic when mixed with proper amounts of a soap such as sodium ricinoleate. 

Soap does not affect all toxins in the same manner, however. While diphtheritic, 
tetanic, and streptococcic toxins and some of the snake venoms are detoxified by the 
soaps of the unsaturated fatty acids, other toxins are activated by these soaps. 
Botulinum toxin, for example, is rendered more toxic when treated with soap. Green 
and Stoesser' have recently shown that the toxicity of a poisonous mushroom — 
Amanita phaUoides — is increased many fold by treatment with sodium ricinoleate. So 
active is this soap upon the Amanita toxin that its effect is apparent in the animal 
body when injected as much as seventy-two hours in advance of the toxin. This ob- 
servation will no doubt stimulate further investigation in the field of toxicology. 

Superficial examination suggests that toxins which are not detoxified by soaps 

' Larson, W. P., and Nelson, E. N.: Proc. Soc. Exp. Biol, and Med., 21, 27S. 1924. 

" Carmichael, E. B.: Jour. Phann. b" Exp. Therap., 31, 445. 1927. 

3 Green, R., and Stoessner, A. V.: Proc. Soc. Exp. Biol, and Med., 24, 913. 1927. 



W. p. LARSON 185 

are toxic when introduced into the digestive tract. Carmichael,' however, has found 
an exception to this rule in ricin, which he found was readily detoxified by sodium 
ricinoleate. 

The bile soaps undoubtedly play an important role in detoxifying the bacterial 
toxins of the intestinal tract. The intestinal tract is essentially a thirty-foot culture 
tube which harbors a large variety of bacteria, many of which, growing under favor- 
able conditions, undoubtedly secrete toxins. Had nature not provided an efficient 
detoxifying mechanism in the form of the bile soaps, the host probably would not 
have survived. Since toxins which have been detoxified with soaps are antigenic, it 
seems reasonable to assume that through absorption of such detoxified toxins man 
and the lower animals acquire a relative immunity to many of the pathogenic bacteria 
to which they are daily exposed. 

Within certain limits the reaction between soap and toxin is quantitative.^ In a 
study published from our laboratory it was shown that the toxicity of soap-toxin 
n-uxtures depends more upon the concentration of the toxin than the actual amount 
present. It was found, for example, that guinea pigs will tolerate an L+dose of 
diphtheritic toxin in a i per cent solution of sodium ricinoleate provided the total 
volume injected was at least 5.4 cc. Animals injected with this amount of toxin in a 
I per cent soap solution, but in smaller volumes, died in periods of time varying in- 
versely with the volume given. If, on the other hand, the toxin concentrations were 
kept constant while varying the soap concentration, it was found that the animals 
would tolerate an L+dose of toxin in 3.57 per cent solution of sodium ricinoleate in 
a total volume of 1.4 cc. 

Tables I-III, which are taken from an article by the writer and collaborators in 
the Colloidal Symposium Monograph, Volume 3,^ illustrate this point. 

These data show that there is a quantitative relationship in the reaction between 
toxin and soap. The reaction is evidently not a true chemical union since in such 
case soap which had reacted with one toxin should fail to react with others. This, 
however, is not the case, as it has been found that, within limits, several toxins may 
be detoxified with the same soap solutions. Furthermore, if the reaction between 
soap and toxin were a chemical union, all toxins reacting with a given soap would 
necessarily be considered identical chemically, and possibly antigenically as well. 
Such a contention would not be tenable. Wells' found that whenever chemical differ- 
ences can be shown between two antigens they invariably differ antigenically. 

Table III shows the effect of dilution upon a soap-toxin mixture which is nearly 
neutral. Moderate dilution causes it to become non-toxic, while further dilution 
causes it to become extremely toxic. 

Dissociation of diphtheritic toxin-soap mixture upon dilution may be observed 
in mixtures which have stood for periods of time up to about ten days. Mixtures 
which have stood for longer periods are not readily dissociated, but are nevertheless 
antigenic. It is believed the data given in the tables support the theory cited else- 
where that toxin exists as molecular aggregates. These aggregates are dispersed by a 

' Carmichael, E. B.: Proc. Soc. Exp. Biol, and Med., 24, 5. 1927. 

2 Larson, W. P., Halvorson, H. O., Evans, R, D., and Green, R. G,: Colloid Symp. Mono., 3, 
152. 1925. 

3 Wells, H. G.: Chemical Aspects of Immunity. 1925. 



1 86 



SURFACE TENSION OF THE MENSTRUUM 



soap solution of proper concentration. After dispersion the toxin is rendered non- 
toxic by adsorption about the soap molecule. 

TABLE I 
Effect of Varying the Concentration of the Toxin Using i Per Cent Soap 



Guinea Pig No. 



3 
4 
5 
6 

7 
8 

9 



Cone, of 

Toxin, L + 

per cc. 



•715 
•417 
. 264 
.185 
.158 
•135 
.119 
• 105 
.096 



Dose, L + 



Conc. of 

Soap in Per 

Cent 



Total V^olume 
Injected, 



7-4 
8.4 
9.4 



Result 



Died in 3 days 
Died in 3 days 
Died in 3 days 
Died in 6 days 

Lived 

Lived 

Lived 

Lived 

Lived 



TABLE II 

Effect of Varying the Concentration of the Soap 



Guinea Pig No. 



Cone, of 

Toxin. L-|- 

per cc. 



Dose, L + 



Total Volume 
Injected, 



Cone, of 

Soap in Per 

Cent 



Result 



2 
3 
4 

S 
6 

7 
8 

9 



71S 
715 
715 
715 

715 
715 
715 
715 
715 



1-4 
1-4 
1-4 
1-4 

1-4 
1-4 
1-4 
1-4 
1-4 



■715 
1-413 
2.14 
2.86 

3-57 
4.28 
S.oo 

571 
6.42 



Died in 3 days 
Died in II days 
Died in 28 days 
Showed paralysis in 
40 days. Lived 

Lived 

Lived 

Lived 

Lived 

Lived 



TABLE III 
Effect of Dilution upon a Soap-Toxin Mixture 



Guinea Pig. 
No. 


Toxin, 
cc. 


4 Per 

Cent 

Soap 

Sol., ce. 


H,0, 
cc. 


Total 
Vol. In- 
jected, 
ce. 


Cone. 
Tox., 

L+ per 
cc. 


Cone. 

Soap, 

Per 

Cent 


Dose, 

L + 


Result 


I 

2 

3 

4 


•4 
•4 

•4 
•4 


I.O 
I.O 

1.0 

I.O 


0.00 
1. 00 
2.00 
3.00 


1-4 

2-4 

3-4 
4-4 


■715 
.416 
.300 
.227 


2.86 
1.66 

1. 18 

0.90 


I 
I 
I 
I 


Died in 41 days 

Lived 
Died in 3 days 
Died in 2 days 



In view of the fact that a number of bacterial toxins, bacteria, and filterable 
viruses' are detoxified by soap, without destruction of their antigenic properties,^ 
it should be possible to develop this principle to the point where it would be practi- 
cable to immunize against several infections in the same injection. 

' McKinley, J- C., and Larson, W. P.: Proc. Soc. Exp. Biol, and Med., 24, 297. 1927. 
' Larson, W. P., and Eder, H.: Jour. Amer. Med. Assoc, 86, 998. 1926. 



W. p. LARSON 187 

The mechanism of the action of soaps upon toxins provides interesting specula- 
tion since some toxins are neutraUzed while others are rendered more active. A theory 
on the basis of the charge carried by the soap, on the one hand, and the toxins, on the 
other, may supply a partial explanation of the phenomenon. If it be assumed that 
soap and diphtheritic toxin are oppositely charged they will attract each other and 
enter into some relationship which we have chosen to term an "adsorption reaction." 
If, as may be the case with the mushroom toxins, they bear the same charge, the 
tendency would be toward dispersion with resultant increased toxicity of the toxin. 
If we accept the theory of dispersion as the mechanism by which the toxin is ren- 
dered more toxic, it will be necessary to assume further that the mushroom toxin 
normally exists not only as free toxin molecules, but in molecular aggregates as well, 
otherwise there would be no dispersion. 

If this assumption, for which there is some experimental support, be accepted, it 
may be assumed further that all toxins may exist in the form of molecular aggregates, 
and that such aggregates act as a unit, or as a single toxin molecule. In the light of 
this theory, toxins which are charged oppositely to the soap would be dispersed by 
being attracted to the soap and condensed — adsorbed — about the soap molecule. The 
like charged toxins, on the other hand, would be repelled by th» soap, and in this way 
the molecular aggregates dispersed. Halvorson and Green have shown by mathe- 
matical deductions that the charge on the surface of a particle or molecule must be 
considered as a part of the surface energy. It is not contended that the charge of the 
molecules is the only force which brings about the reaction between the toxin and soap 
molecules. It is undoubtedly only one factor in the interplay of the forces of surface 
energy. 



CHAPTER XII 

OXIDATION-REDUCTION POTENTIALS OF DYE SYSTEMS 
AND THEIR SIGNIFICANCE IN BACTERIOLOGY 

W. MANSFIELD CLARK' 
Hygienic Laboratory, Washington, D.C. 

INTRODUCTORY 

Various attempts have been made to trace the history of our knowledge of the 
decoloration of dyes by bacteria. But let it be remembered that the preparation of 
several of the older dyes involved fermentative reduction and that the origins of these 
preparative processes are lost in antiquity. The Chinese, importing synthetic indigo 
from America, now avoid the necessity of the fermentative splitting of the glucoside 
of the indigo plant, but still employ the fermentative vat process they imported from 
India in ancient days. So it was that the decoloration of indigo and also the decolora- 
tion of other dyes, during the events known as "fermentations," were common knowl- 
edge during the period when the theory of fermentation took shape. 

Had a century or two in either direction separated the rise of bacteriology from 
the rise of modern chemistry, certain important aspects of fermentative dye de- 
coloration might not have been burdened with a nomenclature which somewhat ob- 
scures their significance. However, the technical and physiological importance of 
oxygenation had already established a dominating point of view at the time the 
theory of the vat process took form. It was the period in which great classes of chemi- 
cal transformations were formulated in terms of the gain or loss of oxygen. As carbon 
dioxide is a "higher" oxide of carbon than carbon monoxide, the process 

2CO-fO.-^2CO. 

was naturally called "oxidation," while the reverse process resulting in the reduction 
of degree of oxidation was called "reduction." So when the cloth came from the indigo 
vat and was blued obviously by atmospheric oxygen, an oxidation process was said 
to have occurred, and by the same token the decoloration was regarded as a reduction. 

The fact that organic chemistry represents the molecule of indigo as differing 
from that of indigo white by the loss of two hydrogen atoms does not perturb the 
formalist. Nothing is simpler than to assume the hydrogen to be removed by com- 
bining with oxygen to form water. But when oxidation of indigo white is accom- 
plished by halogens, ferric salts, and other "oxidizing" reagents the postulated chem- 
istry of the reaction is very complex. 

It is not the purpose of this chapter to review either the chemistry of dyes, the 
theory of their oxidative-reductive transformations, or the details of the uses to which 
these processes are put in bacteriology. There will be described briefly a picture which 
is useful. 

' Now at the School of Medicine, Johns Hopkins University, Baltimore, Md. 

i88 



W. MANSFIELD CLARK 



189 



ELEMENTARY OXIDATION-REDUCTION THEORY 

It has been found that a few oxidation-reduction processes occur in such a manner 
that states in their equihbria may be defined very accurately by certain electrical 
measurements. For instance, let a solution of hydrochloric acid be divided as shown 
in Figure i with a liquid connection made narrow to isolate approximately the parts. 
In one side place a platinum electrode and let there be one atmosphere pressure of 
hydrogen. In the other side place a platinum electrode and a mixture of ferric chloride 
and ferrous chloride. These salts dissociate to furnish the ions Fe+++ and Fe++. If 
the electrodes are placed in metallic connection, a current of electrons will flow in the 
connection from the "hydrogen electrode" to the electrode in the iron solution, and 
this current will be accompanied by the transformation of hydrogen to hydrogen ions 
on the one side (an oxidation) and the transformation of ferric to ferrous ions on the 
other side (a reduction). If the electromotive force of this cell is nicely balanced by an 
external electromotive force, there is at- 
tained a close approach to the operation of 
the cell under conditions of maximum 
work, and there can be applied the ther- 
modynamic equation for the change in free 
energy of the cell process. This free energy- 
change can be factored into two parts: 
The quantity of electricity determined by 
the number of chemical equivalents and 
the intensity factor or electrical potential. 
The latter, as measured by a potentiometer 
under the conditions specified above, is a 
measure of the intensity with which hydro- 
gen, at one atmosphere pressure, re- 
strained by the given hydrion concentra- 
tion, tends to transform the given iron solution in the direction of complete 
reduction. 

The measurements are of the intensity factor of a free energy-change, and in the 
present state of our knowledge we must say that it is a matter of good fortune that 
the iron and several other oxidation-reduction systems are susceptible to study by 
this method. It furnishes no secure evidence of mechanism, but it may be inferred 
from facts which we shall not stop to review that the essential process is one of elec- 
tron exchange. We may imagine that the ferric-ferrous system is exchanging elec- 
trons not only within the system itself but also with the electrode. With a fixed ratio 
of ferric and ferrous ions the electrode will receive a characteristic charge. In like 
manner, many dye systems such as methylene blue-methylene white give stable and 
characteristic electrode potentials. 

It is therefore a convenience to assume that relative electron-escaping tendencies 
determine the ability of one system to reduce another. We shall see presently how, 
in accordance with this orienting assumption, the hydrogen exchange is taken care of. 

For the sake of definiteness let the hydrogen half-cell be that in which the hydro- 
gen is at one atmosphere and the hydrogen-ion concentration is one normal and let 




IQO 



OXIDATION-REDUCTION POTENTIALS 



too 



1' 



'o Oxidation 

15 SO 



0.50 



0.45 



0.40 



0.35 



0.30 



Eh 




0^5 



O.Z0 



0.15 



0.10 



0.05 



25 50 75 

% Reduction 

Fig. 2. — Relation between percentage reduction and elec- 
trode potential (Eh) for different oxidation-reduction systems. 



the potential of this half- 
cell be considered zero. 
This half-cell will be con- 
sidered the standard. There 
may now be substituted for 
the ferrous-ferric system one 
or another of several other 
oxidation-reduction sys- 
tems. If a half -cell contain- 
ing each system in turn be 
joined with the standard 
hydrogen half-cell and the 
potentials of the cell at 
different stages in the re- 
duction of the system be 
measured, the results when 
shown graphically will pro- 
vide a picture like that of 
Figure 2. An oxidant in- 
volving two electrochemical 
equivalents permol for com- 
plete reduction will give a 
curve of the slope of B or 
C. All the dye systems to 
be described furnish the 
two-equivalent slope. A 
system involving one equiv- 
alent gives a shape like that 
of curve A. Any such 
curve is best characterized 
by its center point — that of 
50 per cent reduction or 
oxidation of the system, the 
potential of which furnishes 
a convenient reference 
point with which to describe 
quantitatively the relative 
oxidation or reduction in- 
tensity of the system. Each 
system has such a character- 
istic point under a given set 
of conditions to be described 
later. If it be negative to 
the characteristic potential 
of another system, the first 
is reducing with respect to 



W. MANSFIELD CLARK 



191 



the second. If it be positive with respect to a second, the first system is oxidative 
with respect to the second. 




60 to KX) 



Fig. 3 



In practical applications the capacity factor must not be forgotten. Thus the 
ferrous-ferric ion system has an oxidative potential relative to the system methylene 



192 OXIDATION-REDUCTION POTENTIALS 

blue-methylene white; but if too little ferric iron is used to oxidize completely methy- 
lene white, there will be left ferrous iron as the only form of iron and a mixture of 
methylene white and methylene blue. If excess ferric is used, all the methylene white 
will be oxidized to methylene blue, and there will be left a mixture of ferrous and 
ferric ions. In the first case the potential will lie on the methylene blue — methylene 
white curve; and in the second case it will lie in the ferrous-ferric curve. 

Figure 3 represents a series of actual measurements with sulphonates of indigo. 
The experimental data are shown by the centers of circles, and the theoretical form- 
curve is shown in each case by the line to which the loci of the experimental data evi- 
dentally conform. This theoretical curve is determined by 

„ _,, - Total reductant 

£ = £0-0.03 log Total oxidant 

Since all the systems to be described conform to this equation when the hydrion con- 
centration of the solution is fixed and known, and differ only in the characteristic 

T^, rr^ 1 1 -r • • 1 ,-1 1 Total rcductaut . 
constant L'o, 1 able I, givmg values for the term 0.03 log -. —, — — for various 

percentages of reduction, will be found useful. 

Now it is found that a system which has been studied in a solution of constant 
hydrogen-ion concentration and which has been found to give a curve such as that of 

Figure 3 will give the same form of curve 

77 77 at another value of pH, but the value of 

. C. .. . C . E'o will depend on the value of pH. Thus 

H:C •C:N::C •C:H ^t^q whole curve will be displaced in the 

\ ':'' C : S : C C :: N : R Potential scale. 

R ! N . C ^ . " •*• : + Why is this displacement? 

C *•* C Consider the case of methylene blue- 

g g methylene white and the conventional 

^ ^ . , , . electronic structure of a thiazine shown 

riG. 4. — Conventional electronic structure . , , . , 

of a thiazine. by Figure 4. The terminal substituted 

amino group at the right is polar and in 
methylene blue is a very strong base. The opposite dimethyl amino group in methy- 
lene blue is very weak. When the compound is reduced, the polarity of the one 
group is destroyed and this group becomes weakly basic. The other group in the 
now symmetrical structure is enhanced in strength. At the same time the bridging 
nitrogen fixes a hydrion. These changes make themselves felt in the thermodynamic 
measurements of free energy-change because as the several groups ionize, a virtually 
new oxidant or a new reductant is formed, each differing from the parent by the 
energy of ionization. Putting the matter another way, we may say that the covering 
or uncovering of points by hydrogen ions or hydroxyl ions alters the ease of escape- 
ment and acquirement of electrons. It is for this reason that the hydrion concentra- 
tion must be kept rigidly constant if we are to obtain titration curves such as those 
of Figure 3. 

Remembering that if the hydrion concentration is constant we shall always ob- 
tain a curve of the form shown in Figure 3, our interest centers upon the displacement 



W. MANSFIELD CLARK 



193 



TABLE I 
Relation of Percentage Reduction to Potential at Constant pH Determined by 

Ei, = E'o- 0.03006 log [|^ at 30° C. 

(Values Rounded to Nearest Millivolt) 



Reduction 



Per Cent 

I 

2 

S 

10 

IS 

20 

25 

30 

35 

40 

45 

50 



-0.03006 log 



[So] 



Volts 

+ 0.060 
.051 
.038 
.029 
.023 
.018 
.014 
.Oil 

.008 

.005 

+ -003 

+ o . 000 



Reduction 



Per Cent 

55 

60 

65 

70 

75 

80 

85 

90 

QS 

98 

99 



—0.03006 lof; 



fSo) 



Volts 

— o . 003 

.005 

.008 

.oil 

.014 

018 
.023 

.029 

.038 
.051 

— 0.060 



TABLE 11 

E'o Values for Several Oxidation-Reduction Indicators, 30° C. 
(Values Rounded to Nearest Millivolt) 









i> 






rt , 


cd 














« 


V 


c 






§3 

j= 


C 

-C 


■3 




c 




■a 


■§ 


pH 


c 


XI 

a 
■3 

5 

a 


c 


J3 

a. 
"3 

•S 

H 




j3 

a. 
"3 

H 



3 
5 

c 
>1 


3 

5 

c 




a. 
"3 
in 

Vo 

■og 

il 


V 

.si 
•S 

9^ 


c 
D. 

a-o 
— s 

.a D. 




_5J 


c 

"o 
c 

c 
II 


■3 
c 

C. 

is 

S " 




-a 
c 


c 


-a 
c 


u 

s 


I 


Z£a 


i5 


c 




-a 
c 

m 


c 
6 


E 


50 


— O.OIO 


0.032 


0.065 


0. lOI 


0. 221 


0. 262 




0.33s 
■ 322 
• 307 


0. ^66 


0-335 
.320 

•307 






5.2 


.022 


.020 


.053 


.088 


. 208 


•249 
.236 








352 
339 






5-4 


• 034 


+ .008 


.041 


.077 


. 196 




'f 




* 


* 


^.6 


• 045 


— .004 


.020 


.066 


. 184 

•173 
.162 


.223 
. 210 
.196 






.292 

• 277 

.261 




325 
310 

295 


•293 
.281 
.27c 






5.8 


.057 

.069 


.016 
.028 


.017 
+ .006 


.056 
.047 












6.0 





183 


0.301 




6.2 


.oSi 


■039 


— .006 


•039 


•151 


.181 




171 


■ 245 




279 


•259 


.288 




6.4 


.092 


.051 


.017 


•031 


.141 


.166 




1 59 


.228 




263 


.249 


•275 


* 


6.6 


. 104 


.061 


.027 


.024 


■132 


.150 




147 


. 212 




247 


.240 


. 262 




6.8 


.114 


.072 


•037 


.017 


■123 


•134 




13s 


. 196 




232 


.232 


.248 




7-0 


•125 


.081 


.046 


.011 


•IIS 


.119 




123 


.181 




217 


. 224 


•233 


0.248 


7-2 


•134 


.091 


•055 


+ .004 


.108 


.103 




III 


.166 




203 


.217 


.21S 


• 235 


7-4 


•143 


.099 


.062 


— .002 


. lOI 


.088 




099 


•152 




189 


. 210 


.203 


.221 


7-6 


•152 


.107 


.070 


.008 


.094 


•073 




087 


. 138 




175 


. 204 


.187 


.208 


7.8 


.160 


.114 


.077 


.014 


.088 


.060 




074 


•125 




162 


0.197 


.170 


•193 


8.0 


.167 


.121 


■083 


.020 


.082 


.046 




0O2 


.112 




150 




•15s 


.178 


8.2 


.174 


.127 


.090 


.026 


• 075 


•034 




049 


•09Q 




137 




•139 


.163 


8-4 


.180 


■134 


.096 


.032 


.o6q 


.021 




026 


.087 




125 




.124 


.148 


8.6 


.187 


. 140 


.102 


.038 


.063 


+ .010 




023 


•075 




113 


t 


. 109 


•133 


8.8 


•193 


. 146 


.108 


.044 


■057 


— .002 


+ 


010 


.063 




lOI 




• 095 


•117 


90 


-0.199 


-0.152 


— 0.114 


— 0.050 


0.051 


— 0.012 


— 


003 


0.051 





089 




0.082 


0.103 



* Unstable in this region of pH. 



t Decomposes in this region of pH. 



194 



OXIDATION-REDUCTION POTENTIALS 



of the curve as a whole when pH is changed. In Figure 5 is added the new dimension 
pH, the case illustrated being that of 2-6 dibromo indophenol (or 2-6 dibromo ben- 
zenone indophenol). At a given level of pH the familiar curve relating percentage 
oxidation (or reduction) to potential is similar to one in Figure 3. If any fixed per- 
centage oxidation is carried through changes of pH, the curve is one of those shown 
running from the upper right-hand to the lower left-hand part of the figure. For con- 
venience the curve of 50 per cent oxidation is chosen for use in two dimensional charts. 




Fig. 5. — Isometric drawing showing the surface descriptive of the system of which 2-6 dibromo 
indophenol is the oxidant. The co-ordinates are percentage oxidation, pH, and electrode potential. 

In Figure 6 are several such curves showing the potentials of half-reduced systems 
at different levels of pH. Curves 1-4 are respectively those of the mono-, di-, tri-, 
and tetra-sulphonates of indigo; 5 that of methylene blue, 6 that of Lauth's violet, 
the remainder being curves of indophenols. 

In Table II are given the potentials of half-reduced solutions of several indicators 
at intervals of 0.2 pH unit. This table may be used in conjunction with Table I as 
follows. 

Suppose a culture known to be at pH = 7.2 has reduced methylene blue to the ex- 
tent of 90 per cent. Table II shows that the potential of a 50 per cent reduced methy- 
lene-blue solution is + .004 at pH 7.2. Table I shows that the 90 per cent reduced solu- 
tion is .029 volts more negative. Hence the culture has a reduction intensity wliich 



W. MANSFIELD CLARK 



195 



may be described as — .025 volts. If the culture maintains its pH value at 7.2 and then 
reduces indigo disulphonate to the extent of 85 per cent, the tables show that the re- 




J3 O 



•5 s-s 






c 
o 

f S 

~ Q. 

•£ c 
» O 



3 10 vO t— On 






-=2 S o JiJ-^ 



^ rt 



g 

E o o o o 

oj be 60 bO bO 



e ^ i3 



duction intensity may be described as —.157 volts. But if at 85 per cent reduction of 
indigo disulphonate pH should have changed to 6.6, the potential would be —.081. 



196 OXIDATION-REDUCTION POTENTIALS 

Thus definite numerical values may be given to the reduction intensities of bacterial 
cultures. 

OXIDATION-REDUCTION AND ANAEROBIOSIS 

Now consider Figure 6 in more detail. The zero of potential at pH = o is the 
arbitrary zero of the normal hydrogen electrode. As pH increases the potential of the 
hydrogen electrode becomes more negative and follows the straight line at the left. 
Parallel to this, and about 1.23 volts more positive, is the theoretical line of the normal 
oxygen electrode, an impracticable electrode. Now reverse the point of view. Con- 
sider a reducing agent capable of decomposing water with the liberation of hydro- 
gen at one atmosphere. It should give a hydrogen electrode potential. Consider 
an oxidizing agent capable of decomposing water with the liberation of oxygen at 
one atmosphere. It should give an oxygen electrode potential. But the hydrogen 
electrode at any given hydrion concentration becomes more positive by about .03 
volt for each power of 10 by which the hydrogen pressure (in atmospheres) is 
lowered. Likewise, the ideal oxygen electrode should become .015 volt more 
negative for each power of 10 by which the oxygen pressure (in atmospheres) is 
lowered. Thus if the oxygen pressure is lowered to io~^^ and the hydrogen pressure 
to lo"^'' atmosphere, an oxygen electrode and a hydrogen electrode should give a po- 
tential on the line of Figure 6 marked H2 io~^^, O2 lo"''^ Conversely, a half-reduced 
indophenol system (lying near this line) should indicate a hydrogen or oxygen partial 
pressure of the values indicated, if a state of equilibrium is attained. The foregoing 
condition is extremely important. As a matter of fact, many of the indophenols are 
kept reduced by living cells even though oxygen is bubbled through the suspension. 
This fact alone is sufficient to suggest not only the relative inertness of atmospheric 
oxygen but also the caution to be observed in applying equilibrium data to kinetic 
affairs. It reveals at once the difficulty in obtaining an end-point indicator for oxygen 
which can be rationally formulated by methods comparable with those used in acid- 
base titrations. 

It was not long after the advent of anaerobic culture of bacteria that Gunning 
criticized the methods, claiming that they were not adequate to remove the last traces 
of the oxygen he thought necessary to life. Finally, he appeared before the French 
Academy to claim that by a better approach to strict removal of free oxygen he had 
succeeded in stopping bacterial growth. Needless to say, the claim has received little 
support; but what interests us now is Pasteur's reply to Gunning. He stated that he 
was convinced of the fact of anaerobiosis because he had observed bacteria to thrive 
in solutions which maintained indigo in the reduced state. 

If it can be shown that in any given case conditions are favorable to the attain- 
ment of true equilibrium, calculation shows the following: In a reduced solution of 
indigo disulphonate at pH = 7.o, the potential should be less than —.125 — .060 = 
— .185 (Tables I and II). From the formula 

— .185 = 1 .23— .06 pH+ .015 log Po.. , 

Po2=io~''''-5 atmosphere. Since a gram mol of oxygen contains about 10'' discrete 
molecules and occupies about 24 liters at one atmosphere, the foregoing condition 



W. MANSFIELD CLARK 197 

would leave one discrete molecule of oxygen in a cube the order of magnitude of which 
would be about a million meters to the edge. Therefore, if an approach to an equi- 
librium condition can be shown to be reasonably attained, Pasteur's intuition is 
established as correct and anaerobiosis is an indisputable fact. 

Since many dye systems give very satisfactory electrode potentials, it is an easy 
matter to study with this method the kinetics of dye reduction. It has also been possi- 
ble to determine by the method outlined the dissociation constants of acidic or basic 
groups in dyes of importance to the art of staining. But even in the absence of the 
dye, Gillespie (1920) found definite drifts of potential in bacterial cultures. Further 
work on this was reported by Clark (1920), and was taken up again by Cannan, 
Cohen, and Clark (1926). 

More detail concerning the theory, the measurement of dye systems, and applica- 
tions to various subjects of interest to bacteriology will be found in the following 
papers by Clark and his co-workers:' 

STUDIES ON OXIDATION REDUCTION 
I. Introduction, Pub. Health Rep., 38, 443. 1923. (Reprint No. 823.) 
II. "An Analysis of the Theoretical Relations between Reduction Potentials and pH," 
ibid., p. 666. 1923. (Reprint No. 826.) 

III. "Electrode Potentials of Mixtures of i-Naphthol-2-Sulphonic Acid Indophenol and 
the Reduction Product," ibid., p. 933. 1923. (Reprint No. 834.) 

IV. "Electrode Potentials of Indigo Sulphonates, Each in Equilibrium with Its Reduction 
Product," ibid., p. 1669. 1923. (Reprint No. 848.) 

V. "Electrode Potentials of Simple Indophenols, Each in Equilibrium with Its Reduction 

Product," ibid., 39, 381. 1924. (Reprint No. 904.) 
VI. "A Preliminary Study of Indophenols: (A) Dibromo Substitution Products of Phenol 
Indophenol; (B) Substituted Indophenols of the Ortho Type; (C) Miscellaneous," 
ibid., p. 804. 1924. (Reprint No. 915.) 
VII. "A Study of Dichloro Substitution Products of Phenol Indophenols," ibid., 40, 649. 

1925. (Reprint No. looi.) 
VIII. "Methylene Blue," ibid., p. 1131. 1925. (Reprint No. 1017.) 
IX. "A Potentiometric and Spectrophotometric Study of Meriquinones of the p-Phenylene 

Diamine and Benzidine Series," Supplement No. 54 to ibid. 1926. 
X. "Reduction Potentials in Cell Suspensions," Supplement No. 55 to ibid. 1926. 
XI. "Potentiometric and Spectrophotometric Studies of Bindschedler's Green and 
Toluylene Blue," Supplement No. 61 to ibid. 1927. 

Other papers on the subject are referred to in reviews by Clark^ and by Conant.' 
A review of particular biochemical interest is that of Joseph and Dorothy Needham.'' 

' Clark, W. M., Cohen, B., Gibbs, H. D., Sullivan, M. X., Cannan, R. K., and Phillips, M. 

* Clark, W. M.: C/rew. i?CT., 2, 127. 1925. 

J Conant, J. B: ibid., 3, i. 1926. 

^Needham, J. and D.: Protoplasm, i, 255. 1926. 



CHAPTER XIII 
ANAEROBIOSIS 

IVAN C. HALL 

University of Colorado Medical School 

BIOLOGICAL SIGNIFICANCE OF ANAEROBIOSIS 

Few biological discoveries of more fundamental significance have been made than 
when Pasteur' in 1861 proved the existence of micro-organisms that are able not only 
to live in the complete, or nearly complete, absence of atmospheric oxygen, but which 
are actually unable to multiply at all under ordinary atmospheric conditions, and re- 
quire, if not an absolute elimination of free oxygen from their environment, at least a 
marked reduction in oxygen tension. 

Pasteur was brought to this discovery during his famous researches on fermentation, 
by the observation that certain motile germs in a cover-slip preparation became non-motile 
on approaching the edge of the cover. Having noted that many products were formed in 
the so-called "lactic" fermentation, such as butyric acid, mannitol, alcohol, carbon dioxide, 
and hydrogen, in addition to lactic acid, he was led to investigate the possibility of a special 
butyric ferment. This he decided was an infusorian, the Vibrion butyrique, living only in 
the absence of free oxygen, a conclusion in error so far as concerns the animal nature of the 
organism, but none the less important in its implications and its bearing on the old problem 
of abiogenesis. It had been generally assumed that air was necessary for the existence of all 
living things. But Pasteur revealed that the growth of certain micro-organisms is not only 
not inhibited but is actually dependent upon the practically complete exclusion of air. 

Pasteur^ called these organisms "anaerobes," as distinguished from "aerobes," which 
grow in the presence of air; later^ three groups were distinguished: obligate anaerobes, obli- 
gate aerobes, and facultative aerobe-anaerobes. Holding that all anaerobic life depends upon 
an ability to satisfy oxygen requirements by sugar cleavage, Pasteur was led to define 
fermentation as life without air. While the production of "ferments" or enzymes is by no 
means restricted to the anaerobic micro-organisms, as Pasteur believed, both the most 
actively fermentative and the most actively putrefactive bacteria are found within this 
group. They are therefore of great importance in the chemical cycles of nature for the 
mineralization of organic matter, including processes useful to man, such as the destruction 
of dead bodies of plants and animals, and the formation of useful chemical solvents (butyl 
alcohol and acetone), as well as processes which man would like to avoid, notably food 
spoilage. Some of the obligate anaerobes are dangerous pathogens, producing malignant 
edema, gaseous gangrene, tetanus, botulism, syphilis, and other diseases in man and animals. 
The toxins of tetanus and botulism are the most powerful bacterial poisons known. 

The first pathogenic anaerobe, Vibrion septique, was announced by Pasteur and Joubert 
in 1877 as the cause of a septicemia {charbon symplomaliquc, symptomatic anthrax, or 

' Pasteur, L.: Compl. rend. Acad, de sc, 52, 344 and 1260. 1861. 
^ Pasteur, L.: ibid., 56, 416 and 1192. 1863. 
i Pasteur, L.: Eludes siir la Bicre. 1876. 

198 



IVAN C. HALL 199 

blackleg) in cattle which they distinguished from anthrax.' Discoveries of other pathogenic 
anaerobes have continued from that day until the present, and probably will continue for 
many years, for this field is still a fertile one. No obligately anaerobic plant pathogens have 
been discovered, however. 

The best-known obligate anaerobes are found among the sporulating bacilli, whose 
properties and activities have been summarized by von Hibler,^ by Jungano and Dis- 
taso,^ and more recently by various French,'' English, ^ Italian,^ and American^ investi- 
gators. The frequent occurrence of gaseous gangrene during the war, due to the close 
contact of the soldiers with fertilized (infected) soil, and the extensive uses of high 
explosives which carried soil particles into deep wounds, served greatly to arouse new 
interest in wound infections due to the sporulating anaerobes. During the war also, 
botulism became a conspicuous problem in the United States, possibly due, in part, 
to an unusual increase in the volume of crude home-canning, although the first cases 
of botulism were recognized before the war and there is good clinical evidence that 
botulism has occurred in the United States as in European countries for many years* 
and a few of the outbreaks were due to commercially canned rather than home-canned 
products.^ Important obligate anaerobes occur also among the cocci,'" the actino- 
myces," the strep tothrices," and the spirochaetes.'^ 

What degree of oxygen-tension reduction can be tolerated by higher animals and 
plants is a problem only recently solved and one which has important practical bear- 
ings in the case of man, in connection with aviation. '^^ Bunge'^ pointed out that various 

' Pasteur, L., and Joubert: Bull. Acad, de med., Paris, 6, 781. 1877. 

^ von Hibler, E.: Untersiichimgen iiber path. Anaeroben. 1908. 

3 Jungano and Distaso: Les Anaerobies. Paris: Masson, 1910. 

■'Weinberg, M., and Seguin, P.: La Gangrene gazeiise. Paris: Masson, 1917. 

5 Adamson, R. S.: /. Path. 6° Bad., 22, 345. 1919; Medical Research Committee: Reports on 
the Anaerobic Infections of Wounds and the Bacteriological and Serological Problems Arising Therefrom. 
1919. 

^Desderi, P.: Infezione da germi anaerobi per feritd in guerra. Torino, 1919; Fasiani, G. M.: 
Bolletino delV Institute Sieroterapico Milanese, No. 3. Oct., 1921. 

' Simonds, J. F.: Monograph of the Rockefeller Institute for Medical Research, No. 5. 1915; Heller, 
H.: J. Infect. Dis., 27, 385. 1920; J. Bact., 6, 445, 521. 1921; Kahn, M. C: /. Med. Research, 43, 
155. 1922; J. Infect. Dis., 35, 423. 1924; Kendall, A. I., Day, A. A., and Walker, A. W.: ibid., 30, 
141. 1922; Hall, I. C: ibid., p. 445. 1922. 

^ Dickson, E. C: Monograph of the Rockefeller Institute for Medical Research, No. 8. 1918. 

' Dubovsky, B. J., and Meyer, K. F.: /. Infect. Dis., 31, 501. 1922; (see also numerous papers 
by Meyer and collaborators in succeeding numbers of J. Infect. Dis.); Geiger, J. C, Dickson, E.G., 
and Meyer, K. F.: Pub. Health Bull. 12'j. 1922. 

'"Hall, I. C., and Howitt, B.: /. Infect. Dis., 37, 112. 1925; Prevot, A. R.: Ann. deVInst. 
Pasteur, 3g,4ij. 1925; Schwartz, O. H., and Dieckmann, W.J. : Am. J. Obsi. b'Gynec., 13, 467. 1927. 

" Lieske, R.: Morphologie und Biologie der Strahlenpilze (Actinomyoetes). Leipzig: Borntraeger 
Bros., 1921; Naeslund, C.: Acta path, el microbiol. Scandinav., 2, no. 1925. 

'^ Plant, R.: Centralbl.f. Bakteriol., Abt. I, Orig., 84, 440. 1920. 

'^ Noguchi, H.: J. Exper. Med., 14, gg. 1911; 15, 81 and 466. i9i2;27, 667. 1918; 28,559.1918. 

'^ Schneider, E. C.: Am. J. Physiol. ,34, i and 29. i9i4;36,38o. 1915; also editorialin /./l.Af .^., 
88, 1805. 1927. 

'sBunge, G.: Ztschr.f. phys. Chemie, 8, 48. 18S3; 12, 565. 1888; 14, 31S. 1890. 



200 ANAEROBIOSIS 

invertebrates such as intestinal worms and mud-dwelling organisms probably live 
without free oxygen owing to the presence of numerous micro-organisms which reduce 
the oxygen supply practically to zero, and raised the question as to how the muscles 
of such organisms secure their energy. 

According to Weinland,' intestinal worms effect a true fermentative process under 
anaerobic conditions, as foUows: 

4C6H,.06-9CO.-f sQH.oO.+gH, . 

Piitter^ kept leeches alive ten days without oxygen. During the interval hydrogen was 
set free with a primary rise in CO2 output followed by a fall. 

Packard^ showed that the resistance of Fundulus embryos could be increased either by 
the injection of sodium bicarbonate or of certain carbohydrates. There is a possible analogy 
in Packard's reasoning to that of Pasteur which suggests that carbohydrates probably 
present in the organic slimes at the bottom of lakes make anaerobic life among some animals 
as well as bacteria possible there. 

Juday^ has noted that while the water from near the bottom of a lake may contain 
considerable oxygen during the winter months, in summer two factors operate to reduce the 
oxygen content to so low a value that it cannot be determined, namely, first, decaying 
organic matter, and second, lack of convection currents due to the warm and therefore lighter 
layer of water at the surface. In the slime collected from Lake Mendota, Wisconsin, various 
protozoa as well as higher invertebrates such as rotifers, worms, molluscs, crustaceae, and 
insect larvae were found frequently. None of the protozoans pumped from the bottom into 
anaerobic containers was in any way abnormal or inactive. Juday^ recently recorded an 
obligately anaerobic ciliate resembling Enchelys. On the other hand, according to Juday 
and Wagner, "No fish has ever been found which leads either an active or passive life in 
water that is free from dissolved oxygen."* Therefore, while the depths of a given lake may 
present suitable temperature conditions for the implantation of trout, and the surface layers 
an ample oxygen and food supply, the separation of these factors by stratification of the 
water during the summer months may prevent stocking and even account for the death of 
many fish in a well-stocked lake. Birge and Juday^ state that most fish require an oxygen 
content in the water of at least 2 cc. per liter (N.T.P.), whereas many protozoa and inverte- 
brates live in water containing less than o.i cc. oxygen per liter. 

In the tissues of higher animals many of the processes may be considered as essentially 
anaerobic. Bayliss^ referred to lactic acid as the product of anaerobic change in frog muscle, 
CO2 as that of aerobic change, and Shelford has pointed out that "high respiratory quotients 
of various animals are further evidence of anaerobic respiration. "« 

' Weinland, E.: Z/5c/?r./. 5/0/., 42, 55. i9oi;43, 86. 1902; 45, 113 and 517. 1904148,87. 1906. 
^ Putter, A.: Zlschr. allgem. Physiol., 6, 217. 1907; 7, 16. 1907. 
3 Packard, W. H.: Am. J. Physiol., 15, 30. 1905; 18, 164. 1907. 
^ Juday, C: Tr. Wisconsin Acad. Sc, 16, 10. 1909. 
5 Juday, C: Biol. Bull., 36, 92. 1919. 

* Juday, C, and Wagner: Tr. Wisconsin Acad. Sc, 16, 17. 1909. 
7 Birge, E. A., and Juday, C: ibid., p. i. 1908. 

' Bayliss, W. M.: Principles of General Physiology (4th ed.). London: Longmans & Co., 1924. 
'Shelford, V. E.: chap, ii in Ward, H. B., and Whipple, G. C: Fresh Water Biology. New York: 
Wiley, 1918. 



IVAN C. HALL 201 

Thus the problems of anaerobiosis are not limited to a single group of bacteria 
but are shared by several groups, by protozoa, various invertebrates, and even by the 
tissues of warm-blooded animals. While some of the invertebrate metazoa may live 
as facultative anaerobes during all or part of their existence, the condition of obligate 
anaerobiosis seems to be limited to single-celled micro-organisms, particularly the 
bacteria and protozoa. Whether any of the ultra-microscopic viruses are obligately 
anaerobic must await their certain culture in artificial media. It is of interest to note 
that no obligately anaerobic yeasts or molds are known. 

MECHANISM OF BACTERIAL ANAEROBIOSIS 
PHYSICO-CHEMICAL CONCEPTIONS 

Pasteur's explanation of anaerobiosis was that the organisms secure their oxygen 
through the fermentation of carbohydrates. Certainly the growth of some obligate 
anaerobes is dependent upon the presence of fermentable carbohydrates. This seems 
to be the case with the actively saccharolytic species, B. butyricus, B. mnltifernientans, 
B.fallax, and B. sphenoides, none of which possesses sufficient lytic ability against ni- 
trogenous compounds to hydrolyze even gelatin. Certain other species, e.g., B. welckii, 
B. chauvoei, B. septicus, B. novyi, and B. tetanomorphiis, grow, but poorly, except in the 
presence of fermentable carbohydrates; these split gelatin and peptones but not native 
proteins. But the actively proteolytic bacteria, such as B. bifermentans, B. tyrosino- 
genes, B. aerofoetidus, B. botulinus, and B. sporogenes, grow quite heavily in sugar-free 
media tliough not so heavily as in media containing fermentable carbohydrates 
(mono-saccharides). Still other species, B. tetani and B. putrificus, though unable to 
ferment any carbohydrate, still grow anaerobically ; both are mildly proteolytic. From 
these considerations we may conceive that obligately anaerobic bacteria derive the 
oxygen assumed to be necessary in their metabolism not only through the hydrolysis 
of carbohydrates but also of nitrogenous compounds. 

Unless there is some essential difference in the mechanism of anaerobic growth of 
facultative and obligate anaerobes, something may be learned about the latter by 
studying the former. Pasteur^ showed that the products of fermentation of molds 
and yeasts vary according to whether they are provided with oxygen, thus Mucor 
racemosiis in the open air transforms glucose into CO2 and H2O, but in the absence of 
air produces alcohol and CO2; in other words, oxidative processes fall short under an- 
aerobic conditions. 

Until a few years ago, Hoppe-Seyler's theory of biological oxidation^ was most widely 
accepted. According to him, fermentation results in the liberation of nascent hydrogen 
which combines with atmospheric oxygen, forming water (H2+02 = H20-|-0), setting free 
nascent oxygen which is directly responsible for oxidation in the protoplasm. But this theory 
could not account for anaerobic respiration where oxidations occur in the practically com- 
plete absence of free oxygen. Armstrong^ has shown that in ordinary oxidations free oxygen 
does not unite directly with carbon to form carbon dioxide or with hydrogen to form water, 
but any substance that is to be oxidized is first hydroxylated, atmospheric oxygen acting 

'Pasteur, L.: Eludes sur la Bicre. 1876. 

^ Hoppe-Seyler, F.: Ztschr.f. Physiol. Cliemie, i, 121. 1877. 

3 Armstrong, H. E.: Chem. A^eit's, 90, 25. 1904; Tr. Chem. Soc. London, 83, 1088. 1903. 



202 ANAEROBIOSIS 

as a depolarizer to unite with the nascent hydrogen formed from the water, and upon this 
basis Mathews has formulated the following hypothesis: "Certain active particles in the 
protoplasm attack the water which is decomposed into oxygen and hydrogen. The oxygen 
combines with substances of the protoplasm thus oxidizing them; the hydrogen is either set 
free in gaseous form or it is united with atmospheric oxygen to form water, or it combines 
with other substances in the protoplasm."' In the words of Packard:^ "Respiration is the 
dissociation of water with the liberation of hydrogen and the real respiration is brought 
about not by the oxygen of the air but by that of the water. If atmospheric oxygen is present 
it unites with the hydrogen set free from the water, thus acting as a depolarizer. According 
to this theory, aerobic and anaerobic respiration are identical; the only difference is that the 
anaerobic protoplasm is a powerful enough reducing agent to drive the hydrogen out of the 
water and let it escape as free hydrogen." Mathews' suggested that other oxidizing sub- 
stances may replace atmospheric oxygen as a depolarizer and stimulate oxidation in the 
absence of air. This may, indeed, be the role of carbohydrates and of other organic substances 
in anaerobiosis. Theobald Smiths showed that some facultative anaerobes behave as obligate 
aerobes in sugar-free broth, growing only in the open arm of the fermentation tube, while 
they grow in both the closed and open arms in sugar broth, and Maze^ found that while it is 
the oxidation by free oxygen that furnishes the energy necessary to the aerobic multiplica- 
tion of yeasts it is the splitting of sugars into acetic acid that furnishes the energy for their 
anaerobic growth. 

It is by no means certain that obligate anaerobes require an absolute exclusion of 
oxygen for their maximum growth; but certainly the amount necessary to inhibit 
growth is very small, according to Matzuschita,^ for certain anaerobes about 0.0031 
per cent.^ 

Many anaerobes seem to grow best at or near the limit of oxygen tolerance. Engle- 
mann,7 by observation of the chemotactic behavior of motile bacteria with respect to 
the oxygen secreted by a living filament, and Beijerinck,* by observing the levels at 
which bacteria grow in deep media, showed that different anaerobes vary in their 
oxygen requirements. Chudiakow' and Fermi and Bassu'" also concluded that even 
the anaerobic bacteria require some free oxygen, but if this be so the amount is so 
small for the obligate anaerobes that it is practically impossible to measure it. And 
no one has ever been able to establish the minimal oxygen requirement, if there is any 
such thing, for any strict anaerobe. 

Yet the conception of maximal, optimal, and minimal oxygen tensions has a dis- 
tinct place in connection with the "micro-aerophiles" of Beijerinck,* and in the sporu- 
lation of aerobes as shown by A. Meyer." 

■ Mathews, A. P.: Biol. Bull., 8, 331. 1905. ' Packard, W. H.: loc. cit. 

3 Smith, T.: Centralbl.f. Bakteriol., Abt. I, Orig., 18, i. 1895. 

■•Maze, P.: Ann. del'InsL Pasteur, 18, 277. 1904. 

5 Matzuschita, T.: Arch.f. Hyg., 43, 267. 1902. 

' Cf. Clark, W. M.: chap, xii of this volume. 

'Englemann, W.: Botan. Ztng., 39, 442. 1881. 

*Beijerinck, W. M.: Centralbl.f. Bakteriol., Abt. I, Orig., 14, 827. 1893 

»Chudiakow, N.: ibid., Abt. II, Orig., 4, 389. 1898. 
"> Fermi, C, and Bassu, E.: ibid., Abt. I, Orig., 35, 563 and 714. 1904. 
"Meyer, A.: ibid., 49, 305. 1909. 



IVAN C. HALL 203 

Among the micro-aerophilic aerobes are Bad. abortus (Bang),' the fowl diphtheria 
organism of Miiller,^ and certain pyogenic streptococci of Graf and Wittneben.^ Rose- 
now* beUeves that his success in cultivating streptococci with selective pathogenicity 
depends upon varying oxygen pressures. Wherry and his associates^ have concluded 
from experiments with the gonococcus, Entameha buccalis, Leptoihrix innominata, 
C. diphtheriae, and M. tuberculosis, that reduced oxygen tension is more important in 
disease processes than is ordinarily supposed. A slight reduction facilitates the growth 
of the meningococcus according to Cohen and Markle,^ and Cohen and Fleming^ have 
stated that the optimum is represented by an atmosphere of 10 per cent CO2 and 90 
per cent air. But Kohman* has shown that the buffer action of CO2 is more important 
than the slight reduction of oxygen tension, and this viewpoint has been extended to 
other species by Rockwell and his collaborators,' who have shown that carbon dioxide 
is necessary for the growth, not only of many aerobic but also several obligately an- 
aerobic bacteria. 

While some organisms are favored by decreased oxygen, Moore and Williams"* 
found that many aerobic bacteria may be cultivated in nearly pure oxygen. But al- 
though M. tuberculosis and B. pestis were apparently oxyphobic, being completely 
inhibited by 80-90 per cent of this gas, yet Novy and Soule" have demonstrated that 
M. tuberculosis grows readily in concentrated oxygen if sufficient moisture be pro- 
vided. 

The physico-chemical conception holds that obligate anaerobes differ from the 
aerobes by the fact that small amounts of oxygen are inhibitive to their growth and 
toxic to their protoplasm. It is the toxicity of the oxygen which in practical usage 
separates them from the aerobes by their failure to grow on the slanted surface of 
solid media exposed to the air through the cotton plug of a culture tube, 

BIOLOGICAL THEORIES OF ANAEROBIOSIS 

Aerobe-anaerobe symbiosis. — The remarkable fact that obligately anaerobic bacte- 
ria occur abundantly in soil, water, and decaying substances, apparently in contact 
with the oxygen of the air, was perhaps first correctly explained by Pasteur" on the 

' MacNeal, W. J., and Kerr, J. C: /. Infect. Dis., 7, 469. 1910. 

* Miiller, R.: Centralbl.f. Bakteriol., Abt. I, Orig., 41, 515 and 621. 1906. 
3 Graf, H., and Wittneben, W.: ibid., 44, 97. 1907. 

4Rosenow, E. C: J. A.M. A., 65, 1687. 1915; /. Infect. Dis., 32, 41, 72, 144, 384. 1923. 
s Wherry, W. B., and Oliver, W. W.: J. Infect. Dis., 19, 288, 299. 1916; 20, 28. 1917; Wherry, 
W. B., and Ervin, D. M.: ibid., 22, 194. 1918; Wherry, W. B., and Ray, V.: ibid., p. 554. 1918. 

* Cohen, M. B., and Markle, L.: J. A.M. A., 67, 1302. 1916. 
'Cohen, M. B., and Fleming, J. S.: /. Infect. Dis., 23, 337. 1918. 
'Kohman, E. F.: J. Bad., 4, 571. 1919. 

'Rockwell, G. E., and collaborators: J. Infect. Dis., 28, 249 and 352. 1921; 32, 98. 1923; 35 
581. 1924; 38, 92. 1926. 

'"Moore, B., and Williams, R. S.: Biochem. J., 4, 177. 1909; s, 181. 1910; Brit. M. J., 2, 873. 
1909. 

" Novy, F. G., and Soule, M. H.: /. Infect. Dis., 36, 168. 1925. 

" Pasteur, L.: Compt. rend. Acad, de sc, 56, 416-1192. 1863. 



204 ANAEROBIOSIS 

ground that aerobic micro-organisms of various kinds replace the oxygen with 
carbon dioxide sufficiently to permit the symbiotic growth of the obligate anaerobes. 
Pasteur's explanation was accepted by Roux' who utilized the hay bacillus in the cul- 
tivation of Vibrion septique by overlaying the latter in deep agar or gelatin with broth 
cultures of the former, and by Penzo^ who cultivated the "bacillus of malignant 
oedema," symbiotically with Bad. prodigiosum and Bad. proteus in broth and em- 
phasized the great pathogenicity of such cultures. Novy^ did the same thing with his 
B. oedematis maligni II {B. novyi) and added Bad. acidi lactici and a coccus to the list 
of successful symbionts. 

But Kedrowsky^ found that Clostridium hutyricum and B. tetani would grow in 
chloroformed cultures of various aerobes and suggested the production of a reducing 
agent or ferment by these as a more important means of oxygen reduction than that 
proposed by Pasteur. Kedrowsky was unable to secure this theoretical substance free 
from the bacterial bodies by Berkefeld filtration. 

Other investigators do not accept Kedrowsky's hypothesis. Thus Scholtz^ culti- 
vated B. tetani, B. botulinus, "oedem bacillus," and "rauschbrand bacillus" symbioti- 
cally with various living aerobes in broth and in the condensation water of agar slants, 
but failed to secure any growth with chloroformed cultures of aerobes except when 
large volumes were used, in which, as we now know, growth can be easily secured 
without other means of anaerobiosis aside from boiling just previous to inoculation. 

Bienstock^ and Matzuschita' also failed to confirm the idea of a vital ferment, 
and von Oettingen^ found himself at variance both with Pasteur and Kedrowsky be- 
cause he was unable to secure anaerobic growth when aerobes were closed up with 
anaerobes but not in actual contact, i.e., in "separated symbiosis," as he called it. But 
von Oettingen's technique was probably faulty; perhaps his fantastic and complicat- 
ed containers leaked, because separate growth of aerobes and anaerobes can be se- 
cured in devices operating upon this principle, as, for example, those of Salomonson,' 
Zinsser,'" Nichols and Schmitter," Nowak,/^ MacNeal and Kerr,'^ Smith and Fabyan'^ 
Horton,'5 Giltner,'* Wherry and Oliver,'^ and Torrey,'Hhough it must be admitted that 

• Roux, F.: Ann. de Vlnsl. Pasleiir, i, 49. 1887. 

^Penzo, R.: Cenlralbl.f. Bakteriol, Abt. I, Orig., 10, 822. 1891. 

3 Novy, F. G.: ibid., 16, 566. 1894. 

■t Kedrowsky, W.: Ztschr.f. Ilyg., 20, 358. 1895. 

sSchoUz, W.: ibid., 27, 132. 1898. 

''Bienstock: /Ire/;./, ^yg., 36, 335. 1899. 

'Matzuschita, T.: loc. cil. 

^ von Oettingen, W.: Ztschr.f. Hyg. u. Infektionskrankh., 43, 463. 1903. 

'Salomonsen, C. J.: Bad. Technology. 1890. 
'"Zinsser, H.: /. Exper. Med., 8, 542. 1906. 

" Nichols, J. H., and Schmitter, F.: J. Med. Research, 15, 113. 1906, 
" Nowak, J.: Attn. del'Inst. Pasteur, 22, 541. 1908. 
'3 MacNeal, W. J., and Kerr, J. E.: J. Infect. Dis., 7, 469. 1910. 
"•Smith, T., and Fabyan, M.: Centralbl.f. Bakteriol., Abt. I, Orig., 61, 549. 1912. 
■sHorton, G. D.: /. Infect. Dis., 15, 22. 1914. "Wherry, W. B., and Oliver, VV. W.: loc.cit. 
''• Gillner, VV.: Science, 41, 663. 1915. '* Torrey, J. C: /. Bad., 2, 435. 191 7. 



IVAN C. HALL 205 

these have been more useful in the cultivation of micro-aerophilcs than of obligate 
anaerobes. Yet the fact that obligate anaerobes can be cultivated on the surface of 
solid media without actual contact of aerobes is a sufBcient rebuttal of Kedrowsky's 
assumption of a vital ferment and convinces one of the accuracy of Pasteur's original 
hypothesis of oxygen-tension reduction through bacterial respiration as at least one 
of the important factors in aerobe-anaerobe symbiosis. 

Bienstock' later experimented with several anaerobes in symbiosis with a large 
variety of aerobes. Heat-killed cultures of aerobes were also tried, but of these only 
Bad. pyocyancnm in a fibrin medium would support the growth of anaerobes. Con- 
trol cultures in similar media in which Bad. pyocyanemn had not been cultivated 
failed. This exception was disturbing to Bienstock as an apparent confirmation of 
Kedrowsky's view but may be interpreted in the light of present-day information as 
providing a viscous deep medium sufficiently impervious to air for anaerobic growth. 
Proca' has since cultivated B. tetani and B. botulimis in freshly sterilized liquid cul- 
tures of Bad. coll, Bad. typ/iosnm, and Vibrio cholcrae. Solid media failed, however, 
to yield surface colonies, and certain species of aerobes, Staphylococcus dorc and Bad. 
pyocyaneum, seemed unsatisfactory. Such discrepancies are difficult to interpret; one 
cannot help regarding the results as somewhat accidental. 

Many practical considerations in addition to those already mentioned grow out 
of the phenomena of aerobe-anaerobe symbiosis, as, for example, the necessity of 
autoclave sterilization of broth intended for the manufacture of diphtheria toxin,^ the 
inhibition by certain aerobic bacteria of tetanus^ and botulism^ toxin formation, the 
somewhat questionable use of inert aerobes in fermentation tests of anaerobes,^ and 
the use of symbiotic cultures in the isolation of B, putrificus.'' At the present time 
most of the technical applications of aerobic symbionts in the cultivation of 
anaerobes have been supplanted by more precise methods; their significance is mainly 
academic therefore, but important as confirming Pasteur's conception of the life of 
anaerobic micro-organisms in nature. 

Use of animal and plant tissues in the so-called "aerobic culture" of obligate 
anaerobes. — In 1877 Gunning,^ of the University of Amsterdam, studied the growth of 
putrefactive anaerobes in media containing meat and coagulated egg, and Nencki^ 
and his pupil Jeanneret were led into the same problem in studying the activity of 
the pancreas and its ferments in the absence of air. Others, such as Gaffky and Hesse 
(quoted by Novy),'° were interested in the isolation of organisms from infected tissues 
and perhaps did not appreciate that the tissue exerted a favorable influence in an- 

' Bienstock: Ann. de Vlnst. Paslcur, 17, 850. 1903. 

^ Proca, G.: Coinpt. rend. Soc. de bioL, 63, 620. 1907. 

3 Smith, T.: J. Exper. Med., 3, 647. 1898. 

t Francis, E.: Hyg. Lab. Bull., No. 95. 1914. 

5 Hall, I. C, and Peterson, E. C: J. Bad., 8, 319. 1923. 

nVilson, W. J., and Steer, P.: Brit. M. /., 2, 568. 1918. 

'Sturges, W. S., and Rettger, L. F.: /. BacL, 4, 171. 1919. 

* Gunning, J. W.: J.f. prakt. Chemie, 16, 314. 1877; 17, 266. 1878; 20, 434. 1879. 

»Nencki, M.: ibid._ 19, 337. 1879. 

"Novy, F. G., Jr.: /. Infect. Dis., 36, 343. 1925. 



2o6 ANAEROBIOSIS 

aerobic cultivation. In 1890 Tizzoni, Cattani, and Baquis' utilized clotted rabbit blood 
as a means of cultivating B. ieiani, a change in color indicating reduction. Smith^ 
also in 1890 began the use of fermentation tubes for the cultivation of anaerobes from 
infected tissues, and in 1899^ first utilized sterile tissues other than blood to accelerate 
the growth of B. tetani. Smith clearly recognized that the value of tissues lay in their 
reducing action. Von Hibler^ also began the use of sterile raw and coagulated rabbit 
blood and human brain media about the same time, and Kitt^ observed the initial 
growth of the blackleg bacillus in the immediate vicinity of bits of tissue in liquid 
media exposed to the air. But it was a mistake on the part of these writers to considei 
such growth aerobic. 

The use of animal tissues was also developed by Italian investigators, apparently 
without any knowledge of the work already done in other countries, Tarozzi'' pointed 
out that the tissues should be preferably fresh and that heating destroyed their value 
possibly through oxidation of a reducing agent. Much of the Italian literature is in- 
accessible, but Jungano and Distaso^ and, more recently, Novy^ have reviewed it. In 
1905 Ori showed that a vegetable tissue, potato, could be used as well as animal tis- 
sues, and that sterilization in the autoclave did not destroy its value, 

Grixoni cultivated obligate anaerobes in a glycerol extract of liver heated to 55°C, 
without other means of excluding air, and Wrzosek' used kidney, liver, and spleen 
in the culture of certain bacteria from normal organs which would not grow in the 
absence of the tissues. Later Wrzosek'" studied the growth of B. tetani, B. oedematis, 
B. chauvoei, and B. hotulinus in tissue broth and emphasized the importance of his 
supposedly new viewpoint relative to the "aerobic growth of anaerobes." 

Harass" found that sterilization of animal tissues did not destroy their ability to 
support "aerobic" growth of anaerobes in broth. He recognized the desirability of 
demonstrating aerobic growth on the surface of solid media, but all of his efforts in 
this direction were fruitless. Bandini'^ showed conclusively that obligate anaerobes 
such as B. botidinus, B. oedematis, and B. chauvoei would grow well in broth contain- 
ing not only various animal and plant tissues sterilized in the autoclave at i2o°C,, 
but also such supposedly inert substances as coal, charcoal, coke, and iron filings. 

' Tizzoni, G., Cattani, J., and Baquis, E.: Beitr. z. path. Anal. u. s. allg. Path., 7, 597. 1890. 

= Smith, T.: Centralbl.f. Bakleriol., Abt. I, Orig., 7, 502. 1890. 

3 Smith, T.: J. Boston Soc. Med. Sc, 3, 340. 1899. 

■» von Hibler, E.: Centralbl.f. Bakleriol., Abt. I, Orig., 25, 593. 1899. 

sKitt, T.: ibid., 17, 168. 1895. 

^Tarozzi, G.: ibid., 38, 619. 1905. 

7 Jungano and Distaso: loc. cit. 

^ Novy, F. G., Jr.: loc. cit. 

9 Wrzosek, A.: Wien. klin. Wchnschr., 18, 1268. 1905; Centralbl.f. Bakleriol., Abt. I, Orig., 43, 
17. 1907. 

"> Wrzosek, A.: Milnchen. med. Wchnschr., 53, 2534. 1906; Centralbl.f. Bakleriol., Abt. I, Orig., 
44,607. 1907; 53, 476. 1909- 

" Harass, P.: Miinchen. wed. Wchnschr., 53,' 2237. 1906. 

" Bandini, P.: Giorn. R. Acad. de. Med. di Torino, 12, 265. 1906. 



IVAN C. HALL 207 

These he believed to be reducing agents as shown by their decoloration of methylene 
blue in broth, but he also emphasized the necessity of heating just prior to inoculation, 

Liefmann,' Guillemot and Szczawinska/ and Rosenthal all recognized clearly that 
the growth of obligate anaerobes in the presence of such substances is truly anaerobic, 
not aerobic, and agreed with Wrzosek in attributing such growth to the reducing ac- 
tion supposedly possessed by them. 

Hata,'' on the other hand, emphasized the importance of the particulate nature 
of reducing agents and suggested that the best results are to be expected when these 
agents are in a finely divided condition, as finely minced animal and plant tissues, 
or iron filings. Douglass, Fleming, and Colebrook^ objected that organic substances 
can scarcely be expected to retain their chemical reducing action after heating. This 
seems valid. But while a surprisingly large list of organic and inorganic solids may 
be used in broth cultures to provide anaerobic conditions, including ashes, sand, card- 
board, wool, cotton, lint, sponge, charcoal, chalk, cork, cloth, and iron nails, all have 
a more or less porous or particulate consistency, and the degree of success achieved 
with them depends upon this and upon the amount of substance used. Finally, the 
use of small pieces of capillary tubing in broth affords a striking proof of the fact that 
anaerobic growth begins in the interstices of such substances as afford occluded spaces 
into which oxygen diffuses with difficulty after once being driven out by heat. At 
least motile anaerobes find their way into such interstices by reason of a negative 
chemotactic response to oxygen, and once growth starts, the generation of gas im- 
proves the conditions of anaerobiosis through its action in sweeping out oxygen. This 
has been clearly recognized by Novy,^ Wright,^ Wolf, McGill, and Harris^ and others. 

We thus come to the conclusion that physico-chemical factors play an important 
role in the so-called "aerobic cultures" of anaerobes in the presence of plant and ani- 
mal tissues. The action of such tissues is physical to that degree in which they in- 
terfere with the reabsorption of oxygen ; it is chemical to that degree in which actual 
reduction of free oxygen by unheated tissues occurs. It is probable also that certain 
tissues contribute valuable nutrients to the media and that they serve as important 
buffers against unfavorable H-ion concentrations. The Italian work started with the 
biological hypothesis of a reducing ferment, but it ended without proof. And while 
many were able to demonstrate the growth of obligate anaerobes in broth containing 
animal and plant tissues and other particulate substances, no one was ever able to 
secure surface growth of obligate anaerobes in pure culture in contact with air. 

Yet the idea that unheated plant and animal tissues may accelerate anaerobic 

' Liefmann, H.: Munchen. med. Wchnsckr., 54, 823. 1907; Centralbl. f. Bakleriol.. Abt. I.Orig., 
46, 377. 1908. 

^ Guillemot, L., and Szczawinska, W.: Compt. rend. Soc. de biol., 64, 171. 1908. 

J Rosenthal, G.: ibid., 67, 702. 1909. 

iHata, S.: Centralbl. f. Bakteriol., Abt. I, Orig., 46, 539. 1908. 

s Douglass, S. R., Fleming, A., and Colebrook, L.: Lancet, 2, 530. 1917. 

*Novy, F. G.: Centralbl. f .Bakteriol., A.h\..l,Orig., 14, $go. iSgs; Ztschr.f. Ilyg., 17, 2og. 1894. 

7 Wright, A. E.: Lancet, i, i. 1917. 

» Wolf, C. G. L., McGill, C, M., and Harris, J. E. G.: ibid., 2, 787. 1917. 



2o8 ANAEROBIOSIS 

growth through enzymic action cannot be denied; indeed, a most interesting theory 
of anaerobiosis depends upon this conception. 

In 1922 M'Leod and Gordon' discovered that the early death of the pneumococcus 
in cultures was apparently due to the accumulation of peroxide (of hydrogen?), for 
the production of which evidence was found in the green discoloration of "chocolate" 
blood agar, and in the liberation of oxygen from pneumococcus cultures by liver cata- 
lase. Streptococci and other bacteria also produce peroxide, and it was suggested that 
the well-known beneficial action of fresh tissues upon various bacteria is due to the 
destruction of detrimental peroxide by catalase.^ 

NeilP holds that the oxidation and reduction reactions of sterile plant tissues are 
essentially identical with those of pneumococci and anaerobic bacteria and furnish 
the best explanation, thus far, for the beneficial action of raw plant and animal tissues 
upon anaerobic growth. On the other hand, Novy^ holds that "the inability of an- 
aerobes to grow in the air is not due to the hypothetical production of peroxide and to 
the absence of catalase" but "that the fundamental difference between obligative 
aerobes and anaerobes lies in the nature of the respiratory enzymes, which are des- 
ignated as 'aerase' and 'anaerase,' respectively. The potato and the facultative an- 
aerobes possess both types; that present in obligative anaerobes can function only in 
the absence of oxygen, while that of the aerobe can work only in the presence of 
oxygen." All living protoplasm respires, and Novy's conception of the beneficial ac- 
tion of plant tissues upon anaerobic growth dispenses with the peroxide-catalase 
hypothesis and emphasizes the idea that obligate anaerobes grow in the presence of 
raw plant tissues because the latter reduce the oxygen tension, just as aerobic bacteria 
do, below the toxic concentration. 

AEROBIZATION OF THE ANAEROBES 

Many of the investigations just discussed had for their aim the aerobic cultivation 
of the anaerobes with the end in view of a better understanding of the nature of an- 
aerobic growth. A secondary consideration has been in some cases that if means were 
found to cultivate the anaerobes aerobically upon solid media, the technique of isola- 
tion might be simplified. But no one has yet succeeded in securing surface growth of 
any obligate anaerobe in pure culture upon solid media exposed to the air, and the 
best practical definition of obligate anaerobes separates them from aerobes by their 
failure to grow in pure culture on the surface of solid media at normal atmospheric 
pressure. From this standpoint the rather numerous claims of aerobic growth in liq- 
uid media through the addition of chemical reducing agents, symbiotic aerophilic 
micro-organisms, fresh animal or plant tissues, or particulate substances have no 
standing; all of these are really anaerobic cultivations. Even the symbiotic growth of 
aerobes and anaerobes upon the surface of solid media fails to comply with the fore- 
going definition of "anaerobiosis." The same is true of the supposed aerobic culture 

' M'Leod, J. W., and Gordon, J.: Biochem. J ., 16, 499. 1922; J . Palh. 6" Bad , 25, 139. 1922; 
26, 127. 1923. 

^ See chap, xiv of this vohime. 

3 Neill, J. M.: /. Exper. Med., 41, 535. 1925. 

-•Novy, F. G., Jr.: loc. cil. 



IVAN C. HALL 209 

of anaerobes by Larson, Cantwell, and Hartzell' in broth whose surface tension has 
been reduced by castor-oil soap. 

The really important point concerned in the attempts to "aerobize the anaerobes" 
involves the fundamental validity of differentiating micro-organisms according to 
their oxygen relation and the stability of this relation. 

There have been a few efforts to "aerobize the anaerobes" by acclimation to in- 
creasing oxygen tensions. Ferran' claimed that by starting with pure acetylene and, 
as growth began, mixing more and more air, he could accustom the tetanus bacillus 
to growth in air on the surface of broth without morphological change. But the 
formation of a thick pellicle and loss of virulence reported is suggestive of aerobic 
contamination, for the detection of which no special procedures appear to have 
merited description in his brief publication. But even if surface growth of a pure 
culture of tetanus bacillus did occur on the surface of liquid media in a flask, and the 
writer has seen this without loss of toxicity, it could not be considered correctly as 
aerobic in view of the swe^ing out of oxygen from liquid cultures by other gases 
evolved; Ferran's claim fails to meet the requirements of our definition of aerobic 
growth. 

The progressive transformation of strict anaerobes into aerobic micro-organisms 
has received extended attention by Rosenthal,' who held that the distinction between 
aerobes and anaerobes is not fundamental. By successive cultivation in tubes con- 
taining milk of less and less depth or covered with thin layers of lanolin, Rosenthal 
claimed to have cultivated the anaerobic bacilli of botulism, Achalme {B. welchii), 
and Legros, under conditions of partial exposure to air, which had hitherto failed to 
permit growth. No surface colonies on solid media were secured at this time, but 
later surface colonies were obtained with difficulty with the bacillus of Achalme by 
more persistently decreasing the depth of milk. Partial evacuation was also used but 
with somewhat incomplete success. Similar experiments were conducted with Vi- 
brion septiqiie, and Rosenthal soon came to believe that there were three steps in the 
transformation of anaerobes into aerobes as follows: 

First, the organisms lost their oxygen intolerance without alteration of biochemi- 
cal functions (e.g., fermentation, putrefaction) or of biological properties (e.g., vir- 
ulence). Such cultures were said to grow for one generation only upon the surface of 
solid media in pure culture and when transferred to deep media displayed immediately 
their usual characteristics. Repeated transfers in this stage upon solid media were 
impossible. 

Second, after the second or third agar slant the organisms lost their identifying 
biochemical characteristics, but these might be restored in a few generations by re- 
peated transplantation in suitable media under anaerobic conditions. 

Third, cultures lost their identifying chemical properties as well as their patho- 
genicity permanently, though they were morphologically unchanged. 

Reaching the foregoing conclusions in experiments with the bacillus of Achalme, 

• Larson, W. P., Cantwell, W. F., and Hartzell, T. B.: J. Infect. Dis., 25, 41. 1919. 

^ Ferran, J.: Cenlralbl.f. Bakteriol., Abt. I, Orig., 24, 28. 1898. 

3 Rosenthal, G.: Compt. rend. Soc. de biol., 55, 1292. 1903; 60, 828, 874, 928, 957, 1116. 1906; 
61, 48, 211, 326, 440. 1906; 62, 438, 578, 784, 1020, 1066, 1119. 1907; 64, 398. 1908. 



2IO ANAEROBIOSIS 

Rosenthal shortly claimed to have repeated them with the Vibrion septique and sug- 
gested the use of such attenuated cultures of aerobized anaerobes in immunization 
under the name "allobi- vaccination." 

Rosenthal also undertook the progressive anaerobization of B. anthracis, and 
claimed to have accomplished this by the use of deep tubes of media, milk covered 
with cream, media sealed under lanolin, and the use of decreasing air pressures. The 
number of required transfers varied with the germ, with the rapidity of multiplication, 
and with the size of inoculum. But since B. anthracis is well known to be a facultative 
anaerobe, these experiments lacked conclusiveness, and since Rosenthal's main in- 
terest lay in the original problem of aerobizing the anaerobes, he next studied the use of 
decreasing depths of gelatin as formerly of milk, and of tubes of decreasing diameter. 

In 1907 he reported results with the tetanus bacillus finally analogous to those al- 
ready obtained with other anaerobes, but more irregular. In contrast to Ferran, he 
noted an early appearance of involution forms and loss of sporulation and motility. 
All three steps of aerobization, he says, were accomplished. The bacilli were found 
to be shorter, thicker, and less motile than the anaerobic forms. They were gram 
positive and non-sporulating. Bouillon cultures during the first step of aerobization 
produced tetanus in animals. The first stage of aerobization continued through five 
to six generations on slanted agar, and the loss of morphological characters preceded 
that of biochemical activities and toxicity. The beginning of the second stage was 
announced in the tardy liquefaction of gelatin and the third by the complete and ap- 
parently permanent loss of tryptic activity, sporulation, motility, and toxicity. The 
last vestige of the parentage of the aerobized tetanus bacillus, as well as of the Vibrion 
septique, to disappear was the property of specific serum agglutinability. This Rosen- 
thal considered irrefutable proof of the exactness of his researches upon the aerobiza- 
tion of the anaerobes. 

Having created several strains of artificial aerobic micro-organisms from various 
obligate anaerobes, which he called collectively "bacillogenes," specifically "aerovib- 
rion," "aero-bacille de tetanos," etc., Rosenthal next claimed (1907) that a reversal of 
the process restored the "aero-bacilli" of Achalme and of tetanus wholly to their origi- 
nal state of obligate anaerobes, including their lost chemical functions and power of 
sporulation. 

As a fourth step in the aerobization of the anaerobes Rosenthal (1908) claimed 
the transformation of the aerobized Vibrion septique at the limit of its viability into 
an organism resembling the entero-coccus, which he found could be reversed again 
into a typical Vibrion septique. 

These remarkable researches have been discussed at some length because they 
are not widely known and because to my knowledge they have not been confirmed, 
though confirmation seems most desirable if possible. The length of time, over five 
years, consumed by Rosenthal in the pursuit of these experiments, together with the 
logical progress of the results, suggests that a critical study of the problem would be 
worth while. At least one observer, Szczawinska,' failed to corroborate Rosenthal's 
findings and concluded that our present distinction between aerobes and anaerobes 
is a valid one, and is based upon an essential physiological difference in metabolism. 

' Szczawinska, W.: ibid., 69, 15. 19 10. 



CHAPTER XIV 
BACTERIAL OXIDATIONS AND REDUCTIONS 

JAMES WALTER McLEOD 
University of Leeds, Leeds, England 

REDUCTIONS 

Clark^ has pointed out recently that the modern conceptions of reduction involve 
more than the subtraction of oxygen or addition of hydrogen. Much the greater part of 
the literature, however, dealing with bacterial reductions relates to observations of such 
changes. A great deal of this work had already been done by the end of the last cen- 
tury. The literature is considerable, and since a comprehensive summary of it would 
require much space, only the more interesting points and those to which most work has 
been devoted will be considered. Such are the reductions of compounds of nitrogen 
and sulphur, of metallic salts such as those of selenium and tellurium, of a variety of 
dyes, and of certain substances of biological importance, hemoglobin derivatives, 
cystine or complexes containing it, and bacterial toxins. These various reactions have 
been studied for a variety of ends, some on account of their economical importance, as 
the reduction of nitrates and nitrites; others because of their possible value in bacteri- 
ological technique, as the reduction of tellurium salts: others again because of their pos- 
sible significance in explaining the pathogenic action of bacteria, as the reduction of 
oxyhemoglobin; and almost all have been studied with a view to elucidating the in- 
timate metabolism of the bacterial cell and particularly the mechanism of its respira- 
tion. 

The reduction of nitrates and nitrites was fully studied by Maassen^ twenty-five 
years ago. His chief conclusions were that the majority of the bacteria which are 
capable of growing in simple media reduce nitrates to nitrites, that many but not all 
of these destroy nitrites whereas some of the bacteria incapable of reducing nitrates 
can yet destroy nitrites, that most bacteria destroy the nitrite by transforming it to 
NH, which they utilize almost as rapidly as it is formed, but that one group — the true 
denitrifying bacteria, B. fluorescens liquefaciens , and species allied to it — utilize the 
oxygen of the nitrite and give off gaseous nitrogen. Braun and Cahn-Bronner-' refer 
to this last quality in B. pyocyaneus as enabling it to show pseudo-anaerobic growth 
in the presence of nitrate. Certain of the vibrios are among the group of bacteria 
forming nitrite without reducing it further, and it is such of these as also produce indol 
that give the cholera-red reaction. 

Not much of importance has been added to our knowledge of this subject since 
Maassen's work, but some facts have been added with regard to some bacteria which 

'Clark, W. M.: Pub. Health Rep. (U.S. Public Health Service), 38, 443. 1923; cf. chap, xii, 
this volume. 

^ Maassen, A.: Arbeilen a.d. kaiserliclien Gcsundheilsamte, 18, 21. 1902. 

^ Braun, H., and Cahn-Bronner, C. E.: Centralbl.f. Baklen'ol., Abt. I, Orig., 86, 380. 1902. 



212 BACTERIAL OXIDATIONS AND REDUCTIONS 

he had not investigated. Logic' has drawn attention to the fact that the non-mannite- 
fermenting dysentery strains arc distinguished from the mannite-fermenting strains 
by inability to reduce nitrite. Pelz' pointed out that streptococci never reduce ni- 
trates. More attention has been given in recent work to the reduction of compounds 
of S generally in the form of studies of H2S production by bacteria. 

There is general agreement that most bacteria do not reduce sulphates or form 
H2S from taurine (Sasaki and Otsuka/ Myers/ Burger^). Salkowski*" pointed out, 
however, that whatever may occur in cultural experiment, it is certain that sulphates 
are reduced in sewage with H^S formation, possibly under the influence of mixed cul- 
tures, and Beijerinck^ described two anaerobes, capable of reducing sulphates, which 
are found in the mud of Dutch harbor bottoms. 

It is not clear that evolution of H.S from media containing proteins is necessarily 
evidence of the reduction of S-containing compounds. It is certain, however, that the 
most vigorous production of H2S is carried on by bacteria which can be shown in other 
ways to have strong reducing properties as, e.g., B. sporogenes. It has also been shown 
that such bacteria as tend to produce II2S do so more freely if cystine is added to the 
medium (Kondo,^ Sasaki and Otsuka,^ Myers, ^ Burger^), and it is altogether likely 
that reduction of cystine to cystein is a preliminary to evolution of H2S. Burger, how- 
ever, pointed out that nascent H does not liberate H2S from cystine unless in acid 
solution, whereas bacteria normally grow in slightly alkaline solutions. There is, there- 
fore, need for further investigation on this point. 

The statements of the different workers with regard to the capacity of different 
bacteria to produce H2S from cystine and from protein are conflicting, but there is a 
general agreement that most coliform bacteria, including the Proteus and B. fluo- 
rescens liquefaciens groups, have this property. 

It is in connection with the reduction of dyes and biological products such as 
hemoglobin derivatives that most of the work on bacterial reductions has been done. 
Theobald Smith,' who was one of the earlier workers in this field, used methylene blue, 
the dye which has been most used for this purpose, and concluded that all bacteria were 
alike in their powers of redu-^ing it and that reduction took place only when the bac- 
teria were in actual contact with the dye. Both statements were disputed shortly after- 
ward by Muller,'" and although they are generally true in the sense that all bacteria 
are possessed of reducing powers and that this power is especially exercised within the 
body or on the surface of the bacteria, the trend of subsequent work has been to justify 
Miiller's position. Von Liebermann," Wichern,'- and Carapelle''^ all bring forward 

' Logie, W. J.: J. Hyg., 10, 143. iqio; ii, ,36. igii. 
= Pelz., E.: Centralbl.f. Baklerlol., Abt. I, Orig., 57, i. 1910. 
3 Sasaki,T., and Otsuka, I.: Biochem. Ztschr., 39, 208. 191 2. 

■I Myers, J. T.: /. Bad., 5, 231. 1920. s Burger, M.: Arch.f. Hyg., 82, 201. 1914. 

^Salkowski, E.: Ztschr. f. phys. Cheniie, 83, 143. 1913^ 
' Beijerinck, W. M.: Verzamdde Gcscliriflen, 4, 24. 
"^ Kondo, M.: Biochem. Ztschr., 136, 198. 1923. 

'Smith, Theobald: Centralbl.f. BakterloL, Abt. I, Orig., 19, iSi. 1896. 
'" Muller, F.: ibid., 26, 801. 1899. 

" Von Liebermann, Jr., L.: ibid., 51, 440. 1909. " VVichern, H.: Arch.f. Ilyg., 72, i. 1910. 
'^Carapelle, E.: Centralbl.f. Bakteriol., Abt. I, Orig., 47, 545. 1908. 



PLATE I 






A Growth of B. welchii on "Chocolate" Agar 15 Minutes after Removal from Anaerobic Jar. 
b', Growth of BI welchii on "Chocolate" Agar as It Appears Immediately after Removal from 

Anaerobic Jar. 

C, Appearance of Culture of B. ivckhii Grown at Reduced Atmospheric Pressure— 200-300 Mm. 



JAMES WALTER McLEOD 213 

evidence to show that products capable of producing reduction diffuse out from bac- 
teria, and the most convincing proof that the bacterial body is not essential has come 
with the recent demonstration by Avery and Neill' that filtered extracts of pneumo- 
cocci which are protected from oxidation have definite reducing properties. 

Although it is true that all bacteria are alike in reducing methylene blue and the 
same can be said of oxyhemoglobin (Von Liebermann^), there exist considerable differ- 
ences in their activities in this respect and greater differences in respect of other re- 
ductions, e.g., in the reduction of anthraquinone. Bieling^ has shown that meningococ- 
cus, pneumococcus, and B. typhosus are relatively inactive. 

The writer and Gordon'' have shown that when bacteria are grouped according 
to their power of reducing glutathione they fall into the following order of activity: 
streptococci and B. dyscnteriae Shiga, very poor reducers; Staphylococcus aureus 
and pneumococcus, slight reducers, most bacteria of the coliform group, fairly ac- 
tive reducers; anaerobic bacteria, all active and mostly very active reducers. Another 
indicator of reduction less easily reduced than methylene blue — and hence more use- 
ful in demonstrating differences in reducing activities — is hematin as it occurs in the 
heated blood agar or "chocolate" medium. Colonies of anaerobic bacteria are sur- 
rounded on this medium by a pink halo, due to reduction, which fades very rapidly on 
exposure to air. This effect is shown in Plate I, A and B. It can be put to practical 
use in picking out anaerobic colonies in mixed cultures on plates incubated under an- 
aerobic conditions. 

A very interesting application of the bacterial reduction of methylene blue has 
been made by Quastel^ and his collaborators by applying Thunberg's method in 
studying the intimate processes of bacterial metabolism. The bacteria are suspended in 
a buffered solution containing methylene blue, and the acceleration of the reduction 
of the dye in the presence of various products which may occur in culture media or 
arise in the course of the bacterial disintegration of sugars and proteins is investigated. 
The marked powers shown by B. coli for activating the reduction of methylene blue 
by certain sugars and fatty acids, notably formic, lactic, and succinic, and its relative- 
ly slight powers of activating amino acids are among the interesting results obtained. 
One other aspect of the work on the reducing action of bacteria has great interest, and 
that is the suggestion made by Von Liebermann- but followed out with much greater 
precision by Dibbelt,^ that the successful competition of bacteria with the body tissues 
for the available oxygen of oxyhemoglobin may, especially in the more rapid septi- 
cemias, be an important element in the domination of the animal body by the invad- 
ing bacteria. 

' Avery, O. T., and Neill, J. M.: /. Exper. Med., 39, 357. 1924. 

^ Von Liebermann, Jr., L.: loc. cit. 

3 Bieling, R.: Cenlralbl.f. Bakteriol., Abt. I, Orig., 90, 48. 1923; Ztschr.f. Hyg. u. Infekiions- 
krankh., 100, 270. 1923. 

■•McLeod, J. W., and Gordon, J.: Biochem. J., 18, 937. 1924. 

5QuastelJ.H.,andWhetham,M.D.: ibid., ig, ^ig. 1924; /&/(/., pp. 520 and 645. 1925; Quastel, 
J.H.: ibid., p. 641. 1925; Quastel, J. H., Stephenson, M., and Whetham, M. D.: /6/(f ., p. 304. 1925; 
Quastel, J. H., and Stephenson, M.: ibid., p. 660. 1925; Quastel, J. H., and Woolridge, W. B.: 
ibid., p. 652. 1925. 

^Dibbelt, W.: Cenlralbl.f. Bakteriol., Abt. I, Orig., 64, 52. 1912. 



214 BACTERIAL OXIDATIONS AND REDUCTIONS 

Neill' has recently made the interesting discovery that various bacterial hemo- 
lysins which deteriorate rapidly on exposure to air undergo a reversible oxidation and 
that their hemolytic activity can be restored equally well by exposing them to a chemi- 
cal reducing agent or to the reducing action of bacterial suspensions from cultures of 
B. coli or of an anaerobe. 

BACTERIAL CATALASE AND PEROXIDE PRODUCTION BY BACTERIA 

Although the power of bacteria to decompose hydrogen peroxide was observed 
and investigated independently by Gottstein^ and Beijerinck-' more than thirty years 
ago, it has been very little studied by most bacfyeriologists. Lowenstein/ however? 
noticed the absence of catalase in cultures of the tetanus organism in 1903, and Ry- 
wosch and Rywosch^ in 1907 noted that it was absent or present in very slight amount 
in cultures of CI. tetani and CI. botulinum. In 1923 the writer and Gordon/ following 
up earlier work with Govenlock^ on the substances inhibitory to bacterial growth dif- 
fusing into culture media in the vicinity of growths of pneumococci, found that the 
inhibition was mainly due to the formation of H2O2 by the pneumococcus. This re- 
sult has since been confirmed by Avery and Morgan" and by others. The fact that 
some bacteria could produce H2O2 gave an enhanced interest to observations on bac- 
terial catalase and led the writer and Gordon' to investigate a wider range of varieties 
of bacteria than had been previously examined for their catalase effect. This investi- 
gation led to the following classification of the bacteria investigated: 
Group I. The anaerobes, devoid of catalase, extremely sensitive to H2O2, and considered as 

potential peroxide-producers 
Group II. Peroxide-producers, devoid of catalase and only moderately sensitive to H2O2; 

the pneumococci, many types of streptococci, the lactic acid bacteria, and some sarcinae 
Group III. Non-peroxide-producers and devoid of catalase; certain streptococci, dysentery 

bacilli (Shiga type), and some hemoglobinophilic bacteria 
Group IV. Bacteria producing catalase. The great majority of the bacteria capable of 

growing aerobically or both aerobically and anaerobically 

The most vigorous catalase effect is shown by the strictly aerobic bacilli, especially 
those such as B. pyocyaneus which form pigments. Two pathogenic species are also 
outstanding in this respect — the gonococcus and the B. pertussis. Callow'" specially 
investigated anaerobes and found them devoid of catalase activity. Sherman" has 

' Neill, J. M.: J. Exper. Med., 44, 199 and 215. 1926. 
' Gottstein, A.: Virchoic's Arch.f. path. Anat., 133, 295. 1893. 
J Beijerinck, W. M.: N aturwissenschaftUche Rundschau, 8, 671. 1923. 

-i Lowenstein, E.: Wien. kiln. Wchnschr., 16, 1393. 1903. (Quoted by Kluy\'er, A. J.: Ztschr.f. 
phys. Chemie, 138, 100. 1924.) 

sRywosch, D. and M.: Centralhl.f. Bakleriol., Abt. I, Orig., 44, 295. 1907. 
^McLeod, J. W., and Gordon, J.: /. Path, and Bad., 25, 139. 1922. 
'McLeod, J. W., and Govenlock, P.: Lancet, i, 900. 1921. 
* Avery, O. T., and Morgan, H. J.: /. Exper. Med., 39, 275 and 289. 1924. 
»McLeod, J. W., and Gordon, J.: /. Path, and Bad., 26, 326. 1923. 
" Callow, A. B.: ibid., p. 320. 1923. 
" Sherman, J. M.: J. Bad., 6, 379. 1921; 11, 417. 1926. 



JAMES WALTER McLEOD 215 

drawn attention to an apparent exception to the rule that anaerobes are devoid of cata- 
lase in the propionic acid bacteria. These, however, appear to be microaerophiUc 
rather tiian strictly anaerobic. The further investigation of their metabolism may 
throw some fresh light on bacterial respiration. 

OCCURRENCE OF OXIDIZING FERMENTS IN BACTERIA 

Observations on this subject have been scanty. Lehmann and Sano^ claim to have 
demonstrated tyrosinase in three species of bacteria although they found it absent 
in the great majority. A brown discoloration in agar media around a colony of mold 
is not infrequently observed and is probably due to this ferment. Beijerinck^ also 
found tyrosinase in certain vibrios. Roux-^ observed that B. typhosus produced a green 
color in extract of artichokes similar to that produced by laccase. The only other ob- 
servations on the production of direct oxidizing ferments by bacteria that I have been 
able to find are those of Schultze^ and Kramer^ who studied the immediate effect of 
smearing bacteria on agar containing a-naphthol and dimethyl-para-phenylene dia- 
mine. Briefly stated, their results were: no reaction with anaerobes, coccal forms, 
B. influenzae, protozoa, and dysentery bacilli; a positive reaction with most other 
bacteria, especially strong with strict aerobes and pigment-forming bacteria. Stapp,^ 
working with a H2O2 and the benzidin test, found peroxidase reactions in B. colt, 
Staphylococcus, Sarcina, B. prodigiosus, and B. pyocyaneus. The peroxidase was dis- 
tinct from catalase in its greater resistance to heat and different sensitivity to iodine 
and carbon disulphide. He did not find it in streptococcus cultures. In an extremely 
interesting investigation of Fe containing cell pigments related to hemoglobin Keilin^ 
found that these were very widely distributed in nature and that their association 
with a peroxidase reaction was constant. For such pigment he proposed the name 
"cytochrome," and he found it to be present in yeast and in the aerobic bacterium, 
B. subtilis, but absent in an anaerobic bacillus, CI. sporogenes. Callow^ has also inves- 
tigated this subject and found peroxidase in all of a number of bacteria tested, but 
weak in B. acidl lactici and CI. sporogenes. 

OXIDATIONS PRODUCED BY BACTERIA 

The more the subject of oxidation is studied, the more difficult it becomes to draw 
any hard-and-fast line between oxidation and reduction. This is true especially of 
oxidations depending on processes of dehyrogenation and independent of the presence 
of free oxygen such as have been demonstrated by Wieland' to take place under the 
influence of a catalyst which may be palladium black or an oxygen-free bacterial 
powder. The special value of oxygen when taking part in such processes is simply 
that of taking up the discarded H with which it forms a product, H2O, that does not 

' Lehmann, K. B., and Sano: Arch.f. Hyg., 67, 99. 1908. 

^ Beijerinck, W. M.: Verzamclde Geschriften, 5, i. 

3 Roux, M. G.: Compt. rend. Acad, des sc, 128, 693. 1899. 

"Schultze, W. H.: Centralbl.f. BakterioL, Abt. I, Orig., 56, 544. 1910. 

5 Kramer, G.: ibid., 62, 394. 1912. ' Keilin, D.: Proc. Roy. Soc, B, 98, 312. 1925. 

^ Stapp, C: ibid., 92, 161. 1924. * Callow, A. B.: Biochem. J., 20, 247. 1926. 

»Wieland, H.: Berichted. D. Ch. Gesellsch., 46, 3327. 1913; 54. 2353. 1921. 



2i6 BACTERIAL OXIDATIONS AND REDUCTIONS 

interfere with the process of the reaction. A great deal of investigation on this type 
of oxidation has been done by Quastel' and others, and has already been referred to 
when discussing the reduction of methylene blue. The actual point chiefly studied is 
the extent to which various fatty acids, amino acids, and other substances are capable 
of being induced by bacteria to transfer H to methylene blue, i.e., the capacity of 
these substances for oxidation by various bacteria. It is this type of oxidation which is 
apparently responsible for furnishing the energy required by anaerobes or by faculta- 
tive anaerobes growing in the absence of oxygen by fermentative processes. 

Braun and Cahn-Bronner^ have drawn attention to the more complex nutriment 
required by bacteria growing under anaerobic conditions. 

A second type of oxidation is that in which oxygen is used freely and the fatty acids 
which have been present in the medium at the outset, derived from amino acids by 
deamination or from sugars by fermentative reactions, are oxidized to carbonates. 
Ayers and Rupp^ first drew attention to the fact that such oxidations are associated 
with the development of an alkaline reaction, and changes of this kind occurring in 
diphtheria cultures have been the subject of very careful study by Abt and Loiseau^ 
and by Abt.^ The production of alkali by oxidative formation of carbonates has been 
noted in cultures of many varieties of bacteria by Sierakowski,^ and Phelon, Duthie, 
and McLeod^ have drawn attention to its importance in cultures of gonococci and 
meningococci in which it may be responsible for the rapid death of the bacteria. A 
third distinctive type of oxidation is that of the types of bacteria described by Wino- 
gradsky^ as "anorgoxydanten" and by Orla-Jensen' as "autotrophic." These get the 
energy which they require for the assimilation of the atmospheric CO. by oxidation of 
simple elements like S, H, and Fe or compounds such as nitrites, CH^, or NH3, and 
are incapable of utilizing complex organic substances for their nutrition. According 
to Waksman and Starkey,'° only certain S-oxidizing bacteria and the nitrifying bac- 
teria are strictly autotrophic in the sense of inability to utilize any complex organic 
forms of nutritive matter. 

A fourth type of oxidative effect produced by bacteria is that due to the produc- 
tion of H2O2. The association between production of peroxide by bacteria and methe- 
moglobin formation has been specially studied by Neill and Avery" in connection with 
the pneumococcus, and by Valentine'- in connection with the streptococci. The former 
have brought out the very interesting fact that anaerobic extracts of pneumococci 

' Quastel, J. H., et al.: loc. cit. 

= Braun, H., and Cahn- Bronner, C. E.: Centralbl.f. Bakteriol., Abt. I, Orig., 86, i. 1921. 
3 Ayers, S. H., and Riipp, P.: J. Inject. Dis., 23, 18S. 1918. 
* Abt, G., and Loiseau, G.: Ann. Inst, de Pasteur, 39, 114. 1925. 
s Abt, G.: ibid., p. 387. 1925. 

^ Sierakowski, S.: Compt. rend. Soc. de biol., 89, 1371. 1923; Biochein. Ztschr., 151, 15. 1924. 
^ Phelon, H. V., Duthie, G. M., and McLeod, J. W.: /. Path, and Bait., 30, 133. 1927. 
HVinogradsky, S.: Centralbl.f. Balderiol., Abt. II, Orig., 57, 1. 1922-23. 
9 Orla- Jensen, S.: //;/(/., 27, 305. 1909. 

'"Waksman, S. A., and .Starkey, R. L.: J. General Physiol., 5, 285. 1923. Sec chap, xxiv in 
this volume. 

" Neill, J. M., and Avery, O. T.: J. Exper. Med., 39, 757. 1924. 
" Valentine, E.: J. Infect. Dis., 39, 29. 1926. 



JAMES WALTER McLEOD 217 

in the presence of oxygen and catalase produce much more methemoglobin than any 
concentration of H2O2 which is likely to occur on oxygenation of such extracts would 
do, and they suggest the formation of an organic peroxide insensitive to catalase. 

The heated blood agar or "chocolate agar" first introduced by Cohen and Fitz- 
gerald' is an extremely delicate indicator for peroxide formation by bacteria, and by 
its use the interesting fact is established that anaerobes produce an oxidative effect 
similar to that produced by bacteria which can be shown to produce HoOo by the usual 
chemical tests. This effect can be elicited either as a green ring at the upper limit of 
growth of an anaerobe in a deep tube of "chocolate agar" grown at the ordinary 
atmospheric pressure (McLeod and Gordon^) or it may appear as shown in Plate I, C 
as a faint green coloration around the bacterial colonies when the anaerobe is inocu- 
lated as a surface culture and incubated under the greatest tension of oxygen which it 
can tolerate without complete inhibition of growth. 

THE MECHANISM OF BACTERIAL OXIDATION 

It seems likely that all bacteria tend to produce peroxide and that this tendency is 
proportioned to their reducing activities. Those which can be shown to contain cata- 
lase and peroxidase presumably use it in their respiratory processes. It is interesting 
in this connection that Keilin-' found that a reducing mechanism which cannot be 
separated from peroxidase and may well be identical with it, is essential to the func- 
tioning of the respiratory pigment cytochrome. 

A reducing mechanism, functioning possibly in combination with a catalyst con- 
taining the sulphydryl group such as glutathione, may be responsible for anaerobic 
respiration independently of any Fe complex. Whether H2O2 takes any part in res- 
piratory processes of such bacteria as pneumococci is not clear. The lack of catalase 
and the fact that these bacteria resemble the anaerobes in being relatively insensitive 
to KCN (Burnet)'' which paralyzes cytochrome by fixing it in the reduced state 
(Keilin),^ suggest that H2O2 does not at all events function in combination with a 
peroxidase in these bacteria, and possibly is merely a by-product. The very interest- 
ing problem is presented by the Shiga type of dysentery bacilli. These lack catalase, 
ha\^ slight reducing powers and limited fermentative activities, do not produce detect- 
able amounts of peroxide, give no direct oxydase reaction (Kramer), ^ and although 
sensitive to cyanides (Burnet),'' they diverge in their reaction to this substance from 
other bacteria tested. I have not found a record of a determination of their peroxidase 
content. It is clear that none of the mechanisms mentioned could be considered to 
explain the respiratory processes of this type of bacterium. 

It is not unlikely that the dependence of some strictly parasitic bacteria on the 
presence of blood in the media in which they are cultured indicates their poverty in 
peroxidase, which they require and which is normally supplied to them from the tis- 
sues of the host on which they are parasites (Fildes,'' Webster^). 

' Cohen, C, and Fitzgerald, J. G.: Cenlralbl. f. Baklcriol., Abt. I, Orig., 56, 464. 1910. 

' McLeod, J. W., and Gordon, J.: /. Path, and Bad., 26, 332. 1923; ibid., 28, 147. 1925. 

3 Keilin, D.: loc. cit. 

''Burnet, F. M.: /. Path, and Bad., 30, 21. 1927. 

s Kramer, G.: loc. cit. *■ Fildes, P.: Brit. J. Ex per. Path., 3, 210. 1922. 

'Webster, L. T.: Proc. Soc. Rxper. Biol. ^ Med., 22, 139. 1924-25. 



CHAPTER XV 
PROTEIN (NITROGEN) METABOLISM OF BACTERIA 

LEO F. RETTGER 
Yale University 

Although single-celled and among the smallest known living organisms, bacteria 
can effect the most elaborate syntheses, and through a multiplicity of enzymatic and 
catalytic processes reduce to their elements the most complex organic substances: 
proteins, fats, and polysaccharides. Their range of activities may be said to be almost 
unlimited. 

PROTEINS AND THE LIVING CELL 

Protoplasm defies analysis, for when it is subjected to chemical study it is no 
longer protoplasm, but dead tissue. Our knowledge of the internal structure and of 
the chemical composition of the bacterial cell is as yet very meager. However, suf- 
ficient studies have been made of the chemistry of bacteria to leave no doubt of the 
importance of proteins and protein-like substances, and to show that the bacterial 
cell is in this respect not very unlike those of the higher forms of life, both animal and 
vegetable. 

According to the observations of Nicolle and Allilaire,' and of various other in- 
vestigators, organic nitrogenous material (protein) in bacteria greatly preponderates 
over the non-nitrogenous. It has been found, however, that the quantitative chemical 
composition varies markedly among different organisms, and even within the same 
species. 

Cramer^ has shown that the amount of total solids, ash, protein, and non-nitrog- 
enous matter present in bacterial cells varies with the composition of the culture medi- 
um, though not in direct proportion; also with the age of culture, and incubation tem- 
perature. For example, he determined the protein content of the cholera vibrio to 
be 65 per cent of the total solids, when grown in plain infusion peptone broth, as com- 
pared with 45-50 per cent in Uschinsky's medium. 

It appears to be an established fact that different bacterial species or groups 
possess certain chemical entities which distinguish them from all other forms. For in- 
stance, the acid fast group contains a relatively large proportion of ether-soluble, 
fat-like substances, and the different pathogens elaborate intracellular toxins and 
antigenic substances which distinguish them from each other. 

Nicolle and Allilaire^ determined the nitrogen content of certain organisms to be 
as follows (in per cent): V. cholerae, 9.79; Bact. dysenteriae, 8.89; Bad. typhi, 8.28; 
Bad. colt, 10.32; P. vulgaris, 10.73; B. anthracis, 9,22; E. prodigiosus, 10.55; -^•^• 

' Nicolle, M., and Allilaire, E.: Ann. de VInst. Pasteur, 23, 547. 1909. 
* Cramer, E.: Arch. f. Ilyg., 16, 151. 1893; 22, 167. 1895. 
3 Nicolle, M., and Allilaire, E. : loc. cil. 

218 



LEO F. RETTGER 219 

pyocyanea, 9.79; C. diphtheriae, 9.55. These are not very unlike the results obtained 
by Vaughan and Wheeler. 

Quite recently Seibert' isolated from tuberculin a nitrogenous substance in crys- 
talline form which possesses the properties of ordinary protein and which appears to 
embody the active principle of tuberculin, ^ 

The struggle for existence of bacteria means a struggle to furnish for themselves 
the energy and cell substance necessary to multiply and to reproduce their own kind. 
Organic carbon is, as a rule, the source of energy, and is supplied in various forms, as, 
for example, carbohydrates, organic acids, alcohols, CO, CO2, and CH^. The carbon 
of amino acids and acid amides also serves the same purpose. Carbon is necessary also 
to build up organic cell structure, as are nitrogen, hydrogen, oxygen, phosphorus, 
and sulphur. 

The exact nature and composition of proteins generally is still a matter of con- 
jecture. The quantitative determination of the chemical elements in various known 
proteins has revealed some differences, but the following may be taken as a fair aver- 
age: Carbon, 50 per cent; nitrogen, 16 per cent; hydrogen, 7 per cent; oxygen, 22 
per cent; sulphur, 0.3 per cent; phosphorus, 0.4 per cent.^ 

The nitrogen requirements and the means by which these are met constitute a 
fundamental phase of bacterial, as well as all other plant and animal metabolism, and 
the problem of nitrogen supply in the best available form is one that greatly con- 
cerns all living organisms. 

Bacteria may to a large extent be classified on the basis of physiological activities. 
Jensen believes that this is the only logical criterion. We speak of different groups as 
the nitrifying, proteolytic, putrefactive, lactic acid producing, etc., groups, or as those 
of the sulphur or iron bacteria. 

With respect to nitrogen utilization alone bacteria often reveal the widest differ- 
ences, and numerous processes of nitrogen transformations may be observed, even in 
the same organism. The two main processes, however, are those of katabolism or 
analysis, and anabolism or synthesis. As this paper deals with protein metabolism, 
the first of these two processes may be discussed under the following head. 

PROTEOLYSIS AND PUTREFACTION 

Cell metabolism involves a complex system of interchange of chemical substances 
between the cell and the surrounding medium. The work of Lubbert^, Hesse,^ and 
others, and more recently that of Novy,^ has shown that bacterial cells engage in a 
process of true respiration and that a respiratory coefficient may be established for 
different organisms. Gotschlich^ gives the coefficient as 0.71-0.78 (CO2: O2) in the 
absence of fermentable (sugar, etc.) substances. Novy found the coefficient for the 
tubercle bacillus to be 0,836, when grown on glycerol agar. 

' Seibert, F. : Science, 66, 433, 1927. 

^Mathews, A. P: Physiological Chemistry, p. 109. New York, 1922. 

^Liibbert,: Biologische Spaltpilz Uniersuchungen, p. ^S. 1886. 

''Hesse, W.: Zischr. Hyg. u. Infektionskrankh., 15, 17. 1893; 25, 477. 1897. 

5 Novy, F. G.: J. Infect. Dis., 36, 168. 1925. 

^ Gotschlich, E.: Kolle and Wassermann, Handb. d. Path. Mikroorg., i, 99. 1912. 



2 20 PROTEIN (NITROGEN) METABOLISM OF BACTERIA 

While it cannot be so easily proved, interchanges of nitrogenous materials must 
take place in a somewhat analogous manner. Complex nitrogenous compounds must 
be split into their simple ionizable constituents before they become available for cell 
nutrition. In the words of Abderhalden/ ''No cells can directly assimilate and utilize 
foreign food material. The latter must be prepared for the cell (by enzyme action)." 
He likens this transformation to the conversion of a church building into a school- 
house, in which the church must first be reduced to the individual bricks, etc., and 
then built up again into the new structure. 

Abderhalden's statements were directed mainly to animal physiologists. It was 
he who apparently first showed by actual experiment that man and the higher animals 
depend upon the hydrolysis of complex nitrogenous food in the stomach and intestine, 
and a subsequent resynthesis of the digestion products into the tissue and cell sub- 
stances of the body. 

Bainbridge^ and Sperry and Rettger^ demonstrated that not even the most active- 
ly proteolytic aerobes and anaerobes are able to attack native proteins when deprived 
of accompanying enzymes and when the proteins are the only possible source of ni- 
trogen. Sperry and Rettger employed crystallized egg albumin and edestin, in a me- 
dium which furnished all of the other necessary food substances. The addition of a very 
small amount of commercial peptone (mere traces) was sufficient to initiate growth and 
the elaboration of proteolytic enzyme which then reduced the peptone to simple 
products. 

Berman and Rettger^ demonstrated, further, that proteoses likewise are not di- 
rectly available to the bacterial cell, and that they and the higher polypeptides at 
least must first be hydrolyzed by proteolytic or peptolytic enzymes before they are 
made available. They also showed that many bacteria do not produce the enzymes 
necessary for preparing proteoses and the higher polypeptides for use, and that these 
are therefore not utilized at all by these organisms. Bad. coli is one of this group. 

According to E. Fisher and others protein is a complex aggregate of amino acids. 
By the use of strong sulphuric or hydrochloric acid, barium hydrate, superheated 
steam, or of proteolytic enzymes, these structural units can be broken loose from their 
combinations, and thus be identified by appropriate methods as individual amino 
acids. 

Besides the numerous monoamino, mono-, and di-carboxylic acids, and the hetero- 
cyclic acids (tryptophane, proline, histidine, etc.), two well-known diamino-mono- 
carboxylic acids have been isolated from proteins : arginine and lysine. 

The molecular weight of protein is claimed by some to be at least 15,000-18,000. 
If the entire protein molecule were made up of amino acids of the average molecular 
size of alanine (CH^-CH'NHj-COOH), it would be a composite of well over 150 in- 
dividual amino acids or amino acid units. One hundred per cent recovery of amino 
acids from the protein molecule has not been made. Osborne^ derived amino acids from 

' Abderhalden, E.: Cetitralbl.f. BaktcrioL, Abt. II, 37, 280. 1913. 

* Bainbridge, F. A.: /. Hyg., 11,341. 1911. 

3 Sperry, J. A., and Rettger, L. F.: /. Biol. Chem., 20, 445. 1915. 

1 Berman, N., and Rettger, L. F.: J. Bad., 3, 367. 19 18. 

s Osborne, T. H.: Tlie Vegetable Proteins. London: Longmans, Green & Co., 1909. 



LEO F. RETTGER 221 

vegetable protein which represented 60 per cent of the protein molecule. The defi- 
ciency may be partly explained, according to him, by a loss in the process of hydrolysis 
and isolation. It may also be due to the occurrence in the protein of modified amino 
or amide substances which resist isolation and identification. 

Amino acids are crystallizable and, with very few exceptions, quite soluble in 
water. They behave as both weak acids and bases, and form salts with them. 

The primary amino acids react with formaldehyde by a process in which the 
amino groups are condensed and lose their basic character. This constitutes the under- 
lying principle of the Sorensen method of determining the quantity of primary amino 
acids in any given solution, as, for example, peptone broth cultures of bacteria. 

The amino group is readily destroyed by nitrous acid, and by the quantity of ni- 
trogen gas evolved in the reaction the amount of amino acid or acids may be esti- 
mated (Van Slyke method). 

The occurrence of chromatin or nuclear material in the bacterial cell can no longer 
be doubted. Nishimura' and Galeotti^ demonstrated the presence of nucleins in bac- 
teria. The former also isolated the xanthin bases, xanthin, guanin, and adenin. Nucleic 
acids were obtained from the tubercle bacillus by Ruppel,^ from the diphtheria ba- 
cillus by Aronson,'^ and from Bad. coli by Carega.^ More recently Johnson and Brown^ 
isolated thymine and cytosine from the tubercle bacillus as the pyrimidines of the 
nucleic ("tuberculinic") acid of this organism. At about the same time the purin 
bases, guanin and adenin, were demonstrated by Long^ in the tubercle bacillus, 

ANALYTICAL PROCESSES BROUGHT ABOUT BY BACTERIA 

Through the agency of certain enzymes the complex protein molecule is split up 
into its numerous amino acids and perhaps some other nitrogenous constituents. The 
proteolytic enzyme or enzymes concerned differ from ordinary pepsin in that as a rule 
the hydrolysis is carried beyond the proteose and complex polypeptide stage. They 
differ also from trypsin of the animal body in that they act over a wide range of H- 
ion concentration, that is, on the acid as well as alkaline side of pH 7.0. However, the 
hydrolytic products are, at least in a large measure, like those of trypsin, and are 
chiefly amino acids, including ordinary tryptophane (a-amino jS-indol propionic 
acid). 

In this process of proteolysis some of the nitrogenous products are again utilized 
by the organisms to furnish energy for growth and new cell substance. We must as- 
sume that a synthetic process goes on simultaneously with the analytic. 

Further decomposition of amino acids by bacteria is altogether different from what 
takes place in the animal body, and an entirely different group of final cleavage prod- 
ucts is formed. Indol, skatol, and phenol are common among the strictly bacterial 

' Nishimura, T.: Arch. f. Hyg., 18, 318. 1893. 

' Galeotti, G.: Zlschr.f. phys. Chemie, 25, 48. 1898. 

3 Ruppel, W. G.: ibid., 26, 218. 1898. 

■•Aronson, H.: Arch.f. Kinderh., 30, 23. 1900. 

s Carega, A.: Cenlralbl. f. Bakteriol., Abt. I, 34, 323, 1903. 

* Johnson, T. B., and Brown, E. B.: Am. Rev. Tuberc, 7, 285. 1925. 

'Long, E. R.: ibid., 4, 842. 1920. 



222 PROTEIN (NITROGEN) METABOLISM OF BACTERIA 

products. Indol and skatol result from the action of bacteria on tryptophane, and the 
phenol has its origin largely in the tyrosine liberated from the protein molecule. 

Hydrogen sulphide also is a common product of bacterial action on protein and 
sulphur-containing derivatives of protein, and mercaptans (methyl and ethyl sul- 
phide) are liberated in the process of anaerobic decomposition by the putrefactive 
anaerobes. They are derived apparently from the cystin which forms a part of the 
molecule of most proteins. 

The amino acids themselves are physiologically inert. Before they can be of any 
use to the bacterial cell they require further disruption or rearrangement of the ele- 
ments, and this is readily accomplished by bacteria, through enzymes and catalysts, 
presumably within the cell itself. This transformation of the amino acids is one of 
deaminization in which free ammonia is formed as such out of the amino (NH,) 
groups. This process is commonly one of hydrolysis, with the formation of oxy acid, 
as is shown in the following simple equation: 

CH. • NH. • COOH+HOH = NH3+ CH.OH • COOH 
Glycocoll Oxyacetic acid 

Other processes of amino acid change and ammonia liberation are those of reduc- 
tion, oxidation, oxidation and reduction, and decarboxylation, with the formation, 
besides ammonia, of amines (R-NH2), alcohols, lower fatty acids, and CO2, as the 
case may be. 

In this transformation of amino acids by bacteria, products are at times con- 
structed which are toxic to the animal organism. By a process of decarboxylation, 
toxic amines and diamines may be produced, as, for example, tyramine (from tyro- 
sine), histamine (from histidine), and agmatine (from arginine). Other so-called 
ptomaines are neurin and methyl guanidine. However, not so much credence is given 
today to the idea of "ptomaine poisoning" as was done several years ago, and bacterial 
poisons are now generally regarded as the toxic products of specific organisms, like 5ad. 
enteritidis and CI. hotulinum. 

Ultimate products of bacterial protein decomposition, if the process is not inter- 
rupted, are of the simplest character, and may include ammonia, nitrous oxide, nitrate, 
nitrogen, hydrogen sulphide, methane, carbon dioxide, hydrogen, and water. The 
nature of the final products depends, of course, on the amount of available atmospheric 
oxygen. 

The term "putrefaction" has acquired two distinct meanings. In the more general 
usage it signifies decomposition of protein material through bacterial action, as against 
"fermentation" or decomposition of carbohydrates, with or without the formation 
of gas. It has been used by Bienstock' and by Rettger^ in a more restricted sense to 
mean anaerobic decomposition of protein with the production of foul-smelling prod- 
ucts which are characteristic of cadaveric decomposition. 

The same observers have maintained that, while many aerobic and facultative 
anaerobic organisms have the ability to decompose protein, they do so only under 
aerobic conditions, and that real putrefaction is the work of obligate anaerobes. This 

' Bienstock,: Arch./. Hyg., 36, 335. 1899. ^ Rettger, L. F.: /. Biol. Chem., 4, 45. 1908. 



LEO F. RETTGER 223 

property is possessed by certain anaerobes only, among the most important of which 
are CI. sporogenes, CI. putrificum, and CI. aerofetidum. 

Some bacterial species attack casein, without being able apparently to exert any act- 
ion on serum or egg albumin. Certain staphylococcus and streptococcus forms belong 
in this class. Gelatin is frequently liquefied by organisms which are non-pro-teolytic. 
In some instances the gelatin appears to be reduced to the soluble gelatose stage only. 

The decomposition of organic waste is participated in by many kinds of bacteria. 
According to Tissier and Martelly,' and this is a common observation, aerobes and 
facultative anaerobes play an important part by preparing a favorable gaseous environ- 
ment for the proteolytic anaerobes through which rapid destruction takes place. 

INFLUENCE OF CARBOHYDRATE ON METABOLISM 

It has long been known that utilizable carbohydrates retard bacterial proteolysis. 
This principle has in recent years been re-emphasized by KendalP and his associates, 
who apparently coined the statement, "Fermentation takes precedence over putre- 
faction." Their numerous experiments with glucose-utilizing organisms, particularly 
of the coli-typhi-paratyphi group, have lent further support to the limited obser- 
vations of earlier investigators. 

Kendall and his co-workers showed that when glucose-attacking organisms are 
grown in nutrient peptone containing i per cent glucose, very little nitrogen metabo- 
lism is carried on as indicated by ammonia determinations, in comparison with control 
cultures which contained no glucose. This inhibition of proteolysis was explained 
by them to be a sparing action on the proteoses and polypeptides of the medium by 
the glucose. 

Ordinary market milk is prevented from undergoing putrefaction because of the 
lactose and the lactose-utilizing bacteria {Streptococcus lactis in particular) which are 
always present. The lactic acid which is formed, even in small amount, retards the 
development of proteolyzing organisms, as, for example, members of the B. subtilis 
group and the putrefactive anaerobes, and instead of showing evidence of putrefaction, 
the milk becomes more and more acid, and the casein is precipitated as an acid curd. 

Many other examples may be cited, as, for instance, the absence of proteolysis in 
frozen stored eggs which may contain large amounts (10 per cent) of cane sugar. 

It seems to be well established that this inhibition of proteolysis is due to increased 
H-ion concentration resulting from the sugar fermentation. Berman and Rettger^ 
showed that this inhibition may be prevented by the addition of sufficient bufifering 
agent to regulate the H-ion concentration. 

The retardation of proteolysis varies, however, with the different organisms, ni- 
trogenous substances, and carbohydrates employed. For instance, indol production 
by indol-producing strains of Bad. coli in ordinary peptone broth is prevented by the 
addition of from 0.5 to i.o per cent glucose. In the presence of added tryptophane, 
some indol may be formed. When lactose is substituted for the glucose, indol forma- 
tion may be demonstrated readily. Both of these sugars are fermented by Bad. 

' Tissier, H., and MarteUy: Ann. de I'lnst. Pasteur, 16, 865. 1902. 

^ Kendall, A. I., and Farmer, C. J.: J. Biol. Chent., 12, 13, 19, 215, 219, 465, 469; i3, 63. 1912. 

3 Berman, N., and Rettger, L. F.: J. Bad., 3, 389. 1918. 



224 PROTEIN (NITROGEN) METABOLISM OF BACTERIA 

coli, but the greater ease with which glucose is broken up enables the organism to 
draw upon the glucose more easily for most of its carbon supply, and to this extent 
leave the nitrogenous substrates intact. 

Berman and Rettger' and later Slanetz and Rettger- observed that while B. subtil- 
is readily attacks glucose in ordinary broth, the glucose does not prevent or materially 
retard nitrogen metabolism of this organism. In fact, the proteolytic activity seems 
to be accelerated by the glucose. This may be explained by the very active proteolytic 
property of B. suhtilis, and the simultaneous production of acid and alkali in pro- 
portions which tend to neutralize each other, and thus permit proteolysis to continue. 

Other members of the subtilis group, B. cereiis and B. megatherium, are greatly 
retarded in their nitrogen metabolism by glucose, even in the presence of i per cent 
phosphate buffer, and in this respect they resemble Bact. coli, Bad. typhosum, and 
the paratyphoids, to a certain degree. 

PROTEIN SYNTHESIS BY BACTERIA 

Chlorophyll-bearing plants synthesize their cell substance with the aid of the 
sun's energy. Bacteria must obtain their energy by purely chemical action. Syn- 
thesis involves energy utilization. Analysis or katabolism is accompanied by the 
liberation of energy. Carbon plays the chief role in these processes; in some instan- 
ces the necessary energy is provided by the oxidation of nitrogen (nitrification) and 
of sulphur. 

Amino acids serve as a common source of nitrogen for bacteria. The exact nature 
of the process is still little understood, but it must be assumed that it takes place 
in the cell, and that the amino acids are deaminized to furnish ammonia directly, and 
that a very intensive selective action takes place in which the necessary chemical 
elements are used for energy and the building up of the complex cell substance, par- 
ticularly proteins, and whereby those elements or atomic groups which are useless are 
eliminated or rejected and constitute the so-called "metabolic waste products" of 
the bacterial cell. 

Simple amino acids often serve as the only source of organic nitrogen, as, for ex- 
ample, asparagin in the synthetic medium of Jordan^ and the same amino acid in 
Frankel's modification of the Uschinsky medium. Glycocoll and other simple amino 
acids have also been used to furnish the necessary conditions. Thus a single amino 
acid may supply the nitrogen needs for building up the complex nitrogenous substan- 
ces of the cell which themselves contain simple and complex amino acids of almost 
endless description, and the purin bases which characterize the nuclear substance. 

Turin bases serve as sources of nitrogen in some instances. Koser^ found that the 
aerogenes type of the coli-aerogenes group can attack uric acid and hypoxanthine, 
whereas the coli type cannot. There is no reason to assume, however, that these purin 
bases are converted directly into the nuclear substance of the cell. 

On the other hand, there are many organisms which require the most complex 

' Ihid. 

' Manuscript in preparation. 

3 Jordan, E. O.: /. Expcr. Med., 4, 627. 1899. 

* Koser, S. A.: J. Infect. Dis., 23, 377. 1918. 



LEO F. RETTGER 225 

mixture of amino acids; and, again, some cannot be made to grow in any known mix- 
ture of amino acids, but require for their normal development special material like 
hematin (//. influenzae) or substances of unknown composition which are present in 
blood or blood serum (some streptococcus and pneumococcus forms). 

UTILIZATION OF AMMONIA AND ATMOSPHERIC NITROGEN 

Both ammonia production and ammonia utilization are properties common to 
most bacteria, if indeed not all. These processes are frequently carried on simultane- 
ously. The ammonia is apparently utilized directly. 

Numerous organisms will develop abundantly in media in which the only source 
of nitrogen is ammonia, in the form of a soluble ammonium salt. Proskauer and Beck' 
found that good growth of the tubercle bacillus (human type) can be obtained in a 
medium containing ammonium salts of di-basic and tri-basic acids. This observation 
has been verified frequently. Another example is the medium of Ayers and Rupp' for 
the cultivation of the colon-aerogenes group. The nitrogen is supplied here in the 
form of sodium ammonium phosphate. 

Free atmospheric nitrogen can be used by some organisms as a source of nitrogen. 
The conversion of free nitrogen into ammonia for cell use may be demonstrated easily 
by the Azotobacter genus and CI. pastorianum. It appears that non-symbiotic ni- 
trogen fixation among aerobic bacteria is not uncommon; but the action is as a rule 
so weak and variable as to have little practical significance. 

A process of free nitrogen utilization which is of enormous economic importance is 
that of the Rhizobium or root-nodule type of or gSLuism —Rliizob mm radicicola. This 
depends on a close association with the roots of leguminous plants, and takes place 
within the root tubercles of the legumes. Little is known of the chemical process, but 
it would seem that ammonia must be an important intermediate product of me- 
tabolism. 

Denitrification is a property possessed by a considerable number of organisms, 
particularly of soil origin. Complete denitrification results in the formation of nitro- 
gen or of nitrous oxides. Indirect or incomplete reduction of nitrates gives rise to 
nitrites or ammonia. 

Another process engaged in by a very limited number of organisms is that of ni- 
trification. This involves two separate processes: (i) the oxidation of ammonia to 
nitrous acid, accomplished by two highly specialized species, the Nitrosomonas and 
Nitrosococcus of Winogradsky; and (2) the further oxidation of the nitrous acid to 
nitric through the agency of the Nitrobacter of Winogradsky. 

These two processes may be indicated by the following equations: 

(i) 2NH, +30. = 2HNO.-f2HA 

(2) 2HN0.-h 0. = 2HN03. 

While the nitrification plays an important economic role in soil fertility, it would 
seem that the chief immediate benefit which the bacteria themselves derive from it is 

' Proskauer, B., and Beck, M.: Zlschr.f. Hyg. u. Infekiionskrankh., 18, 128. 1894. 
- Ayers, S. H., and Rupp, P.: /. Bad., 3, 433. 1918. 



226 ' PROTEIN (NITROGEN) METABOLISM OF BACTERIA 

the energy supplied by the reactions. Some of the nitrate nitrogen may of course be 
used for cell structure. 

It has been claimed that the entire oxidation process may be brought about by a 
single organism, but evidence on this point is still quite meager. 

The subjects of utilization of free atmospheric nitrogen, and of nitrification and 
denitrification, are dealt with only very briefly in this chapter because they are dis- 
cussed at some length in other chapters of this volume. 



CHAPTER XVI 

THE UTILIZATION OF CARBOHYDRATES BY BACTERIA 
ARTHUR ISAAC KENDALL 

Washington University School of Medicine, St. Louis, Mo. 

Man has been familiar with the results of the fermentations induced in saccharine 
media from the earliest times, but it was not until 1810 that a serious attempt was 
made to reduce the reactions of a fermentation process to a definite, balanced chemical 
equation. In that year Gay-Lussac' published his "Memoire sur la fermentation," 
in which he stated that glucose undergoing alcoholic fermentation passes quantita- 
tively into carbon dioxide and ethyl alcohol according to the following equation : 

CeHx.Oe = 2 C0.+ 2 C2H5OH . 

Notwithstanding the fact that this equation is an impossible one, as is readily 
seen from a consideration of the space formula for glucose, the observation is very 
valuable not only because two of the most important substances produced from the 
fermentation of glucose by yeast are thus early identified, but also because this is 
one of the very first attempts to study a biological reaction in a quantitative way. 
This observation, furthermore, was made at a time when organic chemistry was a very 
immature science and long before the discovery of the yeast plant itself. 

More than half a century elapsed before the detailed study of micro-organisms 
and their products of development was resumed. Meanwhile, the compound micro- 
scope was brought to a state of perfection compatible with accurate observations, and 
a violent controversy centering on the doctrine of spontaneous generation had brought 
to light the part microscopic organisms play in inducing fermentations and putrefac- 
tions in decomposable media. Also new and useful methods for culturing and identi- 
fying these micro-organisms gradually were developed. The famous controversy be- 
tween Liebig and Pasteur, which lasted nearly twenty years in the aggregate, termi- 
nated with the firmly established thesis of "no fermentation without life," which is 
one of the great contributions of Pasteur to microbiology. About two decades later 
Buchner discovered zymase, which in turn opened up a new and very fertile field 
for further exploration. These three great discoveries — first, the chemical balance 
sheet of fermentation; second, the organism that incites fermentation; and third, 
the complex enzymatic nature of the process of fermentation itself — although not 
fully matured even today — mark a new and highly important epoch in the study not 
only of microbiology, but of cellular activity in general. While it is undoubtedly true 
that the nature of the decomposition of carbohydrates by yeast is far better under- 
stood than decompositions induced by bacteria, nevertheless considerable progress 
has been made along bacteriological lines and much valuable information has been 
obtained. 

' Gay-Lussac, L. J.: Ann. chim. et phys., 76, 245. 1810. 

227 



228 UTILIZATION OF CARBOHYDRATES 

As early as 1862 Pasteur' was studying spontaneously developing butyric fermen- 
tation; indeed, he had already isolated and cultivated the anaerobic "vibrios," as 
he called them, which would induce the formation of butyric acid from lactates. This 
is a highly significant phenomenon which has not received the attention it deserves 
even today, inasmuch as it seems to predicate an extraordinary change of the three- 
carbon-atom lactate molecule to the four-carbon-atom butyrate molecule. Pasteur 
also made the highly important discovery that certain microbes attacked the d- and 
/-forms of tartaric acid at materially different rates, thus paving the way for the 
important studies of Fischer and Thierfelder^ upon the isomers of glucose and their 
relations to yeast fermentation. Fischer, whose master-mind opened the great field 
of carbohydrate chemistry to his successors, did not fail to realize the theoretical 
importance of these relations between carbohydrate structure and protoplasmic 
utilizability, and propounded his famous simile of the "key and the lock"^ in explana- 
tion of the reciprocal relations between the two. 

Much additional information about the fermentations of carbohydrates by 
bacteria was afforded by the earlier researches of Nothnagel^ and of Brieger^ who 
isolated and identified propionic, lactic, acetic, and formic acids among the products 
of decomposition of glucose by acetic and lactic fermenting microbes. Perhaps the 
greatest contribution of all, however, was that of Escherich.^ This very careful 
and thorough investigator not only introduced several of the more important intes- 
tinal bacteria into the group of known microbes, but also added much to the tech- 
nique of bacteriology, including the underlying principle of the fermentation tube. 
He also improved the anaerobic method of cultivation, and, perhaps most significant 
of all, started an entirely new chapter in microbic chemistry dealing with the nature 
of the metabolism of proteins, carbohydrates, and fats by a variety of bacteria, acting 
alone and in mixture, which he obtained from the intestinal tracts of young children. 
With the advent of Escherich's important monograph, modern bacteriology may be 
said to have its origin. 

GENERAL CONSIDERATIONS 

With the probable exception of the genera Thiothrix and Beggiatoa, bacteria 
which appear to utilize inorganic sulphur and sulphur compounds and simple am- 
monium salts for energy, 7 require some preformed, that is to say, organic food in 
their dietary. This food is required in fulfilment of two distinct phases in their life- 
history: the structural or anabolic phase and the energy or katabolic phase. The 
structural phase comprises those phenomena which are embraced in the separation 
and maturation of the daughter-cell from the parent-cell, together with those cellular 
losses incidental to the elaboration of soluble enzymes and other elements. The 

' Pasteur, L. : The Physiological Theory of Fermenlation. An excellent translation appears in 
"Harvard Classics," 38, 289-381. 1910. 

^ Fisher, E., and Thierfelder, H.: Ber. d. dent. chem. Gesellsch., 27, 2031. 1894. 

3 Fischer, E.: Ztschr.f. phys. Chemie, 26, 60. 1898. 

" Nothnagel, H.: Ztschr f. klin. Med., 3, 275. 1881. 

5 Brieger, L.: Ztschr.f. phys. Chemie, 8, 306. 1883-84; 9, i. 1885. 

^ Escherich, T.: Die Darmbakterien. Stuttgart. 1886. 

'See Waksman, S. A.: /. Bad., 7, 231. 1922. 



ARTHUR ISAAC KENDALL 229 

energy phase comprises those chemical changes which the mature microbic cell 
induces in its environment in fulfilment of its particular and characteristic chemical 
activity. 

The amounts of substance required for the structural and energy phases respec- 
tively, are very unlike. Fifteen millions of bacteria of ordinary size would scarcely 
balance an ounce weight.' The weight of a simple bacterial cell, therefore, is little 
indeed, and about 85 per cent of this is water. On the other hand, the amount of 
substance transformed for energy by bacterial cells is, or may be, relatively great. 
Hence, the expenditure of foodstuffs in the energy, as contrasted to the structural 
phase, is frequently in the order of hundreds to one. 

Yet it is not a matter of indifference just what the chemical nature of these phases 
is dependent upon. Bacteria, like other known living things, are nitrogen-containing. 
Hence some suitable source of nitrogen must be available for structural needs. On 
the other hand, most bacteria may derive the oxidizable carbon for their energy 
requirement either from compounds containing nitrogen as well as oxygen, as in the 
amino acids and their complexes, or from carbohydrates having a suitable configura- 
tion. Fats on the whole are probably not particularly suited for the energy require- 
ments of most bacteria. 

There is also a group of non-nitrogenous substances, departing somewhat from 
the carbohydrate configuration, as certain organic acids (tartaric, for example), which 
are acceptable sources of carbon for energy by many bacteria. A discussion of this 
problem, however, is without the scope of this chapter, which deals more specifically 
with carbohydrates. 

Carbohydrates frequently have a profound effect upon the character of the sub- 
stances produced by bacterial action. Thus, to cite a few well-known examples:^ 

The diphtheria bacillus growing in a suitable nitrogenous medium produces the 
characteristic, deadly, well-known soluble toxin which makes the organism formid- 
able. If, before the organisms are cultivated, some glucose is added to such a medium, 
the microbe produces considerable amounts of acid, principally lactic, but no toxin 
whatsoever. 

The proteus bacillus produces large amounts of indol, and forms a soluble, proteo- 
lytic enzyme when it is cultivated in a suitable nitrogenous medium. If, however, 
before the organism is inoculated, some glucose is added to the nitrogenous medium, 
it no longer produces indol nor the proteolytic enzyme. It forms considerable amounts 
of lactic acid instead.^ 

In a similar manner the various strains of Bacillus coli produce indol and phenolic 
bodies from the nitrogenous constituents of ordinary nutrient broth, but the addition 
of glucose to such a medium prior to inoculation with the microbe changes it into a 
lactic-acid-producing microbe. These latter observations have some significance in 
the genesis of indol in the intestinal tract. ^ 

It appears, therefore, that the addition of utilizable carbohydrate to nitrogenous 

' Kendall, A. I.: Civilization and the Microbe, p. 17. 1923. 

^ See Kendall, A. I.: Physiol. Rev., 3, 438. 1923. 

i Kendall, A. I., Cheetham, H. C, and Hamilton, C. S.: /. Infect. Dis., 30, 251. 1922. 

^ Kendall. A. I.: Bacteriology, General, Pathological and Intestinal (2d ed.), p. 70. 1921. 



230 UTILIZATION OF CARBOHYDRATES 

cultural media in which bacteria are growing affects the character of the products they 
form very strikingly. In general, it may be said that utilizable carbohydrate protects 
protein from direct bacterial utilization for energy. On the other hand, it must not 
be forgotten that, under certain circumstances, the addition of utilizable carbohydrate 
may indirectly bring about some alteration in the protein constituents of cultural 
media which might not otherwise take place. 

Thus, it appears to be a fact that utilizable carbohydrate, added to cultures of the 
gas bacillus {Bacillus welchii), indirectly leads to the appearance of a histamine-like 
substance, which is not produced by the action of the organism upon the protein 
constituents when carbohydrate is absent. ' It seems not improbable that the organ- 
ism produces an enzyme, of the nature of a carboxylase, which acts upon histidine, 
or possibly a histidine peptide, in accordance with the equation : 

H— C — NH H— C — NH 

II />C-H II ^C-H 

I (Carboxylase) | +CO2 

CH. CH, 

I I 

CH • NH. CH. . NH, 

I 
COOH 

(Histidine) (Histamine) 

Only very small amounts of this histamine-like substance are produced in ordinary 
cultural media, but it is very reactive chemically. One part in five million may cause 
a very definite contraction in a piece of isolated surviving smooth muscle from a 
guinea pig.^ 

The observations of Koessler and Hanke^ suggest that a somewhat similar reaction 
may occur among certain strains of other common bacteria. Finally, it must be borne 
in mind that "resting" bacteria, i.e., actively growing bacteria washed free from 
cultural medium, and suspended in non-nutritive, isotonic solutions containing ap- 
propriate amounts of test substance, can and do bring about chemical changes that 
do not necessarily take place when the organisms are growing freely. Thus, many 
"resting" bacteria will change methyl glyoxal to lactic acid, although this reaction 
does not seem to take place under conditions where the microbes are growing freely. 
Quastel and his associates^ have made several important studies upon this significant, 
but little studied, aspect of bacterial metabolism. 

CARBOHYDRATES AND THE STRUCTURAL REQUIREMENTS OF BACTERIA 
CELL CONSTITUENTS 

The predominance of nuclein compounds in bacteria, described by many investi- 
gators, focuses attention upon the nature of the carbohydrate component. Bendix,^ 

I Kendall, A. I., and Schmitt, O. F.; J. Infect. Dis., 39, 250. 1926. 
^ Guggenheim, M.: Die biogene Amine (II. Aufl.) 1923. 
^Koessler, K. K., and Hanke, M.: /. Biol. Chem., 50, 131. 1922. 
4 Quastel, J., et al.: Biocliem J. 1924-27. (Numerous articles). 
sBendix, E.: Deutsche med. Wchnschr., 27, 18. 1900 



ARTHUR ISAAC KENDALL 231 

studying the chemistry of the cellular substance of the tubercle bacillus, claimed to 
have identified a pentose among the constituents. This, if substantiated, would seem 
to place this organism at least among the plants, because the animal nucleus con- 
tains a hexose.' The presence of cellulose, and of hemicellulose, is still a matter of 
discussion, with the balance of evidence as yet unfavorable, although Emmerling, 
Winterstein,^ and others claim to have detected it in the substance of bacteria. On 
the other hand, chitin, a polymer of glucoseamine^ found in the animal kingdom, 
principally in the carapace of Crustacea, has been reported by several investigators. 
This is quite important if true, because it leaves the bacteria in an ambiguous position 
with reference to the usual chemical concepts of classification — having nuclear sub- 
stance of plant affinity, and at the same time possessing chitin, usually regarded as 
of animal origin, in their cell membranes. 

CAPSULAR CONSTITUENTS 

Certain kinds of bacteria, as pneumococci, pneumobacilli, the gas bacillus (Ba- 
cillus welchii), and several occurring in the soil, are surrounded with mucoid envel- 
opes. Acetic acid precipitates this mucoid substance in many instances, which has 
led to the belief that the material may be true mucin. Recently Dochez, Avery, 
Heidelberger, and others'" have isolated polysaccharides from the pneumococci, the 
Friedlander bacillus, and other organisms which are not true mucins but which are 
precipitated with the homologous-type sera in very high dilutions. These "species 
specific" polysaccharides are very interesting and important both from the stand- 
point of immunity and virulence. 

EFFECTS OF CARBOHYDRATE UPON THE CHEMICAL COMPOSITION OF BACTERIAL CELLS 

Cramer,^ Lyon,^ and others have published analyses which seem to indicate that 
the presence or absence of utilizable carbohydrate in cultural media otherwise of the 
same composition influences the relative amounts of nitrogenous substance, alcohol 
and ether extracts, and ash quite materially. Just what significance is to be attached 
to these data is problematical. They may, however, be of significance in view of the 
fact that Dochez and Avery" found that the yield of the "species specific" polysac- 
charides from type-II pneumococci increased from 3-4 to 35-40 gm. per 300 liters of 
culture when glucose was added to the medium. 

CARBOLIGASE 

Mention should be made at this point of the important observation of Neuberg^ 
that yeast is capable of bringing about syntheses of organic compounds in the presence 

' Jones, W. : Nucleic Acids, pp. 21, 29. 1924. 

^Emmerling, O.: Ber. d. dent. chem. Gesellsch., 32, 541. 1897; Winterstein, E.: Ztschr. f. phys. 
Chemie, 21, 134. 1895-96. 

3 Viehover, A.: Ber. d. dent, botan. Gesellsch., 30, 443. 1912. 

■» See, for an excellent summary, Heidelberger, M.: "Immunologically Specific Polysaccharides," 
Chem. Rev., 3, 403. 1927; Heidelberger, M., and Goebel, W. F.: /. Biol. Chem., 70, 613. 1926; also 
chap. X in this volume. 

5 Cramer, E.: Arch.f. Hyg., 16, 151. 1893. ^Lyon, R. E.: ibid., 28, 30. 1897. 

'Neuberg, C, and Kobel, M.: Handb. d. biol. Arbeiismeth., Abt. IV, p. 625. 1927. 



232 UTILIZATION OF CARBOHYDRATES 

of carbohydrates. Thus, when benzaldehyde is added to a suspension of yeast in 
glucose solution a condensation product of benzaldehyde with acetaldehyde is formed 
which seems to have the formula 

C6H5 • CHOH • CO • CH3 . 

This reaction, according to Neuberg, is brought about by the activity of an enzyme, 
carboligase. Up to the present time similar studies have not been made with bacteria, 
but it is not unreasonable to suspect that somewhat similar enzymatic processes may 
be discovered. 

CARBOHYDRATES AND THE ENERGY REQUIREMENTS OF BACTERIA 
THE SUGAR SERIES, AND DERIVED ALCOHOLS AND ACIDS 

Mention has been made above of the fact that the addition of utilizable carbo- 
hydrates and carbohydrate derivatives of the proper configuration to the nitrogenous 
constituents of cultural media may very materially affect the character of the prod- 
ucts. 

Generally speaking, but with some well-known exceptions, as, for example. 
Bacillus alcaligenes, which apparently does not utilize even glucose for energy, the 
utilizable carbohydrate is burned for energy, sparing to a very considerable degree 
thereby the nitrogenous constituents from bacterial attack.' No authentic instance 
has been recorded in which a microbe utilizes any carbohydrate for energy that will 
not utilize glucose. There are, however, organisms that do not seem to be able to 
use any carbohydrate except glucose for energy. This is reminiscent of the human 
body, which seems to utilize glucose for energy requirements. 

It is rather a striking fact, commented on long ago by Smith,^ that the bacteria 
highly pathogenic for man and for animals are usually less reactive both culturally 
and chemically than the parasitic types. Thus, typhoid, dysentery, diphtheria, and 
tubercle bacilli, as well as meningococci and gonococci, are relatively inert culturally, 
whereas the colon, proteus, and mesentericus groups of bacilli and the staphylococci 
are characterized by considerable cultural reactivity. This is reflected not only in the 
respective changes induced in nitrogenous constituents of culture media, but also in 
the configuration of carbohydrates and carbohydrate-derivatives which these mi- 
crobes can utilize for energy; which they can ferment. The members of the pathogenic 
groups above mentioned can generally utilize glucose, and the closely related hexoses, 
mannose, and fructose and the derived alcohol, mannitol, but cannot apparently 
break down such biose molecules as lactose and saccharose, or utilize starches. 

On the other hand, there are parasitic microbes, as certain members of the 
mucosus capsulatus group, which can ferment a very considerable variety of carbo- 
hydrates, some with 3, 4, 5, 6, 12, and more carbon atoms. The exact explanation for 
this difference in versatility, aside from Fischer's simile of the "key and the lock,"^ 
is still to be revealed. Nevertheless, the remarkable specificity of these relations be- 
tween carbohydrate configuration and protoplasmic utilization is one of the remark- 
able phenomena of biology. 

' See Kendall, A. I.: Physiol. Rev., 3, 438. 1923. 

' Smith, T.: Am. Med., 8, 711. 1904. ^Fischer, E.: he. cil. 



ARTHUR ISAAC KENDALL 



233 



GLUCOSIDES AND GLUCOSEAMINE 

There is a large group of substances possessing in common a carbohydrate 
molecule united to one or more organic compounds, which are resolved into their 
respective components upon hydrolysis with the addition of H and OH ions. These 
are glucosides and carbohydrate ethers of organic acids, alcohols, or ring compounds, 
and they correspond in chemical structure to the well-known methyl glucosides. 
Their significance to the bacteriologist at present lies in the fact that some of these 
have been used from time to time for purposes of separation and of recognition of 
certain groups of organisms, notably the streptococci. 

The relation of two naturally occurring enzymes,, emulsin and maltase, to these 
glucosides is of great significance. The former cleaves many of those glucose-gluco- 
sides which possess the iS-glucose configuration, whereas the latter hydrolyzes many 
glucose-glucosides which possess the a-glucose configuration. This has been very 
properly construed as evidence of the /3 or a linkage between the glucose molecule 
and the associated radicle, alcohol, acid, or otherwise.' 

The relationships between the a and /3 glucose-glucosides are indicated in the 
following diagrams in which "R" indicates the bound radicle: 



H— C— O— R 



H— C— OH 
HO— C— H 
H— C O 



R— O— C— H 

H— C— OH 
HO—C— H 
H-C 



H— C— OH 



H— C— OH 



CH.OH 
(a Type of glucoside) 



CH.OH 

(/S T\'pe of glucoside) 



Those glucosides that are commonly used in the identification of bacteria — 
salicin, amygdalin, and arbutin — together with many others that are occasionally 
utilized for this purpose, are all members of the /3 group, hence, aside from certain 
minor factors involved in the effects of the radicles upon cleavage, one glucoside of 
the /3 type is, or theoretically should be, as informative concerning the enzyme equip- 
ment of the microbe as another. It would appear that the principal biochemical 
information to be elicited from the use of one or several members of the /3-glucoside 
group, therefore, is the presence or absence of an enzyme of the emulsin type. Never- 
theless, some practical differentiations between closely related bacteria seem to have 
been made through the use of some of these /3 glucose-glucosides.^ Little or no atten- 
tion has been paid to a glucosides from this aspect, probably because glucoside deriva- 

' See Armstrong, E. F: The Simpler Carbohydrates and Glucosides (4th ed., 1924), for a fairly 
complete chapter on glucosides. 

* See Gordon, M. H. : Siipp. Ann. Rep. Loc. Gov. Bd., p. 388. London, 1903; Andrews, F. W., and 
Horder, T. J.: Lancet, 2, 708, 775, 852. 1906; Holman, W. L: J. Med. Research, 34 (N. S., 29), 
377. 1916; Kendall, A. I., Day, A. A., Walker, A. W., and Ryan, M.: J. Infect. Dis., 25, 189. 1919. 



234 UTILIZATION OF CARBOHYDRATES 

tives of the a-glucose series have not thus far been obtained from the vegetable king- 
dom. Also, the glucosides derived from sugars other than glucose, d- and /-arbinose, 
/-xylose, galactose, mannose, and fructose do not seem to have been carefully investi- 
gated. 

Glucoseamine, a cleavage product of chitin, as well as mucin from the submaxil- 
lary gland, and from mucous membranes,' seems to have been isolated from the body 
substance of certain bacteria.^ Meyer^ states that this substance may be fermented 
by a considerable number of bacteria, i.e., it may be a source of energy for these 
microbes. In the list is Bacillus proteiis. If indeed it be true that the proteus bacillus 
will ferment glucoseamine, then some evidence will have been produced in favor of 
the glucose rather than the mannose formula for this substance— a point of contention 
at the present time, because Bacillus proteus does not ferment the mannose configura- 
tion. ^ 

THE CARBOHYDRATE FRACTION OF PROTEINS 

Many proteins contain a carbohydrate nucleus in their molecule. Mathews' states 
that egg white contains 0.5 per cent of glucose. Some evidence of the occurrence of 
such a carbohydrate-like substance is afforded by a study of the nitrogenous me- 
tabolism of active cultures of Staphylococcus aureus in suitable nitrogenous media. It 
has been found that the deamination induced by this organism is comparatively 
slight until the Molisch reaction' disappears. Also an acidity develops which increases 
progressively with the persistence of the Molisch reaction. When the Molisch reaction 
disappears, however, usually by the end of the fifth day of growth, deamination 
proceeds rapidly, and concurrently the reaction becomes quite alkaline, due to the 
accumulation of basic products of the nitrogen metabolism of the organism. It seems 
very probable that both the initial acidity and low ammonia formation are associated 
with the utilization of the carbohydrate nucleus of the protein molecule for energy. 
Little nitrogenous change would be expected under these conditions. When the 
carbohydrate is used up, the organism attacks the residual nitrogenous fraction for 
its energy requirements, bringing about the typical evidences of deamination and 
accumulation of basic substances. 

Not many bacteria thus far studied exhibit this phenomenon, however. It seems 
not unreasonable to explain this difference on the basis of the nature of the peptid 
linkages in the protein molecule, which are broken by the staphylococcus in such a 
manner as to liberate the carbohydrate nucleus early in the digestive process, on the 
one hand, and differently cleaved by most bacteria, on the other hand. 

BACTERIAL UTILIZATION OF CARBOHYDRATES 
EFFECT OF CONFIGURATION OF THE CARBOHYDRATE MOLECULE 

Seventy years ago Pasteur^ noticed that the green mold, Pemcillium glaucum 
attacked the d- and /-forms of tartaric acid at materially different rates, utilizing the 
(/-tartaric acid quite rapidly, leaving the /-tartaric acid practically untouched. Not 

' Meyer and Jacobson: Lehrb. d. org. Chem. (II. Aufl., Vol. I, Part 2), 1913. 

' Viehover, A.: loc. cit. ^ Meyer, K : Biochem. Ztschr., 57, 297. 1913. 

^ Kendall, A. I., Cheetham, H. C, and Hamilton, C. S.: loc. cit. 

5 Mathews, A. P.: Physiol. Chem. (2d ed.), 151. 1916. 

* Kendall, A I., and Farmer, C. J.: /. Biol. Chem., 12, 215. 1912. 

7 Pasteur, L.: Compt. rend. Soc. de biol., 46, 615. 1858. 



ARTHUR ISAAC KENDALL 



235 



much attention was paid to this observation until Fischer, the master-chemist of 
the carbohydrates, and his pupil, Thierf elder,' exposed various members of the hexose 
series of sugars to the action of yeasts. They found at once that certain members of 
the glucose series, always with the ^/-configuration, were consistently fermented by 
yeast, whereas other members were equally consistently left unattacked. Fischer 
grasped the meaning of this remarkable relationship between carbohydrate configura- 
tion and the ability of the yeast to utilize certain configurations as a very fundamental 
biological phenomenon. His famous simile of the "key and the lock" is ample 
evidence of his comprehension of the significance of this very important phenomenon. 
He states: "Bei dieser Annahme ware es nicht schwer zu verstehen, dass die Hefezel- 
len mit ihrem asymmetrisch geformenten Agens nur in die Zuckerarten eingreifen 
und garungserregend wirken konnen, deren Geometric nicht zu weit von derjenigen 
des Traubenzuckers abweicht."^ Fischer and Thierf elder also showed that all the 
yeasts they studied fermented all those hexoses of the glucose series that had a com- 
mon enol — namely, glucose, mannose, and fructose. That is to say, their experiments 
showed very clearly that a yeast culture which fermented any one of these three 
hexoses fermented the others as well. Armstrong^ has reaffirmed this conclusion. 

Bacteria are considerably more versatile in their fermentation reactions in the 
aggregate than yeasts, and it is to be deplored that bacteriology had not developed 
far enough when Fischer was studying this profoundly interesting "biochemical 
geometry" to provide him with some of the more active fermenting types to extend 
this highly important field. 

It is now well known that several important groups of bacteria, e.g., members of 
the Bacillus proteus and Vibrio comma group, do not ferment mannose-i at all, 
and several other similar instances are known. Furthermore, such changes of terminal 
groups in the glucose molecule as are shown below alter the utilization of the resulting 
compound for many bacteria, thus:^ 

H 



H— C=0 

1 


HO— C=0 

1 


HO— C=0 


H— C— OH 

1 


H— C— OH 


H— C— OH 

1 


H— C— OH 


H— C— OH 


HO— C— H 


HO— C— H 

1 


HO— C— H 

1 


HOC— H 


H— C— OH 

I 


H— C— OH 

1 


H— C— OH 

1 


H— C— OH 

1 


H— C— OH 

1 


H— C— OH 

1 


H-C— OH 

1 


H— C— OH 

1 


H— C— OH 


H— C— OH 

1 


1 
HOC=0 


H— C— OH 

1 


H 


1 
H 




H— C— OH 

1 
H 


((/-Glucose) 


(J-Gluconic acid) 


(J-Saccharic acid) 


(J-Sorbitol) 



' Fischer, E.: loc. at.; Thierfelder, H.: loc. cit. ' Fischer, E.: loc. cit. 

3 Armstrong, E. F.: op. cit., p. 171. 1924. 

'• Kendall, A. I., and Yoshida, S.: /. Infect. Dis., 32, 355. 1923. 

s For convenience the older formula for glucose is used in place of the closed-chain formula, and 
the acids are not written in the lactone form. 



236 UTILIZATION OF CARBOHYDRATES 

It has been shown' that departure from the glucose configuration renders the 
resulting mono- or di-glucose acid and alcohol progressively less utilizable as a source 
of energy for bacteria. In general, the more fastidious types are the ones which are 
the more readily affected by departures from the simple hexose configuration. One 
of the rather unexpected results which flowed from the study of this phenomenon 
was the failure of members of the Staphylococcus aureus group and Micrococcus tetra- 
genus to utilize either gluconic or saccharic acid. Glycuronic acid was not available 
at the time these studies were made. Similar series were studied with mannose and 
galactose as the starting-point. The members of these series most difficult of utiliza- 
tion again were acids: manno-saccharic and mucic acid. Dulcitol, the hexatomic 
alcohol of the galactose series, is optically inactive through internal compensation. 
It is much less readily utilized for energy by most bacteria, and it possesses, largely 
for this reason, diagnostic value as a reagent for distinguishing certain types of 
organisms, notably many members of the Bacillus mucosus capsulatus group of 
bacteria, which are versatile fermenters. 

Much more study will be required before the significance of the departure from 
the glucose configuration in relation to protoplasmic utihzation is understood, but 
at least one rather striking practical possibility has emerged, namely, the ability to 
identify carbohydrates and their derivatives by the use of microbes. Experiments 
already published^ indicate that not only may certain carbohydrates be detected 
thus by bacterial means, but also mixtures of sugars and their derivatives may be 
identified^ and even measured with considerable accuracy. As little as one-one-thou- 
sandth of a per cent of a sugar in a mixture has thus been detected and estimated,^ 
with considerable precision. Not the least interesting possibility inherent in these 
studies is the bringing together of the field of pure chemistry, biochemistry, and 
bacteriology in the exploration of one of the most fundamental of problems: the 
chemistry of vital processes. 

BACTERIAL FERMENTATION OF CARBOHYDRATES 

The simplicity of structure and the monotony of structure of bacteria has had 
even from the earliest pioneer days a peculiarly directing influence upon the lines of 
development of bacteriology. Coincident with the recognition of the necessity for 
supplemental criteria to the meager anatomical characteristics of microbes, attention 
was early directed to chemical changes they might induce in suitable cultural media 
to afford data upon which to identify them. In this manner Pasteur^ recognized 
butyric acid, and Nothnagel^ and Brieger^ identified lactic, acetic, propionic, and 
formic acids as well as carbon dioxide and hydrogen among the products of fermen- 
tation of sugars by various microbes, and Escherich^ made surprisingly accurate 
balance sheets for the metabolism of some of the more common bacteria. 

Influenced, doubtless, by long familiarity with the yeast plant, by the remarkable 

' Kendall, A. I., Bly, R., and Haner, R. C: /. Infect. Dis., 32, 377. 1923. 

» Kendall, A. I., and Yoshida, S.: ibid., p. 362. 1923. 

3 Kendall, A. I., and Yoshida, S.: ibid., p. 369. 1923, ^ Nothnaf;el, H.: loc. cil. 

■» Kendall, A. I., and Yoshida, S.: ibid., p. 355. 1923. i Brieger, L.: loc. cil. 

5 Pasteur, L.: loc. cil. * Escherich, T.: loc. cit. 



ARTHUR ISAAC KENDALL 237 

work of Fischer upon the structure of carbohydrates, and by the discovery of hexose 
phosphate by Harden and Young,' attention has been paid by later investigators to 
the mechanism of sugar fermentation by yeast. The obvious complexity of the process 
and the relative ease with which suitable amounts of yeast may be obtained have 
together focused attention upon the fermentation activities of the Saccharomycetes 
to the virtual exclusion of the bacteria. 

However, the products resulting from yeast fermentation have at least some quali- 
tative resemblance to those produced by certain types of bacteria and, in the light of 
studies made in the earlier days of bacteriology by Frankland and Frew,^ Frankland 
and Lumsden,5 Harden,^ and more recently by the highly significant work of Neuberg 
and his associates,^ much light has been shed upon some of the bacterial fermentations. 

The Neuberg equations. — Neuberg^ has propounded three principal types of 
fermentation, aerogenic in character, and relating especially to yeast: 

Type I: C6Hu06 = 2CO.+ 2CH3 • CH.OH . 
(Glucose) (Alcohol) 

This is the classical Gay-Lussac equation.^ 

Typell: aH^.Oa + Na.S03+H.O = C3H8O3 + CH3 CHO • NaHS03+NaHC03. 
(Glucose) (Sodium sulphite) (Glycerol) (Acetaldehyde- 

sulphite compound) 

This type is given in the presence of sodium sulphide (Abfangverfahren). The sul- 
phite protects the acetaldehyde from secondary change; the yield of carbon dioxide 
and of alcohol is diminished and the yield of glycerol is increased. 

Type III (in the presence of alkali) is presumed to occur in three stages, as 
follows: 

Type III: (a) C6H:.06 = CH3CHOH • COOH+CH3 • CO • COOH+H, 
(Glucose) (Lactic acid) (Pyruvic acid) 

(b) 2CHs • CO • COOH = 2CH3 • COH+2CO. 

(Acetaldehyde) 

(c) 2CH3 • CHO+H.O = C.,H50H+CH3 • COOK 

(Alcohol) (Acetic acid) 

The completed reaction, therefore, becomes: 

2C6Hx.06+H.O=2CH3 • CHOH • COOH+C.H3OH+CH3 • COOH+2CO.+ 2H. . 

'Harden, A., and Young, W. J.: Proc. Chem. Soc, 21, 189. 1905; Proc. Roy. Soc. (Series B), 
80, 299. 1908. 

^ Frankland, P. F., and Frew, W.: J . Chem. Soc, 61, 254. 1892. 

3 Frankland, P. F., and Lumsden, J. S.: ibid., p. 432. 1892. 

••Harden, A.: ibid., 79, 612. 1901. 

sSee especially Abderhalden, E.: Ilandb. d. biol. Arbeilsmelh., Abt. IV, pp. 565, 593, 615, 625. 
1927; Oppenheimer, C: Handb. d. Bioch. d. Menschen u. Thiere (H. Aufl.), 2, 422. 1924. 

* Neuberg, C, and Hirsch, J.: Biocliem. Ztschr., 100, 304. 1919. 

7 Gay-Lussac, L. J.: loc. cil. 



238 UTILIZATION OF CARBOHYDRATES 

The Cannizzaro reaction in step (c), whereby in the presence of alkaH two molecules 

of aldehyde are reduced and oxidized, respectively, into a molecule each of acid and 

of alcohol, has been termed "dismutation" by Neuberg. 

This third type of fermentation is virtually that worked out by Harden' for B. coli 

many years ago. Harden believed that at least three separate and distinct enzymatic 

processes were involved; the first of these results in the change of glucose to lactic 

acid: 

C6H..06=2CH3 . CHOH • COOH . 

The second was responsible for a second molecule of glucose being broken down into 
a molecule each of alcohol and acetic acid, and two molecules of formic acid: 

C6H:.Oo+H,0 = C.H50H+CH3 • COOH+2H • COOH . 

The third reaction involved the cleavage of formic acid by the enzyme formiase into 
carbon dioxide and hydrogen : 

2H-COOH = 2H.+ 2CO. . 

The production and subsequent cleavage of formic acid by micro-organisms seems 
to be a point of much discussion. Schade- and others believe that the fermentation 
of glucose by yeast involves the production of formic acid, but this view has been 
unacceptable to many subsequent observers, partly on the ground that the yeast 
plant cannot decompose formates with the evolution of gas.^ On the other hand, there 
is much evidence that formic acid is produced under widely differing conditions during 
the utilization of glucose for energy and that this formic acid may or may not be 
decomposed subsequently into CO2 and Hj. The enzyme "formiase" is found in the 
press juice from muscles^ and especially in cultures of certain bacteria. ^ According to 
Clark,* one of the chemical differences between bacteria that produce gas from fer- 
menting sugars, and those that produce acid but no gas under parallel conditions, is 
the presence of formiase in the enzyme armamentarium of the former and its absence 
among the latter. According to this view, most of the common bacteria produce 
formic acid during fermentation. Gas-forming bacteria, as the members of the para- 
typhoid-colon-proteus-mucosus-capsulatus groups, and many anaerobes, which pos- 
sess formiase in their enzyme equipment, are able to transform formic acid more or 
less completely into CO, and H2. Many if indeed not a decided majority of bacteria 
that do not produce gas also produce formic acid, but it accumulates in the culture. 
This is certainly true of the typhoid bacillus,^ cultures of which, grown in the presence 

' Harden, A. : loc. cit. 

2 Schade, H.: Biochem. Ztschr., 7, 299. 1908. 

3 See Thomas, K.: Conipt. rend. Acad, de Sc, 136, 1015. 1903. 

t See Stoklasa; J.: Ber. d. dent. Chem. Gesellsch., 38, 607. 1905; Battelli, F.: Compt. rend. Acad, 
de Sc, 138, 651. 1904. 

s See Pakes, W. C. C, and Jollyman, W. H.: /. Chem. Soc, 79, 386, 459. 1901; Franzen, H., and 
Greve, G.: Ztschr. f. physio! . Chemie, 64, 169. 1910. 

* Clark, W. M.: Science, 38, 669. 1913. 

'See Franzen, H., and Egger, F.: Ztschr. physiol. Chemie, 79. i77- 1912; 83, 226. 1913. 



ARTHUR ISAAC KENDALL 239 

of glucose, are found to be quite rich in formic acid even after forty-eight hours' 
incubation. 

According to Frankland and Frew,' the decomposition of formic acid is more 
complete under conditions where oxygen is rigorously excluded. It is worthy of note 
that the equation involving the cleavage of formic acid to carbon dioxide and hy- 
drogen does not involve the interreaction of H and OH ions; nearly all true enzymic 
processes do. 

Butyric acid fermentation. — Mention has already been made of Pasteur's dis- 
covery^ that certain anaerobic "vibrios" could produce butyric acid from lactates. 
This involves a synthetic process, whereby the four-carbon-atom chain of butyric 
acid is formed from the three-carbon-atom chain of the lactic acid. Neuberg and 
Arinstein^ have studied the butyric acid fermentation induced by Bacillus butylicus 
fitzianns, and find by the Anfangverfahren procedure that both butyric acid and butyl 
alcohol are produced if sulphites are added to the slightly alkaline cultural media. 
Acetaldehyde was also detected in these cultures, and it seems probable that a syn- 
thetic process is involved much like that noted by Pasteur more than sixty years ago. 

Acetic acid fermentation. — The industrial production of acetic acid from alcohol 
has long been practiced, but the process has not been very well understood other than 
that there was an oxidation of the alcohol to the acid. Recently Neuberg and Win- 
disch^ have brought forward evidence which seems to show that the process is some- 
what more intricate than a mere addition of oxygen to the alcohol molecule, thus: 

C,H30Hv C.H.OH. C.H5OH. 

CH3 . CHO*<' CH3 . CHO*<' CH3CHO,* etc. 

CH3 • COOK CH3COOH 

*Cannizzaro reaction. 

It is of great interest to recall that Liebig,^ over fifty years ago, showed that the 
change of alcohol to acetic acid took place in two steps: (i) removal of hydrogen from 
the ethyl alcohol molecule to form acetaldehyde, and (2) the addition of oxygen to 
the acetaldehyde molecule to form acetic acid. 

Glycerol fermentation. — Glycerol is fermented by many bacteria; some, like the 
Shiga type of dysentery bacillus, produce acid from this triatomic alcohol, while 
many members of the mucosus capsulatus group of bacteria evolve gas during its fer- 
mentation. Very little is known as yet of the chemical change involved. 

Voges-Proskauer reaction. — The three equations discussed above represent, as well 
as available information affords, the course of fermentation of simple sugars by the 
majority of ordinary bacteria, with and without gas production. Nevertheless, they 
apparently do not provide a complete summary of the products of fermentation 

• Frankland, P. F., and Frew, W.: loc. cit. 

^ Pasteur, L. : loc. cit. 

3 Neuberg, C, and Arinstein, B.: Blochem. Zlsckr., 117, 269. 1921. 

"i Neuberg, C, and Windisch, F.: Biochcm. Ztschr., 166, 454. 1925. 

5 Quoted by Hofmann, A. W.: Faraday Lecture for 1S75, p. 112. Macmillan and Co., 1876. 



240 UTILIZATION OF CARBOHYDRATES 

produced by some few microbes. Voges and Proskauer' described a color reaction 
that could be elicited in glucose cultures of certain bacteria by the addition of enough 
caustic potash solution to render the reaction of the medium very alkaline. Ordinari- 
ly, the color develops slowly in fermentation media, requiring from twenty-four to 
forty-eight hours to reach its maximum. The color thus evolved was reminiscent of 
the yellowish fluorescence exhibited by alcoholic solutions of eosin. 

Harden and Walpole' studied the reaction chemically and found acetylmethyl- 
carbinol (CHjCO- CHOH- CH,) was present in such media. They discovered, further- 
more, that this compound would not give the color with caustic potash, but that when 
the oxidization product of acetylmethylcarbinol. diacetyl (CH3'CO-CO'CH3) was 
obtained it reacted with some unknown constituent of peptone in the presence of 
caustic potash and gave the color very quickly and intensely. The steps involved 
seem to be: 

1 .C6H,.Oo->CH3 . CO • CHOH • CH3 , 

(Glucose) (Acetylmethylcarbinol) 

2 .CH3 • CO • CHOH • CH3-^CH3 • CO • CO • CH3 , 

(Diacetyl) 

3.CH3 • CO • CO • CH3+Peptone+KOH->Voges-Proskauer reaction . 

Grimbert^ found that certain spore-forming bacteria, as Bacillus suhtilis and 
Bacillus meseiitericus , gave this Voges-Proskauer reaction, and MacConkey^ added 
Bacillus lactis aerogenes, Bacillus cloacae, and certain members of the Bacillus 7nucosus 
capsulatus group to the list. Most strains of Bacillus coll do not give it.^ 

The reaction seems to be elicited quite definitely in cultures of certain bacteria 

which give a negative methyl-red reaction^ (low H-ion concentration) and a gas ratio 

(^O, 2 — ^ 

of -^ = . This includes the lactis aerogenes and mucosus capsulatus groups of 

H2 I 

bacteria in addition to Bacillus cloacae.'' 

Miscellaneous fermentations. — A considerable number of fermentations induced 
by bacteria that have significance in the industries and agriculture have been report- 
ed. Some of these, as the organisms which produce mannitol, together with lactic 
and acetic acids, and carbon dioxide from fructose* are important not only from the 
viewpoint of their relation to the wine, sauerkraut, and other similar industries, but 
also because they provide a starting-point for the study of biologically induced 

' Voges, O., and Proskauer, B.: Ztschr.f. Ilyg. 11. Infektionskranhh., 28, 20. 1898. 

= Harden, A., and Walpole, S. G.: Proc. Roy. Soc. (Ser. B), 77, sqq. 1906; Harden, A.: ibid., 
p. 424. 1906. 

J Grimbert, L.: Compt. rend. Soc. de hiol., 53, 304. 1901. 

■•MacConkey, A.: J. Hyg., 5, 349. 1905. 

sLevine, M.: Official Publication, Iowa Slate College Agrinilt. &" Afecli. Arts, 15, No. 14. 1916. 

'Rogers, L. A., Clark, W. M., and Evans, A. C: /. Infect. Dis., 15, 100. 1914; 17, 137. 1915; 
Perkins, R.: ibid., 37, 232. 1925; Paine, F. S.: J. Bad., 13, 269. 1927. 

' MacConkey, A.: loc. cit. 

8 See Stiles, H. R., Peterson, W. H., and Fred, F. B.: /. Biol. Cliem., 64, 643. 1925. 



ARTHUR ISAAC KENDALL 241 

transformations in the group of the hexoses and related compounds. Others, as 
Bacillus granulohacter pcctinovorum, are notewortliy because they are used on a hirge 
scale to produce acetone and butyl alcohol and other solvents which are indispensable 
to industry.' The consideration of these, and other technical fermentation procedures, 
however, is beyond the scope of this discussion. 

BACTERIAL SYNERGISM 

Ten years ago Castellani and Taylor- called attention to a phenomenon, which, 
while it had undoubtedly been noticed before, had not apparently received the atten- 
tion it deserved. This phenomenon has recently been stated by Fiallos' as follows: 
"Two bacilli, neither of which causes the production of gas in certain compounds, may 
do so when artificially mixed together, provided one of them is capable of producing 
simple acidity (never gas) in these compounds, and the other, though inert to these 
compounds (i.e., produces neither acid nor gas), is capable of producing gas from glu- 
cose." Many observers have studied this phenomenon recently^ and have amply 
confirmed the facts, but the explanation is not agreed upon; also, several different 
types of chemical activity are included in this group of reactions. The most common 
of these is the one described by Fiallos.^ It is quite clear that all of the phenomena 
thus far discovered are of the nature of coupled reactions. 

The following specific instance may be mentioned: a staphylococcus and Bacillus 
paratyphosus acting together produce acid and gas from nutrient lactose-fermentation 
media, although the staphylococcus alone merely produces acid, but no gas from this 
sugar, and Bacillus paratyphosus fails to utilize the sugar at all. Examples might be 
multiplied almost indefinitely, but in each instance it will be found that one of the 
coupled reacting organisms must produce acid but no gas from the carbohydrate; 
the other coupled organism need have no action whatsoever upon the carbohydrate 
but must be capable of producing CO, and H, from formates. All the known bacteria 
that liberate CO, and H2 from formates will also produce gas and acid in the usual 
nutrient glucose media. The addition of nitrates (sodium or potassium nitrate) in 
small amounts will prevent the formation of gas either in glucose solution or in for- 
mate solution. 

Takes and Jollyman^ long ago showed that bacteria which produce gas from 
nutrient sodium-formate media also produce gas from nutrient glucose solutions with 
the exception of many, if not most, yeasts; they also showed the converse to be true 
— namely, that bacteria which do not produce gas from formate do not produce gas 
from glucose. They also made extensive studies of the effect of nitrates upon the 
reaction and found that the nitrates interfere with gas production in accordance with 
the following equation: 

H . COONa+NaN03 = NaHC03-FNaNO. . 

' See, for details, Fred, E. B., Peterson, W. H., and Mulvania, M.: J. Bad., 11, 323. 1926. 
' Castellani, A., and Taylor, F. E.: Brit. M. J., 2, 855. 1917; i, 183. 1919. 
J Fiallos, J. M.: /. Trop. Med., 28, 426. 1925. 

* Much of the later literature is reviewed by Holman, W. L.: J . Infect. Dis., 39, i ^5. 1926. He 
proposed the term "synergism." Cf. chap, viii in this volume. 

sPakes, W. C. C, and Jollyman, W. H. : /. Cliein. Soc, 79, 386, 450. 1901. 



242 UTILIZATION OF CARBOHYDRATES 

It is by no means definitely proved that the mechanism of the reaction in every 
instance involves the formation of formic acid by the fermenting member, but this 
certainly happens in at least some of the synergetic couples tried thus far: in fact, 
formic acid has been separated and measured with considerable precision from several 
of these synergetic reactions. 

This reaction has significance in several fields of bacteriology: It is of prime im- 
portance, as Sears and Putnam pointed out' in the "presumptive test" for B. coli 
in water. It has an important bearing upon the gas formation by intestinal bacteria, 
and it occasionally leads to false conclusions with respect to gas formation in contam- 
inated and in mixed cultures. 

Another type of coupled reaction is one described by Kendall- in relation to gas 
formation in milk by B. coli and an associated, actively proteolytic organism. Under 
normal conditions B. coli does not produce gas in milk, although this medium is rich 
in lactose, but does ferment lactose energetically in nutrient fermentation media with 
a considerable evolution of gas. If, however, B. coli be inoculated into milk together 
with an actively growing, strongly proteolytic strain of microbe which does not fer- 
ment lactose, as, for example, B. mesentericus, gas is formed in considerable amounts. 

Here the explanation seems to depend upon the cleavage of the casein by the 
proteolyte with the liberation of amino acid complexes. These in turn, in some man- 
ner not well understood, cause the colon bacillus to liberate gas in addition to the 
acid which it normally produces in milk. Similar results may be obtained by digesting 
the milk prior to inoculation, with trypsin or by repeated heating of the milk, which 
of course tends to break down the protein, and thus accomplish the same end as the 
cleavage by a proteolyte. 

Coupled reactions are as yet but little studied, but there is little doubt that some 
very important results await discovery in this field of bacterial synergism. 

' Sears, H. J., and Putnam, J. J.: /. Infect. Dis., 32, 270. 1923. 
* Kendall, A. I.: Boston M. b' S. J., 163, 322. 1910. 



CHAPTER XVII 

UTILIZATION OF ALIPHATIC AND AROMATIC 
COMPOUNDS BY BACTERIA 

S. A. KOSER 

University of Illinois 

Investigations of the carbon and nitrogen requirements of bacteria have directed 
attention to the relatively simple compounds of these elements which in many cases 
appear to be readily utilized by the organisms, both as a source of structural material 
for building new protoplasm and also as a source of energy. The utilization of the com- 
moner carbohydrates which are widely used in the ordinary fermentation tests is well 
known. In addition to these, however, there are many other chemical groups which 
are susceptible to bacterial attack. In the amino acids, the purines and extractives, 
the alcohols, and the organic acids or their salts, we have a wide range of compounds 
of definite chemical structure in which the nitrogen or carbon is supplied in a great 
variety of different combinations. 

The utilization of these compounds has been studied in several ways, by qualita- 
tive or quantitative tests for a certain component of a complex medium and by the 
use of isolated compounds in chemically definite media. In the latter case an amino 
acid, for example, may be added to a solution of certain inorganic salts so that it con- 
stitutes the sole supply of available nitrogen. By varying the amino acids or other 
nitrogenous compounds we can determine the ability of the organism to make use of 
certain chemical groupings. Also, by analysis of cultures developing under these con- 
ditions we can determine the products formed and thereby gain an insight into the 
mode of attack and breakdown which the organism employs. It does not necessarily 
follow, of course, that the same type of decomposition will take place under other con- 
ditions or in the presence of other foodstuffs. 

UTILIZATION OF NITROGENOUS COMPOUNDS 

The common observation that most bacteria can develop readily in digests of 
protein material has led to investigation of the availability of single, isolated amino 
acids or other simple compounds of nitrogen. Added impetus has perhaps been given 
to this line of study by the studies on animal nutrition in which it has been shown 
that certain amino acids are indispensable for growth. In considering the nitrogen 
requirements of bacteria the question arose whether certain amino acids are also in- 
dispensable for the growth and reproduction of these forms. Can the inability of 
certain pathogens to develop on ordinary media be explained by the lack of certain 
essential components of nitrogen which are not supplied by the usual laboratory 
media? May different species or groups of bacteria be distinguished from one another 
by the ability of one to utilize certain simple compounds which the other is unable to 
attack? Can the various toxin-producing types make use of simple amino acids and 
if so is the toxin elaborated in their presence? 

243 



244 ALIPHATIC AND AROMATIC COMPOUNDS. 

Investigations along this line have been many and varied, and the results at times 
have been contradictory.' In spite of this, however, certain general conclusions may be 
drawn. A number of the commoner saprophytic types, B. fluorescens, B. pyocyancus, 
B. prodigiosus, B. proteus, B. coli, and probably others, are able to satisfy their ni- 
trogen and carbon requirements when a single mono-amino acid such as alanine is 
added to a solution of certain inorganic salts, though the development under such 
conditions will not be as rapid or luxuriant as in nutrient broth. If a readily available 
source of carbon such as lactic acid, glycerol, or dextrose is also supplied, the value of 
the medium is enhanced and certain organisms which experienced difficulty in appro- 
priating the amino acid for their own uses will speedily break down these more readily 
available sources of carbon and energy, and at the same time derive their nitrogen 
from the amino acid. A surprising number of organisms are able to develop under 
such conditions. Among the better-known forms are the green fluorescent bacilli, B. 
pyocyaneus, B. prodigiosus, B. proteus, the coli-aerogenes group, certain members of 
the paratyphoid group, and a few spirilla. The tubercle bacillus and other acid fast 
types are also able to satisfy their nutritive requirements from very simple sources 
of nitrogen and carbon.^ Most of the pathogens, however, are unable to develop 
under these conditions, and negative results have usually been reported for the ty- 
phoid and dysentery bacilli, B. abortus, B. diplitheriae, the pneumococcus, streptococci, 
and staphylococci of various types, to mention only some of the commoner ones. 

The question of the relative values of the various amino acids may be regarded as 
still an open one, for there is much conflicting evidence. In many instances an or- 
ganism, if able to satisfy its nitrogen requirements from one of the mono-amino acids, 
may be expected to make use of the others, although exceptions to this have been re- 
ported. Lysine, arginine, histidine, and tryptophane are other amino acids which offer 
additional nitrogenous groupings. It has been claimed that they possess an added 
nutritive value in some instances while in others they appear to be of no more value 
than the simpler mono-amino acids. Dipeptides, also, are susceptible to bacterial 
attack^. Glycyl-glycine, glycyl-tyrosine, glycyl-tryptophane, and other combina- 
tions are split by a number of organisms, and the separate amino acids may be re- 
covered if they are not in turn utilized. Finally, in considering the utilization of amino 
acids or related compounds it should not be overlooked that at least certain of them 
have been found to exert a toxic or inhibitory effect upon the growth of some of the 
more "fastidious" bacteria.^ 

The mode of attack and the steps in the breakdown of the amino acids present an 
interesting though a complex problem, for we may have both nitrogen and carbon 

' For reviews of earlier work and bibliography see Gordon, M. H.: J . Roy. Army M. Corps, 28, 
371. 1917; Koser, S. A., and Rettger, L. F.: J. Infect. Dis., 24, 301. 1919; Braun, H., and Cahn- 
Bronner, C. E.: Cenlralbl.f. Baklcriol., Abt. I, Orig., 86, 196 and 380. 1921; Krasnow, F.,Rivkin, H., 
and Rosenberg, M. L.: /. Bad., 12, 385. 1926; den Dooren de Jong, L. E.: Bijdragc tot de kennis van 
lift mineralisaticproccs, pp. 1-200. Rotterdam: Nijgh and Van t)itmar, 1926. 

^ Long, E. R.: Am. Rev. Tiibcrc, 3, 86. 1919; 5, 857. 1922. 

3 Sasaki, T.: Biochem. Zlsclir., 41, 174; 47, 463 and 472. 1912; Otsuka, I.: Physiol. Ahst., 2, 
15. 1917, 

■• Gordon, J., and MrLeod, J. W.: ./. Path. & Bail., 29, 13. 1926. 



S. A. KOSER 245 

supplied in many different groupings. In addition, various methods of decomposition 
are doubtless brought into play by different organisms and certain other factors exert 
a pronounced influence upon the process, such as the supply of oxygen, the tonicity 
of the medium, and the presence of other available nitrogenous and non-nitrogenous 
compounds. 

The initial step in the process of utilization is usually a deamination or decar- 
boxylation. Deamination may be accompHshed in a variety of ways: by reduction 
with the formation of a saturated fatty acid, by oxidation with the formation of a 
ketone acid, and by hydrolysis with the formation of a hydroxy acid. A simple mono- 
amino acid such as alanine may be broken down as follows: 

CHj-CH-NH^-COOH-h H. . . . .CH3-CH.-COOH-1-NH3 

Propionic acid 
CH3.CH-NH..C00H-h O . . . .CH3-CO-COOH+NH3 

Pyruvic acid 
CH3-CH-NH.-C00H-HH,0. . . .CH3-CHOH-COOH-HNH3 

Lactic acid 

The ring structures such as those found in tyrosine, proline, histidine, and trypto- 
phane, though not invulnerable, are more resistant to bacterial attack, and in many 
instances only the side chain is used.' In tryptophane, for example, we have nitrogen 
in the alanine side chain and in the indol ring. The side chain is utilized by many or- 
ganisms while the ring structure is frequently left intact in the form of indol-propionic 
or -acetic acid, skatol, or indol. Another type of change has been described by Raist- 
rickj^" who found that histidine was converted to urocanic acid, an unsaturated acid, 
by a number of organisms. 



HC 
\ 



N— C • CH. • CH • NH. • COOH N— C • CH = CH • COOH 



/ 
\ 



+NH3 



N— C N— C 

H H H H 



Decarboxylation of an amino acid results in the formation of an amine by the 
loss of CO2: 

CH3-CH-NH.-C00H — > OTrCH.-NH.+ CO, . 

Ethyl amine 

The ethyl amine formed from alanine may in turn be hydrolyzed to form ethyl alcohol 
and ammonia: 

CH3-CH.-NH.+H.0 > CH3CH.OH4-NH3. 

Other amino acids may be changed in a similar way. Histidine is converted into 
histamine, lysine yields cadaverine, and tyrosine yields tyramine.^ It is of special in- 

• Raistrick, H.: Biochem. J., 13, 446. 1919; Raistrick, H., and Clark, A. B.: ibid., 15,76. 1921; 
Hanke, M. T., and Koessler, K. K.: J. Biol. Ckem., 50, 131. 1922; Long, E. R.: Am. Rev. Tiiberc, 
5, S57- 1922. 

-' Raistrick, H.: Biochem. J., 11, 71. 191 7. 

^ Hanke, M. T., and Koessler, K. K.: /. Biol. Chcm., 39, 539. 1919; 50, 131, 1922; 59, 835. 1924. 



246 



ALIPHATIC AND AROMATIC COMPOUNDS 



terest that certain of these amines are significant from the pharmacological stand- 
point. 

In addition to the amino acids, other compounds may be considered briefly. The 
purines and their oxidation products, the pyrimidines such as cytosine, uracil, and 
thymine, the extractive bodies, creatine, creatinine, carnosine, and others present 
another series of nitrogenous compounds. The utilization of these bodies by various 
types of micro-organisms has not been studied so extensively as that of the amino 
acids, and our knowledge concerning them is rather fragmentary. Hypoxanthine, 
xanthine, and uric acid contain nitrogen only in the purine ring. Evidently nitrogen 
in such form is of no value to a great many organisms, for many of those which are 
able to make use of isolated amino acids are unable to develop when the foregoing 
purines are supplied as the sole source' of nitrogen. On the other hand, allantoin, 
which is an oxidation product of uric acid and contains a free amino group, frequently 
yields results quite similar to an amino acid.' In a similar way, it might be expected 
that the amino purines, adenine and guanine, could be utilized by a number of or- 
ganisms which are unable to make use of nitrogen in the purine ring. 

NON-NITROGENOUS COMPOUNDS 

Of the non-nitrogenous organic compounds which are used by micro-organisms 
the best known are the carbohydrates, higher alcohols, and organic acids. Only the 
last group will be dealt with here since the others are treated elsewhere in this volume'. 
The structure of some of the commoner organic acids is shown below : Here we have 

Formic H-COOH Malic CHOH-COOH 



Acetic CH,-COOH 
Propionic CH3-CH.-C00H 

n-Butyric CH3-(CH.).-C00H 

Glycollic CH.OH-COOH 

Lactic CH3 • CHOH • COOH 

Pyruvic CH3-C0-C00H 

Oxalic COOH 

COOH 

/COOH 
Malonic CH2 

\COOH 

Succinic CH.-COOH 

I 
CH.-COOH 

Maleic CH-COOH 
Fumaric || 

CH-COOH 

■ Koser, S. A., and Rettger, L. F. : loc. cit. 



CH.-COOH 

Tartaric CHOH-COOH 

I 
CHOH-COOH 

Glutaric CH.-COOH 

I 
CH. 

CH.-COOH 

Adipic CH.-COOH 

(CH.). 

I 
CH.-COOH 

Citric CH.-COOH 

C(OH) • COOH 

CH.-COOH 

CH-COOH 
C6H,(0H)-C00H 



Benzoic 
Salicylic 



- Chapter xvi. 



S. A. KOSER 247 

the fatty-acid series and a few of their hydroxy derivatives, some members of the 
dicarboxyUc and tricarboxyHc series, and aromatic acids containing the phenyl radical. 
The outline will serve to show the wide variety of chemical groupings which are pre- 
sented. 

In the presence of a suitable source of nitrogen many of these acids or their salts 
are broken down and utilized, though a few appear to be relatively resistant to bac- 
terial attack. Benzoic and salicylic acids, in which the carbon is contained in the car- 
boxyl group and the benzene ring, appear to present a formidable obstacle to most 
organisms, and very few types are able to make use of them. Oxalic acid with two 
carboxyl groups joined directly is also resistant to most bacteria. Whether this is due 
to an inability of the organism to make use of this type of structure or whether it is 
due to the poisonous character of the compound is not clear. When the two carboxyls 
are linked by a methyl group, as in malonic acid, the compound is attacked by certain 
organisms which are unable to make use of oxalic acid, for example, some members 
of the alcaligenes group and the coli-aerogenes group. Succinic acid appears to lend 
itself still more readily to bacterial attack, surprising as this may seem, for it is rela- 
tively resistant to chemical oxidizing agents. Many of the other acids are readily 
utilized by the commoner saprophytic bacteria and molds, and in some cases by 
pathogenic types.' Lactic acid, for example, when supplied as the only source of car- 
bon and with a suitable inorganic nitrogen compound will support development of the 
coli-aerogenes group, the alcaligenes group, the green fluorescent bacilli, and even cer- 
tain members of the paratyphoid group. 

A comparison of the structural formulas of these acids with their utiHzation by 
various types or groups of organisms presents an interesting field though it is impossi- 
ble to review the results at length here. Suffice it to say that a slight change in the 
structure of a compound or even the presentation of a different isomer may alter 
materially its availability for certain organisms though perhaps not for others. 
Furthermore, we are not always able to predict the utilization of a compound by 
micro-organisms from a consideration of their behavior toward a similar chemical 
grouping in another compound. 

As might be expected, the utilization of organic acid salts is influenced markedly 
by the oxygen supply. Under aerobic conditions many bacteria will develop in a much 
simpler medium than they require for anaerobic life. Coliform organisms can develop 
in a simple inorganic salt medium with ammonium lactate under aerobic conditions, 
while under anaerobic conditions other substances such as a fermentable sugar are 
required. Supposedly, the sugar is required for anaerobic growth because it can be 
disintegrated without the intervention of oxygen in such a way as to supply energy, 
whfle this is not possible with salts of lactic acid.^ 

In the breakdown of the organic acids akaline end-products are frequently 
formed. These are usually carbonates and bicarbonates. The decomposition of the 

■ den Dooren de Jong, L. E.: loc. cil.; Avers, S. H., Rupp, P., and Johnson, W. T.: U.S. Depl. 
Agr., Bull. 782. IQ19; Koser, S. A.: J. Bad., 8, 493. 1923; Brown, H. C, Duncan, J. T., and Henry, 
T. A.: J. Hyg., 23, i. 1924. 

^ Braun, H., and Cahn-Bronner, C. E. : loc. cit.; Stephenson, M., and Whetham, M. D. : Biochcm. 
J ., 18, 498. 1924; Quastel, J. H., and Stephenson, M.: ibid., 19, 660. 1925. 



248 



ALIPHATIC AND AROMATIC COMPOUNDS 



calcium and sodium salts of formic acid represents a simple case and may be shown 
as follows: 

Ca(COOH).+H30 = CaC03+CO.+ 2H, , 

Na(COOH) +H.0=NaHC03+H, . 

The manner of decomposition of other organic acids has been studied in some in- 
stances. A review of much of the older work is given by Harden.' More recently the 
decomposition of citric acid has been studied by Brown, Duncan, and Henry, ^ and 
fumaric, succinic, and pyruvic acids by Aubel' and Quastel.'' One example of the type 
of breakdown accomplished by bacteria may be given, namely, the decomposition of 
fumaric acid, an unsaturated dicarboxylic acid, by B. pyocyaneus.^ The main course 
of fermentation is believed to proceed as follows with acetic acid and carbon dioxide 
as the final products: 



COOH COOH 

1 1 


COOH 


CH +0 COH 

II ->ll 


CO 


II -^11 ■> 
CH CH 
1 1 


CH3 

1 


COOH COOH 


COOH 


Fumaric 





COOH 



COOH 



CO +0 CH, 

I +C0. > +C0. 

CH, 



Pyruvic 



It has been suggested that the utilization of certain of the simpler aliphatic or 
aromatic compounds might be used as a basis for the separation of different species 
or groups of bacteria. A few of the more recent suggestions will serve to illustrate the 
point. In a study of the alcaligenes group, Ayers, Rupp, and Johnson^ used organic 
acid salts as test substances instead of the usual carbohydrates which in many in- 
stances are not attacked by this group of bacteria. It has been further shown by Ayers 
and Rupp7 that sodium hippurate is hydrolyzed to benzoic acid and glycocoU by 
hemolytic streptococci of bovine origin but not by those of human origin. 

The availability of uric acid for certain members of the coli-aerogenes group has 
been used to distinguish the coli from the aerogenes-cloacae subgroup.^ When given 
this compound as the only source of nitrogen, B. aerogenes and its allies are able to 
split the purine ring whereas B. coli lacks this ability. A somewhat similar distinction 
has also been made, based on the utilization of citric acid by this group of organisms.'' 
When this acid in the form of its sodium, potassium, or ammonium salt is supplied as 
the sole source of carbon, fecal strains of B. coli are unable to develop. They apparent- 
ly lack the ability to make use of the citrate radical while closely related types of soil 

' Harden, A.: Thorpe's Diclionary of Applied Chemistry, pp. 502-36. 191 2. 
' Brown, H. C, Duncan, J. T., and Henry, T. A.: loc. cil. 
3 Aubel, E.: Bull. Soc. Chim. Biol., 6, 288. 1924. 
^Quastel, J. H.: Biochem. J., 18, 365. 1924; 19, 641. 1925. 
5 Ibid. 

* Ayers, S. H., Rupp, P., and Johnson, VV. T.: loc. cit. 

7 Ayers, S. H., and Rupp, P.: ./. Infect. Dis., 30,388. 1922. 

* Koser, S. A.: ibid., 23,377. 191S. ' Koser, S. A.: ./. Bact., 8,493. 1923. 



i 



S. A. KOSER " 249 

origin — aerogenes and others — are able to break down the citrate and to appropriate 
it for their own needs. The salts of organic acids have also been suggested for sepa- 
rating different types within the paratyphoid group. By the use of citrate, d-, 1-, and 
m-tartrate, fumarate and mucate, certain distinctions are brought out.' These may 
be especially useful in this or other groups of organisms where sugar fermentations 
at times fail to differentiate certain serologically well-defined types. 

A consideration of the salient points in the utilization of aliphatic and aromatic 
compounds brings out in a striking manner the ability of many bacteria to satisfy 
their food and energy recjuirements from the simpler chemical compounds. Many bac- 
teria, like plants, are able to build up their own protoplasmic structure from a variety 
of non-nitrogenous organic substances and ammonia. One is impressed by the wide 
range of chemical groupings which may be broken down and appropriated by the 
micro-organisms for their own needs. Nitrogen may be utilized in various forms: as 
an inorganic ammonium compound and in the amino, amide, or imino groups. Partic- 
ularly striking is the ability of some organisms to tear apart complex ring structures 
such as the imidazole or purine rings. Others apparently are not so well equipped with 
the necessary tools for making use of such structures and are compelled to satisfy 
their needs by the utihzation of a side chain while the ring structure is left intact. 
Whatever the form of nitrogenous organic grouping attacked, ammonia is usually 
liberated. After separation of the nitrogen in this form the carbonaceous residue, an 
organic acid or perhaps an alcohol, may be further used by the organism. It may be 
oxidized to supply energy, or perhaps rearranged and combined with ammonia for 
synthesis of the organism's own particular protoplasm, or if a more readily available 
source of carbon is present to satisfy these purposes, it may remain as an end-product 
of metabolism. 

' Brown, H. C, Duncan, J. T., and Henry, T. A.: loc. cit. 



CHAPTER XVIII 

GAS METABOLISM OF BACTERIA 

M. H. SOULE 

Hygienic Laboratory, University of Michigan 

INTRODUCTION 

It is questionable whether any gas other than oxygen is absolutely necessary for 
the activities of the cell. The fixation of atmospheric N2, the oxidation of H2S to S 
and H2O, etc., are activities independent of the main function of respiration. The 
working hypothesis has been laid down that CO2 is essential as a stimulus for the 
growth of organisms. It will be shown later that this hypothesis probably holds true for 
all organisms just as this gas is required for the life of the higher animals. It is not 
utilized as a source of food or energy but it maintains a physico-chemical equilibrium 
within the cell. 

The study of gas changes produced by micro-organisms is of rather recent date. 
The combustion theory of respiration initiated by Lavoisier' was extended to all kinds 
of animals by Spallanzani,^ and was developed by Pasteur (1859, 1861 ff.) in his work 
on fermentations. This was followed by many isolated studies such as those of Buch- 
ner^ on the Fitz bacillus and of Escherich^ on B. coli and B. lactis aero genes. Hesse^ 
demonstrated CO2 production and O2 absorption by eight organisms which were not 
of the aerogenic type. These results were questioned at first by Scheurlen,^ who 
denied that bacteria, like animals, could respire and, with Buchner, he thought that 
CO2 was the result of the action of acids on the carbonates of the medium. Eventu- 
ally, however, he confirmed Hesse's work by finding that every one of one hundred 
and forty-one strains of bacteria which he examined produced CO2. In the meantime, 
Winogradsky^ had conclusively proved that certain bacteria fix atmospheric nitrogen 
— a fact indicated many years before by the work of Berthelot^ on soils. The pres- 
ence of CH4, N2, H2, and H2S in intestinal gases had been reported by various ob- 
servers as mentioned by Escherich,' but exact quantitative determinations of the gas- 
eous metabolism of the organisms producing these substances have not been made. 

The early methods employed in the study of gas changes by bacteria were some- 

' Lavoisier, A. L.: Hist, et Mem. de I'Acad. de sc. Paris, 17S0; Mem., Annee, p. 185. 1777. 

* Spallanzani, L.: Dissertazioni Varie, Memorie sidla Respirazione, Vol. 2. 1826. 

3 Buchner, E.: Zlschr.f. phys. Chemie, 8, 367-90. 1884. 

■f Escherich, T. : Die Darmbahlerien des Sdiiglings und ihrc Beziehiingcn zur Physiologic der 
Verdauung, pp. 128-33. Stuttgart: Enke, 1886. 

s Hesse, W.: Ztschr. f. Hyg. u. Infeklionskrankh., 13, 17-37; 183-91- 1S93; 25,477-81. 1897; 
Arch. f. Hyg., 2S, S07 -11- 1897. 

^ Scheurlen, E.: Arch. f. Hyg., 26, 1-29. 1S96; Inkrnat. Beilr. 2; inn. Med. zur Feicr jo jdhrigen 
Gehurtstages E. von Leyden, 2, 205-7. Berlin: Hirschwald, 1900. 

' Winogradslcy, S.: Compt. rend. Acad. Sc, 116, 1385. 1893. 

SBerthelot, M.: ibid., 85, 178. 1877. » Escherich, T.: loc. cit. 

250 



M. H. SOULE 251 

what complex and often gave very inexact results. In 1913 the literature was re- 
viewed in detail by Frieber' and, during the following year, by Rogers, Clark, and 
Davis^ who contributed a valuable supplement to Frieber's study; in both papers the 
untrustworthy nature of the recorded bacteriological gas analyses was deplored and 
new methods were suggested. In each of these investigations the metabolism of B. 
coli was studied. This germ, ordinarily considered an aerobe, was grown in broth, 
in vacuo, under anaerobic conditions, and the gases formed were analyzed. Anderson^ 
modified the technique of Rogers and his co-workers and made accurate quantitative 
estimations of the gaseous components evolved by several strict anaerobes. The pro- 
cedure at best has a very limited application. 

METHODS 

As already stated, the methods which have been employed in the study of gas changes by 
bacteria as a rule have been somewhat complex. It is beyond the scope of this article to re- 
view these earlier investigations on gas metabolism. The recent studies of Novy, Roehm 
and Soule^ have resulted in methods which are of general applicability. They are equally 
suitable for the study of the respiration of organisms when grown on solid or liquid media, 
in tubes, flasks, or on plates, either in an atmosphere of air or in varying concentrations of 
O2, CO2, N2, or other gases. The technique not only permits of accurate manometric observa- 
tions over extended periods of time, but also allows the easy withdrawal of samples of the 
contained gases for the purpose of analysis. These methods have been utilized in the study 
of a large number of organisms, including various protozoa as well as plant tissue. For full 
details the reader is referred to the original memoirs. 

It is essential in the study of gases to be able to observe the pressure changes which take 
place within the respiratory chamber. A manometer usually not only reveals whether an or- 
ganism is alive and growing, but also indicates the point when growth or respiration ceases. 
Some investigators have made use of ordinary manometers attached to their culture flasks. 
The instrument devised by Barcroft^ is well known and, as modified by Brodie,^ has been 
used for diverse respiration studies such as those on excised tissues by Warburg^ and others. 

It is desirable to have a manometer which is highly sensitive and yet independent of 
variations in atmospheric pressure. These conditions are realized in the compensation mano- 
meter. This instrument is shown in Figure i, attached to a glass-capped //-tube of about 
loo-cc. capacity; in Figure 2 it is connected to a Novy jar. For details of construction and 
calibration the original paper should be consulted. 

When an experiment is in progress the apparatus is kept at constant temperature and 
cocks I and 3 on the manometer remain closed. The system is not subject, therefore, to 
changes in barometric pressure, and any variation in the mercury level is due entirely to the 
gas exchange of the germs. This is extremely important when an experiment extends over a 
period of time. 

The purpose of stopcock 2 is twofold: (i) to shut off the manometer when the gas pres- 

' Frieber, W.: Centralbl. f. Baktcriol., Abt. I, Orig., 69, 437. 1913. 

^Rogers, L. A., Clark, W. M., and Davis, B. J.: /. Infect. Dis., 14, 411-75. 1914. 

3 Anderson, B. G.: ibid., 35, 213-81. 1924. 

'' Novy, F. G., Roehm, H. R., and Soule, M. H.: ibid., 36, 109-67. 1925. 

5 Barcroft, J.: Ergehn. der Physiol., 7, 772-75. 1908. 

^ Brodie, T. G.: /. Physiol., 39,391-96. 1909-10. . ■; ■',*■ / 

> ■ -^ ^ t\ "^ 
7 Warburg, O.: Biochent. Ztschr., 100, 230-70. 1919; 1923; 1924. / .;*. < ■ 

«*••'*- <*■ 
BRA 

'Was*' 



252 



GAS METABOLISM OF BACTERIA 




Fig. I. — Manometer and support with h-tuhe 




Fig. 2. — The Novy jar as a respiratory chamber, attached to a manometer 



' M. H. SOULE 253 

sure is likely to exceed the capacity of the U-tube; and (2) to enable one to withdraw gas 
from the culture tube without sucking over the mercury column. Likewise, when evacuating 
the culture tube with the object of refilling it with fresh air, or with O2, CO2, etc., this cock 
must be closed. At all other times it is kept open. 

Stopcocks I and 3 are used to equilibrate the mercury levels at the beginning of a test, 
after which both are closed. By means of tailcock 3 a sample of the gas content in the cul- 
ture tube or jar is easily withdrawn for analysis. It is also the means by which the gas within 
the culture apparatus is evacuated and replaced with fresh air, or with any desired gas. 

At this place it may be well to indicate the ease with which desired gas tensions may be 
approximated by the use of an evacuating apparatus which consists of a Chapman water 
pump, a vertical manometer, and a Woulff bottle. One tubulure of the latter is closed with a 
rubber stopper through which is inserted one of the main arms of a three-way cock. The 
midarm of this cock, horizontal in position, is joined to a glass Y-shaped connector with cocks 
on each arm. The upper main arm is attached to the descending limb of a vertical mano- 
meter. Another tubulure is connected with the pump by means of a three-way tailcock. This 
serves to disconnect the latter when the desired negative pressure has been obtained. 

If it is desired to introduce into a jar a certain percentage of CO2, for example, 40 per cent, 
this can be readily done. Assuming the barometer to read 750, and the thermometer 22.3°, 
the corresponding aqueous tension is 20 mm. of mercury. Hence 5 — / = 750— 20 = 730 
mm. and 40 per cent of this = 292. This value represents the partial tension of the gas to be 
admitted, provided it is dry. Connection is made to the jar by way of one arm of the Y; the 
cock on the other arm is closed. The pump is started and evacuation continued until the 
manometer registers 292 mm. The three-way cock is now turned so that the connection to 
the Woulff bottle is closed, and pure CO2 (from a tank or generator)' is run in through the 
other arm of the Y-connector until the manometer reads zero. By means of the foregoing 
procedure, it is possible to introduce into the jar any desired amount of a given gas or various 
mixtures of gas, and with care the desired tensions may be easily obtained within one-half 
of I per cent. For exact work a sample of the gas should always be withdrawn and analyzed. 
Satisfactory manometric readings may be obtained with culture tubes, but they are not suit- 
ed for the withdrawal of 10-20 cc. of gas for analysis. 

There is another important consideration, viz., the necessity of providing for a sufficient 
volume of O2 to permit the full growth of the organisms; for example, it was found that the 
human variety of the tubercle bacillus required at least 500 cc. of air to produce a rich growth 
in a tube. Hence if the //-tube, with a volume of 100 cc, is used, little or no visible growth 
will occur. For this reason it is preferable to make use of the large chamber or jar (Fig. 2) of 
known volume which can easily be made gas tight. These jars can be obtained in two sizes 
which have approximate air capacities of 2,400 and 3,400 cc, respectively. 

Culture tubes, flasks, or Petri plates may be placed in the jar which can then be used with 
air or filled with any desired gas. In order to develop quickly the full aqueous tension in the 
container it is necessary to place a few drops of distilled water on the floor of the jar. 

Rubber tubing is not desirable for connecting the respiratory chamber to a manometer. 
It is well known that CO2 diffuses through a rubber tube, and without doubt it would be 
preferable to have all-glass connections. However, a satisfactory connection can be made by 
bringing the end of the manometer into contact with the glass stopper of the respiratory 
chamber by means of the special No. 25 rubber stopper (Fig. 2). Negative or positive pres- 
sures of 200 mm. or more have held for weeks. 

The gases may be analyzed by any one of the many standard methods. The modified 

' Gases procured in ordinary commercial tanks must be purified before use or the percentage of 
foreign substances determined and allowed for in the calculations. 



254 



GAS METABOLISM OF BACTERIA 



Henderson-Haldane gas apparatus' is extremely accurate, and has proved very satisfactory. 
Figure 3 shows the method of connecting the gas burette with cock 3 of the manometer for 
the purpose of withdrawing a sample of the gas for analysis. The gas sample is then measured 




Fig. 3. — The modified Henderson-Haldane apparatus on stand with adjustable platforms to 
hold the manometer and culture tube or Novy jar. The gas sample is drawn from cock 3 on the 
manometer, through the capillary connector, into the burette. 



and analyzed. The CO2 is determined by absorption with KOH after which the O2 is re- 
moved by alkaline pyrogallate. If combustible gases are present, a known amount of O2 is 
now mixed with the sample, the gas passed into the combustion chamber, and the platinum 
wire heated to a dull-red glow to give a temperature which is sufficient for the ignition of H,. 
To determine hydrocarbons, such as methane, the wire must be raised to a bright red so that 

' Novy, F. G., Roehm, H. R., and Soule, M. H.: loc. cit. 



M. H. SOULE 255 

they are burned to CO2 and H2O. By this method the gaseous components may be deter- 
mined with an accuracy of 0.02 per cent. 

The analysis of the gas over the culture does not give the total gas change since a con- 
siderable and varying amount of CO. is taken up by the medium. The CO2 may be present 
in mere physical solution, or, reacting with NajCOj, NaiHPO^, Na of proteins, it may form 
NaHCO,; or combining with the NH3 and amines made by microbic action, it may yield cor- 
responding carbonates. A culture of B. subtilis growing on 10 cc. of plain agar may produce 
enough alkali to bind 15 cc. of CO2. 

When working with broth cultures, Van Slyke's' apparatus for the determination of CO2 
in blood plasma can be used. Rogers and his co-workers, in their study of the colon bacillus, 
removed the dissolved CO2, but not the fixed CO2, by evacuation. Obviously these methods 
cannot be used when working with solid media. Satisfactory determinations of the total dis- 
solved CO2 can be made in liquid or solid media by the aeration method which consists in 
passing COi-free air through the acidified culture medium and then into known amounts of 
a standard solution of Ba(0H)2. The medium can be kept at 90° C. to liquefy the material 
if a solid substrate, such as agar, is used. The addition of the acid liberates the combined 
CO2. After sufiicient aeration the tubes containing the standard solution are placed in ice 
water, and the excess of alkali subsequently titrated with standard HCl. The number of 
cubic centimeters of N/io hydroxide neutralized by the CO2 from the medium multiplied by 
1. 1 1 29 gives the volume of CO2 in the medium in cubic centimeters at 0° C. and 760 mm. 

The determination of the CO2 content of a blood-agar medium requires a slight modifi- 
cation of the foregoing procedure. It is impossible to liquefy the medium, since the blood co- 
agulates at a relatively low temperature, thus making it difficult to secure proper aeration of 
the material. The medium is, therefore finely comminuted with a flattened glass rod, and, 
after adding the H2SO4, is aerated at a temperature of about 40° C. 

RESPIRATORY QUOTIENTS 

By respiration in its widest sense must be understood all those processes in the 
cell whereby the potential energy stored up in chemical compounds of high complexity 
is set free to furnish the energy required by an organism for its vital activities. The 
object is effected by processes of oxidation; the result is the production of energy with 
the formation of simple chemical substances such as H2O and CO2. The fact that man 
and animals consume O2 and return CO2 was shown by Lavoisier in 1777. Dulong,^ 
continuing the work, found a difference in the volume of CO2 returned per volume of 
O2 consumed in dogs, rabbits, and fowls. He suggested that this might be due to a 
difference in the character of food. Dulong's suggestion was substantiated by Re- 
gnault and Rieset.^ The value obtained, by dividing the volume of CO2 produced by 
the volume of O2 consumed, was later designated as the "respiratory quotient." The 
ratio of this exchange is the same whether the volumes are expressed in cubic centi- 
meters, or percentage, or as millimeters of pressure. 

On the assumption that the oxidation is completed to CO2 and H2O, the theoreti- 
cal respiratory quotient of a carbohydrate is i.o, of a fat 0.71, and of a protein 0.8. 
Thus, in the case of glucose, we have the equation 

C6Hi206+602 = 6C02+6H20 . 

' Van Slyke, D. D.: J. Biol. Chcm., 30, 347-68. 1917. 
= Dulong, M.: Ann. d. chim. et d. phys. (3), i, 440. 1841. 
sRegnault, V., and Reiset, J.: ibid., 26, 299. 1849. 



256 GAS METABOLISM OF BACTERIA 

It follows, therefore, that the respiratory quotient = ^p>^ ~^^i- I^ the respiratory 

quotient is high, carbohydrate is supposedly being oxidized in the body; and, if low, 
fat is undergoing change. 

The respiratory quotients of micro-organisms, as a rule, have been computed 
from the analysis of the gases over the cultures, but the values thus obtained can at 
best be considered only approximate. The direct use of analytical data, uncorrected 
for the changes in pressure, is wrong, since it gives an apparent respiratory quotient 
which is usually higher than the real quotient. A further and common error is the 
failure to determine the amount of CO2 dissolved, free or chemically combined in the 
medium. Another point which must be considered is the period of incubation, which, 
with some organisms, may be without appreciable, effect; with others, on account of 
secondary changes in the medium such as decarboxylation, a longer period may re- 
sult in an increased CO, production, and hence in a higher final value. 

In Table I are presented, by way of illustration, the results of a determination of 
the respiratory cjuotient of B. suhtilis when grown on i per cent agar containing 5 per 
cent glycerol. 

A jar (Fig. 2) received two inoculated tubes (20X150 mm.), very loosely plugged, 
each containing 10 cc. of the medium; also one open tube with 10 cc. of boiled dis- 
tilled water, the latter to supply the recjuisite aqueous tension. In order to hasten the 
production of this tension five drops of water were placed on the bottom of the jar. 
Thereupon the jar was closed, clamped, and attached to a manometer and placed in 
the hotroom at 32° C, with stopcocks i and 3 closed. It was equilibrated four hours 
later. 

In ten days the manometer showed a pressure of — 20 mm.; on the thirteenth day 
it had reached — 21, and remained at that point until the nineteenth day when its con- 
tents were analyzed. It will be seen from the table that the O2 was practically all gone, 
and evidently this state had been reached about the tenth day. The culture in the 
two tubes produced 233 cc. of CO2 and consumed 275 cc. of O2 at 0° and 760 mm. The 
close approximation of the respiratory quotient thus obtained with the theoretical 
oxygen quotient of glycerol should be noted. 

The errors often seen in the published work on gas metabolism are brought out 
in Table I. Thus, the calculated apparent respiratory quotient is the one usually 
given and is incorrect since it is based on data uncorrected for temperature and pres- 
sure changes and does not include the dissolved CO2. The calculated, real respiratory 
quotient, corrected for changes in volume, does not include the dissolved CO2. The 
corrected, real respiratory quotient is based on all of the CO, produced and all of the 
O2 consumed, corrected for changes in pressure, and converted to standard conditions. 

The value of the manometric reading as a check on the analysis is apparent. 
When such agreement does not exist, it shows that an error is present which may be 
in the analysis of the control or of the culture. Apart from this, the error may be due 
to formation of a gas, such as N2, by the culture. The manometer is therefore a serv- 
iceable indicator of the production of such a gas or of products which have high 
vapor tensions. 

The respiratory quotients for a number of organisms arc presented in Table II 



M. H. SOULE 



257 



To avoid repetition, it may be well to state that in each of these experiments the pro- 
cedure was essentially the same. Since the protozoa studied do not live in the absence 

TABLE I 

Showing Calculation of Results in Determination of Corrected Real Respiratory 
Quotient for B. subtUis Grown on Glycerol Agar, 2 Tubes, 19 Days 



Percentage 


Control 


Culture 




CO, 


0.03 
20.93 


18.17 
0. 05 


Apparent gain CO, = 18. 14 
Apparent loss 0, = 20. 88 


0. 




COo+0, 


20.96 
79.04 


iS. 22 
81.78 


Apparent loss = 2.74 
Calc. apparent manometer = — 19. 24 mm. 


Na 




100.00 


100.00 


Calc. apparent resp. quot. = 0.869 


Corrected analysis:* 

CO. 

O2 


17.561 
0.048 


Real gain CO2 = 17.531 
Real loss O2 = 20 882 






C0,+0, 


1 7 . 6og 
79.04 


Real loss = 3- 351 
Calc. real manometer =—23.5 mm. 


N. 




96.65 


Corr. observed manometer = — 21.6 mm. 
Calc. real resp. quot. = 0.8395 




1700 
3-74 


Bar. at equilibration = 738 mm. 
Aqueous tension at s-° C. = 35. 674 


Volume in manometer arm 


\'olume of agar 20 

\ olume of water 10 


1703-74 
57 


B-T = 702.326 
Calc real manometer = 23.53 


B-b-T = 678.796 


Actual air volume, 


/ 


1646. 74 





F„ = 

Cc. at 0°, 760 mm. 

CO2 in 2 agar tubes 
COj in water tube 



V 



B-T 



1+0.003665/ 760 



= 1362 . 03 cc. a t 0,° 760 mm. 



= 2.398 
= 0.674 



241.249 
Corr. real resp. quot. = = o. S482 



CO2 in 2 agar controls 

CO2 dissolved 
CO2 gaseous 

Total CO2 
O2 loss 





3 


072 


= 





60 


= 


2 


472 


= 


238 


777 



284. 419 



Resp. quot. of glycerol 
Per cent dissolved CO2 
Tension dissolved CO2 
Tension gaseous CO2 
CO2 quotient 



= 0.857 
= o. 1875 

1.32 mm 
= 127. 50 mm 
= o.oio 



241.249 
284. 419 



* Obtained by multiplying the foregoing culture values by the nitrogen factor -^ = o.g665. 

01.70 

of blood, no attempts were made to determine quotients for these organisms on blood- 
free media. 

A comparison of the quotients determined by experiment with the theoretical 
values given in the last line is of special interest. In every case, excepting the diph- 



2S8 



GAS METABOLISM OF BACTERIA 



theria bacillus, the addition of glucose to the medium increased the quotient, showing 
that when glucose is present it is utilized, more or less completely, by the organisms 
as a source of energy. 

If only glucose were oxidized the quotient would be i.o. It will be seen from Table 
II that this value is very closely approximated by several organisms. On the other 
hand, it is also clear that some combustion of the proteins or amino acids in the medi- 
um is taking place at the same time that glucose is being oxidized and that, as a result, 
the observed quotient represents the sum of all of the oxidations. 

Since the quotient for protein is 0.81, it follows that complete combustion of a 
mixture of protein and glucose would yield a value intermediate between 0.8 and i.o, 
depending upon the relative amounts burned. 

Furthermore, the utilization of any free amino acids that may be present in the 
medium may influence the quotient to a marked extent. Thus, the c^uotient for gly- 
cine and asparagine is 1.33, while for alanine it is 1.0 and for leucine it is 0.80. Hence, 

TABLE II 
Average of Corrected Real Respiratory Quotients Obtained in Jar Experiments* 



Organism 


Plain Agar 


Glycerol Agar 


Glucose 
Agar 


Serum 
Agar 


Blood 

Agar 


Glucose 
Blood Agar 






0.856(13) 
■ 903 ( 9) 
. 843 ( 8) 
. 850 ( 4) 
• 802 ( 4) 


0.992 (3) 
1.036 (3) 
1.278(9) 

0.972 U) 
0.906 (4) 


0.904(3) 
.852(4) 
.874(8) 








o.888( 4) 
.912 (10) 

.84i( 5) 
.921 ( 4) 






B subtUis 






Glanders bacillu'^ 


0.848(4) 
. 942 (4) 




Diphtheria bacillus 








.822(4) 
.875(4) 
. 868 (4) 
.862(4) 
0.81 


0.938(3) 


L. tropica 










0.951 (3) 


Z. infantum 










1 . 000 (4) 












0.968(4) 


Theoretical value 


0.81 


0.857 


I. 000 


0.81 


I. 000 



* The figures in parentheses give the number of experiments. 

depending upon which amino acid is being utilized, the observed respiratory quotient 
will be greater or lower than 1.0. 

Another factor which tends to raise the respiratory quotient above the theoretical 
value is the decarboxylation of amino and other organic acids, because CO2 is liberated 
without oxygen consumption. 

As shown in Table II, the quotient for B. suhtilis, when grown on glucose agar, is 
unusually high. This may be due solely to the two factors just mentioned. On the 
other hand, it is possible that a slight reaction, in the nature of alcoholic fermenta- 
tion, produces an additional excess of CO2 sufficient to give a quotient of 1.4 or even 
higher. 

It appears that glycerol is an important source of energy. The energy thus ob- 
tained permits complete utilization of such nutrient substances in the medium as are 
needed by the organism. It is worthy of note that the human \-ariety of the tubercle 
bacillus, which is unable to grow on plain agar, multiplies readily on glycerol agar and 
gives a respiratory cjuotient corresponding closely to the theoretical value. 

On plain agar, serum agar, and blood agar the organisms obtain the required en- 
ergy by breaking down protein matter or by utilizing the free amino acids present. 



M. H. SOULE 



259 



As a rule, the observed quotient is in excess of the theoretical value (0.81), and the 
reason for this must be sought in the factors already discussed. 

THE GAS EXCHANGE 

The jar method serves not only to determine the respiratory quotient but also to 
supply data as to the extent of the gas changes brought about by the culture in a 
single tube. With 10 cc. of medium in a tube the surface area represents about 15 
sq.cm. of culture. Undoubtedly, it would be preferable to have the exact weight of 
the organisms which have developed in a tube in order to make comparisons of the 
gas exchange of various germs and the different media. It is more important, how- 
ever, to determine the total dissolved CO2 in the medium, and to do this the weight 
of the growth must be sacrificed. 

Out of a large mass of data, much as yet unpublished, the values given in Table 
III have been selected. They show the volumes of CO2 produced and of O2 consumed, 
per culture tube, by a number of organisms. In the last column of the table is given 
the volume of air corresponding to the Oo consumed. It will be seen from these values 
that many of the organisms can and do require relatively large amounts of air. 

It has been shown in Table II that an organism when grown on a medium which 
contains no sugar yields a respiratory quotient of less than i.o. This is because of the 
fact that the volume of the O2 consumed is greater than that of the CO2 produced. A 
glance at Table III will serve to bring out this difference in the gas exchange. Failure 
to appreciate the nature of the chemical change involved has led some writers to mis- 
interpret the meaning of this difference. The fact that the volume of CO2 produced 
is less than that of the O2 consumed does not mean that the difference represents the 
volume of O2 assimilated or retained by the organism. The difference represents the 
volume of oxygen used to oxidize hydrogen to form water. 

It will be noted on reference to Table III that 10 gm. of sterile raw potato con- 
sumed the oxygen present in 552 cc. of air in ten days. These data are included be- 
cause of the importance of this plant tissue in anaerobic culture. Novy' has furnished 
conclusive evidence that it is this marked gas exchange that favors anaerobic growth 
by the rapid and complete removal of the oxygen that may be present. 

INCREASED AND DIMINISHED OXYGEN TENSIONS 

The first real tests of the influence of high partial pressures of oxygen on pure cul- 
tures of micro-organisms were made by Moore and Williams.^ In their work slanted 
tubes of agar inoculated with the test cultures were placed in a bell jar containing 
varying percentages of pure oxygen. Samples of the gas in the jar were withdrawn for 
analysis at the beginning of the experiment and at various intervals during the incu- 
bation period. The net result of these investigations, which were extended by Adams^ 
working in the same laboratory, was that out of twenty-six cultures tested in partial 
pressure of 500 mm. or more of oxygen, the growth of only the tubercle bacillus and 
B. pest is was inhibited. These organisms had not been killed, as subsequent incuba- 

' Novy, F. G., Jr.: /. Infect. Dis., 36, 343-82. 1925. 

' Moore, B., and Williams, R. : Biochem. J., 4, 177-90. 1909. 

^ Adams, A.: ibid., 6, 297-313. 1911. 



TABLE III 

Gas Exchange per Tube of Some Organisms Grown at 37° C.; Cc. at 0°, 760 Mj 



Organisms and Media 



Human tubercle bacillus: 

Plain Agar 

Glycerol agar 

Glucose agar 

Serum agar 

Bovine tubercle bacillus: 
Plain agar 

Glycerol agar 

Glucose agar 

Serum agar 

Diphtheria bacillus: 
Plain agar 

Glycerol agar 

Glucose agar 

Blood agar 

Glanders bacillus: 



Plain agar. . . . 
Glycerol agar. 
Glucose agar . 
Blood agar . . 



Hay bacillus, 32° C.: 
Plain agar 



Glycerol agar . . 
Glucose agar . . . 
Serum agar . . . . 

Tr. lewisi, 31° C.: 
Blood agar 



Glucose blood agar. 

L. tropica, 31° C.: 
Blood agar 



Glucose blood agar. 
Raw potato, 34° C.: . . . 









Corresponding 


No. Days 


CO. Made 


O2 Lost 


Air Volume 

at ,3 7°C. 






48 




57 




ri2 
I27 


334 


124 


149 


874 


/26 


26. 


50 


293 


185 


66 


70 


410 


28 


21 


23 


135 


/28 


21 


22 


118 


\56 


24 


29 


170 


/28 


89 


100 


S86 


I56 


155 


174 


1020 


/28 


104 


98 


579 


\56 


108 


105 


616 


/28 


19 


22 


118 


\S6 


28 


33 


193 


/ 7 


16 


17 


99 


\28 


19 


22 


129 


/ 7 


21 


26 


152 


l2I 


27 


33 


193 


/ 7 


6 


7 


41 


I28 


20 


23 


134 


/ 7 


18 


19 


III 


\28 


20 


22 


129 


/ 7 


17 


21 


123 


I21 


21 


25 


146 


/ 7 


88 


102 


598 


121 


141 


164 


962 


/7 


54 


55 


322 


l2I 


122 


126 


739 


[ 7 


38 


45 


264 


I21 


38 


44 


258 


/ 7 


66 


72 


409 


156 


84 


85 


483 


/ 7 


76 


89 


50s 


1 20 


205 


244 


1386 


7 


74 


62 


352 


1 7 


6S 


75 


426 


I21 


142 


159 


903 


/ 7 


2 


3 


16 


I28 


44 


49 


277 


/ 7 


9 


10 


56 


\2I 


59 


63 


354 


/ 7 


12 


15 


85 


[2& 


34 


.'.8 


212 


/ 7 


25 


2() 


147 


l2I 


5g 


<).■; 


354 


! 3 


70 


6() 


396 


1 10 


94 


96 


552 



M. H. SOULE 261 

tion of the test cultures in air permitted excellent growth. However, Adams found 
later that actinomyces and mycetoma were actually killed by exposure to the high 
tensions of oxygen. 

Recently Karsner, Brittingham, and Richardson' streaked the surfaces of agar 
plates with organisms suspended in salt solution. The plates were then stacked in a 
jar, the atmosphere of which was replaced with high oxygen tensions at normal baro- 
metric pressure. The growth of B. proteus, hemolytic streptococci, B. typhosus, and 
B. mucosus capsulatus, four of the nine cultures tested by this method, was inhibited 
by concentrations of over 80 per cent. Novy and Soule^ found that the human and 
bovine strains of the tubercle bacillus were stimulated by tensions of 40-60 per cent 
oxygen, but concentrations of 80 per cent or higher gave partial inhibition. No gas 
other than CO, was produced under these conditions, and the respiratory quotients 
were identical with the values given under ordinary air tensions. The observation 
was made by Adams,-' and confirmed by Novy and Soule, that the colonies of the 
tubercle bacillus developing in concentrations of oxygen above 80 per cent were iso- 
lated and heaped up. It appeared that relatively few organisms were able to grow 
under this extreme condition. The limiting tension for the growth of L. tropica'^ and 
Tr. lewisi was found to be in the neighborhood of 60 per cent. Cleveland^ subjected 
several species of parasitic and free living protozoa to atmospheres of pure oxygen 
under high pressure, and found that exposure for forty minutes was usually sufficient 
to kill the organisms. 

In making tests to determine the inhibiting tensions of oxygen it is of course nec- 
essary to have a large volume of the gas available at the experimental pressure. This 
will make it impossible for the initial growth of a few resistant cells to lower materi- 
ally the tension to a concentration that will be easily tolerated by the other organisms 
planted, and thus lead to erroneous conclusions. Conceivably, the initial growth in a 
small container may result in the production of sufficient CO2 to counteract the in- 
hibiting action of the remaining oxygen. 

Just how high tensions of oxygen inhibit the growth of organisms it is not possible 
to state. It may be supposed that the oxidative changes within the cells are increased 
to a point at which the vitality of the organism becomes exhausted. This may imply 
inhibition or even destruction of a respiratory enzyme; or, it may mean the formation 
of oxidative products which are directly injurious to the cell. 

Of more interest, perhaps, than the action of bacteria under increased oxygen 
tensions is the growth of organisms under conditions where the oxygen pressure is 
below that normally found in the air.^ 

In order to answer the question as to what the effect of decreased oxygen tension 
may be on a given organism, it is obviously necessary to expose the organisms to 
known concentrations of this gas. This can be effected by placing the inoculated agar 

' Karsner, H. T., Brittingham, H. H., and Richardson, M. L.: J. Med. Research, 44, 83-88. 1923. 

= Novy, F. G., and Soule, M. H.: ./. lufecl. Dis., 36, 168-232. 1925. 

3 Adams, A.: loc. cit. " Soule, M. H.: J. Infect. Dis., 36, 245-30S. 1925. 

s Cleveland, L. R.: Biol. Bull., 48, 455-68. 1925. 

* For a discussion of anaerobiosis, cf. chap, xiii in this volume. 



262 GAS METABOLISM OF BACTERIA 

tubes or preferably agar plates in containers the atmospheres of which may be varied. 
It is essential that the absolute amount of oxygen which is present, at the tension 
under consideration, be sufficient to meet the requirements of the organism. These 
conditions may be illustrated by the following experiment of Novy and Soule: 

It had been determined from a study of the respiratory quotients that the human tuber- 
cle bacillus, in order to produce a rich growth, must be provided with about loo cc. of O2 or 
about 500 cc. of air. With this fact in mind, they grew this organism on agar slants in con- 
tainers which progressively increased in size as the tension of oxygen in the confined gas de- 
creased. Atmospheres of 6, 3, i, and 0.5 per cent were provided in containers of 2,000, 3,300, 
7,700, and 20,000 cc. respectively. Each receptacle had previously received one freshly in- 
oculated agar slant. After drawing a sample of the gas for analysis each chamber was placed 
at 37° C. The experiment was conducted over a period of six weeks with analyses at frequent 
intervals. At the end of this time all of the oxygen in each container had been consumed and 
CO2 produced. The respiratory quotients were very nearly the same as those obtained under 
ordinary aerobic conditions. An excellent though varying growth was present in each tube, 
conclusive evidence that the tubercle bacillus can grow in the culture tube under any de- 
crease in the oxygen tension and that it can utilize this element to the last free molecule. The 
only limit imposed is the size of the container which, for reasons stated, must be of such 
capacity as to provide approximately 100 cc. of oxygen. It was plainly evident that respira- 
tion ceased when the oxygen was gone, as there was no increase in the CO2 content of the 
jars after this time. The growth of an aerobic organism under diminished oxygen tensions 
is of necessity slower than under ordinary air conditions. 

Several other aerobic organisms have been investigated by the same methods, and all 
have given similar results. It may be tentatively concluded that a true aerobic organism can 
utilize free oxygen under any diminished tension, but a visible growth will be obtained only 
when the absolute volume of this element is present in the amount required under ordinary 
air conditions. 

INCREASED AND DIMINISHED CO^ TENSIONS 

Mention was made earlier in this paper that all growing organisms utilize oxygen, 
whether free or combined, and liberate CO2. Some writers have felt that this waste 
product plays an important role in the metabolism of bacteria, and that it is required 
in certain tensions for the growth of the cells. Ample proof has been advanced to 
show the favoring influence of the presence of this gas when making primary isola- 
tions from infected material, but after the organisms have become adapted to culti- 
vation on artificial medium the beneficial action of added CO, is lost. 

Many organisms have been grown in the presence of high tensions of CO2, and by 
way of illustration one of the experiments of Novy and Soule with the tubercle bacil- 
lus may be cited: 

Freshly inoculated glycerol agar slants were placed in jars of about 2,000-cc. capacity; 
the atmospheres of the jars were adjusted to tensions of 30, 40, 50, and 60 per cent CO2 with 
an adequate supply of oxygen present. The jars, after the analyses of the gas contents, were 
incubated for fifty-nine days and then samples of the gases were withdrawn and analyzed. 
Excellent cultures were obtained in each tube. The data showed that the growth of the or- 
ganisms had altered the original composition of the gas by utilizing the oxygen and increas- 
ing the CO2 tension. Calculations of the respiratory quotients demonstrated that the pres- 
ence of the high CO2 tensions had no intluence on the respiratory relations. In another ex 



J 



M. H. SOULE 263 

periment, 90 per cent CO, and 10 per cent O2 were used with good results. The high tensions 
of CO2 necessarily produced marked changes in the acidity of the medium, but it was of 
extreme interest to note the rich growths obtained under such conditions. In general, the 
protozoa are unable to tolerate tensions of over 50 per cent of this gas. 

Of more interest, perhaps, than the action of high tensions of CO2 on the growth 
of bacteria is the question of the absolute necessity of this gas for germ life. In all 
probability a certain tension of this gas is required for the growth of some if not of 
all organisms, and if this partial pressure is not available, the germs may die. Such a 
concentration must be extremely small. From the fact that many of the reported ex- 
periments designed to test this point cannot be successfully repeated by independent 
investigators it seems probable that factors other than the presence or absence of free 
CO2 have influenced the results. 

The removal of the free CO2 as fast as it is formed by a rapidly growing culture pre- 
sents many difficulties. Any method used must of necessity be a continuous one to 
prevent, if possible, the accumulation of the respired gas in close proximity to the 
cells. In addition, the procedure must alter only the concentration of this gas, for if 
changes are produced other than in the tension of CO2 the effects noted cannot con- 
sistently be attributed to the presence or absence of this substance. 

The problem has been attacked in two ways: (i) the freshly inoculated media 
(broth, agar slants, or agar plates) are placed in close proximity to an alkaline ab- 
sorbent such as KOH to absorb the CO2 as fast as formed; (2) a rapid stream of CO2- 
free air is passed over the cultures to remove mechanically the free CO2 respired by 
the cell. In some of the reported experiments the two methods have been combined. 

The second method would seem to offer the most interesting possibilities, and it 
has been extensively used, but the results, in so far as inhibition is concerned, have 
been uniformly disappointing. If a stream of air is passed over an agar slant by lead- 
ing the air inlet to the base of the agar, the passage of the air quickly desiccates the 
medium. Novy and Soule found that, if the vapor pressure of the aerating gas was 
adjusted to prevent the drying out of the medium, it was impossible to inhibit the 
growth of the human tubercle bacillus, B. suhtilis, or other organisms. In fact, the 
passage of 500 cc. per minute of COz-free air over the surface of freshly inoculated 
agar slants or Kitasato plates gave better growth than in controls under ordinary con- 
ditions. Analyses of loo-cc. samples of the gas after it had passed over the cultures 
gave negative tests for CO2. However, these investigators found that by using 
extract agar pH6.o in place of the usual beef infusion agar PH7.4 in their COi-free 
aeration experiments, it was possible to inhibit the growth of, andeventually kill, 5. 
typhosus, B. sultilis, and other organisms. 

The first method, in which alkaline absorbents are present to remove the respired 
CO2, has received a great deal of consideration. Moore and Williams' placed soda 
lime in the bell jars to remove the CO2 when testing the growth of the tubercle bacil- 
lus in high oxygen tensions. Wherry and Ervin^ connected freshly inoculated tubes 

' Moore, B., and Williams, R.: loc. cit. 

^ Wherry, W. B., and Ervin, D. M.: J. Infect. Dis., 22, 194-97. 1918. 



264 GAS METABOLISM OF BACTERIA 

of the tubercle bacillus to containers of Ba (0H)2 and found that the organisms failed 
to grow. From this they concluded that free CO2 was essential for growth. These au- 
thors explained the lag period in the development of cultures as a quiescent interval 
during which the CO2 was accumulating to the optimum concentration. 

Rockwell' studied the gaseous requirements for the growth of various bacteria 
and, from a large series of investigations/ has come to the conclusion that all bacteria, 
yeasts, and molds require free CO2 for their growth and that the gas is used as a source 
of carbon. He inoculated the surface of agar plates and incubated them over alkali; 
in many of his experiments the growth of the organisms was inhibited. The lack of 
growth was not due to the drying of the medium as the use of more vigorous dehydrat- 
ing agents such as HjSO^, CaCL, and glycerol permitted excellent growth despite 
marked desiccation. The negative results, i.e., the cases in which no inhibition was 
evidenced over alkali, were explained by the lack of control of all or any one of five 
factors, which are: the presence of carbohydrate in the medium, the protein content 
of the medium, the amount of inoculum, the acidity of the medium, and the concen- 
tration of salt. The conclusion that free CO2 is utilized as a source of carbon by all 
organisms is not warranted from the data presented. In fact, the work on respiratory 
quotients already mentioned proves that such an utilization does not take place. 

A more plausible theory of the influence of COj on the growth of organisms is 
that developed by Novy and Soule, but as yet unpublished. In an attempt to ac- 
count for the conflicts in the existing data, an extensive series of investigations has 
been carried out. From these experiments, and based on the fact that bacteria can 
grow luxuriantly on agar surfaces in the absence of free CO2 when this substance is 
removed by aeration, and that it is impossible to inhibit the growth of micro-organ- 
isms in a liquid medium by either of the methods mentioned, the conclusion has been 
reached that it is the intracellular CO2 which plays the important role in the life of the 
germ. If this intracellular CO2 is removed by exposure to alkali, or by other means, 
inhibition, and even death, may result. Any method that will maintain the intracellu- 
lar CO. equilibrium will prevent or overcome the inhibiting action of alkali and permit 
growth to take place. 

GAS METABOLISM UNDER ANAEROBIC CONDITIONS 

No phase of the gaseous metabolism of bacteria has been more thoroughly inves- 
tigated than the growth of organisms under anaerobic conditions. The bacteria of the 
colon group were early recognized as gas-producers, and because of their importance 
have received considerable attention. It was early recognized that CO2 and H2 pre- 
dominate in the gases produced under anaerobic conditions. Although CH^ has been 
reported as a by-product of the anaerobic growth of B. coli, there is no evidence that 
such is the case. It is relatively simple to determine the ratio of H2 and CO2 produced 
when only traces of other gases are formed. An alkali is introduced into the liquid 
trapping the gas; the CO, present unites with this substance leaving the hydrogen 
intact. By measuring the total volume of gas formed and the decrease in volume due 

' Rockwell, G. E.: ibid., 28, 352-56. 1921. 

' Rockwell, G. E., and Highbcrger, J. H.: ibid., 40, 438-46. IQ27. 



M. H. SOULE 265 

to the removal of the CO,, one obtains the ratio of the gases present. Smith' used the 
fermentation tube to differentiate three groups of organisms on the basis of the ratios 

TJ CO 

r7\ • (Subsequent workers have usually preferred the ratio-jy^ .) 



Croup I: 


co; 


Group II: 


CO. 


Group III: 


CO. 



I 

Keyes- reviewed the literature of gas analysis and elaborated on the limitation of the 
Smith tube. The two chief objections to its use are: (i) CO. is very soluble in the 
medium and a large percentage of that substance actually produced does not appear 
as a gas. Hence an analysis of the supernatant atmosphere does not give a true indi- 
cation of the volume of gas produced nor a true ratio between CO. and the other gases. 
(2) The CO. diffuses from the closed arm through the medium to the open arm and is 
lost. For these reasons the open fermentation tube has been discarded for use in exact 
Cjuantitative work. 

Rogers, Clark, and Davis,^ following Keyes, made an exhaustive study of the gas 
production by members of the colon group. Their cultures were grown in a vacuum, 
and the dissolved as well as the supernatant gases were estimated. They made no 
attempt to determine the fixed CO., which, however, must have been appreciable in 

CO, 

amount in spite of the final acidity of the medium. The " ratio in two hundred 

H. 

and sixteen determinations varied from 0.95 to 2.72. In almost all of the analyses 

there was found to be a small but appreciable amount of "residual gas" which they 

called N.. The average amount found for the colon group was about 0.7 per cent of 

the total gas. 

The majority of the quantitative data from studies made on strict anaerobes are 
open to the criticism emphasized before; the workers analysed only the surface gases. 

Keyes and Gillespie-" employed the exact vacuum method previously described 
by Keyes, in studying cultures of B. welchii. Bushnell^ presented the gas changes pro- 
duced by a saprophytic anaerobe. He removed the dissolved gas by evacuation but 
made no attempt to estimate the fixed CO2. A more thorough investigation of the 
gaseous metabolism of strict anaerobes was made by Anderson^ who studied twenty- 
five strains of anaerobic organisms by a modification of the methods of Rogers, Clark, 
and Davis. 

■ Smith, T.: Cenlralbl.f. BaklcrioL, Abt. I, Orig., 18, 1-9. 1895. 

^ Keyes, F. G. : /. Med. Research, 21, 69-82. 1909. 

3 Rogers, L. A., Clark, W. M., and Davis, B. J.: loc. cit. 

^ Keyes, F. G., and Gillespie, L. J.: J . Biol. Cheni., 13, 291. 1913. 

s Bushnell, L. D.: /. Bad., 7, 373-403. 1922. ^ Anderson, B. G.: loc. cit. 



266 



GAS METABOLISM OF BACTERIA 



Some of the data presented by Anderson are given in Table IV. The general pro- 
cedure was as follows: 300 cc. of medium were inoculated in each case; the culture 
flasks were evacuated, sealed, and incubated. After seven days the resulting gases 
were removed by evacuation, measured, and analyzed. No evidence of the presence 
of NH3, N2O, CO, CH4, or other carbonaceous gas was found. The odorous gas 
fraction was composed mostly of H2S. This substance may have been present in con- 
centrations of 2-3 per cent, but the methods used for its determination were too un- 
satisfactory to give precise data. 

When glucose was added to the peptone broth, B. botulinus, B. sporogenes, and 
B. welchii utilized the carbohydrate with a marked increase in the total volumes of 

TABLE IV* 

CO2 

Showing Gas Production at 0° C, 760 Mm., and --- Ratios; Cultures Grown in 300 Cc. of 

Broth in vacuo, 7 Days; Medium: 2 Per Cent "Difco" Peptone and 2 Per Cent 
"DiFCo" Peptone plus i Per Cent Glucose 



Organism 


Medium 


Volume 

in Cc 

of Gas 

Produced 


CO. % 


H. ro 


N> % 


Total % 


Ratio: 

ca 


B. botulinus 97 

B. hotulimis 97 


2% peptone 
/2% peptone + 
\i'^/V^- glucose 

2% peptone 
/2'''^ peptone+ 
\i% glucose 

2^'c peptone 
J2% peptone-|- 
\i% glucose 

2% peptone 
/2% peptone-t- 
\i% glucose 


70 


82.0 
75-5 


13 6 

24.0 


41 

0.7 


99-7 
100. 2 


6.0 
31 




B. sporogenes 46 


118 
430 


89.5 
82.2 


3-5 
16.8 


6.0 
0.9 


99 
99.9 


25.6 
4-9 


B. sporogenes 46 




B. welchii 157 


42 

645 


259 
42.3 


64- 5 

39-2 


9-5 
18,0 


99 
99-5 


0. 40 


B. welchii 57 


1.08 






B. histolvlicus W.V 


70 
63 


92.5 
90.7 


0.0 
0.99 


7.0 
7-9 


99- 5 
99.6 




B. kislolylicHs W.V 























* Assembled from Anderson's Tables 2,A and sB. 

gas produced. B. histolyticus did not decompose glucose, hence its presence had no 
effect on the quantity of gas produced. By providing only a very small amount of 
protein material it was possible to force this organism to utilize glucose, but a poor 
growth was obtained. 

The accurate detection and estimation of such gases as CH^, CO, etc., produced 
by pure strains of organisms has not been reported. CH^ has been detected and esti- 
mated in cultures of B. lactis aerogenes (Baginsky),' in the gaseous effluent from sewage 
tanks, and in intestinal gases. 

The evolution of large quantities of gases other than CO2 and H, has been reported 
when special media have been used for the growth of organisms. The addition of NO/ 
and NO3' to the nutrient substance frequently results in a marked evolution of N,. 
However, such reactions have not been thoroughly investigated. Such problems, and 
the interesting question of gas production by so-called "bacterial synergism," offer 
new and fascinating fields for investigations. 

' Baginsky, A.: Zlschr.f. phys. Chemic, 12, 434-62. 1888. 



M. H. SOULE 267 

It is apparent that relatively simple apparatus and methods are now available 
for the exact determination of the gaseous metabolism of bacteria. When applied to 
aerobic forms, the amount of O2 consumed and of CO2 produced can be readily as- 
certained. By contrast, the anaerobic organisms yield relatively large volumes of 

CO2 
gases which consist essentially of CO2 and H2. The hydrogen quotientj-r^r^ , may pos- 

sibly be useful as a basis of classification. 



CHAPTER XIX 
ENZYMES OF BACTERIA 

SELMAN A. WAKSMAN 

New Jersey Agricultural Experiment Station, New Brunswick, N.J. 

DEFINITION AND PROPERTIES OF ENZYMES 

Pasteur was the first to establish the difference between "formed" or "organized" 
ferments, which bring about various reactions, such as alcoholic and acid fermenta- 
tions, through the action of the living cell, and "unorganized ferments," such as 
diastase, pepsin, etc., later designated by Kiihne as "enzymes," which are active in the 
absence of living cells. A distinct difference was thus made between "metabolic" 
processes and "enzymatic" processes, or between a ferment as an organized living 
cell and an enzyme as a product of the cell, active inside or outside of the cell. How- 
ever, further studies, especially the contributions of Buchner on the intracellular 
enzymes bringing about alcoholic and acid fermentations, finally established the fact 
that no essential difference exists between enzymes and ferments. 

An enzyme is a catalytically active substance which is produced by living cells 
and the action of which is independent of the life-processes of the cell. Enzymes are 
capable of accelerating the rate of chemical reactions and remain themselves un- 
changed. Seemingly the enzyme does not enter into the reaction itself. In certain 
cases enzymes may accelerate the reaction in one direction more than in another, as 
in the following illustration: 

2H.O-f0.^2H.O. 

when only the reaction from right to left is accelerated by catalase. 

Enzymes act specifically, i.e., they act upon substances of definite structural and 
stereoisomeric configuration. This specificity of enzymes is independent of the degree 
of their purity. However, it has not been demonstrated as yet that enzymes are 
definite chemical substances; no enzyme has been isolated in a pure state. Enzymes 
must be defined upon the basis of their action rather than their chemical nature. 

Enzymes possess certain physical properties, such as being apparently amphoteric 
electrolytes and colloids, enabling them to form absorptive compounds; they also 
possess definite structures which give them a specific affinity to the substrates upon 
which they act. 

NATURE OF ENZYMES 

The nature of the structure of enzymes is still imperfectly understood, especially 
for a number of enzymes not yet separated from the cell constituents, notably the 
proteins, with which they are bound in the cell. It is possible to obtain aqueous solu- 
tions of certain enzymes, such as amylase (diastase), protease, and invertase (sac- 
charase). It has been assumed (Fodor) that yeast invertase is identical with a carbo- 
hydrate yeast gum but the removal of this gum does not seem to affect the activity 

268 



1 



SELMAN A. WAKSMAN 269 

of the enzyme; the same is true of Fischer's idea that enzymes possess a protein 
nature, the work of Willstiitter having established that some enzymes at least (lipase, 
peroxydase, invertase) can be purified to such an extent that the protein reactions 
disappear completely. Some enzymes were obtained practically free from iron and 
phosphorus. Nitrogen seems to be an essential constituent, the purest invertase con- 
taining 13 per cent and peroxydase 9.37 to 13.37 pcr cent nitrogen. According to 
Euler, this is proof of the protein nature of enzymes. According to Willstatter, the 
proteins are merely impurities which cannot be readily removed. 

Enzymes are thus considered' to be composed of a specifically active group and 
a colloidal carrier, the latter varying in its nature but essential for the stability of the 
active group. A theory has also been advanced^ that various enzymatic reactions 
brought about by bacteria are primarily due to polarizations of substrate molecules 
induced by electric fields which characterize particular centers of cellular and intra- 
cellular surfaces, these being "active centers"; one active center may be able to 
activate various substrates, the structure of a molecule influencing its activation. 

CLASSIFICATION 

According to Neuberg and Oppenheimer, enzymes can be divided into two large 
groups: (i) Hydrolases, which bring about hydrolytic decomposition. There is only 
a very small gain of energy in these reactions. Here belong the esterases which act 
upon fats and esters, the carbohydrases which act upon carbohydrates, the proteases 
which act upon proteins, the amidases which act upon the amide and amino groupings. 
(2) Desmolases, which influence the bonds of atoms. They break the carbon chain 
with the liberation of free energy. They are the enzymes of respiration and of metabo- 
lism. Here belong those enzymes which bring about the various oxidation and reduc- 
tion processes, the fermentation reactions (anaerobic), as well as the important 
enzymes zymase and catalase. 

FORMATION AND SECRETION OF BACTERIAL ENZYMES 

The enzymes of bacteria have been very insufficiently studied. In most instances, 
analogies must be drawn with animal and plant enzymes, or enzymes of other micro- 
organisms. The recent investigations of Meyerhof and others on respiration and 
fermentation have shown that such analogies are usually justified. 

A bacterial cell is able to elaborate at least two distinct types of molecules, name- 
ly, highly specialized molecules which exhibit enzymatic behavior, and non-enzymatic 
substances; these together make the protoplasmic and histological cell structures, 
and are inseparably connected with one another.^ 

Many enzymes are produced in an inactive condition, namely, as proenzymes or 
zymogens, and are changed into the active form by an activator. This activator may 
be merely an acid or an alkali necessary to adjust the reaction to the optimum for 
the particular enzyme, as in the case of pepsin or lipase. In some instances, electro- 
lytes (NaCl) are essential, anions being active in the case of diastase, and cations such 

•Willstatter, R.: J.Chem. Soc, p. 1374. 1927. 

^ Quastel, J. H., and Wooldridge, W. R.: Biochem. J., 21, 1224. 1927. 

3 Quastel, J. H., and Wooldridge, W. R.: ibid. 



270 ENZYMES OF BACTERIA 

as calcium being essential for the action of tryptases and thrombase. The zymogen 
may be a mixture of an enzyme with an inactivating agent or paralyzer; activation 
then consists in the destruction of the paralyzer. An activator for enzyme A may 
be the paralyzer for enzyme B, which acts destructively upon A, as in the action of 
tryptase of yeast upon zymase. The proteolytic enzyme of the pancreas acts only 
upon peptone, fibrin, casein, and protamines, but not upon true proteins; when 
activated by enterokinase, it can act also upon these. This is, however, not a trans- 
formation of a zymogen into trypsin, as previously assumed, but a stoichiometric 
and reversible combination of trypsinogen and kinase to give a new enzyme possessing 
new characteristics. 

The presence of specific nutrients in the medium regulates the quantitative forma- 
tion of specific enzymes. Thus, the presence of starch will increase the amylolytic 
power; the presence of proteins, the proteolytic power, etc. The digestive enzymes 
are usually secreted readily into the medium and are found among the so-called 
extracellular or exo-enzymes, since the colloidal nutrients (celluloses, starches, pro- 
teins) cannot enter the cell; the enzymes of metabolism (respiratory enzymes, 
zymases, etc.) are found among the so-called endo-cellular or endo-enzymes. The 
same enzyme may be in a biological sense both an exo- and an endo-enzyme, the 
difference between these two enzymes being one of degree rather than of kind. In 
some cases, organisms can be cultured so that they produce a certain enzyme which 
they do not form otherwise, as in the formation of a zymase capable of decomposing 
galactose into alcohol. The nature of nutrition influences very markedly the nature 
and abundance of the different enzymes. This is especially true of bacteria and other 
micro-organisms. 

PREPARATION AND PURIFICATION OF ENZYMES 

The bacterial enzymes which are secreted into the medium can be obtained by 
filtering the culture free from cells or by using the whole culture as the enzyme 
preparation. Certain enzymes are obtained only by the destruction of the cell. A 
slight injury of the cell, resulting in a change in permeability, may be sufficient to 
allow some of the enzymes to be secreted readily into the surrounding medium. This 
can be accomplished either: (i) by changing the surface tension of the cell, (2) by 
bringing about autolysis of the cell, (3) by drying the cells first, then following by a 
brief autolysis, or (4) by breaking up the cells by mechanical means, as in the use of 
the Buchner press. The nature of the treatment will depend more upon the nature of 
the cell than upon the nature of the enzyme. Some enzymes have as yet not been 
separated from the cell substance, as in the case of lipase of seed or bacterial zymase. 

There are various methods available for the separation, purification, and concen- 
tration of enzymes. None of these has resulted, however, in the preparation of an 
enzyme in a pure state. The processes of purification consist in removing as nearly 
as possible all extraneous matter. Frequently some of the enzyme itself is lost in the 
process. Since enzymes are very labile, their separation from the accompanying pro- 
teins, carbohydrates, and salts is accomplished by physical processes of adsorption, 
elution, and precipitation rather than by chemical processes such as salt formation. 
The enzyme is first brought into solution, using as a solvent water, dilute salt or very 
dilute acid and alkali solutions. A phosphate buffer solution of a definite pH has been 



SELMAN A. WAKSMAN 271 

used lately with great success. The solution of the enzyme is filtered through paper 
or through a Charnberland or other convenient filter. Salts can also be removed by 
dialysis. The enzyme can be precipitated from solution by alcohol or by a definite 
concentration of a sulphate, preferably ammonium sulphate. The use of different 
concentrations of salt frequently permits the separation of one enzyme from another. 
The precipitation is followed by selective adsorption and elution of the enzyme. Posi- 
tively charged aluminum hydroxide and negatively charged kaolin have been used 
extensively as adsorbing agents. 

The microbial cell begins to liberate the enzyme into solution only after its death, 
as in the case of yeast. The autolysis of the cells or the enzymatic degradation of the 
protoplasm of the microbial cell is accompanied by the passing of the various enzymes 
into solution. Gentle autolysis accompanied by adsorption processes resulted in the 
liberation of much more active enzymes than previously obtained.' 

For the concentration of bacterial enzymes, the following method has been rec- 
ommended:^ Pure cultures of bacteria are grown in i liter portions of nutrient media 
for three days. The culture is then treated with an aqueous emulsion of mastix and 
acidified with acetic acid. The emulsion is prepared by dissolving mastix in alcohol 
and diluting with an equal volume of water. The precipitate which is formed as a 
result of treatment of the culture is allowed to stand twenty-four hours and is then 
filtered off. The mastix is removed from the precipitate by dissolving with alcohol 
and ether. The residual precipitate contains the bacterial enzymes. 

MEASUREMENT OF ENZYME ACTION 

An enzyme is characterized not by its structure, which is so far unknown, but 
by its activities. The methods of demonstrating enzyme action are based either upon 
the disappearance of the specific substrate or the formation of reaction products. The 
action of diastase (amylase) upon starch is measured, on the one hand, by the dis- 
appearance of starch, as shown by the iodine reaction, or by a change in the colloidal 
condition of the starch, and, on the other hand, by the formation of dextrins and 
maltose, etc. Unfortunately, we are dealing in most instances with mixtures of en- 
zymes and not with pure enzymes. Hence it is frequently difficult to determine just 
which products are formed by one enzyme and which are acted upon by another. 

INFLUENCE OF ENVIRONMENTAL CONDITIONS 

Enzymes are influenced in various ways by physical and chemical factors, which 
either favor (activate) or injure (paralyze) their action. 

Increasing temperatures, up to a certain region, bring about an increase in en- 
zyme action, according to the laws of chemical kinetics; however, even at relatively 
low temperature an injurious action sets in. Most enzymes are very sensitive to 
temperatures above 70° C, even during a very brief period. Temperatures above 
45° C. prove injurious after a prolonged period of action. The temperature coefficients 
(Arrhenius' constant A) become smaller with increasing temperatures, finally be- 
coming negative; different enzymes, however, behave differently. The optimum tem- 

' Willstatter, R.: op. cit., p. 1359. 1927. 

' Schierge, N.: Biochem. Ztschr., 179, 248. 1926. 



272 



ENZYMES OF BACTERIA 



perature for most enzymes is between 35° and 50° C. The destructive temperature 
was defined by Euler as that temperature at which the enzyme is reduced to 50 per 
cent of its strength in one hour. This temperature also varies for various enzymes, 
some bacterial proteases resisting for a brief time even boiling temperature. 

Sunlight is injurious to enzymes in aqueous solution but not in a dry condition, 
the ultra-violet rays being especially injurious; the action of visible rays is inap- 
preciable. The destructive action of an electric current is proportional to the current. 

The influence of reaction of the medium upon enzyme action should be measured 
in the actual concentration of hydrogen ions. The optimum reaction varies consider- 
ably for the various enzymes, as shown in Table I. 

TABLE I 



Enzyme 



Lipase 

Lipase 

Invertase. . 
Invertase . . 
Invertase. . 
Invertase. . 
Diastase. . . 
Diastase. . . 

Urease 

Tryptase. . 
Proteases. . 
Proteases. . 
Tyrosinase . 
Catalase. . 



Source 



Streptococcus 
Pneiimococciis 
Yeast 

Asp. ory-.ae 
PeniciUiinn 
Bacteria 
Asp. oryzae 
File imococciis 



Bacteria 

Yeast 

Yeast 



Optimum 
pH 



4-4 
0-5 



9 

7 

4 

5 

50 

7.0 

4.8 

7.0 

7-2-7-5 
7.2 

6.7-8.5 
6.0 

6.5 
7.0 



Author 



Stevens 

Avery 

Sorensen 

Kuhn 

Josephson 

Avery, Stevens 

Sherman 

Avery 

Lcivgren 

E. Meyer 

Abderhalden and Fodor 

Dernby 

Raper and Wormall 

Michaelis, Sorensen 



The influence of pH upon the action of the enzyme may depend upon its purity. 
A purified enzymatic preparation may show a different optimum reaction than an 
impure preparation.' 

It is frequently difficult to differentiate between the injury caused to the enzyme 
itself (usually irreversible) and the influence upon the reaction velocity of the process 
(usually reversible). This is especially true of the action of salts upon enzymes. Salts 
of heavy metals, especially Hg, Ni, Co, Zn, Ag, and Au, are distinctly injurious, the 
action being reversible, because of the formation of a complex between the enzyme 
and the cation; when the metal is removed, as by treatment with KCN or with 
neutral salts, the activity is frequently restored. 

ENZYMES AS ANTIGENS 

The problem of the ability of enzymes to bring about the formation of antibodies 
attracted at one time considerable attention. There is no doubt that on the injection 
of enzymes antibodies of a specific nature are formed. This phenomenon can be used 
to separate not only different enzymes, but also enzymes of different origin. However, 
in view of the fact that each enzyme consists of an active substance possessing a 
definite structure and a colloidal carrier, and in view of the fact that the nature of this 



Willstatter, R.: op. oil., p. 1359. 1927. 



SELMAN A. WAKSMAN 273 

carrier varies with the source of the enzyme, the nature of the antibody varies accord- 
ingly. Enzymes are bound by the specific substrate much as antigen is bound to 
antibody. In the first case, however, the binding is followed by catalytic action, the 
substrate is decomposed, the binding is dissolved, and the enzyme is again liberated 
to combine with more substrate; the combination of antigen-antibody is permanent, 
except under the influence of special factors such as specific precipitating antibodies 
for the proteins contained in the antigen or the antibody carriers, alkaline sugar and 
salt solutions, etc' 

It has been recently suggested- that the whole system of specific antibody forma- 
tion can be readily explained by enzyme action. The introduction of complex foreign 
proteins into the animal body leads to a series of hydrolyses, whereby the complex 
proteins are broken down into simpler components, similar to the processes taking 
place in gastro-intestinal digestion. Some of the cleavage products presumably have 
the same specificity as the original proteins. The hydrolytic processes are followed 
by processes of synthesis, coagulation, conjugation, and adsorption between the for- 
eign proteins or their cleavage products and normal humoral or cellular components. 
Some of these resulting products may have a specific protective action for the body 
as a whole; others may increase specific susceptibility; still others may be non-specific 
or even inert. 

OCCURRENCE OF BACTERIAL ENZYMES 

Esterases are enzymes which hydrolyze esters into fatty acids and alcohols; those 
enzymes which hydrolyze true fats into glycerol and higher fatty acids are usually 
referred to as lipases; the enzymes which hydrolyze esters of lower fatty acids are 
frequently spoken of as butyrases. Lecithinase (lecithase), or the enzyme which acts 
upon lecithin and phosphatides; chlorophyllase, the enzyme acting upon chlorophyll; 
cholesterinase, acting upon cholesterin ester, and the enzymes which hydrolyze 
esters of phosphoric acid (phytase, nucleases) and of sulphuric acid (sulphatase), 
belong to this group. 

A number of bacteria, including B. prodigiosus, B. pyocyaneus, B. fliwrescens, 
Staph, aureus, B. typhosus, B. tuberculosis, and other acid fast bacteria were found 
to form lipase readily. •^ Lower esterases are produced by a number of bacteria includ- 
ing B. typhosus.'^ 

Carbohydrases.— The enzymes which hydrolyze polysaccharides are frequently^ 
divided into two groups: (i) The polyases acting upon the complex polysaccharides. 
These include amylase (diastase) which acts upon starch and glycogen, cellulase 
which acts upon cellulose, inulinase (inulase) acting upon inulin, cytases (hemicellu- 
lases), including lichenase, which act upon various hemicelluloses, pectinase (pectase) 
and gelase capable of hydrolyzing pectins and agar-agar, respectively. (2) The 
hexosidases acting upon di- and tri-saccharides. These include the fructosidases, 

' See chap. Ixx in this volume. 

^ Manwaring, W. H.: J. Immunol., 12, 177, 1926; Scient. Month., p. 362. Oct., 1927. 

^ Eijkman, C: Centralbl. f. BakterioL, I, 29, 841. 1901; ibid , 35, i. 1903; Michaelis, L., and 
NJakahura, Y.: Ztschr.f. Immunitdtsforsch. n. ex per. Therap., 36, 449. 1923. 

■• Kendall, A. I., and Simonds, J. P.: /. Infect. Dis., 15, 354. 1914. 

^ Oppenheimer, C: Lehrhuch der Enzyme. Leipzig, 1927. 



274 ENZYMES OF BACTERIA 

which hydrolyze fructosides, such as sucrose, raffinose, gentianose, and stachyose; 
the a-glucosidases, which hydrolyze maltose and trehalose; the /3-glucosidases, which 
act upon cellobiose, gentiobiose, and others; galactosidases, acting upon lactose and 
melibiose. 

The various carbohydrases are produced abundantly by bacteria and other micro- 
organisms. However, considerable specificity exists among the enzymes of different 
organisms. Cellulase is produced by very few groups of bacteria, namely, those which 
are capable of utilizing complex celluloses as sources of energy; this is an endoenzyme, 
although it may also diffuse outside of the cell.^ Hemicellulase or cytase is distributed 
more abundantly, due to the fact that many more types of bacteria are capable 
of attacking hemicelluloses and utilizing them as sources of energy. However, 
neither of these enzymes can be obtained in any great abundance from bacterial 
cultures. 

Amylase (diastase) is produced both by a greater number of organisms and in 
considerably larger amounts. It is sufficient to mention the bacteria of the B. siibtilis- 
mesentericus group, which are even utilized for producing the enzyme on a commercial 
scale. B. anthracis, B. tuberculosis, V. cholerae, pyogenic streptococci, etc., B. coli, 
B. pneumoniae, and various other bacteria produce only traces of this enzyme.^ This 
enzyme can be best demonstrated by growing the bacteria upon a starch agar plate, 
then covering the plate with a dilute solution of iodine in potassium iodide. 

Gelase is produced by some bacteria.^ Pectinase is produced by B. carotovorus 
and other bacteria. 

Saccharase or invertase is produced by various bacteria, including B. subtilis, B. 
mesentericus , B. megatherium, B. fluorescens, B. pneumoniae, hemolytic streptococcus, 
butyric acid bacteria, etc. The most important source of invertase among micro- 
organisms is found, however, among the yeasts. Maltase and lactase are also pro- 
duced by a number of bacteria. These also are formed in greater abundance by yeasts 
and fungi than by bacteria. Emulsin formation has been demonstrated for a number 
of bacteria,^ especially among representatives of the colon-typhoid group. The prop- 
erty of forming this enzyme is frequently utilized for the specific differentiation of 
bacteria. In the decomposition of glucosides in various natural fermentations (indigo, 
flax, etc.), bacteria play a prominent role. 

Nucleases, or the group of enzymes which hydrolyze the nucleic acids, are also 
found among the bacteria.^ 

The decomposition of the protein molecule to its simplest constituents is carried 
out by three distinct groups of enzymes, namely, (i) the proteases, which hyrolyze 
true proteins to proteases and peptones; (2) the peptidases or ereptases which hydro- 

' Kellerman, K. F., McBeth, I. G., Scales, F. M., and Smith, N. R.: Centralbl.f. BakterioL, II, 
30, 502. IQ13; Pringsheim, H.: Ztschr. f. phys. Clieiiiic, 78, 226. IQ12. 

^ Fermi, C: Ccntralbl. f. Baklrn'oL, 12, 713-15. i8q2; ibid., I, 40, 1S7. 1905. 

^ Biernacki, W.: //'/</., II, 29, 166. igii. 

^ Weintraub, A.: //)/(/., I, Orig., 91, 273. 1924; Fermi, C, ami Montesano, G.: ibid., 15, 72.' 
1894. 

5 Schittenhelm, A., and Sthroeter, F.: Ztschr. f. phys. Chemie, 39, 203. 1903; 40, 62, 70; 41 
284; 57, 21. 1908. 



SELMAN A. WAKSMAN 275 

lyze proteoses, peptones, and various other polypeptides to amino acids; (3) the 
amidases which attack the amino acid molecule with the formation of ammonia. 

Bacteria vary greatly in their ability to form proteolytic enzymes. Some form 
true proteases, similar to the animal trypsin; others are unable to form enzymes which 
act upon true proteins, but form ereptases. Although the bacterial proteases are 
usually classified with animal trypsin, due to the fact that they are sensitive to acids 
and that they act best at neutral or alkaline reactions, they are not identical with 
this enzyme.' Some bacteria produce both trypsin-like and erepsin-like enzymes.^ 
The formation of proteolytic enzymes by bacteria in culture media is independent 
of the presence of proteins in the medium, the gelatin-liquefying enzyme being pro- 
duced by various bacteria in simple, non-protein media just as abundantly, although 
not quite so rapidly, as in complex protein media. The reaction of the medium affects 
enzyme formation only in so far as it influences the growth of the bacteria.^ 

Bacteria vary also greatly in their capacity to form autolytic enzymes. The ca- 
pacity of bacteria to produce proteolytic enzymes has frequently been used for diag- 
nostic purposes. Here belong the various tests of liquefaction of gelatin, coagulated 
egg-albumen and blood serum, milk coagulation and clarification, etc. Of special 
interest in this connection is the role of proteolytic enzymes in the formation 
of toxins by bacteria. Some investigators'' consider bacterial toxins to be not spe- 
cific secretory products of the metabolism of the particular organisms, but biochemi- 
cal transformation products of the constituents of the medium, as a result of the 
transformation of the nutrients by bacterial enzymes. The primary non-toxic trans- 
formation products give rise, by secondary fermentative decomposition, to very 
toxic transformation products which may become non-toxic as a result of further 
decomposition. They are not formed in the cell, since autolyzed cells are only 
slightly toxic and are not regularly produced in the culture when amino acids are 
present, but are more frequently produced from albumoses and peptones. 

Hemolysin, or the enzyme which "dissolves" red blood cells, is formed by a num- 
ber of bacteria including B. pyocyaneus, B. tetani, B. coli, staphylococci, and strepto- 
cocci. This enzyme was first considered to be of the nature of a proteolytic enzyme, 
but more recent studies have shown these enzymes to be distinctly different. ^ No 
definite relationship has been established between the hemolytic power of an organism 
and its pathogenicity. 

The bacteriolytic enzyme of certain bacteria is closely related to the proteolytic 
and hemolytic enzymes. Pyocyanase, or the bacteriolytic enzyme of B. pyocyaneus, 
has been studied most extensively. This enzyme or enzyme-like substance is rela- 
tively thermostabile, resisting heating at 100° C. for thirty minutes. 

Rennet (lab), the enzyme responsible for milk coagulation, is produced abundant- 
ly by bacteria, such as B. prodigiosus, B. pyocyaneus, B. fluorescens, B. amylobacter, 

' Dernby, K. G.: Biochem. Ztschr., 126, 105. 1921; Dernby, K. G., and Blanc, T-: J- Bad., 6, 
419. 1921. 

^ Corper, H. J., and Sweany, H. C: /. Biol. Chem., 29, xxi. 1914; J. Bad., 3, 129. 1918. 

3 Jordan, E. O.: Biol. Studies, Pupils of W. T. Sedgwick, p. 124. Boston, 1906. 

'' Dernby, K. G., and Siwa, S.: Biochem. Ztschr., 134, i. 1922. 

5 McNeil, A., and Kahn, B. L.: /. Immitnol., 3, 295. 1918; Orcutt, M. L., and Howe, P. E.: 
J. Exper. Med., 35, 409. 1922. 



276 ENZYMES OF BACTERIA 

V. cholerae, etc' It is formed by bacteria on casein-containing and casein-free media, 
and is readily secreted by the cells into the surrounding medium. At first it was 
thought to be identical with protease but was later found to be a different enzyme. 

Among the amidases and deaminases, or the enzymes which hydrolyze the amides 
and amino acids with the formation of ammonia, it is sufficient to mention urease, 
histozyme, asparaginase, arginase, and purinamidases. Urease is produced by various 
bacteria in considerable amounts. The formation of the enzyme is greatly influenced 
by the composition of the substrate. Histozyme hydrolizes hippuric acid into ben- 
zoic acid and glycocoll and is formed by various fungi and bacteria. 

A detailed review of the various theories of oxidation-reduction is given else- 
where.^ It is sufficient to call attention here to the occurrence of some of the enzymes 
of bacteria responsible for such reactions. A typical oxidation-reduction reaction can 

be illustrated as follows: 

R.CHO H._R.CH.OH 
R.CHO"^0 "R.COOH 

This reaction can also be represented as follows: 

CH3-CHO-fHa=CH3-CH.OH 
CH3 • CH<^Q^ - H3 = CH3 • COOH 

One molecule is oxidized and the other is reduced. In the foregoing illustration both 
molecules ("donator" and "acceptor") are the same. The oxidative phase can be 
considered as one of dehydration and the reducing phase as one of hydration. It is 
possible, however, that the acceptor and donator of hydrogen are two distinctly 
different substances, as in the reduction of methylene blue in fresh milk in the presence 
of formaldehyde; the latter is the hydrogen donator and the methylene blue the 
acceptor; nitrate may also act as an acceptor and sulphhydryl group (R.SH) as a 
donator. The process of dehydration is oxidation and the hydration is reduction. 
The enzyme which is responsible for the splitting off of the carboxyl group is car- 
boxylase. No atmospheric oxygen is introduced in the oxido-reductase and carboxyl- 
ase reactions. The same is true of catalase, which brings about the decomposition of 
H2O2 to water and oxygen. 

According to Bach and Warburg, direct oxidases, which are capable of activating 
atmospheric oxygen, are to be distinguished from oxido-reductases (perhydridase). 
Peroxidases, or the enzymes which are active in the presence of peroxides, and 
zymases, or the enzymes of fermentation, are also included in this group. 

Respiration, consisting in the absorption of oxygen and the liberation of CO2, is 
replaced under anaerobic conditions by fermentation or intramolecular respiration. 
Both result in the liberation of energy. An anaerobe can be grown in the presence of 
oxygen provided the medium has a proper reduction potential.^ 

' Loeb, A.: Centnilhl.f. BakterioL, I, 32, 471. 1902; Gorini, C: ibid., II, 8, 137. 1902; ibid., 24, 
369, 470. 1915; ibid., 26, 195, 223. 1917; ibid., 55, 240. 1920. 

^ See Schoen, M.: Le Problcme des fcrmcnialions. Paris: Masson et Cie, 1926; Nord, F. F.: 
Client. Rev., 3, 60. 1926; also chaps, xii, xiv, in this volume. 

^ Quastel, J. H.: Biocheni. ./., 18, 365. 19^4; 19, 304, 660. 1925. 



SELMAN A. WAKSMAN 277 

Zymases are produced in abundance by a number of bacteria, especially those 
forming lactic and acetic acids. The reductases produced by bacteria have been 
studied extensively in connection with the Schardinger reaction or the reduction of 
dyes in milk. Among the oxidases' which are produced by bacteria, it is sufficient 
to mention tyrosinase, luciferase, and various phenolases. 

Catalase is produced abundantly by various aerobic bacteria as well as by bacteri- 
al spores, the time of maximum formation of the enzyme depending upon the nature 
of the organism and composition of medium. Anaerobic bacteria produce practically 
no catalase; this accounts for the sensitiveness of these organisms to H2O2 and to 
oxygen. A definite relation was found' to exist between the reducing power of bac- 
teria and their capacity for forming peroxide, anaerobes tending to produce peroxide 
in the presence of oxygen. The formation or presence of catalase prevents the accumu- 
lation of the peroxide and injury to the growth of the bacteria. Hence the ability of 
an organism to produce catalase may account for its ability to grow in an atmosphere 
containing free oxygen.^ 

IMPORTANCE OF ENZYMES IN LIFE-PROCESSES 

Most of the nutrients which are available to bacteria, both in artificial culture 
media and under natural conditions, are not in a form that can be utiHzed by these 
organisms for metabolic purposes. These nutrients must first undergo a series of 
changes, which are largely brought about by means of enzymes produced by the 
bacterial cells. These changes include the processes of decomposition, which result 
in the transformation of the complex organic substances into simpler compounds, and 
the processes of oxidation which lead to the liberation of large quantities of energy. 
In addition to these changes, which result in the degradation of the complex organic 
substances, the synthesizing activities of the cell (as well as most processes of reduc- 
tion) are also carried out by means of enzymes. The simpler substances formed from 
the complex organic materials by processes of hydrolysis, oxidation, and reduction 
are utiHzed as building stones for the synthesis of bacterial protoplasm and other 
complex organic materials. 

Since bacteria vary in the nature of the nutrients which they can utilize and in 
the conditions under which these nutrients are utilized, the nature of the enzymes 
produced by the different organisms will also vary. 

The enzymes do not increase the energy content of the system nor do they influ- 
ence the equilibrium conditions. However, they do influence the rapidity with which 
the equilibrium is reached or the time required to carry out a certain process. 

GENERAL REFERENCES 

Bayliss, W. M.: The Nature of Enzyme Action. 5th ed. London, 1925. 
Euler, H. V.: Chemie der Enzyme. Part I. 3d ed. 1925; Part II, Abt. i and 2. 2d ed. 
Munchen. 1922-27. 

' F"elton, L. D.: J. Exper. Med.. 38, 291. 1923; /. Inject. Dls., 34, 407. 1924. 

^ M'Leod, J. M., and Gordon, J.: J. Path. &° Bad., 26, 326, 332. 1923; 38, 147, 155. 1923. 

^ See chap, xiv in this volume. 



278 



ENZYMES OF BACTERIA 



Oppenheimer, C: Die Fermente und Hire Wirkimgen. sth ed. Leipzig, 1924-27. 

Waksman, S. A., and Davison, W. C: Enzymes, Properties, Distribution, Methods and Appli- 
cations. Baltimore, 1926. 

Waldschmidt-Leitz, E.: Die Enzyme. Braunschweig, 1926. 

A detailed review of the formation of enzymes by bacteria is given by: 

Fuhrmann, F.: Vorlesungen iiber Bakterienenzyme. Jena, 1907. 

Waksman, S. A.: Enzymes of Microorganisms. Abstr. Bact., 6, 265, 331. 1922. 

Kruse, W.: Allgemeine Mikrobiotogie. Leipzig, 1910. 



CHAPTER XX 
SYNTHETIC CULTURE MEDIA 

H. W. SCHOENLEIN 
Detroit, Mich. 

Synthetic culture media such as are used in the bacteriological laboratory may be 
defined strictly as those substrates which contain only ingredients of known composi- 
tion and purity, and prove useful in the cultivation of micro-organisms. In some 
cases, the term is used more loosely to include certain media in which the ingredi- 
ents are of known or specified purity, though not necessarily of known composition. 
Occasionally the term is (incorrectly?) used for certain media containing materials 
of unknown or indefinite composition.' 

Synthetic media may therefore be grouped under the following headings: 

A. Synthetic media in the strict sense 

B. Pseudo-synthetic media 

1. Media in which there are present compounds of more or less uncertain chemical com- 
position, but of known purity. For example, the exact chemical composition of certain 
of the proteins is unknown, although they may be secured in a condition of high purity. 
In such media the materials of unknown composition are nutrients. 

2. Media in which all nutrients are of known composition, but in which the non-nutrients 
may be of unknown or indefinite composition. Agar, for example, may be quite com- 
pletely freed from materials which might serve as nutrients. Its exact chemical com- 
position is somewhat indefinite, and it is not itself a nutrient for most micro-organisms. 
Its inclusion in the medium is therefore frequently not regarded as preventing such a 
medium being classed as synthetic. 

It is commonly agreed that a medium is non-synthetic if it includes plant or 
animal extracts, digests, infusions, or tissues. 

In general, the media used by the earlier workers in bacteriology were non- 
synthetic; most of the synthetic media have been suggested within the last three 
decades. A few were noted as useful before 1900. Probably the first to be described 
was that of Cohn.^ This investigator developed a synthetic medium containing 
ammonium tartrate which he found to be particularly useful for the cultivation of 
fluorescent bacteria. Other early synthetic media were proposed by Naegeli,^ Fermi,^ 
Uschinsky,5 Beijerinck/ and Winogradsky.'' Synthetic media are listed by Krasnow, 

' The so-called Waksman's synthetic acid agar contains peptone, an indefinite mixture' of un- 
known composition. 

^ Cohn, Ferdinand: Beitr. z. Biol. d. Pflanzen, i, 127. 1875. 

^Naegeli,: C. Untersuchungen iiber niedere Pilze aus dent Pflanzenphysiologischem Instihit in 
Miinchen. 1882. 

■'Fermi, C: Arch.f. Hyg., 14, i. 1892. 

sUschinsky, N.: Cenlralbl.f. Bakteriol., I AbL, 14, 316. 1893. 

'^Beijerinck, M. W.: ibid., II Abt., i, i. 1895. ? Winogradsky, S.: ibid., II Abt., 2, 425. 1896. 

279 



28o SYNTHETIC CULTURE MEDIA 

Rivkin, and Rosenljerg' in nine groups, based upon the types of organisms for which 
the media were designed. A review of the bacteriological Hterature up to 1925 shows 
that approximately twenty-five hundred different combinations, which may be classi- 
fied as synthetic media in the broader sense, have been described. It is manifestly 
impossible here even to outline a classification of these media, and certainly im- 
practicable to discuss formulas in detail. It is the purpose of this article briefly to 
outline the newer knowledge relative to the uses to which synthetic media may be 
put, and the precautions to be used in the development of a satisfactory or "op imum" 
medium. 

Probably the outstanding advantage of a synthetic medium over a non-synthetic 
medium is the ease of duplication. It is only when culture media are identical in 
composition that the cultural characters of two organisms can be compared or the 
results of studies by different observers on the same organism satisfactorily evaluated. 
Synthetic media are essential in many cases to a determination of the exact nutrient 
requirements of an organism, and often constitute the most satisfactory substrates 
to be used in the isolation and identification of the various products of metabolism. 
They lend themselves to the determination of ion effects or the necessity for the pres- 
ence of bios, hormones, auximones, or other growth accelerants. In general, it is ad- 
visable, when possible, to use synthetic media for studies of the chemical composition 
of the cells of micro-organisms or of their products. Particular care is needed in testing 
the purity of constituents when it is desired to determine the ability of organisms to 
synthesize vitamines, bios, etc. 

A satisfactory synthetic medium should include: 

a) All elements essential to normal cell metabolism 

b) The elements combined into compounds which may be utilized by the cell 

c) Compounds which will act as buffers, poisers, etc., useful in that they tend to maintain a 
suitable hydrogen-ion concentration, oxidation-reduction potential, etc. 

d) Compounds, elements, or ions not strictly nutrients but which may exert a stimulative 
action 

e) All compounds and elements adjusted in concentration to give optimum growth conditions 

ELEMENTS ESSENTIAL FOR THE GROWTH OF MICRO-ORGANISMS 

The presence of an element, even in considerable amounts, in the cell of a micro- 
organism is not sufficient to prove it essential. It has been repeatedly determined that 
the chemical composition of the cell of a micro-organism may be markedly influenced 
by the composition of the medium in which it is grown. Experimental evidence based 
upon cultural tests is therefore necessary to determine the essential or the nonessen- 
tial character of a particular element. 

Apparently all cells contain proteins, nucleo-proteins, and water. The elements 
always found in these compounds, elements certainly essential to cell life, are carbon, 
hydrogen, oxygen, nitrogen, phosphorus, and probably sulphur. In addition, potas- 
sium, chlorine, and iron are generally regarded as necessary. It is common to in- 
clude in the list sodium, calcium, and magnesium. It may be shown, however, that 
with certain micro-organisms, at least the last-named elements are either nonessen- 
tial or need to be present only in traces. 

' Krasnow, F., Rivkin, H., and Rosenberg, M. L.: J. Bad., 12, 385. 1926. 



H. W. SCHOENLEIN 281 

COMPOUNDS OF ELEMENTS ESSENTIAL FOR GROWTH 

It is necessary in a synthetic medium not only that the essential elements be 
present, but that they be present in types of compounds which may be utilized by the 
cell. Of special importance are the compounds which contain the elements phos- 
phorus, carbon, or nitrogen. 

Apparently phosphates constitute the source of jihosphorus for practically all 
micro-organisms. Salts of phosphoric acid are therefore almost universally added to 
synthetic media. They are useful not only as nutrients but also as buffers. 

Synthetic culture media may be sharply differentiated into two groups based upon 
the form in which carbon is supplied. Certain types of organisms are capable of 
synthesizing organic compounds from carbon dioxide or the carbonates, while others 
require carbon in "organic" form. 

Those micro-organisms which are capable of utilizing carbon dioxide, or the 
carbonates, must in the synthesis of cell-carbon compounds reduce the carbon partial- 
ly. Those bacteria which bring about this change have been termed "oligocarbo- 
philous" (Beijerinck) or "autotrophic." For their cultivation, synthetic media are 
commonly employed. 

The reduction of carbon dioxide and the synthesis of organic carbon compounds 
are endothermic processes, and require available energy. Apparently two sources of 
such energy have been utilized by different t^-pes of autotrophic organisms. The 
radiant energy of sunlight is absorbed by the pigments of certain types of cells, and is 
used as an energy source. Such organisms are said to be "photosynthetic." Among 
the bacteria most of the autotrophic forms are "chemosynthetic." These secure the 
needed energy by bringing about chemical changes (oxidations) in elements or their 
compounds. 

Synthetic media for the growth of photosynthetic organisms have been found 
suitable for cer ain of the green algae (Chlorophyceae), blue-green algae (Schizo- 
phyceae), and attempts (largely unsuccessful) have been made to secure such a 
medium for certain of the true sulphur bacteria containing bacteriopurpurin. For the 
group last named a suitable synthetic medium is highly desirable, as the metabolism 
is poorly understood. For such would be necessary a medium relatively rich in carbon 
dioxide or carbonates and a sufficient concentration of hydrogen sulphide to stabilize 
the oxidation-reduction potential at a point suitable for growth of these micro- 
aerophiles. All of these strictly photosynthetic forms require exposure to light. 

Synthetic media for the cultivation of chemosynthetic micro-organisms have been 
elaborated for each of the important known groups. The first were those developed 
by Winogradsky' for the culture of the so-called "nitrifying bacteria." Essential to 
the growth of the organisms which oxidize ammonia to nitrites (Nitrosomonas) was 
found to be the presence of a suitable concentration of an ammonium salt (usually 
ammonium sulphate), carbon dioxide or carbonates, a high buffer content to prevent 
the solution from becoming acid,^ and a sufficient supply of free oxygen. Buffer and 
carbonates are usually supplied by adding an excess of magnesium carbonate. Simi- 

' Winogradsky, S.: Attn. deVInst. Pasteur, 4, 213; 4, 577. 1890. 

'If the reaction be written as (NH^), 804+40, = 2HN03-fH,S044-2H,0, it is evident that 
from each molecule of the neutral salt there are developed three molecules of acid. The necessity for 
the presence of an ample base reserve is evident. 



282 SYNTHETIC CULTURE MEDIA 

larly, the essentials for the growth of Nitrobacter (oxidizing nitrites to nitrates) are a 
suitable salt of nitrous acid (usually potassium nitrite), carbon dioxide or carbonates, 
and a satisfactory buffer. 

Numerous synthetic media have been developed for the group of organisms ca- 
pable of securing "growth energy" for the assimilation of carbon dioxide by the oxida- 
tion of sulphur or its compounds. Those organisms which have been cultured belong 
to the genus Thiobacillus of Beijerinck. One species, Thiobacillus denitrificans Bei- 
jerinck,' secures its "growth energy" for the assimilation of carbon dioxide under 
anaerobic conditions by the simultaneous reduction of nitrates to free nitrogen and the 
oxidation of elementary sulphur to sulphuric acid. Essential to such a medium are 
therefore sulphur (elementary), nitrates, carbonates (or carbon dioxide), and a 
suitable buffer. , 

Other species of the genus Thiobacillus oxidize sulphur or its compounds under 
aerobic conditions, and a variety of synthetic media have been devised for their study. 
These contain either elementary sulphur or reduced sulphur in the form of thio- 
sulphates. Carbon is derived from carbon dioxide or the carbonates, and nitrogen 
usually from ammonia. 

Synthetic media for the growth of certain of the autotrophic iron bacteria 
(Chlamydobacteriales) have been developed by Migula^ and Schorler.^ Apparently in 
all cases there must be provided a soluble salt of ferrous iron or manganous man- 
ganese, carbon dioxide or carbonates, ammonia, and oxygen. It is claimed that 
"growth energy" is secured by the oxidation of the metallic ion. 

Certain bacteria (as members of the genus Hydrogenomonas) develop in a 
synthetic culture medium supplied with gaseous hydrogen, oxygen, carbon dioxide, 
and a suitable source of nitrogen (as ammonia)."* 

Other organisms have been described which develop on a similar culture medium 
in which the hydrogen is replaced by carbon monoxide as does Carboxydonionas 
oligocarbophila described by Beijerinck and Van Delden,^ or by methane {Methano- 
monas methanica) J' 

Non-autotrophic (better, non-oligocarbophilous) organisms require for their 
growth the presence of organic (carbon) compounds. Apparently in all cases studied 
these carbon compounds serve at least three functions: (o) they may be oxidized in 
whole or in part with the freeing of energy of use to the cell, {h) they may be used in 
the synthesis of protoplasm or essential cell parts, or (c) they may be stored (after 
more or less modification) as reserve food. Carbon compounds which have been used 
in the preparation of synthetic media are very numerous. Such synthetic media may 
conveniently be classified on the basis of the source of the nitrogen supplied. 

The nitrogen-fLxing bacteria are frequently cultured on synthetic media; in most 
cases the "energy source" supplied is a carbohydrate or a polyatomic alcohol. The 

' Beijerinck, M. W.: Centralbl.f. Bakteriol., II Abt., ii, 587. 1904. 

' Migula, M.: Arbeiten aus den bakt. Inst, dcr techn. Hochschide zu Karlsruhe, i, 235, 238. 1S94. 

3 Schorler, B.: Centralbl.f. Bakteriol., II Abt., 12, 691. 1904. 

4 Kaserer, H.: ibid., 16, 681-96. 1906. 

s Beijerinck, M. W., and Van Delden, A.: ibid., 10, 33-47. 1903. 
' Sohngen, N. L.: ibid., 15, 513-17. 1906. 



H. W. SCHOENLEIN 283 

sole source of nitrogen is the nitrogen gas of the atmosphere. Such synthetic media 
are generally used for the cultivation of Azotobacter and Rhizobium. 

Synthetic media to which nitrogen compounds are added may be divided into (a) 
those in which the nitrogen is supplied as ammonia, or some closely related amine, 
amino-acid, etc., from which ammonia is produced by hydrolysis; (b) those contain- 
ing nitrites or nitrates; and (c) those containing other nitrogenous compounds such 
as cyanogen, ring compounds, etc. Ability to utilize these various forms of nitrogen 
constitutes an excellent criterion for the differentiation of micro-organisms. Most 
micro-organisms are able to utilize ammonia; a smaller proportion nitrates; and fewer 
yet, other compounds. 

DETERMINATION OF THE OPTIMUM MEDIUM 

It is sometimes advisable to determine the concentration of each of the various 
ingredients which will produce a medium optimum for the growth of a particular 
micro-organism. Such media have been frequently elaborated by plant physiologists 
for the cultivation of plant seedlings. Several investigators have used a somewhat 
similar technique for the development of a medium for bacteria. 

It should be emphasized that there can scarcely be such a thing as an optimum 
medium in the broad sense for any organism. The addition of one ingredient may 
alter the optimum concentration of another. For certain materials the optimum con- 
centration is a function of the temperature; in other cases it varies with hydrogen-ion 
concentration. 

It is evident that the term "optimum" as applied to a medium can mean only 
those concentrations of a given number of ingredients which will give maximum 
growth with other environmental influences fixed. In the development of such an 
optimum medium it is usually advisable to determine the materials which must be 
present for growth, and vary their concentrations one at a time. Such a method, for 
example, has been used by Fulmer, Nelson, and Sherwood,' in the development of 
their Medium E for the growth of a yeast. It was found that cane sugar, K2HPO4, 
NH4CI, CaCL, and CaC03 were necessary for growth. The exact concentration of 
each which would give a maximum rate of growth per cell during the logarithmic 
growth period was determined. It is of interest to note that the optimum concen- 
tration of certain of the ingredients named varied little or not at all with temperature. 
This was not true of the concentration of the ammonium salts; this varied with the 
temperature, that is, for each temperature there was a concentration of ammonia 
which gave maximum growth. 

Many studies have been made for the selection of optimum media by other meth- 
ods. A notable example of such is the effort of Krasnow, Rivkin, and Rosenberg-' who 
studied the suitability of some 671 different synthetic media to support continuous 
growth of streptococci without success. 

Much interest has been manifested since the work of Wildiers' on the study of the 
continued growth of organisms in synthetic media. This author stated that yeasts 

• Fulmer E. I., Nelson V. E., and Sherwood, F. F.: /. Am. Chem. Soc, 43, 191-99. 1921. 
' Krasnow, F., Rivkin, H., and Rosenberg, M. L.: /. Bact., 12, 385. 1926. 
3 Wildiers, E.: Cellule, 18,313. 1901. 



284 SYNTHETIC CULTURE MEDIA 

would not grow when seeded in small numbers in a synthetic medium, but required 
the presence of a growth stimulant, bios. For a summary of the literature on this 
topic, the reader is referred to the excellent review by Tanner, Devereux, andHiggins.' 

For a synthetic medium to be wholly satisfactory, it should support growth when 
continuous transfers of small inocula are made. 

Certain other difEculties are inherent in the preparation of synthetic media. Par- 
ticular care must be used in most cases to adjust suitably the buffer content. In many 
cases distilled water may prove troublesome, for traces of metal from containers may 
exert a marked inhibitory action; in other cases they may stimulate growth. Distilled 
water made from city supplies which are chlorinated may contain sufficient chlorine 
to influence results markedly. In many cases it is necessary to guard against the toxic 
effects of one ion by the addition of another which antagonizes it. A comprehensive 
review of the influence of ions on microbial physiology has been given by Falk.^ 

'Tanner, F. W., Devereux, E. D., andHiggins, F. M.: /. Bad., 11,45-64. 1926. See also chap, 
xxxvii in this volume. 

^ Falk, I. S.: Absi. Bad., 7, ^^i 87, 133. 1923. 



CHAPTER XXI 

DETERMINATIONS OF THERMAL DEATH-TIME 

J. RUSSELL ESTY 
Research Laboratories, National Canners Association, San Francisco 

Studies on thermal death-points date back to 1745, when Needham showed that 
boiling an infusion of mutton gravy and soup from seeds to kill all living things and 
sealing it hermetically did not prevent "spontaneous generation" since some flasks 
became cloudy. About 1760 Spallanzani, believing that Needham had not heated the 
bottles long enough or plugged them tightly enough, and after heating an extensive 
series of flasks containing an infusion of peas and almonds, recommended boiling for 
three-quarters of an hour. In 1861 Pasteur demonstrated that heating at 110° C. 
(230° F.) under pressure may be necessary to prevent "spontaneous germination." 
In 1870 Ferdinand Cohn showed that irregular results following heating were due to 
the survival of spores which, according to later workers, were able to resist varying 
degrees of heat. 

DESTRUCTION OF SPORES 

Bredfeld' reported that spores of B. subtUis required 3 hours at 100° C. 
(212° F.) or 5 minutes at 110° C. (230° F.) to kill them. Arloing, Cornevin, and 
Thomas^ found that spores of B. chauvoei, if dried, resisted boiling for nearly 2 hours, 
whereas in the moist state they did not resist boiling for more than 2 minutes. In this 
connection Zettnow's^ observations on the resistance of spores to dry heat are of inter- 
est. He reported viable organisms in lime paste from a sugar factory after heating for 
30 minutes at 3io°-32o° C. (590°-6o8° F.). These organisms grew readily at 37° C. but 
did not grow at s8°-59° C. He also claimed that spores from the lime paste which had 
been dried on silk threads survived from 20 to 25 hours in live steam. On retesting 
the original material, which had been stored in a paper carton in a sealed room for 
six months, he observed that the organisms were killed below 199° C. (390.2° F.). Sim- 
ilar material, secured from twelve places in the same sugar factory about a year later, 
was found to be sterile after 30 minutes' exposure at 220° C. (428° F.). 

An extensive literature dealing with this subject has accumulated, and it is impos- 
sible in this chapter to review adequately many important papers. The reader is, there- 
fore, referred to the bibliographies and summary which have been prepared byMagoon.-i 

Morrison and Tanner^ have compiled the thermal relations and thermal death- 
points of a large number of thermophilic spore-bearing organisms which have been 
described by various investigators. They show a wide variation, some being destroyed 

' Cited by Morrison, L. E., and Tanner, F. W.: J. Bad., 7, 358. 1922. 
^Ibid., p. 359. 1922. 

^Zettnow, E.: Cenlralhl.f. Bakteriol., Abt. I, Orig., 66, 131. 1912. 
^Magoon, C. A.: J. Bad., 11, 253. 1926. 
'Morrison L. E., and Tanner, F. W.: op. cit., 7, 346-53. 1922. 

285 



286 DETERMINATIONS OF THERMAL DEATH-TIME 

in a few minutes at temperatures below ioo° C, while others, notably certain soil or- 
ganisms, B. cylindricus and B. tostus, studied by Blau,' are reported to have with- 
stood heating in boiling water at ioo° C. for ig hours but were killed in 20 hours. 
They^ have also determined the thermal death-point of the spores of several aerobic 
thermophilic bacteria from water. 

Spores of certain obligate thermophiles were found by Bigelow and Esty^ to with- 
stand boiling at 100° C. in corn juice, pH 6.1, for 21 hours. More recently the same 
strains, when heated in a phosphate solution, pH 7.0, survived 45 hours' continuous 
boiling at 100° C. but were killed in 46 hours. This group of organisms (group 100) 
had been isolated by Cameron and Esty^ as the causative spoilage agent from certain 
understerilized canned foods. As such, the foregoing findings are not only of academic 
interest but are also significant when adequate sterilizing processes are considered. 

Numerous articles have been published on the subject of thermal death-points, 
and in many of these a definite time and temperature have been reported for specific 
organisms. However, this time- temperature relation has been frequently based upon 
the study of a single strain or merely a few superficial tests without taking into con- 
sideration certain fundamental biological principles involved. As a result, one is con- 
fronted with many conflicting statements which make it extremely difficult, if not 
impossible, to interpret discordant observations. In many cases it is necessary to 
make additional tests before the results can be evaluated and safely applied to all 
life-processes. 

EXPERIMENTS WITH B. TUBERCULOSIS 

Take, for example, the problem of establishing a safe standard for the pasteuriza- 
tion of milk. This involves a study of the heat resistance of pathogenic organisms 
which are present in milk, particularly B. tuberculosis, B. typhosus, and the patho- 
genic streptococci. As regards the tubercle bacillus, at least twenty-five reports were 
published from 1883 to 1906. 

It appears from the results reported^ that under certain conditions the heat re- 
sistance of B, tuberculosis may vary from i4o°-i56° F. for 15 minutes, i4o°-i55° F. 
for 20 minutes, and at 140° F, from 10 minutes to 6 hours, and at 212° F. from less 
than 30 seconds to 3 hours. 

When we consider these variable results in the light of the apparently successful 
time and temperature requirements for pasteurization that were in effect in 1923 in 
one hundred of the larger American cities (142° F. for 30 minutes was required for 
pasteurization in forty of these cities and the maximum in any case was 145° F. for 
30 minutes),* it is evident that some of these observations on the heat resistance of the 
tubercle bacillus are either in error or are not applicable to the commercial process. 
In this connection it should be borne in mind that heat-resistance data obtained from 

' Blau, O.: Cenlralbl.f. Bakteriol., Abt. II, 15, 97. 1Q05. 

^ Morrison, L. E., and Tanner, F. W.: Bot. Gac, 77, 2, 171. 1924. 

3 Bigelow, W. D., and Esty, J. R.: /. Infect. Dis., 27, 602. 1920. 

•• Cameron, E. J., and Esty, J. R.: ibid., 39, 2, 89. 1926. 

5 U. S. Pub. Health Bull. 147, Part X, Table I, p. 129. 1925. Also cf. chap, xxxii in this volume. 

* Secured from data collected in an unpublished survey by the American Public Health Associa- 
tion and the U.S. Public Health Service {Am. J. Pub. Health, p. 375. 1927). 



J. RUSSELL ESTY 287 

arbitrary laboratory tests apply only to the conditions under which they were made. 
They are valuable in establishing certain fundamental principles, but in making defi- 
nite regulations for commercial practice the work must be done under actual existing 
conditions. 

THE DESTRUCTION OF SPORES OF CI. tetani 

The variations in the heat resistance of the spores of CI. tetani may be cited as 
another example. Some of the published reports may be briefly summarized as fol- 
lows: 

Kitasato/ who isolated the bacillus, stated that the spores survived moist heat at 80° C. 
(176° F.) for I hour but were destroyed in steam (100° C.) (212° F.) in 5 minutes. Vaillard 
and Vincent^ observed that the spores resisted 80° C. for 6 hours, 90° C. for 2 hours, 100° C. 
for from 3 to 4 minutes, were not always destroyed in 5 minutes, but never resisted more than 
8 minutes. Levy and Bruns^ report that CI. tetani spores were not destroyed in 8| minutes 
at 100° C. and that few survived 15 minutes, but they were killed in 30 minutes. Anderson' 
reports the resistance of one strain isolated from commercial gelatin as 20-30 seconds at 
100° C. Tuck'5 claims that no spores of tetanus can resist boiling over 20 minutes. Falcioni" 
impregnated gelatin with spores of tetanus grown in agar or broth for 10 to 1 2 days and found 
that these spores survived 2\ hours but not 3 hours in steam (100° C.) in 2, 5, and 10 per cent 
gelatin. Smith's" experiments showed that tetanus spores survived 100° C. for 20 minutes 
regularly, 40 minutes usually, and 60 minutes occasionally. In one case cultures contained 
viable spores after 70 minutes at 100° C. Von Hibler^ observed variations in the heat resist- 
ance of seven different strains and found that in one case spores survived 2\ hours at 100° C. 
Becker^ reported the death-point of two strains as 2 and 3 hours, respectively, in boiling water 
when heated in brain or alkaline reaction. 

Based on a study of twenty-four strains of CI. tetani, Esty and Meyer'" found the heat 
resistance of tetanus spores to vary at 100° C. from 15 to 90 minutes, the average survival 
time being 25 minutes. At 105° C. the heat resistance varied from 3 to 25 minutes and showed 
an average survival time of 9.2 minutes. The spores were produced either in pea peptic- 
digest broth, brain medium, double-strength veal-infusion peptic-digest gelatin, or casein 

M 
broth, and were heated in a — phosphate solution, pH 7.0, with the exception of those 

produced in the brain medium. 

These results indicate, as in the case of B. tuberculosis, that strains of CI. tetani exist, 
spores of which vary widely in their resistance to moist heat. Smith," in reviewing the liter- 
ature on the thermal death-point of tetanus spores, comments as follows: "It would be 
necessary to know the tendency to spore formation in different media and at different 

■ Kitasato, O.: Ztschr.f. Hyg., 7, 225. 1889. 

^ Vaillard, L., and Vincent, H. : Ann. de I'lnst. Pasteur, 5, i. 1891. 

3 Levy, E., and Bruns, H.: Grenzgeb. d. Med. in Ckir., 10, 235. 1902. 

1 Anderson, J. F.: U.S. P.H. and Mar. Hosp. Service, Hyg. Lab. Bull. p. 1902. 

5 Tuck, G. L.: /. Path fir Bad., 9, 38. 1903. 

^ Falcioni, D.: Ann. d'ig. sperimentale, 14, 319. 1904. 

7 Smith, T.: J. A.M. A., 50, 929. 1Q08. 

* von Hibler, E.: Untersuchungen iiber die pathogenen Anaeroben, p. 211. Jena: G. Fischer, 1908. 

9 Becker, L.: Centralbl.f. Bakteriol., Abt. I, 84, 71. 1920. 

'<• Esty, J. R., and Meyer, K. F.: /. Infect. Dis., 31, 650. 1922. 

" Smith, T.: op. cit., p. 932. 1908. 



288 DETERMINATIONS OF THERMAL DEATH-TIME 

temperatures; the age of the culture at which spores are ripe and, therefore, most resistant; 
the reaction of the medium in which the spores are boi!ed or steamed, because all of these 
variable factors have probably entered into the experiments. In the case of nutrient gelatin, 
which has been a favorite medium, I find sporulation very feeble and involution forms com- 
mon. It is quite probable that such cultures would resist boiling but feebly. The use of 
dextrose in fairly large amounts, such as one or two per cent, has been frequent among 
bacteriologists, although this amount is inimical to rapid spore formation." 

THE DESTRUCTION OF THE SPORES OF CI. botuUnum 

Equally interesting on account of the many discrepancies in the published data are 
the recent heat-resistance studies on the spores of CI. botulinum. 

Van Ermengem' stated that the spores of this bacterium are considerably less heat 
resistant than are those of the other anaerobic bacilli known to the bacteriologists of his 
time. They were destroyed at 80° C. for 30 minutes, 85° C. for 15 minutes, or by boiling for 5 
minutes. Von Hibler^ reported many years later varying resistance to heat, based on a study 
of three strains of CI. botnlimim produced in different media. In one case a 4-day brain cul- 
ture survived 3 hours at 100° C. (212° F.). 

Burke^, using ten American strains (probably CI. parabotnlinum Bengtson),'' concluded 
that autoclaving at 15 lb. (121.3° C.) for 10 minutes is insufficient to destroy the more 
resistant spores of this anaerobe. Thom, Edmondson, and Giltner^ tested the heat resistance 
of cultures of the Boise asparagus strain and found that they survived 100° C. for i hour but 
failed to grow in subcultures from tubes heated for 2 hours. In a series heated under pressure, 
growth occurred in 50 per cent of the tubes autoclaved at 10 lb. for 15 minutes, but no growth 
was observed after 15 lb. for 15 minutes. Weiss* found that the Boise strain survived 210 
minutes at 100° C. (212° F.) but was killed in 240 minutes, survived 16 minutes at 105° C. 
(221° F.), but was killed in 24 minutes and in 3 minutes at 120° C. (248° F.). Working with 
sixteen strains he concluded that it is evident that under the best conditions for survival 
the most resistant spores of CI. hotulinuni will be destroyed within 5 hours at 100° C, within 
40 minutes at 105° C, and within 6 minutes at 120° C, whereas the greater number of them 
will not survive 3 minutes. 

Dickson and his associates' report marked variations in the heat resistance of forty 
American strains showing a survival period varying from 30 to 375 minutes at 100° C. Table 
I gives the maximum thermal death-times for spores heated in sealed tubes in different media 
and at different temperatures. The times include a lag period of from 2\ to 4 minutes in each 
case, 

Esty and Meyer, and Esty,^ found the spore resistance of 112 strains of CI. bolidinum to 
vary from 3 to 75 minutes at 105° C. The maximum survival times of such spores heated in a 
phosphate solution, pH 7.0, was found to be 330 minutes at 100° C. (212° F.), no minutes 
at 105° C. (221° F.), 33 minutes at 110° C. (230° F.), 11 minutes at 115° C. (239 F.), and 4 
minutes at 120° C. (248° F.). 

' Van Ermengem, E.: Ilandb. der path. Mlkroorg. (2d ed.), 4, 909-38. Jena, 1912. 

= von Hibler, E.: loc. cii. ^ Burke, G. S.: J. A.M. A., 72, 88. 19 19. 

4 Bengtson, I. A.: Hyg. Lab. Bull. 136. 1924. 

sThom, C, Edmondson, R. B., and Giltner, L. T.: J. A.M. A., 73, 907. 1919. 

6 Weiss, H.: /. Infect. Dis., 28, 70. 1921. 

7 Dickson, E. C, Burke, G. S., Beck, D., Johnston, J., and King, 11.: J.A.M.A., 79, 1239. 1922; 
Dickson, E. C, Burke, G. S., Beck, D., and Johnston, J.: /. Infect. Dis., 36, 472. 1925. 

8 Esty, J. R., and Meyer, K. F.: loc. clt.; Esty, J. R.: Am. J. Piih. Health, 13, 108. 1923. 



J. RUSSELL ESTY 



289 



Tanner and McCrca' also noted variations in the resistance of different strains under 
controlled conditions. Spores in sealed tubes exhausted to 17 mm. were destroyed within 5 
hours at 100° C, 2 hours at 105° C, i^ hours at iio°C., 40 minutes at 115° C, and 10 
minutes at 120° C. A longer time was required for spores of the same age in open tubes than 
in tubes exhausted and sealed. 

VARIATIONS IN THERMAL DEATH-TIMES 

From the foregoing statements, which are typical examples of all thermal death- 
point studies, there appears to be considerable variation in the results obtained by 
different investigators on the same group of organisms. The question arises, Why do 
such differences exist and how can they be interpreted? Several explanations may be 
offered to account for these discrepancies. 

Recent researches have shown that the accurate determination of the thermal 
death-point of bacteria depends on the careful consideration of several very important 
factors. It is not, as it was formerly considered, a simple procedure of subjecting the 

TABLE I 



Medium 




Time 


IN Minutes At 




100° C. 


107° C. 


iiS°C. 


118° C. 


121° c. 


Oil-stratified broth 

Plain broth . 


375 
255 
225 
285 


85 
45 
25 
55 


43 
8 
6 

II 


26 


23 

5 
4 
8 


Agar 


Brain 



micro-organisms, vegetative or spore, to heat and then removing a loopful or small 
portion of the treated material to various sorts of media to determine whether or not 
the organisms had been killed. Moreover, there is no one time and temperature com- 
bination which alone may be defined as the thermal death-point, as the time varies 
with the temperature at which the organisms are heated. There is, however, a definite 
time-temperature relationship, the time decreasing as the temperature increases. In 
view of this, the term "thermal death-time"^ probably more appropriately designates 
this relation than "thermal death-point." 

It must be emphasized that the resistance of bacteria to moist heat is not a con- 
stant but a variable influenced by certain conditions. From previous statements con- 
cerning the spore resistance of CI. tetani and CI. boiidinmn, it is apparent that the 
heat resistance of spores of different strains of the same organism fluctuates within 
wide limits, even with identical conditions of cultivation, spore production, and heat 
treatment. Similar variations have been noted with vegetative cells, and the following 
examples may be cited. Based upon a study of 174 cultures of B. coll, Ayers and 
Johnson^ found that all the cultures survived heating for 30 minutes at 51.7° C, 
54.59 per cent at 60° C, 6.89 per cent at 62.8° C, 0.57 per cent at 65.6° C, and 

' Tanner, F. W., and McCrea, F. D.: /. Bad., 8, 269. 1923. 

2 Mentioned for the first time by Bigelow, W. D.: J. Infect. Dis., 29, 528. 1921, 

3 Ayers, S. H., and Johnson, W. T., Jr.: /. Agric. Research, 3, 401. 1915. 



290 DETERMINATIONS OF THERMAL DEATH-TIME 

o.o per cent at 68.3° C. On retesting the 6.89 per cent, or 12 cultures, the following 
variations were noted in a two-tube series heated at 62.8° C. for 30 minutes: 

Positive Positive 

First test 12 Fourth test 6 

Second test 4 Fifth test 9 

Third test 8 Sixth test o 

Thus the results show that cultures of B. coli heated for 30 minutes at 62.8° C. may or 
may not survive, owing in all probability to the resistance of a few cells. Gage and 
Stoughton' also noted that the temperature at which final sterilization occurred with 
cultures of B. coli varied from 60° to 95° C. in 18 different tests, while the thermal 
death-point of the majority lay between 50° and 55° C. in 14 tests and 55°-6o° C. in 
4 tests. 

Any statement on the heat resistance of an organism must of necessity apply to 
the strains tested, since it is possible that a study of a larger number of strains might 
give entirely different results. It has been noted that sometimes conditions may de- 
velop which affect the resistance of unknown types or even those already tested. 
Variants may be produced under certain conditions of growth or storage, the spores 
of which may exhibit varying resistance to heat. As to the comparative resistance of 
types A and B, CI. bokdinum, Esty and Meyer^ found the average survival time of 
the spores of 78 type A strains to be 40.1 minutes at 105° C, whereas the average 
survival time of 30 type B strains at the same temperature was 23.7 minutes. Al- 
though this shows that spores of type B are apparently less heat resistant than those 
of type A, yet from the data presented in the same paper it is evident that under 
exceptional circumstances strains and spores of a resistance equal to that recorded 
for type A strains may be encountered in nature or may be produced artificially in the 
laboratory. Dickson et al.^ also observed that in general the average resistance of 
type A strains was somewhat higher than that of the type B strains, although the 
maximum survival time of a type B strain (No. 10) when heated in oil-stratified broth 
at 121° C. was found to be the same as that obtained in any of the tjqje A strains sub- 
jected to the same temperature. He therefore concluded that many more tests must 
be made before one may assume that type B strains of CI. botulinwn are consistently 
less resistant to heat than type A strains. 

Not only the strains and the types but many other factors enter into the produc- 
tion of resistant spores which may influence the resistance to heat. The composition 
and the acidity of the media, the temperature at which the cultures are incubated, and 
the anaerobic conditions employed are of primary importance. Attention has already 
been directed to certain statements by Smith'' with respect to the sporulation of CI. 
tetani. According to Esty and Meyer, ^ CI. bokdinum spores formed in certain media 
were usually of a low resistance. In such cases sporulation was feeble. Von Hibler's^ 
observations that "shyly sporulating" species in ill-adapted media produce spores 

' Gage, S., and Stoughton, G. V. E.: Technol. Quart., 19, 41. 1906. 

^ Esty, J. R., and Meyer, K. F.: loc. cit. 

3 Dickson, E. C, Burke, G. S., Beck, D., and Johnston, J.: loc. cit. 

■* Smith, T.: op. cit., p. 932. 1908. s von Hibler, E.: Joe. cit. 



J. RUSSELL ESTY 291 

which would scarcely be differentiated from granular forms of vegetative bacilli have 
a direct bearing on this aspect of the problem. Furthermore, spores generated in dif- 
ferent flasks of the same medium, inoculated with equal amounts of the same stock 
culture, and incubated for the same period show striking differences in heat resistance. 
Although the most resistant strains of CI. bokilinum behave fairly consistently, yet 
in the most suitable medium they may exhibit a low degree of heat resistance. No 
explanation can be offered for this peculiar behavior. These differences are independ- 
ent of the final reaction of the culture fluid within the range of pH 6-8, the numbers 
and the structure of the spores, as demonstrated by Burke's staining reaction.' 
However, a well-buffered medium which furnishes abundant food material for "pro- 
gressive growth" produces spores of average or low resistance. Kronig and PauP have 
noted that the same food does not always induce the production of spores of equal 
resistance. 

Von Hibler^ found that the spores from a single species were very constant in heat 
resistance if they were not too young or too old or had not been exposed too long to incubator 
temperatures or in an acid medium. Weiss'* and Esty and Meyer^ found young spores to be 
the most heat resistant. Dickson et al.^ report that the age of botulinum spores which have 
been kept at room temperature in the culture medium in which they developed does not 
materially influence the resistance after they reach an equilibrium. They found that the 
22-day-old spores were considerably less resistant than those in a culture 66 days, but that be- 
tween 66 and 315 days the resistance was practically constant. Magoon,^ working with spores 
of B. mycoidcs grown on sand moistened with broth, concluded that the degree of resistance is 
influenced by age, temperature, humidity, and possibly other factors. He states that the 
maximum resistance to heat develops under conditions of moderate temperature and hu- 
midity and is probably reached by the time the spores are 60 days old. Furthermore, the 
change in resistance took place slowly when spores were dry and cold, while a low tempera- 
ture accompanied by high humidity resulted in the development of resistant spores. 

Esty and Meyer* observed that the dehydration of moist spore suspensions of CI. 
botulinum lowered the heat resistance, although this remained constant over a period of 347 
days. In fact, perfectly dry spores were preserved equally well at 37° C, room temperature, 
or 20°C. indefinitely, and as such were well suited for comparative studies. 

Convincing experimental evidence indicates that some of the variations in heat resistance 
are due to the chemical composition of the solutions in which the organisms are heated, pH 
and other so-called "extrinsic" factors. For instance, in some cases vegetative forms and 
spores were heated in the medium in which they were produced, in others a portion of the 
suspension was transferred to fresh culture media or to different solutions, while in others 
the organisms were washed and then resuspended in the test solution. Striking variations 
have been noted in the resistance of spores heated in different solutions, and Table H^ 
illustrates the results obtained with peptone solutions as compared to a phosphate mixture. 

In view of these and numerous other observations, the spore material should be concen- 
trated by centrifuging and the sediment washed and resuspended in a well-buffered solution, 
preferably a phosphate mixture of a neutral reaction for comparative heat-resistance tests. 

' Burke, G. S.: J. Infect. Dis., 32, 6, 433. 1923. 

2 Kronig, B., and Paul, T.: Ztschr.f. Hyg. u. Infektionskrankh., 25, i. 1897. 

3 von Hibler, E.: loc. cit. 6 Dickson, E. C, et al.: loc. cit. 
" Weiss, H.: loc. cit. 7 Magoon, C. A.: loc. cit. 

5 Esty, J. R., and Meyer, K. F.: loc. cit. » Esty, J. R., and Meyer, K. F.: loc. cit. 



292 



DETERMINATIONS OF THERMAL DEATH-TIME 



The phosphate mixture is easily duplicated and undergoes practica^y no change during heat- 
ing. Experience has shown that the resistance of spore suspensions can be readily standard- 
ized on this basis, and this method is highly recommended. 



INFLUENCE OF THE HYDROGEN-ION CONCENTRATION 

The effect of the hydrogen and hydroxyl-ion concentration on the heat resistance 
of spores of CI. botulinum is shown in Chart L' In the tests illustrated by the chart, 
carefully washed spores had been suspended in two different series of solutions of vary- 
ing pH values: {a) phosphate mixture series f — Na2HP04 and KH^POJ varying 

N 
from pH 3.5-8.8; (//) double-strength veal infusions to which varying amounts of 

hydrochloric acid, citric acid, and sodium hydroxide had been added. It will be seen 

TABLE II 
Heat Resistance or CI. botulinum Spores in Various Solutions 





Resistance at 100° C. in Minutes* 




pH 


-1- 


- 


Phosphate solution 

Peptic-digest broth 

Double-strength veal infusion 

2 per cent Aminoids 


6.92 
7.00 
6. 70 
5.65 
5-65 
7.40 
7.08 
7-45 
7-45 


IIO 

120 
90 
60 
60 
40 
30 
20 
20 


120 

130 

100 

65 


2 per cent Difco peptone 


65 
50 
35 
25 
25 


2 per cent Witte's peptone 





* The figures given in the -|- column are observed survival times and those in the — column destruction times. 

that the maximum resistance in the phosphate series is slightly on the acid side (pH 
6.3-6.9), and in the veal-infusion series the influence of the hydrogen-ion concentra- 
tion on the heat resistance is striking below pH 6.0 and above pH 9.0. 

Chart I also shows graphically the spore resistance of two dilTerent suspensions of 
CI. bohdinum in juices of different varieties of canned food. The results differed funda- 
mentally from the phosphate and veal-infusion series, indicating that other factors to- 
gether with the hydrogen-ion concentration affect the thermal death-time. For ex- 
ample, spores heated in ripe ohves with a pH 7.93; corn, pH 6.35; and spinach, 
pH 5.05 exhibited approximately the same resistance, while in asparagus with a pH 
5.25 and 5.55 sterilization was more readily accomplished. In food juices with a pH 
value below 4.5 the hydrogen-ion concentration had a decided influence on the de- 
struction of CI. botulinum. The results by Weiss^ are also included in Chart I by a 
broken line, and although the spores appear to be less heat resistant, the .same rela- 
tionship exists. Similar observations, many of which are unpublished, have been 
made by the writer and his associates with thermophilic organisms both in phosphate 
solutions and food juices. 

' Ibid. 

^ Weiss, H.: op. cil., 29, 362. 1921. 



J. RUSSELL ESTY 



293 



Whitworth' states that the heat resistance of B. anthracis is influenced by the hy- 
drogen-ion concentration and the composition of the medium in which they are sus- 

. M . 

pended. Anthrax spores suspended in - phosphate mixtures show greater resistance 

at 85° C. than those in broth or in broth to which 3 per cent gelatin has been added. 

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Resistance or B. botulinus Spores in Phosphate Mixtures, Veal Infusion of Varying pH 
Values by the Addition of Acid or Alkali, and Food Juices. 

He also noted that the optimum pH was about 7.0 at 85° C, and that the decrease of 
resistance is more marked on the acid side. 

Another factor which influences the resistance has been frequently overlooked. 
Esty and Cathcart^ found that heating unbuffered solutions in soft-glass tubes changes 
the hydrogen-ion concentration. Mixtures of Na2HP04 and KH2PO4 in hard-glass 
tubes were not altered when subjected to high temperatures, although prolonged 
heating in soft-glass tubes dissolved alkali in excess of the amount which can be con- 
trolled by the buffer salts. 

' Whitworth, S. H.: Thesis, Inst. Veterinary Pathology, University of Zurich. Jan., 1924. 
^ Esty, J. R., and Cathcart, P. H.: /. Infect. Dis., 29, 29. 1921. 



294 DETERMINATIONS OF THERMAL DEATH-TIME 

The hydrogen-ion concentration of food juices heated in soft-glass changed less 
than in hard-glass tubes, due largely to the fact that the reaction of the food juices 
generally decreases during heating. In the case of corn juice, with an initial pH of 
6.0 inoculated with 22,000 spores of a thermophilic organism, the destruction times at 
100° were 25 hours in soft-glass and 21 hours in hard-glass tubes. Although no general 
statement can be made regarding the relative merits of these two types of glass in 
heat-resistance tests, yet the influence should be determined for each solution. It is 
just as important, however, to know the hydrogen-ion concentration of the solutions 
during the entire heating period. 

INFLUENCE OF NUMBERS OF BACTERIA 

Harrison and Hood' observed that the thermal death-time depended on the 
presence or absence of clumps. Spores from filtered material having a thermal death- 
time ranging from i to 10 minutes in boiling water survived from 30 minutes to 3 
hours if unfiltered. 

The decided influence of numbers on the heat resistance of bacteria has been 
pointed out by several investigators.^ However, this is observed only when dilutions 
of a given suspension are used in the tests. In fact, owing to the remarkable varia- 
bility of the resistance of spores of the same strain produced under identical condi- 
tions, cultures containing less than a million may be more resistant than others con- 
taining several billion spores. 

Our observations have consistently shown that the larger the number of spores in a 
given suspension the longer the time necessary to destroy them. This statement is 
supported by experiments involving an extensive series of packs of canned foods,-' 
both artificially and naturally contaminated with varying numbers of spoilage organ- 
isms. It was shown that the greater the contamination, the heavier the spoilage losses 
from understerilization. This appears to be contrary to the statement made by Hast- 
ings, Fred and Carroll to the effect that "certainly one million spores should include 
all grades of heat resistance." 

EFFECTS OF SALTS AND OTHER SUBSTANCES 

The heat resistance is also affected by the presence of varying amounts of sodium 
chloride in the heating medium. The thermal death-time of CI. botulinmn was not 
reduced until the salt concentration reached 8 per cent, and a very decided de- 
crease was noted with 10 and 20 per cent solutions of sodium chloride. The addition 
of 0.5 and I per cent sodium chloride made the destruction more difficult than in a 
2 or 3 per cent concentration.^ Viljoen,^ studying the effect of different amounts of 
sodium chloride on thermophilic spores, reports a protective influence up to a con- 
centration of ^-Tyh per cent. A 4 per cent solution showed either no effect or was only 

'Harrison, F. C, and Hood, E.: Proc. Roy. Soc, Canada, 17. 1923. 

' Bigelow, W. D., and Esty, J. R.: loc. ciL; Esty, J. R., and Meyer, K. F.: loc. cit.; and Weiss, 
H.: op. cit., 28, 70. 1921. 

3 Esty, J. R.: Canning; Age, 5, 179-81, 236. 1924; Cameron, E. J., Williams, C. C, and Thomp- 
son, R. J.: article to appear in early number of J. Bad. 

"Hastings, E. G., Fred, E. B., and Carroll, W. R.: Centralbl. f. BakterioL, Abt. H, 67, 165. 
1926. 

sEsty, J. R., and Meyer, K. F.: loc. cit. H^iljoen, J. A.: J. Infect. Dis., 39,286. 1926. 



J. RUSSELL ESTY 



295 



slightly toxic. The highest protection occurred from i to 2^ per cent. He also ob- 
served that the resistance of the total number of spores is increased in a i| per cent 
salt solution, and that the spores were destroyed according to a logarithmic law. Un- 
published data on the heat resistance of thermophilic organisms and non-spore- 
formers show that different concentrations of sugar, either alone or in combination 
with salt, also influence the thermal death-time. 

The studies of Dickson and his associates' definitely indicate a protective in- 
fluence when spores are heated in a medium stratified with oil. Their data leave no 
doubt that CI. botuliniim spores heated and incubated in broth, covered with a thin 
layer of mineral oil, exhibited thermal death-times which were very much greater 
than those ordinarily recorded in identical broth without the covering of ofl. Table 
III iflustrates their results, the data having been selected by the writer from Table 

III, page 479, of their article.' 

TABLE III 





Thermal Death-Time in Minutes Of 


°c. 


Strain No. lo 


Strain No. 58 




Oil Stratified 


Not Stratified 


Oil Stratified 


Not Stratified 


100 


300 
50 

23 


240 

35 

7 

4 


28s 
60 

43 
23 


255 

45 

8 

S 


107 

"5 

121 



The increased resistance is much more apparent when the spores are heated at 
higher temperatures (115° and 121° C). According to Dickson, the spores immersed 
in oil are exposed to the maximum heat for a shorter time than those suspended in 
broth and, therefore, may not receive the same "sterilizing effect." Although the 
exact mechanism of this protective influence of oil on the resistance of botulinum 
spores is not understood, yet it is now appreciated that these observations have a di- 
rect bearing on the processing time of all foods which contain oil, as was first pointed 
out by Dickson. 

EFFECTS OF DORMANCY 

Another feature which deserves consideration in the interpretation of heat resist- 
ance data is the dormancy factor characteristic for certain organisms. The germina- 
tion of heated spores of many bacteria when transferred to a favorable environment 
occurs promptly, whereas with some other organisms, notably CI. botulinum, the ger- 
mination of the surviving cells may be markedly delayed even under ideal conditions 
of subcultivation. Dickson and his associates' report the following maximum dor- 
mancies for heated spores of CI. bctulinum: 

37 months for spores which were heated and incubated in oil-stratified broth' 
22 months for spores which were heated and incubated in broth without oil 
II months for spores which were heated and incubated in agar 

' Dickson, E. C, Burke, G. S., Beck, D., and Johnston, J.: loc. cit. 

■ ^ According to Dickson, since the publication of the foregoing data, observations have been made 
at frequent intervals on the germination time of spores in the oil-stratified bath series. These show 
to date a dormancy of sixty-six months for cultures incubated one month at 37.5° C. and then held 
at room temperature. 



296 DETERMINATIONS OF THERMAL DEATH-TIME 

Esty and Meyer' report germination of subcultures prepared from heated suspensions 
after an incubation of 378 days at 37° C. The cultures produced a virulent toxin and 
presented typical morphological and biological characteristics. They also' observed 
that subcultures prepared from diluted unheated or slightly heated spore suspensions 
(80° C. for I hour or 100° C. for 2 minutes) exhibited the phenomenon of retarded 
germination. Similar findings have been recorded by Burke.^ In view of this, the 
dormancy of unheated and heated suspensions must be determined for each organism. 

Thermophilic organisms and many aerobic types^ germinate readily even when 
only a few viable cells are present. In fact, germination of heated spores of thermo- 
philic organisms occurs from 48 to 120 hours when cultures are incubated under 
optimum conditions. 

It has previously been suggested that another reason for irregularities and vari- 
able results may be unsatisfactory technique. The method by Bigelow and Esty^ has 
been used with minor modifications generally by research workers of this country. 
This method deserves wider recognition and, therefore, is briefly described. 

A definite amount of a uniform, preferably strained or filtered, suspension is 
inoculated into a series of sterile glass tubes (hard or soft) of standard size, approxi- 
mately 7-mm. inside diameter with i-mm. thickness of wall. The inoculated tubes are 
sealed in an oxygen flame, preheated in boiling water and then completely immersed 
in an electrically heated oil bath so adjusted and controlled as to maintain a constant 
uniform temperature throughout. The preheating and an initial rise in the tempera- 
ture of the bath of from 2° to 3° during the first 2 minutes' exposure of the tubes 
reduces the lag period to approximately 3 minutes. The bath is so operated that at 
the end of the lag period the temperature reaches the desired degree and thereafter 
remains constant. 

At definite periods tubes are removed, cooled immediately in a bath of ice water 
and the entire content subcultured in a favorable medium, if not already in suitable 
material, and incubated to determine survival. 

Weisss modified the foregoing procedure by using 6"Xf" test tubes, in which he 
found that a lag of about 30 minutes raised the inside of the tubes from room tempera- 
ture to 99° C. (with a bath at 100° C), of 15 minutes to 105° C. and 8 minutes to 
120° C. It is evident that in the tests reported by Weiss, 3 minutes at 120° C, the 
spores were destroyed during the period of lag when the temperature inside the tube 
was still rising and long before the bath temperature was attained. He also modified 
the method of subculturing: A loopful of the heated material was inoculated into 
meat infusion glucose agar tubes, which were finely layered with paraffin oil and 
incubated at 37.5° C. 

Magoon** used thin-walled capillary tubes and introduced them into the culture 
tubes for sterility tests. 

' Esty, J. R., and Meyer, K. F.: loc. cit. 

' Burke, G. S.: J. Infect. Dis., 33, 274. 1923. 

3 Bigelow, W. D., and Esty, J. R.: loc. cii.; Esty, J. R., and Williams, C. C: ibid., 34, 516. 1924. 

" Bigelow, W. D., and Esty, J. R.: loc. cit. 

5 Weiss, H.: loc. cit. 'Magoon, C. A.: loc. cit. 



J. RUSSELL ESTY 



297 



Dickson and Burke' adopted the so-called "sealed-tube technique" to exclude 
all possibility of contamination and to obtain accurate information as to the inci- 
dence of "skips" (bacterial growth occurring in tubes which have been heated for a 
longer period than those which frequently remain sterile). The majority of their final 
tests on the heated resistance of CI. hotulinum spores were made in sealed tubes 
heated and incubated in i per cent glucose peptic digest broth (pH 7.0-7.4) covered 
by a thin layer of mineral oil. For comparison a smaller series was run in glucose 
broth without oil, glucose agar, and brain medium. Heating records show a lag period 
of from 2^ to 4 minutes at each temperature. 

With this technique, Dickson^ et al. demonstrated "skips" in all the media and 
at all the temperatures at which the spores were heated. 

Using the Bigelow and Esty^ method to determine the spore resistance of numer- 
ous strains of the common aerobic soil and water organisms, facultative and obligate 
thermophiles, CI. botidimmi, and allied anaerobes, "skips" as noted by Dickson 
were frequently observed by the writer. Cultures heated for periods up to and includ- 
ing a certain time showed growth uniformly when incubated in a favorable environ- 
ment, but for longer periods the results were irregular. These observations indicated 
that spore suspensions in a single-tube series occasionally contained viable organisms 
that had been heated for a considerable time in excess of those in which uniform 
growth had been observed. To explain these irregularities and to determine more ac- 
curately the actual resistance of organisms to moist heat, Esty and Williams^ modified 
the original method by subjecting a large number of tubes, from twenty-five to thirty, 
instead of one or two, each containing the same suspension, to various temperatures. 
This procedure conclusively showed that these "skips" are due to the variable re- 
sistance of individual spores in any suspension. They occurred in cultures containing 
different numbers of spores and to some extent in all media. Table IV^ gives typical 
results of tests on twenty-five to thirty tubes each containing the same spore suspen- 
sion, all heated alike, as compared to those obtained when single tubes were treated. 
It will be seen that spores in a phosphate solution pH 5.1 in single tubes were viable 
for 18 minutes at 110° C. (230° F.), while even after 12 minutes, one of twenty-six 
tubes failed to show growth. In other words, 96.4 per cent of the tubes showed viable 
organisms after 12 minutes and 64.3 per cent after 15 minutes; and although a single 
tube indicated complete destruction in 20 minutes, yet 39.4 per cent of the tubes gave 
growth by the multiple method. 

Chart II illustrates graphically the results obtained with phosphate solutions, 
pH 7.09 and 6.2, corn juice 6.1 and pea juice 5.74, as given in Table IV. A straight 
line is drawn as nearly as possible through the experimentally determined points. 
These graphs are suggestive of logarithmic relations similar to those found in the 
mortaHty curves of bacteria generally and in thermal death-time relations as pointed 

' Dickson, E. C, and Burke, G. S.: Proc. Soc. Exper. Biol. &° Med., 19, 99. 1921. 

= Dickson, E. C, Burke, G. S., Beck, D., and Johnston, J.: loc. cit. 

3 Bigelow, W. D., and Esty, J. R.: loc. cit. 

" Esty, J. R., and Williams, C. C: loc. cit. 

sEsty, J. R., and Williams, C. C.: /. Infect. Dis., 34, 518. 1924. 



298 



DETERMINATIONS OF THERMAL DEATH-TIME 



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J. RUSSELL ESTY 



299 



out by Bigelow/ They are not parallel to each other, indicating that solutions of dif- 
ferent chemical composition bear no definite relationship to the heat resistance of 
bacterial cells. However, the two phosphate solutions appear to be related, and this is 



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confirmed by other results which are given in this same article.^ Data secured in this 
manner makes it possible to establish standard type curves for a given number of 
spores in a given solution by determining percentage survivals for at least four well- 
selected times on a large number of tubes (one hundred to three hundred each time). 
Such a curve can be used as a type to determine the effect of different numbers of 
spores in the same solution or the influence of varying hydrogen-ion concentration in 

J Bigelow, W. D.: /. Infect. Dis., 29, 528-36. 1921. 
' Esty, J. R., and Williams, C. C: ibid., 34, 516, 1924. 



300 DETERMINATIONS OF THERMAL DEATH-TIME 

phosphate solutions by constructing a curve through the percentage survival points 
that have been experimentally determined. 

In general, the percentage survival greatly decreases as the heating progresses, 
and the results are uniform in proportion to the number of tubes used. The larger the 
number, the closer the range obtained in duplicate sets. However, for all practical 
purposes it is believed that twenty-five to thirty tubes of the same suspension heated 
for at least four different times will give reliable and fairly consistent information. 
Single-tube results are valuable in preliminary tests to locate the range within which 
all but the occasional spore is destroyed. A few tubes, all heated alike, determine the 
approximate percentage survival and serve to establish the range of maximum re- 
sistance. The actual death-time of the more resistant spores can be determined only 
by heating a large number of tubes. This method has a direct bearing on all steriliza- 
tion processes and permits of the accurate determination of thermal death-times.' 

' For a discussion of other aspects of this subject, cf. chaps, vi, xxii, and xxxii in this volume. 



CHAPTER XXII 

THE STANDARDIZATION OF DISINFECTANTS 
AND ANTISEPTICS 

GEORGE F. REDDISH 

Baltimore 

DISINFECTANTS 

In 1881 Koch' produced the first satisfactory method of comparing the germicidal 
efficiency of disinfectants. Sternberg and Klein, Castro and Wynter-Blyth, had al- 
ready made a distinct step forward by their use of pure cultures. The test devised 
by Koch is known as the "thread method"; spores of B. anthracis were used as the 
test organism. The time necessary to kill spores dried on a silk thread was used to 
indicate the germicidal value of the substances tested. Kronig and PauP modified 
Koch's method by using small garnets instead of silk thread; this had certain definite 
advantages in that much less of the germicide being tested was carried over into the 
subculture medium. 

The Rideal- Walker method^ was the first really precise test for standardizing dis- 
infectants and is the basis for all standard procedures used up to the present. In this 
test the following factors are controlled: time, age of culture, choice of medium and 
its acidity, temperature of medication and incubation, control of resistance of test or- 
ganisms, specification of a distinct species of test organism, proportion of culture to 
disinfectant, and the use of a definite standard germicide as control. Space will allow 
but brief mention of the outstanding points in this test (192 1 revision). 

The culture medium is composed of 2 per cent Liebig's meat extract, 2 per cent Witte's 
peptone, and i per cent sodium chloride in distilled water, adjusted to a reaction of -I-1.5. 
Five-tenths of a cc. of a 24-hour culture of B. typhosus in this broth is added to 5 cc. of the 
diluted disinfectant at 15° to 18° C. and transferred with a 4-mm. loop into 5 cc. of similar 
broth at 2.5-, 5-, 7.5-, and lo-minute intervals and incubated at 37° C. for 48 hours. Only 
one phenol control is used and this dilution must kill the test organisms in 7.5 minutes but 
not in 5 minutes. A phenol coefficient is calculated by dividing the dilution of disinfectant 
which kills the test organism in 7.5 minutes but not in 5 minutes by the dilution of phenol 
which gives the same result. This figure is called the "Rideal-Walkcr coefficient."-* 

The Hygienic Laboratory method-^ differs from the Rideal -Walker test in the fol- 
lowing respects (1921 revision): 

The culture medium contains 0.3 per cent Liebig's meat extract, i per cent Armour's 

' Koch, R.: Mitth. a. d. kais. Gsandhlsamle, i, 234. 1881. 

^ Kronig, B., and Paul, Th.: Zlschr.f. Hyg. u. Infectionskranhh., 25, r. 1897. 

3 Rideal, S., and Walker, J. T. A.: /. Roy. San. Inst., 24, 424. 1903. 

"Rideal, S., and Walker, J. T. A.: Approved Technique of the Rideal-Walker Test. London: 
H. K. Lewis & Co., 1921. 

^ Hygienic Laboratory Bull. S2 . hY)x\\, igii; Reprint 675, Pub. Health Rep., ^6, 1559, 1921. 

301 



302 DISINFECTANTS AND ANTISEPTICS 

peptone, and 0.5 per cent sodium chloride in distilled water with a reaction between pH 6.0 
and 7.0; the Hopkins strain of B. typhosus is used as the test organism; the temperature of 
medication is 20° C; o.i cc. of culture is added to 5 cc. of diluted disinfectant; transfer is 
made into subculture broth at 5, 7.5, 10, 12.5, and 15 minutes; a special spiral loop is used; 
six phenol dilutions are used in each test instead of one; the phenol coefficient is calculated 
from the weakest dilutions of disinfectant and phenol killing the test organism in 5, 10, and 
15 minutes, the average from these three being taken and the figure obtained called the 
"Hygienic Laboratory phenol coefficient."' 

The "Lancet" method, but little used in America, is also based on the Rideal- 
Walker test, dififering from it in features somewhat similar to the Hygienic Laboratory 
method. The use of B. coli instead of B. typhosus as a test organism constitutes one 
of the chief points of difference between the Lancet method and the other standard 
procedures. 

In 1 91 8 a committee on standardization of disinfectants appointed by the Lab- 
oratory Section of the American Public Health Association reported a new method 
for obtaining a phenol coefficient for disinfectants.^ In this "Report" the committee 
modified the Hygienic Laboratory test in such a way as to get at least theoretically 
more accurate results. Since this method has not come into practical use in this coun- 
try a discussion of the procedures will not be given. However, one suggestion was 
made which is of interest at this time, namely, that phenol coefficients against dis- 
ease-producing bacteria other than B. typhosus should be determined. Since the ma- 
jority of bacteriologists conducting phenol coefficient tests agree that a phenol coef- 
ficient against one organism only is not of much value, further work on the use of dif- 
ferent organisms seemed desirable. 

The writer^ suggested that representatives of the various groups of pathogens 
should be used as test organisms for determining the germicidal efficiency of disin- 
fectants. The organisms proposed were: B. typhosus (representative of the gram 
negative, non-sporing bacilli); M. aureus (representative of the suppurative group, 
and also gram positive cocci) ; B. diphtheriae (representative of granular gram positive 
group, and diphtheroids) ; B. tuberculosis (representative of the acid fast group) ; Dip. 
pneumoniae (representative of the encapsulated, gram positive cocci); Strep, he- 
molyticus (representative of the septicemic pathogens, scarlet-fever organisms, ery- 
sipelas, etc., gram positive chain-forming cocci). 

At that time the writer made a thorough study of the resistance of M. aureus to 
phenol and outlined a method for the use of this organism in disinfectant testing. In 
1927 the writer^ followed out this plan and presented a method for testing disinfect- 
ants in which these various test organisms would be used. At this time detailed pro- 
cedures for B. typhosus and M. aureus were given and tentative procedures for the 
other pathogens were outlined. Objections to the Rideal- Walker and the Hygienic 
Laboratory tests were discussed in detail. Experience with both of these methods has 
shown weaknesses and inconsistencies which are responsible for inaccuracies. Both 

' Reprint 675, Pub. Health Rep., 36, 1559. 1921. 

2 "Report of the Committee on Standard Methods of Examining Disinfectants," Am. J. Pub. 
llcilth, 7, 506. 1918. 

3 Reddish, George F.: Am. J. Pub. Ilealth, is, 534- 1925; ibid., 16, 283. 1926. 
"• Reddish, George F.: op. cil., i6, 283. 1926. 



GEORGE F. REDDISH 



303 



methods also require a greater amount of time and material than the results warrant. 
This led to a phenol-coefficient test using B. typhosus as a test organism in which the 
best features of the Rideal- Walker and Hygienic Laboratory tests were retained and 
their worst features eliminated. In this way a very simple test was obtained which 
gave consistent results with a minimum amount of time and material.' 

The methods recommended by the writer^ and as approved by the American Pub- 
lic Health Association are given below: 

B. TYPHOSUS 
Medium 

Liebig beef extract or Lemco 5 g. 

Peptonum siccum (Armour) 10 g- 

Sodium chloride 5 g. 

Distilled water i ,000 cc. 

Place the ingredients in about 1,000 cc. of distilled water. Boil for 30 minutes and then 
filter. Add enough sodium hydroxide to bring the broth to a pH of 6.8 with brom thymol 
blue. Add enough water to make the total volume 1,000 cc. Run into test tubes, placing 
10 cc. in each tube. Sterilize at 15-pound pressure for 30 minutes. 

Stock culture. — The stock culture is transferred at least once a month on beef-extract 
agar slants pH 7.0-7.5, and kept at room temperature in tubes plugged with cotton. The 
agar should be made as follows: 

Percentage 

Liebig beef extract 0.5 

Armour's peptone i . o 

Sodium chloride 0.5 

Agar 1.5 

It is transferred in the foregoing broth, incubating at 37° C, on three consecutive days before 
using in the test. Fresh broth culture is started from agar-slant stock culture each month. 

Organism. — -The organism used in the test is a 22-to 26-hour culture of B. typhosus 
grown in the specified medium at 36°-38° C. This culture must be capable of resisting dilu- 
tions of phenol ranging from 1-90 to i-ioo for at least 5 minutes at 20° C, and must be killed 
in 15 minutes by dilutions of phenol ranging up to and including 1-90. 

In order to obtain the desired results, it is necessary to make daily transfers; however, 
the Sunday transfer may be omitted and the Tuesday culture will still be satisfactory. 

Temperature. — The mixture of culture and diluted disinfectant must be held at 20° C. 
during the test. 

Phenol. — The phenol used must meet all the requirements of the United States Pharma- 
copoeia and, in addition, must have a congealing point (point of constant temperature on 
cooling) not below 40° C. If the congealing point of the available phenol is slightly below 
40° C. the phenol may be distilled and the middle third of the distillate collected. If the con- 
gealing point of this portion is not below 40° C, it is suitable for use. 

Prepare a 5 per cent (by weight) stock solution of the phenol and standardize it by titra- 

' This method, as well as the procedures which are given below, was approved by the Standard 
Methods Committee of the Laboratory Section of the American Public Health Association at the 
fifty-fifth annual meeting, 1926. 

^Reddish, George F.: op. oil., 17, 320. 1927. 



304 



DISINFECTANTS AND ANTISEPTICS 



tion with deci-normal bromine water as described under "Phenol" in the United States 
Pharmacopoeia (loth rev.), or with sodium bromide and bromate solution as described by- 
Francis Sutton.' Preserve it in 200-cc. amber-colored tightly stoppered bottles, protected 
from the light. 

Proportion of culture to disinfectant. — Five-tenths cc. of unfiltered culture is added to 
S cc. of disinfectant. 

Inoculation loops. — A 4-mm. loop of platinum wire, United States 24 standard gauge, 
i| inches long, is set into any suitable holder, such as aluminum or glass rod about 0.5 cm. in 
diameter. 

Incubation. — Subcultures are incubated for 48 hours at 37 °C. 

Dilutions. — Any series of dilutions which the operator considers advisable may be used, 
but these dilutions must be made accurately and, within the limitation of the test, must show 
the maximum dilution capable of killing B. typhosus in 10 minutes. The method of making 
the dilutions also is left to the operator, as it is presumed that he has sufficient training to do 
this accurately. 

Seeding tiibcs. — Lipped test tubes 5XI inches, plugged with cotton, as used in the 
Rideal-Walker method, or test tubes of even larger size, according to the preference of the 
operator, may be used for medication. 

Subculture tube racks. — The subculture tube racks specified in Hygienic Laboratory Bull. 
82 are convenient. 

Method of conducting the test.- — Any number of dilutions up to ten may be used. Of these 
dilutions two must be reserved for the phenol control. Dilutions of 1-90 and i-ioo phenol 
are arbitrarily chosen for this purpose. These are accurately made from the stock 5 per 
cent solution of phenol. 

At intervals of 30 seconds, 0.5 cc. of culture is run into 5 cc. of each dilution from a 5-cc. 
pipette. Care must be taken not to contaminate the sides of the tubes with the culture and 
be sure that no cotton threads adhere to the open ends of the seeding tubes. After inoculating, 
thoroughly sterilize in the flame the ends of the tubes where the pipette has touched. 

At intervals of 5, 10, and 15 minutes transfer from each dilution to the specified broth. 
This will allow 30 seconds between transfers if ten dilutions are used. Incubate the tubes 
48 hours at 37° C. and read results. If there is any doubt that the growth in the subcultures 
is B. typhosus, this can be confirmed by agglutination tests. 

Calculation of coefficient. — Divide the greatest dilution of the disinfectant capable of 
killing B. typhosus in 10 minutes but not in 5 minutes by the phenol dilution which should 
do this and divide these figures one into another. In order not to convey a false idea of the 
accuracy of the method the coefficient is calculated to the nearest o.i point if under i.o, to 
the nearest 0.2 point if between i and 5.0, to the nearest 0.5 point if between 5 and 10, and 
to the nearest i.o point if between 10 and 20. For example, if results are read as follows: 

DISINFECTANT 



Dilution 



1-300. 
1-350 
1-400 
1-450 



5 Min. 



10 Min. 



15 Min. 



+ 
+ 



Sutton, Francis: A Systematic Ildndbook of Volumetric Analysis (nth ed.), pp. 404-5. 1924. 



GEORGE F. REDDISH 

PHENOL 



305 



Dilution 


S Mill. 


10 Mill. 


15 Min. 


1-90 

I-IOO 


+ 




+ 




+ 



If the reading is as follows: 



350-^90 = 3-89 
Coefficient is 3.8 

DISINFECTANT 



Dilution 



1-30 

1-35 

1-40 

1-45 



S Min. 



+ 
+ 



10 Min. 



+ 
+ 



15 Min. 



+ 



PHENOL 



Dilution 


S Min. 


10 Min. 


51 Min. 


T— no 




+ 




+ 





I-IOO 










then estimate the dilution of the disinfectant killing in 10 but not in 5 minutes as 1-37.5 
and the phenol as 1-95 (37.5-^-95 = 0.395 or 0.4) giving a coefficient of 0.4. 

In the method just described the figure obtained is named the "B. typhosus phenol 
coefficient." When any other organism is used, the coefficient could then be named 
after the organism employed. The coefficient so named will be of service in making 
specifications for any soluble disinfectant; it is far easier to name a certain figure than 
it is to enter into a cumbersome, minute description of what is desired, as would be the 
case if all coefficients were entirely abolished. 

M. AUREUS 

When M. aureus is substituted for B. typhosus some changes in the dilutions of the 
phenol control are found necessary. The same medium and general technique have been 
found to be satisfactory, but the organism is much more resistant to phenol. At 20° 
C. it should survive a 1-70 dilution of phenol for ten minutes, and may or may not 
be resistant to a 1-60 dilution for five minutes. In routine work both dilutions may 
be used as controls, and the results obtained are used in calculating the M. aureus 
phenol coefficients. Thus in the following example the figure used as divisor would 
be 65. 



Phenol 


5 Min. 


10 Min. 


15 Min. 


1—60 




+ 




+ 





1-70 


+ or 





3o6 DISINFECTANTS AND ANTISEPTICS 

If the results were as follows, 60 would be the figure employed. 



Pheno! 


S Min. 


10 Min. 


IS Min. 


1 — 60 

1-70 


+ 
+ 



+ 




+ 



Only resistant cultures are admissible in the test, for the use of weak strains leads 
to erroneous conclusions.' As old-stock strains rarely meet these requirements, it is 
necessary to secure a freshly isolated culture. After isolation, if directions already 
given for B. typhosus are followed, the resistance to phenol should be maintained for 
at least a year. 

B. DIPHTHERIAE 

A preliminary comparison of freshly isolated strains of this organism with a stock 
culture of Park No. 8 showed that all strains used possessed about equal resistance 
to phenol. Since Park No. 8 proved so satisfactory as a test organism in a few prac- 
tical tests and in repeated tests with phenol, it was selected as our stock organism for 
this purpose, and so long as it continues to retain resistance comparable to that ex- 
hibited by new isolations, it will be satisfactory as a test organism. 

The method used at the present time is: 

Three or four slants of Loeffler's blood serum, made from Difco dehydrated Loeffler's 
blood serum, are streaked heavily with B. diphtheriae and incubated at 37° C. for 24 hours; 
this is repeated for three consecutive days. The growth is then taken up in sterile saline and 
made up to a density corresponding to nephelometer 2. Five-tenths cc. of this suspension is 
then added to 5 cc. of the diluted disinfectant at 20° C. following the technique given for B. 
typhosus and Staph, aureus. After 5, 10, and 15 minutes, one loopful of the mixture of culture 
and diluted disinfectant is transferred to the Loeffler's slant, first dipping it into the liquid 
at the base of the medium and then streaking over the slant. Another transfer is then made 
from the liquor of the first tube and streaked on to a second tube of LoeflSer's medium. In 
this way further dilution of the disinfectant carried over is assured. Subculture tubes are 
then incubated at 37° C. for 48 hours, when they are observed for typical colonies of B. 
diphtheriae. The slants showing growth from the most concentrated dilutions are con- 
firmed by stained microscopic smears. Two phenol controls, i-ioo and 1-120, are included in 
each test. B. diphtheriae under the conditions outlined above should not be killed by i-ioo 
phenol in 5 minutes, nor by 1-120 in 15 minutes. 

A "B. diphtheriae phenol coefhcient" can then be calculated in the usual way from 
the data obtained with the disinfectant and phenol if desired. 

B. TUBERCULOSIS 

Specimens of sputum from clinically active tuberculosis are pooled and evenly 
mixed by prolonged shaking with broken glass. Stained smears of this material should 
show one or more tubercle bacilli in nearly every field of the microscope or an average 
of one to the field. 

Four 5-cc. portions of the pooled sputum are then measured out into sterile rubber- 
stoppered bottles, and equal quantities of disinfectant in saline dilutions of 1-25, 1-50, 
I-IOO, and 1-200 added, so that the final dilutions are as follows: 

'Reddish, George F.: op. cit., 17, 320. 1927. 



GEORGE F. REDDISH 307 

1. Sputum 50 per cent, disinfectant present in 1-50 

2. Sputum 50 per cent, disinfectant present in i-ioo 

3. Sputum 50 per cent, disinfectant present in 1-200 

4. Sputum 50 per cent, disinfectant present in 1-400 

The mixtures are thoroughly shaken at frequent intervals, and at the end of 30 minutes 
each disinfectant mixture is injected in 2-cc. amounts into the right inguinal region of each of 
two guinea pigs. With frequent shaking in the following interim, two more pairs of guinea 
pigs are injected similarly at the end of an hour. Each pig in the first group receives 0.04 cc. 
of disinfectant, in the second group 0.02 cc, in the third group o.oi cc, and in the fourth 
group 0.005 cc. of disinfectant. 

Four control pairs of guinea pigs are then inoculated with 2-cc. amounts in like manner 
with pooled sputum alone in the following dilutions: i-i, i-io, i-ioo, and 1-1,000. After 64 
days all surviving animals are chloroformed and necropsied. The results given are based on 
gross pathological changes only. Histological examination in every case might give positive 
results in some of the cases reported as negative. It should be pointed out that in this experi- 
ment the sputum and disinfectant were very intimately mixed, in a manner which cannot be 
approximated in the ordinary use of disinfectants. 

PNEUMOCOCCUS AND HEMOLYTIC STREPTOCOCCI 

The pneumococcus Type II and hemolytic streptococci may be handled similarly 
when used as test organisms in examining disinfectants. They require about the same 
conditions for optimum growths, and the same technique and culture media may be 
used for both. The technique used for these two organisms is briefly as follows: 

If the strain is from a blood-agar plate culture from fresh pathological material, transfer 
from an isolated colony into lo-cc glucose broth of the following compositions: i per cent 
Armour's peptone, 0.5 Liebig's beef extract, 0.5 per cent sodium chloride dissolved in dis- 
tilled water and to which i per cent glucose is added; the final reaction after sterilization 
must be between pH 7.2 and 7.4. Incubate at 37° C. for 24 hours, and then transfer o.i cc. 
of the culture to 10 cc. of plain broth of the foregoing composition without glucose, but with 
the same reaction, pH 7.2-7.4. Incubate at 37° C. for 24 hours and then transfer with a 4-mm. 
platinum loop into another similar tube of plain broth and then carry out the test as outlined 
for B. typhosus, using glucose broth for subculture medium. The subcultures in glucose 
broth are incubated at 37° C. for 48 hours. Comparisons between the killing strength of the 
disinfectant being examined may be made with phenol in the usual manner and the results 
indicated as the "hemolytic streptococcus phenol coefficient" and the "pneumococcus phenol 
coefficient." Two phenol controls are included in each test. The hemolytic streptococcus 
should not be killed by 1-90 in 5 minutes, nor by i-iio in 15 minutes, while the penumococ- 
cus should not be killed by i-ioo phenol in 5 minutes nor by 1-120 in 15 minutes. Results 
with streptococcus strains especially indicate that a somewhat more concentrated dilution 
of phenol might be selected, but in order that the limits may not be too exacting, the more 
dilute solutions are suggested. It is certain that if cultures are killed by the dilutions of phe- 
nol here given, they wiU not be acceptable as test organisms. 

ANTISEPTICS 

Antiseptics are substances which, when applied to micro-organisms, will render 
them innocuous, either by actually killing the organisms or by preventing their growth, 
according to the character of the preparation and the method of application.' For 

'Reddish, George F.: Drug Markets, 20, 495. 1927; /. Am. Phar. Assoc, 16, 501. 1927. 



3o8 DISINFECTANTS AND ANTISEPTICS 

testing the efficiency of substances for use as food preservatives, or for the preserva- 
tion of protein and carbohydrate materials, methods may be employed which prove 
or disprove the capacity of such substances to prevent the growth of bacteria. Anti- 
septics for use in treating or counteracting infections or for ridding the skin and mu- 
cous membranes of pathogenic micro-organisms should be tested in a manner which 
simulates as far as practicable the conditions under which these reagents are used in 
practice. When antiseptics, such as the ordinary licjuid preparations, are used in such 
a manner that short-time contact only is assured, the germicidal test such as described 
above for disinfectants is used. However, a temperature of 37° is employed instead of 
20°. In this case M. aureus' is the test organism, and the ability to kill it in 5 minutes 
is the criterion of the efficiency of such liquid antiseptics. 

The following types of preparations are examined by what may be called the "fil- 
ter-paper method." The method, briefly, is as follows: 

Number 2 Whatman Filter paper is cut into pieces about 0.5 cm. square, placed in a 
test tube, plugged with cotton, and sterilized in the hot air oven at not over 170° C. The 
desired number of these sterile squares are then immersed in a 24-hour broth culture of the 
test organism, M. aureus. These paper squares, impregnated with the culture of M. aureus, 
are then fished out with a sterile culture wire (bent at the end) and transferred to the anti- 
septic. They are kept immersed in this preparation for 5 and 15 minutes, and they are 
then transferred to a tube of broth (10 cc.) of the composition given above. By shaking 
thoroughly at intervals over a period of 5 to 10 minutes, the excess of the antiseptic is washed 
off from the paper squares. The pieces of paper are then fished out and transferred to another 
tube of broth (10 cc.) and incubated at 37° C. for 48 hours. The tubes are then observed for 
growth. 

Preparations that may be tested by this method are antiseptic oils, antiseptic 
powders, antiseptic toothpaste, antiseptic dyes, shaving cream, and antiseptic soaps. 
In the case of soaps, a thick lather must be made before the filter-paper squares are 
used. Lozenges can also be tested in the same way after a concentrated solution has 
been made. 

Antiseptic salves and ointments, however, must be examined by a special pro- 
cedure which is, briefly, as follows: 

M. aureus of normal resistance is grown at 37° C. in the broth described above and trans- 
ferred in this medium for three consecutive days. One-tenth of a cc. of a i-ico dilution of 
this culture is added to 15 cc. melted nutrient agar contauiing 2 cc. normal sterile blood 
serv.m at 45° C. (1.5 per cent agar in the broth), the culture thoroughly mixed in the agar 
and poured into a sterile p^tri dish and allowed to cool at room temperature. As soon as 
this inoculated agar has hardened, the salves and ointments, previously melted at 37° C, are 
streaked over a small surface of the inoculated agar and the plate, inverted, incubated at 37° 
C. for 48 hours. A vaseline control streaked in the same way is also included in this test. 
After being incubated it will be noted that colonies of M. aureus grow immediately adjacent 
to the vaseline control and even under it. There is no active ingredient in pure vaseline 
which will prevent the growth of M. aureus. However, in effective antiseptic salves and 
ointments a part of the active ingredients contained in them is absorbed into the agar 
and prevents the growth of the organisms.^ Therefore, the plate will show a clear zone around 

' The M. aureus used must exhibit resistance to phenol as indicated above. 
' The organisms in the clear zone surrounding the antiseptic preparations are not only inhibited, 
but usually actually killed except near the edge, as may be proved by subculture into broth. 



GEORGE F. REDDISH 309 

the antiseptic salve or ointment which is in marked contrast to the turbidity of the surround- 
ing medium caused by the heavy growth of the organism. 

In treating infected surfaces v^^ith preparations of this nature it is necessary that the 
active ingredients leave the inert base and become free to surround the infective or- 
ganisms. It is only in this way that the preparation will be of benefit in preventing 
the growth of and even killing these micro-organisms. Serum agar simulates fairly 
closely the conditions met with in skin and wounds. It is permeable, semi-solid, iso- 
tonic, and constitutes a valuable laboratory means of approximating the conditions, 
found in human and animal tissues, at least so far as the preparations under consider- 
ation are concerned. 

In testing bunion pads, corn plasters, surgical dressings, and powders for wound 
dressings for which antiseptic claims are made, the procedure just given is employed. 
These preparations are placed on top of a poured serum agar plate containing M. au- 
reus, and incubated at 37° C. for forty-eight hours. The antiseptic in such products per- 
meates the agar medium and prevents the growth of the test organism. The amount 
of antiseptic contained and its solubility, together with the ease with which it leaves 
the base, are factors in determining the width of the clear zone around these prepa- 
rations. 

In these tests effort has been made to simulate practical conditions of disinfection 
and antisepsis as closely as possible. In an arbitrary test it is difficult to duplicate 
exactly the conditions found in practice, but results obtained by these methods give 
a fair indication of the efficiency of the preparations tested. It is very important that 
the method of testing should suit the preparation being examined. 



CHAPTER XXIII 

NATURE, DISTRIBUTION, AND FUNCTIONS OF SOIL 
MICRO-ORGANISMS 

SELMAN A. WAKSMAN 
New Jersey Agricultural Experiment Station, New Brunswick, N.J. 

THE SOIL POPULATION 

Practically every lump of soil, however small in size, contains numerous micro- 
organisms, varying both in kind and activities. The microscopic plant world is repre- 
sented in the soil by (i) the algae, including blue greens, grass greens, and diatoms; 
(2) the fungi, including the actinomyces and yeasts; and (3) the bacteria, including 
cocci, bacilli, and spirilla. The microsporic animal world is represented in the soil by 
the numerous protozoa, including various amebae, flagellates, and ciliates, by the 
nematodes, the rotifers, insects, myriapods, etc. 

These various micro-organisms are found in greatest abundance in the upper soil 
layers, the numbers diminishing with depth of soil. Well-aerated soils, such as culti- 
vated field and garden soils, may contain organisms which are quite different from 
those found in water-logged soils, such as the peat bogs and acid forest soils. The dif- 
ferences in the flora and fauna of various soils are frequently both qualitative and 
quantitative in nature. The reaction of the soil, the amount of organic matter, soil 
temperature, and moisture, are among the factors which modify considerably the 
nature and distribution of the soil population. 

Methods have been developed which enable us not only to count the different 
organisms present in the soil but also to isolate them from the soil, separate them from 
one another and study, under controUed laboratory conditions, their physiological 
activities. This is of great importance, since it enables us to determine the probable 
role of these various organisms in soil processes. One must keep in mind, however, 
the fact that these numerous micro-organisms act in the soil not in pure culture but 
in associations. The activities taking place in the test tube or flask, under artificial 
laboratory conditions, especially in the case of cultures long kept in cultivation, may 
or may not take place in the soil, in the presence of numerous antagonistic and associa- 
tive influences from other organisms. In the soil, the products of metabolism of one 
group of organisms frequently serve as nutrients for other organisms. The substances 
formed as a result of these activities under natural conditions may be altogether dif- 
ferent from those produced in pure culture in the laboratory. 

NUMBERS OF MICRO-ORGANISMS IN SOIL 
METHODS OF STUDY 

The methods used in the study of soil micro-organisms vary considerably, depending 
chiefly upon the nature of the organisms under consiileration. For the determination of the 
numbers of bacteria and fungi, direct microscopic and cultural methods are available. For 

310 



SELMAN A. WAKSMAN 311 

the determination of the numbers of protozoa, algae, and specific physiological groups of 
bacteria, the dilution method has to be employed. The nematodes and other worms can be 
counted directly in soil, by using a magnifying glass and special methods of suspension. The 
study of biochemical activities can be carried out either in the soil itself or by the use of 
pure and mixed cultures of organisms, which have been separated from the soil, under con- 
trolled laboratory conditions. 

Most of the bacteria live in or upon the surface of the colloidal material which surrounds 
the inorganic soil particles. It is frequently very difficult to remove the cells from the dead 
organic and inorganic matter and separate them from one another. As a result of this and 
also because many of the soil bacteria do not develop upon ordinary agar media, the determi- 
nation of the number of soil bacteria by the plate method is far from sufficient to give an 
idea of the abundance of the soil population. The direct microscopic method is more accurate 
for the determination of the abundance of micro-organisms in the soil. This method consists 
in spreading a definite amount of a dilute suspension of soil in water upon a definite area of 
a glass slide, drying, covering with a dilute solution of agar or gelatin, fixing in alcohol and 
staining with a solution of rose bengal or erythrosine in 5 per cent phenol solution. The 
bacteria are stained red, while the inorganic particles and the dead organic matter is either 
not stained at all or is stained only faintly. Only the bacteria, the spores of fungi, and proto- 
zoan cysts can thus be counted. The living protozoa, as well as some of the mycelium of 
fungi and actinomyces, are largely destroyed in the process of staining. It is also somewhat 
difficult to distinguish, by this method, the living from the dead bacteria, the active from 
the inactive forms. This method, having the great advantage of giving the absolute number 
of bacteria present in the soil, has the disadvantage that it does not lend itself to determina- 
tions, except within certain broad groups, of the specificity of the relative groups of organisms 
in the soil. The presence and abundance of fungi in the soil can be determined by adding a 
drop of methylene blue to a suspension of soil in water placed upon a slide and examined 
directly. 

The cultural methods which are used for the determination of the numbers of micro- 
organisms in the soil are divided into two categories: (i) the plate method, and (2) the dilu- 
tion method. 

1. The plate method has been used most commonly. It consists in diluting the soil with 
different amounts of sterile tap water, then plating out i-cc. portions of the final dilutions, 
using an appropriate agar or gelatin medium. The plates are incubated for a period of time 
ranging from two to fourteen days, then the number of colonies developing on the plate is 
determined. This method has a number of very serious disadvantages, which make it almost 
worthless as far as accurate information concerning the actual abundance of soil organisms 
is concerned. There is no single medium yet devised which allows the development of 
all soil organisms. The anaerobic bacteria do not develop at all or only to a very limited 
extent. The autotrophic bacteria, including the nitrite- and nitrate-forming, sulphur-oxi- 
dizing organisms, etc., do not develop at all. Many of the cellulose-decomposing bacteria do 
not grow on common media; the nitrogen-fixing forms may grow only to a limited extent, 
etc. However, as far as the organisms that are capable of developing on the particular 
agar medium are concerned, the plate count gives just as accurate results as the direct mi- 
croscopic examination. The justification for the use of the plate method is that it permits 
determination of at least the relative abundance of those organisms which are capable of 
developing upon the particular medium. 

2. The dilution method consists in diluting the soil with sterile water, then adding i-cc. 
portions of the final dilutions to a medium (liquid or solid) which contains a particular 
pabulum favorable for the development of the specific organisms. Thus the number of urea- 



312 



SOIL MICRO-ORGANISMS 



decomposing organisms is determined by adding i-cc. portions of several of the final dilutions 
to flasks of urea medium; cellulose-decomposing organisms in media containing cellulose as 
the only source of energy; nitrogen-fixing organisms in media containing no combined 
organic or inorganic nitrogen, but containing a source of energy, such as glucose or mannitol; 
protozoa on media favorable for the development of bacteria and protozoa, etc. If i cc. of a 
dilution of i : 100,000, or i gm. of soil diluted with 100,000 cc. of water, gives no growth, 
while a dilution of i : 10,000 gives growth, the number of specific organisms per i gm. of 
soil is estimated to be between 10,000 and 100,000. On repeated determination, using nar- 
rower dilutions, closer results can be obtained. This method is very convenient not only for 
determining the abundance of specific organisms, but also for their isolation from soil, and 
separation from other forms. By selecting carefully a series of dilutions and using two plates 
with agar medium or two tubes or flasks with liquid medium for each dilution, incubating 
for seven to twenty-eight days, then determining the number of positive and negative 
growths, a more or less accurate picture of the abundance of specific protozoa in the soil is 
obtained. 

TABLE I 

Numbers of Micro-organisms in Soil, by Direct Microscopic Method 



Type of Soil 


Depth 


Numbers of Bacteria 


azotobacter 
Cells 


Pieces of Fungus 


Cocci 


Ba:illi 


Mycelium 


Forest 


[Surface 
] 10 cm. 
[20 cm. 
/Surface 
\io cm. 
[Surface 
] 10 cm. 
[20 cm. 


1,379,000,000 
99 I , 000 , 000 
281 ,000,000 
870,000,000 
569,000,000 
519,000,000 
407,000,000 
269,000,000 


1,212, 000 , 000 
466 , 000 , 000 
169,000,000 
376,000,000 
106,000,000 
192,000,000 
153,000,000 
139,000,000 


I , 000 , 000 
3 1 , 000 , 000 


47,000,000 
34,000,000 
7 , 000 , 000 
5 , 000 , ooc 
3 , 000 , 000 
3 , 000 , 000 
I 9 , 000 , 000 
3 , 000 , 000 




Brown loam 

Sandy soil 


84 , 000 , 000 

I , 000 , 000 

79,000,000 

23,000,000 

8 , 000 , 000 



DISTRIBUTION OF MICRO-ORGANISMS IN SOIL 

The numbers of various micro-organisms found in a definite quantity of soil de- 
pend upon the nature of the soil, depth of soil, season of year, state of cultivation, and 
a number of other factors. By the use of the direct microscopic method of Wino- 
gradsky, Richter found the numbers shown in Table I in i-gm. portions of different 
soils. 

By the use of the plate method, the number of bacteria found in the soil is con- 
siderably less, due to the various limitations of the method. The numbers vary from 
two or three hundred thousand to a hundred million cells per gram of soil. The or- 
ganisms developing on the plate consist of actinomyces (10-40 per cent of the col- 
onies), non-spore-forming bacteria (50-80 per cent of the colonies), and spore-forming 
bacteria (3-10 per cent of the colonies). 

The numbers of fungi found in the soil by the plate method range from 10,000 to 
1,000,000 per gram. Acid soils as well as aerated soils rich in organic matter have the 
highest number of fungi. The most important limitation of the plate method in this 
connection is the lack of differentiation between the numbers of spores and vegetative 
mycelium found in the soil. Yeasts are rare in soil, occurring somewhat more abun- 
dantly in acid soils, in vineyards, and in orchards. 



SELMAN A. WAKSMAN 313 

The protozoa occur in the soil in numbers ranging from a few hundred to over a 
million per gram. The flagellates and amebae are most abundant; the ciliates are less 
abundant. Nematodes occur in hundreds of millions per acre of soil. The numbers of 
earthworms and insects also run up to millions per acre. 

The maximum number of organisms is found either at or just below the surface of 
the soil; then the number diminishes rapidly, so that at a depth of 18-30 inches 
humid soils contain only very few bacteria and fungi. In arid soils, however, the mi- 
croflora and microfauna are distributed throughout much greater depths, frequently 
to 10 feet or more, due to better aeration and to a more uniform distribution of the 
soil organic matter. The numbers of micro-organisms in the soil vary with the differ- 
ent seasons of the year, and frequently they may fluctuate even daily, under field con- 
ditions. There is a rapid increase in the number of organisms in the spring, followed 
by a drop in the summer, then by another increase in the fall and a second drop in the 
winter. The summer drop is a result of a lack of sufiicient moisture and the winter 
drop because of low temperatures. 

ISOLATION AND CULTIVATION OF SOIL MICRO-ORGANISMS 
METHODS OF ISOLATION 

For the isolation of the great majority of soil micro-organisms, the same general 
methods are employed as for the isolation of bacteria, yeasts, and fungi from any other 
substrate such as milk, water, sewage, foodstuffs, etc. However, as a result of the fact 
that the soil harbors a number of specific organisms, which are not found readily in 
most of the other substrates and which do not develop at all on most of the common 
media, special methods must frequently be employed. Various enrichment or elective 
methods have been utiHzed by Beijerinck and others for the isolation of organisms 
from soil. These methods consist in preparing a specific medium which allows the 
development of one particular organism in preference to others. Such a medium is 
inoculated with soil, and, after successful growth has been obtained, transfers are made 
to fresh lots of the same medium, with the result that all contaminants, or those 
organisms which are not able to grow on the particular medium or find conditions un- 
favorable for their development, are gradually eliminated. Final isolation and purifi- 
cation of the specific organism is accomplished by the use of special plate methods or 
by employing high dilutions. A number of bacteria have been isolated from the soil by 
the use of this method. It is sufficient to mention the symbiotic and non-symbiotic 
nitrogen-fixing bacteria, the sulphur-oxidizing, the urea-decomposing, the sulphate- 
reducing, the cellulose-decomposing, the hydrogen bacteria, the methane bacteria, 
and a number of others. 

Instead of using liquid media to bring about the development of the specific or- 
ganism, Winogradsky suggested the employment of silica gel media, freed from all 
traces of impurities, and containing the specific nutrient, which can be acted upon only 
by the specific organism which is to be isolated. For example, silica gel plates con- 
taining the necessary minerals, a source of nitrogen, and some ground cellulose on its 
surface will allow the growth of practically pure cultures of cellulose-decomposing 
bacteria, when inoculated with a particle of soil containing these organisms. When a 
silica plate, containing mannitol or glucose as the only source of carbon and no traces 



314 SOIL MICRO-ORGANISMS 

of combined nitrogen is inoculated with particles of soil, the development of the aer- 
obic nitrogen-fixing organism Azotobacier will take place. This organism is frequently 
accompanied by the anaerobic nitrogen-fixing BacL amylohacter. The isolation of the 
nitrite- and nitrate-forming bacteria and a number of other organisms that are highly 
selective in their nutrition can be accomplished by the use of this method. 

Those organisms, especially the fungi, actinomyces, yeasts, protozoa, and many het- 
erotrophic bacteria that develop readily upon nutrient agar or gelatin plates can be 
most readily iso'ated from the respective colonies upon the plates. The protozoa 
have so far not been isolated in cultures free from bacteria (with certain exceptions, 
which are still a matter of dispute); however, they can be isolated free from other 
protozoa and can even be grown on media containing only dead bacterial cells. 
There are indications that some protozoa at least may be grown on purely inorganic 
media. The algae can be isolated on media (agar, siHca gel, sand) containing the nec- 
essary minerals and a source of nitrogen; they are cultivated in the light, since they 
utilize the energy of the sun. 

NUTRITION OF SOIL MICRO-ORGANISMS 

The soil micro-organisms can be generally divided, on the basis of their nutrition, 
into two groups: (i) The autotrophic organisms, or those which obtain their energy 
from the oxidation of inorganic elements (S2, H2), inorganic compounds (NH3+, NO2-, 
S2O3 ~,H2S), simple compounds of carbon (CO, CHJ ; carbon dioxide of the atmosphere 
is used as a source of carbon for structural purposes. The nitrite- and nitrate-forming, 
the sulphur-oxidizing, the hydrogen, methane, and iron bacteria belong to this group. 
All these organisms obtain their energy chemosynthetically. The algae, or the chlo- 
orophyll-bearing microscopic plants, which obtain their energy photosynthetically, 
are also autotrophic organisms. (2) The heterotrophic organisms, or those that ob- 
tain both their energy and carbon from complex organic compounds. 

There are very few autotrophic bacteria that are obligate in nature; most of them 
are facultative, being capable of obtaining their energy also from various organic 
compounds.' 

The heterotrophic micro-organisms include a great many forms, which vary con- 
siderably both in their morphology and physiology. A number of organisms which 
are very specific in their nutrition are found among the bacteria. The fungi, actino- 
myces, and protozoa are all heterotrophic. Some of the bacteria are aerobic; others 
are anaerobic. Some are capable of utilizing the required nitrogen in the form of gas- 
eous atmospheric nitrogen; others require combined nitrogen, either in an inorganic 
or in an organic form. Some require celluloses as the only source of energy; others 
can grow on a great variety of organic compounds. Some decompose paraffins, phe- 
nols, fats, lignins; others prefer proteins and carbohydrates. 

The composition of the media, to be used for the cultivation of these numerous 
micro-organisms, is so selected as to have it adapted to the food requirements of the 
particular organisms. Liquid and solid, organic and inorganic, media are employed. 

' See chap, xxiv in this volume. 



SELMAN A. WAKSMAN 315 

FUNCTIONS OF SOIL MICRO-ORGANISMS 
DECOMPOSITION OF ORGANIC MATTER 

Considerable quantities of organic matter of plant and animal origin are con- 
stantly introduced into the soil, in the form of stable manures, green manures, plant 
roots, plant stubble, and various waste products as well as certain organic fertilizers. 
The decomposition of these materials is one of the most important functions of the 
soil organisms. Various bacteria, fungi, protozoa, nematodes, rainworms, and other 
invertebrate animals take an active part in this process. Some macerate the organic 
matter mechanically and remove certain constituents for their own nutrition; others 
decompose a large part of the organic matter, but may leave certain constituents 
undecomposed. Some attack only certain specific ingredients of the organic matter. 
All of them build up cell substance, thus tending to replenish the supply of organic 
matter in the soil. 

The organic matter which is commonly added to the soil consists of water-soluble 
constituents including sugars and amino acids, of pentosans and other hemicelluloses, 
of true celluloses, of lignins, proteins, fats and waxes, tannins, pigments, etc., and of 
ash. Among the fungi, for example, the Mucorales can attack the water-soluble con- 
stituents, the proteins, and certain hemicelluloses, but not the celluloses, the lignins, 
the cutins, and the tannins. Other fungi, like various species of Aspergillus, Penicil- 
lium, Trichoderma, Fusarium, Cephalosporiiim, can readily attack the celluloses and 
hemicelluloses, but not the lignins and the cutins. Some of the wood-destroying fungi 
can decompose the lignins in preference to the celluloses, while others are unable to 
decompose the lignins but can decompose the celluloses. Most of the heterotrophic 
bacteria, including the numerous spore-forming and non-spore-forming bacteria and 
cocci, attack only the water-soluble constituents, the proteins, and the hemicellu- 
loses. A few highly specific organisms are able to decompose celluloses. Certain an- 
aerobic bacteria decompose celluloses with the formation of organic acids (acetic 
butyric), alcohols (butyl, ethyl), and gases (hydrogen, methane, carbon dioxide). 
The aerobic bacteria decompose the celluloses largely to carbon dioxide and water, 
with the synthesis of considerable protoplasm and frequently with the formation of 
slimy substances or gums. The decomposition of lignins by bacteria is still un- 
decided. 

The decomposition of organic matter in soil by the mixed-soil population of pure 
cultures of organisms can be followed by three distinct methods: 

I. By determining the evolution of carbon dioxide, which is the most important final 
product in the metabolism of all aerobic micro-organisms. The more easily the organic 
matter is decomposed, the larger is the amount of CO2 given off both under controlled labora- 
tory conditions and in the field. Soil itself will give off a constant stream of carbon dioxide, 
the actual amount depending on the temperature, moisture, and reaction of the soil and the 
content of organic matter. Under normal temperature, practically all the CO2 is given off 
as a result of the decomposition of the soil organic matter by micro-organisms. The amount 
of CO2 given off by the soil itself must be subtracted from the CO2 given off from the soil 
which has received the particular organic substance, the difference being an index of the 
decomposition of the latter. 



3i6 SOIL MICRO-ORGANISMS 

2. By determining the changes in the nitrogen content. When the organic materials are 
rich in nitrogen, their decomposition will be accomplished by a rapid evolution of ammonia, 
which is a waste product in the metabolism of micro-organisms. This ammonia will be rapidly 
changed, in well-aerated soils, to nitrates. The determination of the formation and accumu- 
lation of ammonia and nitrate nitrogen can serve as an index of decomposition of the organic 
matter. However, with a low nitrogen content, especially when the organic matter contains 
I per cent or less of nitrogen, the decomposition processes are always accompanied by the 
assimilation of the nitrogen which has been made available from the decomposition of the 
soil organic matter, or of the nitrogen which is present in the artificial medium. This nitrogen 
is required by the micro-organisms for the synthesis of their cell substance. The lower the 
nitrogen content of the decomposing organic matter, the greater will be the need for addi- 
tional nitrogen to enable the organisms to decompose the organic matter added. There is a 
very definite relation between the organic matter which is decomposed and the nitrogen (as 
well as phosphorus) which is required by the organisms for the synthesis of their cell sub- 
stance. 

3. The most accurate method of following the decomposition of organic matter by soil 
micro-organisms is the complete analysis of this organic matter at the beginning and at the 
end of the decomposition process. This method requires considerable time and a very careful 
technique. It involves the determination of the various constituents of the organic matter, 
especially the celluloses, pentosans, lignins, proteins, starches, sugars, fats, and waxes. 

The decomposition of natural organic matter added to the soil results in the for- 
mation of soil organic matter or "humus." This so-called "humus" consists of certain 
constituents of the natural organic matter added to the soil and which resist decom- 
position, namely, the lignins, the cutins, the fats and waxes, the tannins; of certain 
synthesized substances, namely, the living and dead cells of the soil micro-organisms; 
and of a number of substances which are still undergoing decomposition. When one 
hundred parts of organic matter are composted in the manure heap or are allowed to 
decompose in soil, there will be left, after two to six months, depending on the acidity, 
moisture, temperature, and nature of micro-organisms active in the process, nature 
of organic matter, etc., forty parts of organic matter. The sixty parts that have de- 
composed comprise practically all the pentosans, celluloses, and proteins. The forty 
parts remaining comprise largely the lignins and other resistant constituents and syn- 
thesized cell substance. The residual organic matter will tend to be uniform, as far 
as the content of nitrogen and phosphorus is concerned. The organic matter of the 
soil is more or less constant in composition, containing carbon and nitrogen in a 
definite ratio which approaches 10: i. The exact nature of this organic matter is still a 
matter of dispute. 

TRANSFORMATION OF NITROGEN IN SOIL 

When proteins are acted upon by micro-organisms, they are first broken down to 
amino acids, and these are sooner or later decomposed with the liberation of a part of 
the nitrogen as ammonia. A part of the nitrogen will be used by the organisms for 
the synthesis of their own cell substance, giving rise again to proteins and other com- 
plex nitrogen compounds. Since the carbonaceous substances are used by the or- 
ganisms as sources of energy, and since the assimilation of nitrogen depends on the 
amount of energy available, the liberation of nitrogen as ammonia will depend on the 
ratio of carbon to nitrogen in the compound or substance which is decomposed by the 



SELMAN A. WAKSMAN 317 

micro-organisms. Since the carbon content of natural organic materials, including 
carbohydrates and proteins, ranges between 40 and 50 per cent (with the exception 
of lignins, which contain 63-64 per cent carbon, but which are not readily available 
as sources of energy), and since the nitrogen content varies, we may conclude that 
the greater the percentage of nitrogen in an organic substance, the more rapid is the 
liberation of its nitrogen in the form of ammonia. The lower the nitrogen content 
and the higher the carbohydrate content of the organic matter, the more delayed will 
be the process of liberation of nitrogen as ammonia. 

This process of formation of ammonia can be carried out by practically all soil 
fungi and actinomyces, and by a large number of bacteria. Some of the bacteria, 
especially many spore-forming anaerobes and aerobes, are very active in the decom- 
position of native proteins, while other bacteria, especially certain non-spore-forming 
organisms and cocci, cannot attack native proteins, but act readily upon protein de- 
rivatives, such as the various amino acids. The non-protein nitrogenous substances, 
such as urea, uric acid, hippuric acid, xanthine, hypoxanthine, etc., are also decom- 
posed by various soil bacteria and fungi, and the nitrogen is sooner or later converted 
to ammonia. 

The ammonia thus liberated may (i) either be assimilated by soil micro-organ- 
isms, in the presence of undecomposed carbonaceous materials and changed back 
into proteins; (2) or be used by higher plants as a source of nitrogen; (3) or be ab- 
sorbed by the colloidal soil substances and held as ammonia; (4) or be acted upon 
further by other micro-organisms and changed into nitrates. The last process is 
carried out by two groups of autotrophic bacteria,' one changing the ammonia to 
nitrous acid and the other changing the nitrous acid to nitric acid. The latter is 
immediately neutralized by the soil bases and changed to nitrates. 

The nitrates may again (i) either be used by soil micro-organisms, in the pres- 
ence of available energy material, and changed into microbial proteins; (2) or be ab- 
sorbed by the roots of higher plants and used for plant nutrition; (3) or be leached 
out from the soil; (4) or be reduced by various bacteria to nitrites, to ammonia, to 
atmospheric nitrogen and simple gaseous oxides of nitrogen. The last process is carried 
out by various micro-organisms, under partial anaerobic conditions in the absence of 
atmospheric oxygen, when the nitrates are used as sources of oxygen. The reactions 
may also take place in the process of assimilation of nitrates as sources of nitrogen. 

The nitrogen which is changed into the gaseous form is lost as far as the supply 
of combined nitrogen in the soil is concerned. However, certain micro-organisms are 
capable of utilizing the gaseous nitrogen and "fix" it in the soil, thus making it again 
available for plant growth. This process of fixation of nitrogen is carried out either 
symbiotically or non-symbiotically. In the first instance, the bacteria {B. radicicola 
Beij.) can grow in association with leguminous plants, forming nodules on their roots, 
the plants supplying the bacteria with the necessary energy and the bacteria "fixing" 
the gaseous nitrogen, changing it into forms available for the leguminous plants. In 
some instances bacteria form nodules on the leaves of plants. The non-symbiotic ni- 
trogen-fixing bacteria, namely, the aerobic species of Azotobacter and other bacteria 
and the anaerobic species of Clostridia {B. amylobacter), do not need any host plants, 

' Described in chap. xxiv. 



3i8 



SOIL MICRO-ORGANISMS 



but are capable of using gaseous atmospheric nitrogen in the presence of available 
energy. Soil algae, fungi (with the possible exception of some mycorrhiza fungi), and 
actinomyces are unable to fix atmospheric nitrogen. 

True proteins are again synthesized by various soil organisms (including algae, 
fungi, actinomyces, and bacteria) and by higher plants, all using the nitrogen in the 
form of ammonia or nitrate ; proteins are also produced by the nitrogen-fixing organ- 
isms. These proteins may again be transformed when the bacteria, algae, and fungi 
are used as food by the protozoa and other invertebrate animals. 

The processes of nitrogen transformation can be schematically summarized as 
follows: 

Proteins 



Heterotrophic 
organisms 



Amino acids 




Ammonia 



Ammonia 



nous micro-organisms 

Proteins 

Ammonia Ammonia 



Autotropliic 
algae and 
bacteria 



Proteins 



Heterotrophic fungi 
and bacteria in presence 
of available energy 



Higher plants 



Autotrophic 
Nilrosonwnas 



Proteins 



Proteins 



Nitrites 



i 



Autotrophic 
Nitrobacter 



Nitrates 



Various bacteria 
nd fungi 



Proteins 




Proteins 



Proteins 



Leached out 



Nitrites, ammonia, 
oxides of nitrogen 
(N,0, NO, NA). 
gaseous 
nitrogen 



Nitrogen-fixing 
bacteria 



Proteins 



TRANSFORMATION OF MINERALS IN THE SOIL 

Among the various mineral elements which are of importance in the growth of 
plants and which are acted upon in one form or another by micro-organisms, the fol- 
lowing may be included: sulphur, phosphorus, potassium, calcium, and iron, and to a 
less extent, magnesium, zinc, and manganese. 



SELMAN A. WAKSMAN 319 

In the decomposition of proteins, the sulphur is liberated first as cystin and thio- 
compounds, then as hydrogen sulphide. The latter can be used as a source of energy 
by certain specific bacteria which oxidize the sulphide first to elementary sulphur, 
then to sulphuric acid. Elementary sulphur is also oxidized by certain specific bacteria 
to sulphuric acid. Thiosulphates are oxidized to sulphates and thionates. Some bac- 
teria are capable of reducing the sulphates to hydrogen sulphide, using the sulphate 
as a source of oxygen. In the metabolism of heterotrophic organisms only a small 
amount of sulphur is required. This is usually assimilated in the form of sulphate or 
organic sulphur. 

When insoluble phosphates are introduced into the soil they are made readily 
soluble by interaction with carbon dioxide as well as with the various organic and 
inorganic acids produced by the soil micro-organisms. When a mixture of sulphur 
and insoluble phosphate (the tri-calcium form) is added to soil or to a culture solution 
and inoculated with the proper bacteria, the sulphur will at first be oxidized to sul- 
phuric acid. This acid interacts immediately with the tri-calcium phosphate, changing 
it to di-calcium, then to mono-calcium phosphate and finally to phosphoric acid. Phos- 
phorus is a very important element in the nutrition of micro-organisms, and con- 
siderable quantities of it are utilized in the synthesis of the microbial protoplasm. It 
has been found that there is a definite ratio between the nitrogen and phosphorus 
content of bacterial cells such as Azotohacter, This led to the introduction of a meth- 
od for determining the amount of available phosphoric acid in a given soil, when a 
certain amount of soil is used as the exclusive source of phosphorus in a medium in 
which nitrogen fixation takes place. From the amount of nitrogen fixed, the amount 
of available phosphate is calculated. 

Potassium compounds are changed in the soil in an entirely diflferent manner from 
the phosphorus compounds. This, as well as the fact that potassium is not as indis- 
pensable to all micro-organisms as phosphorus, makes the role of micro-organisms in 
the transformation of potassium in soil somewhat different. The small amounts of 
potassium added to the soil in organic combinations are liberated when the organic 
matter is decomposed, some of it being again assimilated. Small amounts of potas- 
sium may also be liberated from the insoluble silicates when these interact with the 
various organic and inorganic acids formed by soil micro-organisms. 

Calcium is used to a limited extent as a nutrient by soil micro-organisms. It is 
of greater importance in the neutralization of the acids produced by the various or- 
ganisms; the reaction of the soil can thus be kept at or near neutrality, a reaction 
which is essential for the growth of some very important groups of soil micro-organ- 
isms. The formation of nitrates from ammonium salts would soon come to a stand- 
still if it were not for the presence of calcium which neutralizes the acids formed. The 
growth of some nitrogen-fixing bacteria takes place within a narrow range of reaction 
optimum; this can be attained by the application of calcium in the form of its car- 
bonate or oxide. Azotobacter, for example, will not develop in soil or in solution at 
a pH of less than 6.0. The formation of acids by bacteria, notably nitrous, nitric, 
and sulphuric, and their leaching out from the soil, in the absence of growing plants, 
also leads to considerable losses of calcium from the soil. 

Magnesium plays a role similar to calcium although it is likely to be more toxic 



320 SOIL MICRO-ORGANISMS 

than calcium in high concentrations. Magnesium is also somewhat more essential in 
the growth of certain organisms, like the fungi, than calcitjm. 

The presence of iron, even if only in very small quantities, is essential for the 
growth of all micro-organisms. Some organisms refuse to grow when a medium having 
an alkaline reaction is sterilized under pressure, due to the precipitation of the iron. 
Iron, in the form of ferrous carbonate, can be used by certain specific bacteria as a 
source of energy; the iron is thereby precipitated in the form as ferric hydroxide. 
However, precipitation of iron may also take place as a result of the chemical inter- 
action between the iron salt and products of microbial metabolism.^ 

SOIL MICROBIOLOGICAL ACTIVITIES AND SOIL FERTILITY 

Soil fertility or the ability of the soil to support the growth of higher plants de- 
pends to a large extent upon the activities of micro-organisms. 

One of the primary functions of these organisms is the mineralization of the or- 
ganic matter which is constantly added to the soil in the form of plant residues, in- 
cluding roots, stubble, weeds, and the tree products, green manures, stable manures, 
various organic fertilizers. These are decomposed, whereby most of the carbon is 
given off as CO2, the minerals and nitrogen are made again available for plant growth 
while a part of the organic matter which is more resistant to decomposition remains 
in the soil, thus tending to improve the soil as a physical and chemical medium for 
plant growth. When environmental conditions do not favor the activities of most of 
the micro-organisms, as in the case of water-logged soil, the organic matter tends to 
accumulate, giving rise to organic soils, such as peats. When conditions favor the 
activities of micro-organisms, as in well-cultivated and limed soils, organic matter is 
rapidly decomposed and the soil becomes depleted. This depletion is partly compen- 
sated for by the residues of the crops grown on the soil. 

Fixation of nitrogen is carried on in the soil to a limited extent by chemical proc- 
esses. The symbiotic bacteria, in the presence of the host plants, and the non- 
symbiotic bacteria, in the presence of available sources of energy, bring about a con- 
siderable fixation of atmospheric nitrogen. This valuable element, which is constant- 
ly removed from the soil by the growing crops, is thus, at least partly, replaced in the 
soil. 

In view of the fact that numerous micro-organisms carry out their varied activi- 
ties in the soil simultaneously, stimulating or injuring the growth and activities of 
one another, the resultant processes may often be unfavorable for plant growth. It 
has been suggested that the limitation of soil fertility, or the inability of the soil to 
support a better growth of cultivated plants, and frequently actual soil exhaustion, 
are due to the destruction of one group of soil micro-organisms by another. This 
theory was based on the following facts and assumptions: (i) Bacteria are largely 
responsible for the liberation of plant nutrients from soil organic matter. (2) Protozoa 
feed largely upon bacteria in the soil. (3) Treatment of soil with volatile antiseptics 
or with steam or dry heat makes the soil considerably more fertile. (4) These treat- 
ments result in an initial reduction in the numbers of bacteria followed by a very 
considerable increase greatly in excess of the numbers present in untreated soil. This 

' See chap, xxiv in this volume by R. L. Starkey. 



SELMAN A. WAKSMAN 321 

increase in bacteria is accompanied by a considerable increase in the evolution of 
ammonia (and carbon dioxide). (5) These treatments result in the complete or al- 
most complete destruction of the protozoa. The natural assumption was that protozoa 
by destroying the bacteria in the soil are responsible for the limitation of the fertility of 
the soil. The destruction of the protozoa by partial sterilization of soil results in the 
removal of the limiting agents and thus leads to an improvement of soil fertility.' 

However, further investigations have shown that many other organisms, espe- 
cially the fungi, actinomyces, nematodes, and other invertebrates, are also active in 
the transformation processes in the soil and are also influenced very markedly by 
partial sterilization of soil. 

The soil is a complex medium, harboring, too, many organisms that are responsible 
for numerous processes. By modifying the conditions of the soil, as by drainage and 
aeration, liming, and addition of available energy, in the form of organic matter, we 
can modify markedly not only the nature of the population, but even its activities, 
and direct them in such a manner as to have the results beneficial to the growth of 
cultivated crops. 

The following references may be consulted for more detailed analyses of soil mi- 
crobiology: 

Russell, J.: Tlie Microorganic Population of the Soil. Longmans, Green & Co., 1924. 
Waksman, S. A.: Principles of Soil Microbiology. Baltimore: Williams & Wilkins Co., 1927. 
Waksman, S. A., in collaboration with Barthel, Chr., Cutler, D. W., and Bristol-Roach, 

B. M.: Methoden der mikrobiologischen Bodenforschiing. Abderhaldcii's Handbiich dcr 

biologischen Arbeitsmethoden. Abt. XI, Tail 3. 1926. 

' For a more extensive discussion of micro-organisms in relation to soil fertility, see chap, xxvi 
by J. G. Lipman in this volume. 



CHAPTER XXIV 
AUTOTROPHIC BACTERIA 

ROBERT L. STARKEY 
New Jersey Agricultural Experiment Station, New Brunswick, N.J. 

Most of the lower forms of life, including the filamentous fungi, protozoa, and 
most of the bacteria, derive the energy for their metabolic processes as well as the 
carbon used for synthesizing their cells from complex organic compounds such as 
carbohydrates, fats, proteins, degradation products of these, or other compounds of 
the aliphatic and cyclic series. Such organisms are termed "heterotrophs." 

A typical reaction by which such organisms may obtain energy for their nutrition 
may be represented as follows:' 

I. CeH^Oe (solid)+60. (i atm.)=6C0. (i atm.)+6H,0 (liquid) 

AF298= —689,800 calories^ 

From this reaction the complete oxidation of i gm. molecule or 180 gm. of glucose 
liberates 689,800 calories which may be available to the organism bringing about such 
a reaction. 

Organisms effecting the synthesis of their own organic compounds from inorganic 
substances are termed "autotrophs." Of this group by far the most conspicuous forms 
are the higher plants which contain chlorophyll. Among the microscopic forms of this 
group would be included the smaller algae. Organic matter is produced within the 
cells of such organisms by photosynthesis. In this reaction the sunlight furnishes the 
necessary energy for the elaboration of organic compounds from carbon dioxide. 

There is another distinctive group of autotrophs which may live in the absence 
of light. They are generally microscopic and are indistinguishable from other bacteria 
in their morphological characteristics. These autotrophic bacteria are distinguished 
from the heterotrophic forms in that they have the specific ability of obtaining energy 
for their metabolism by the oxidation of certain inorganic substances. Autotrophic 
bacteria differ from the autotrophic plants in that this energy, derived from the oxida- 
tion of inorganic materials, is utilized by the bacteria for the reduction of carbon 
dioxide to organic compounds. In the group of sulphur bacteria are found representa- 
tives of forms which appear to be intermediate between the autotrophic bacteria and 
the chlorophyllous plants. These are the purple bacteria which require both hydrogen 
sulphide and light for their development. It would appear that their nutrition is 
dependent upon both a photosynthetic reaction and an oxidation of an inorganic 
substance. 

'Baas-Becking, L. G. M., and Parks, G. S.: Physiol. Rev., 7, 85-106. 1927. 

^ AF,58 refers to the free energy decrease or the maximum amount of useful work ol)tainable from 
the process at 25° C. or 298° absolute temperature. The data reported in these pages on free energ>' 
were all obtained from Baas-Becking and Parks {loc. cit.). 

322 



ROBERT L. STARKEY 323 

Obligate autotrophic bacteria and facultative forms existing under autotrophic 
conditions show certain distinctive physiological characteristics: 

1. They thrive on strongly elective, purely mineral media containing the specific in- 
organic oxidizable substances. 

2. Their existence is dependent upon these substances which are oxidized in the per- 
formance of the life-processes of the organisms. 

3. These oxidation processes furnish the only source of energy for the organisms. 

4. They require no organic nutrients as sources of energy. 

5. They use carbon dioxide (dissolved) as their exclusive source of carbon. This is re- 
duced by means of the energy obtained from the oxidation of the inorganic foods.' 

The number of known organisms which live as obligate autotrophs is quite small. 
Included in this group are nitrifying bacteria and some of the sulphur bacteria and 
iron bacteria. There appears to be a greater abundance of forms which can live as 
facultative autotrophs. These may either derive their energy from the oxidation of 
inorganic substances and reduce carbon dioxide for synthesizing their organic struc- 
tures or they may derive their energy, like the heterotrophs, from purely organic 
substances. The facultative autotrophs have representatives in the groups of sulphur 
bacteria, iron bacteria, and hydrogen bacteria. The transformations produced by 
autotrophic bacteria may be represented by the following reaction: 

IL NH4++iiO. = NO.-+H,0+2H+(io-8) 

AF,98= —66,500 calories 

The oxidation of i gram molecule or 18 grams of ionized ammonia to ionized nitrous 
acid results in the liberation of 66,500 calories. 

This group of organisms, called "autotrophic bacteria" (more accurately, auto- 
trophic micro-organisms lacking chlorophyll), is composed of forms varying greatly 
in morphological appearance as well as cultural habits. Some are minute single cells 
identical in appearance with the Eubacteriales. Some are large multicellular fila- 
mentous forms many micra in diameter. Some appear to be closely related to the 
algae in morphology (shape and complex mode of division) and may even contain 
pigments (as the bacteriopurpurin of the purple sulphur bacteria) which function in 
photosynthetic processes. These organisms compose a physiological group and are 
not distinctive morphologically from other bacteria. The known autotrophic bacteria 
may be classified as follows: 

A. Bacteria which oxidize compounds of nitrogen 

a) Oxidize ammonia to nitrite (Nitrosomonas, Nitrosococcus) 

b) Oxidize nitrite to nitrate {Nitrohacter) 

B. Bacteria which oxidize sulphur or compounds of sulphur 
a) Simple bacteria (genus Thiohacillus) 

I. Strictly autotrophic 
{a) Aerobic 

(i) Develop at reactions close to neutrality (species Th. thiopanis Beijerinck) 
(2) Develop under very acid conditions (species Th. ihiooxidans Waksman 
and Joffe) 
{b) Anaerobic (species Th. dcnitrificans Beijerinck) 

• Winogradsky, S.: Centralbl.f. Baktcriol. Abt. II, 57, 1-21. 1922. 



324 AUTOTROPHIC BACTERIA 

2. Facultative autotrophic 

(a) Facultative anaerobic (species of Trautwein) 
b) Higher bacteria (complex in morphology) 

1. Colorless (includes the genera Beggiatoa, Thiothrix, Thioploca, Achromatium, 
Thiophysa, Thiovulum, and Thiospira) 

2. Pigmented-red or purple bacteria (includes the genera Thiocystis, Thiocapsa, 
Thiosarcina, Lamprocystis, Thiopedia, Amocbohader , Thiothece, Thiodictyon, Thio- 
polycoccus, Chromatium, Rhabdochromatium, Thiospirillum, Rhodocapsa, Rhodo- 
thece) 

C. Bacteria which oxidize ferrous or manganous compounds 

a) Simple bacteria 

1. Long excretion filaments (genus Gallionella) 

2. Coccoid or oval shapes in masses (genera Siderocapsa and Sideromonas) 

b) Filamentous bacteria (genera Leptothrix and Crenothrix) 

D. Bacteria which oxidize hydrogen 

Winogradsky was the first to recognize the existence of bacteria with autotrophic 
habits. The first to be studied were certain of the higher sulphur bacteria' and later 
iron bacteria^ and nitrifying organisms.^ 

BACTERIA WHICH OXIDIZE COMPOUNDS OF NITROGEN 

Two distinct reactions are performed by organisms of this group: (i) oxidation 
of ammonia to nitrite {N itrosomonas and Nitrosococcus) and (2) oxidation of nitrite 
to nitrate {N itrobacter) , The transformation proceeds according to the following 
reactions: 

III. NH4++i|0. = NO.-+H.O+2H+ (10-8) 

AF298= —66,500 calories 

IV. N0.-+§0. = N03- 

AF298= 17,500 calories 

These reactions are produced by different bacteria, and no single species is known 
which can oxidize ammonia completely to nitrate.'' These organisms, called "nitri- 
fiers," are strict autotrophs and are unable to exist in the absence of their specific 
sources of energy: ammonia for the nitrite formers and nitrite for those which oxidize 
nitrite to nitrate. The organisms are non-sporulating cocci or short rods. They are 
very widely distributed in nature, occurring in practically all arable soils and in many 
bodies of water. These organisms are of particular importance in their natural habitat 
since they appear to be the agents primarily responsible for the formation of nitrate 
which is so generally utilized by higher plants as the source of nitrogen. It is an 

•Winogradsky, S.: Botan. Zeit., 45, 489-507, 513-23. 529^39, 545-^59. 569-76, 585^94, 606-10. 
1S87; Bcilrdgc ziir M or phol ogie mid Physiologic der Balder icn. Ziir Morphologic und Physiologic dcr 
Schwefelbakkricn. 120 pp. Leipzig: Felix, 1888. 

» Winogradsky, S.: Bolan. Zeit., 46, 261-70. 188S. 

3 Winogradsky, S.: Ann. del'Inst. Pasteur, 4, 213-31, 257-75, 760-71. 1890. 

"t See, however, Kaserer, IL: Zlschr. f. d. landiv. Versuch. in Oesler, lo, 37. 1907 (Centralhl. f. 
Bakieriol., Abt. II, 20, 170. 190S); Sach, J.: ibid., 62, 15-24. 1924. 



ROBERT L. STARKEY 325 

aerobic process requiring abundance of free oxygen and carbon dioxide.' The limiting 
reactions for the process are close to neutrality' (in solution cultures pH 7.4—8.4 for 
Nitrosomonas and pH 6.5—10.3 for Nitrobacter. Much wider ranges of activity are 
observed in natural habitats.) Their discovery and isolation by Winogradsky was the 
climax to a long series of investigations aiming to explain the mechanism of nitrate 
formation in soils. 

BACTERIA WHICH OXIDIZE SULPHUR OR COMPOUNDS OF SULPHUR 

The organisms concerned in these transformations represent a variety of forms 
concerned in many different types of reactions, some of which appear to be among the 
most unique physiological processes for deriving functional energy. They are of con- 
siderable importance in the oxidation of natural sulphides in waters, and may be 
active agencies in the transformation of inorganic sulphur compounds originating 
from organic combinations in decomposition processes.'' 

The known organisms included as the "simple sulphur bacteria" are all non- 
sporulating small rods. Some are obligate autotrophs and others facultative; some 
are aerobic, some facultative, and others obligate anaerobes. Among the aerobic 
forms are two very distinct organisms, both obligate autotrophs, one of which de- 
velops at reactions close to neutrality and the other under very acid conditions. 

The first — Thiobacillus thiopariis Beijerinck^ — occurs very , widely in soils and 
natural bodies of water. It oxidizes thiosulphate (NajSiOj), tetrathionate (Na2S406), 
sulphur, and sulphide. Some of the reactions may be explained as follows: 

V. 3Na.S203+50. = 2Na.S04+Na3SA 

AF298= — 260,000 calories (approximate) 

VI. S+i|0.+H.0 = H.S04 

A F298 = — 1 1 8, 500 calories 

As a result of the growth of this organism, precipitated sulphur is formed outside 
of the cells. It is generally believed that this arises as a product of the fundamental 
reaction such as expressed by Beijerinck: 

VII. 2Na.SA+02 = 2Na.S04+2S 

'Winogradsky, S., and Omeliansky, W.: Cenlralbl. f. BaktcrioL, Abt. II, s, 329-43, 377-87, 
429-40. 1899; Godlewsky, E.: ibid., 2, 458-62. 1896; Meyerhof, O.: Archiv.f. d. ges. Physiol., 164, 
353-427; 165, 229-84. 1916; 166, 240-80; 1917. Bonazzi, A.: /. Bact., 6, 479-99. 1921; 8, 343-63. 
1923; Gibbs, W. M.: Soil Sci., 8, 427-81. 1919. 

^ Gaarder, T., and Hagem, O.: Bergens Mus. Aarhok, No. 6, i-^i. 1920. See also Meyerhof, O.: 
loc. ciL; Meek, C. S., and Lipman, C. B.: /. General Physiol., 5, 195-204. 1922. 

3 Diiggeli, M.: Neiij. d. Naturfors. Gescll. Zurich, No. 121. 43 pp. 1919; Bavendamm, W.; Die 
farblosen und rotcn Schwefelbakterien. 156 pp. Jena: Gustav Fischer, 1924; Waksman, S. A., J. Bad., 
7, 231-56. 1922; Baas-Becking, L. G. M.: Ann. Bol., 39, 613-50. 1925. 

* Nathansohn, A.: Mitt. a. d. zoolog. Slation Neapel, 15, 655. 1902 {Cenlralbl. f. Bakleriol., Abt. 
II, II, 109. 1904); Beijerinck, M. W.: Arch. d. Sci. Exacles Nat. Haarlem (2d ser.), 9, 131-57; 
Cenlralbl. f. Bakleriol., Abt. II, 11, 592-99. 1904; Jacobsen, H. C: Folia MikrobioL, i, 487-96. 
1912; 3, 155-62. 1914; Waksman, S. A.: 5oi7 5'«., 13, 329-35. 1922; /. .4 gr. i?e^., 24, 297-305. 1923; 
Kilpatrick, M., Jr., and M. L.: J. Am. Ghent. Soc, 45, 2132-35. 1923. 



326 AUTOTROPHIC BACTERIA 

Since the sulphur is precipitated outside of the cells it seems likely that it is produced 
by some secondary reactions independent of the vital reactions of the cells. 

The bacterium developing under acid conditions — Thiobacilliis thiooxidans Waks- 
man and Joffe' — is distinctive in that it is able not only to tolerate but to produce 
higher concentrations of acid (also H+) than any other living organism yet known.^ 
Further, this acid is mineral and not organic. Its growth is inhibited in alkaline solu- 
tions but may not be injuriously affected in soils at neutral or alkaline reactions. 
In solutions, growth is most rapid at the very acid range of pH 2.0-3.0. It may fur- 
ther produce from 5 to 10 per cent (2N) sulphuric acid from the oxidation of elemen- 
tary sulphur. It oxidizes sulphur and thiosulphate to sulphate quantitatively with 
the accumulation of no intermediary products in purely inorganic media. The oxida- 
tion of sulphur may be explained by reaction VI, but the thiosulphate oxidation may 
occur as follows: 

VIII. Na.SA+H.O+20. = Na.S04+H.S04 

This reaction was also suggested by Nathansohn for Th. thioparus, but since the 
medium did not become acid, he believed that reaction V better explained the process. 
With thiosulphate as the source of energy for Th. thiooxidans, some sulphur may be- 
come precipitated as the oxidation proceeds but this is further oxidized to sulphate. 
This sulphur probably originates from some secondary reactions of the sulphuric acid 
with the thiosulphate. 

Th. thiooxidans was obtained from composts of sulphur, soil, rock phosphate, 
and was probably introduced with the sulphur. It has been noted in soils about 
sulphur mines but has not been found generally in soils which have not received 
applications of sulphur.^ Its distribution and importance under natural conditions is 
but little known. It is an obligate autotroph and fails to develop in the absence of 
its specific energy source in the form of sulphur or incompletely oxidized compounds 
of sulphur. 

Another of the morphologically simple sulphur bacteria is an anaerobic form — 
Thiobacillus denitrificans Beijerinck.^ It grows only in the absence of free oxygen and 

' Waksman, S. A., and Joffe, J. S.: Science (N.S.), 53, 216. 192 1; Proc. Soc. Ex per. Biol, b' Med., 
18,1-3. 1921; /. £/o/. CAem., 50, 35-45. 1922; /. 5ac/., 7, 239-56. 1922; Waksman, S. A.: Soil Sci., 
329-36. ig22; J. Bact., 7, 602-16. 1922; Waksman, S. A., and R. L. Starkey: Proc. Soc. Exper. Biol. 
&" Med., 20, 9-14. 1922; J. General Physiol., 5, 285-310. 1923; Starkey, R. L.: /. Bad., 10, 135-63, 
165-95. 1925. 

' Of interest may be the early observation of Preyer: Silzmii^ab. Bcr. Med. Gcs. Natiir. Hcilkiinde 
in Bonn, pp. 6-9. i866 (Bayliss, J. M.: Principles of General Physiology, p. 359. Longmans, Green & 
Co., 1924). He found in a large mollusc a salivary gland which produced sulphuric acid to a strength 
of 4-5 per cent. 

3 Joffe, J. S.: New Jersey A gr. Exper. Sia., Bull. 3^4. 91pp. 1922. See, however, Brown, H. D.: 
J. Am. Soc. Agrori., 15, 350-82. 1923; Jensen, H. L.: Centralhl. f. Bakieriol., Abt. II, 72, 242-46. 
1927. 

■t Beijerinck, M. W.:Arch. d. Sci., E.xactes el Naliir. Haarlem (2d ser.), 9, 131-57- 1904; Ccnlralbl. 
f. Bakieriol., Abt. II, 11, 592-99. See, however, Beijerinck, M. W.: Proc. Kon. Akad. v. Wetenschap- 
pen, Amsterdam, 22, 899-908. 1920; Gehring, A.: Ccnlralbl. f. Bakieriol., Abt. II, 42, 402-38. 1915; 
Lieske, R.: Jahr.f. Wiss. Bolan., 49, gi-127. 1911. 



ROBERT L. STARKE Y 327 

will not develop upon organic materials. It oxidizes sulphur, thiosulphate, and other 
compounds of sulphur to sulphate, obtaining oxygen from nitrates by such a reaction 
as the following: 

IX. 5Na.S A+8KN03+NaHC03 = 6Na.S04+4K.S04+4N.+ 2C0.+H,0 

AF298 = — 893 ,000 calories 

The organism has been found widely distributed in waters and soils. The results of 
Lieske indicate that the reaction does not proceed directly to sulphate since other 
sulphur compounds were present in the media at certain stages of growth. However, 
none of these intermediary products accumulated. It is likely that in the oxidation of 
sulphur or sulphur compounds by other sulphur bacteria the process is not as simple 
as is generally assumed. 

An organism obtained by Trautwein' showed characteristics similar to Th. 
denitrificans but differed in that it could exist either aerobically or anaerobically and 
either as an autotroph or heterotroph. In the presence of nitrate it developed as an 
anaerobe, and in the absence of organic compounds could obtain energy from the 
oxidation of sulphide, sulphur or thiosulphate, but the last was the best source of 
energy. Like the other forms, it has been found in soils, sewage, and fresh water. 

Autotrophic bacteria were first recognized by Winogradsky' in his studies of the 
more complex sulphur bacteria, some of which are pigmented. These forms are char- 
acterized by intracellular globules of amorphous sulphur. They develop as obligate 
autotrophs and utilize hydrogen sulphide as a source of energy which is oxidized in at 
least two separate stages, first to sulphur and then to sulphate. The reactions may 
be represented as follows:-' 

X. H.S (aq.)+^0. = H.04-S 

AF2y8= —41,500 calories 

XI. S+i^O^+H.O^H.SO^ 

AF298= — 118,500 calories 

In the presence of a continuous supply of sulphide the organisms always contain 
globules of sulphur. However, if the sulphide is all removed, the globules of sulphur 
are oxidized to sulphate and disappear from the cells which then die. The oxidation 
of both the sulphide and sulphur proceeds simultaneously in the presence of sulphide. 
For the colorless organisms free oxygen and carbon dioxide are indispensable. ^ 

These higher sulphur bacteria have been found extensively in both fresh and salt 
waters but not in soils. The pigmented forms frequently develop to such an extent 
as to lend very striking red colors to the waters. 

Two different pigments have been obtained from the bacteriopurpurin of the purple 
(red) sulphur bacteria.^ One, a green pigment unlike chlorophyll, is called "bacterochlorin" ; 

' Trautwein, K.: Cenlralbl.f. Bakkriol., Abt. II, 53, 513-48. 1921; 61, 1-5. 1924; see also Klein, 
G., and Limberger, A.: Biochem. Ztsclir., 340, 473-83. 1923. 

^ Loc. cit. 

3 See, however, Baas-Becking, L. G. M.: Ann. Hot., 39, 613-50. 1925. 

1 Keil, P.: Beitr. z. Biol. d. Pflanzen, ii, 335-72. 1912. 

s Molisch, H.: Die Piirpurbaclerien nach ncite Untcrsiichungen. 95 pp. Jena: Gustav Fischer, 
1907; Buder, J.: Jalir.f. Wiss. Botan., 58, 525-628. 1919. 



328 AUTOTROPHIC BACTERIA 

the other, a red pigment of the nature of a carotin, is called "bacterioerythrin." The pig- 
mented bacteria differ in their nutrition from the colorless forms in that they require light 
in addition to hydrogen sulphide and carbon dioxide, but only little or no free oxygen.' Some 
photosynthetic reaction appears to be associated with the utilization of the inorganic energy, 
but the action may be explained in at least two ways. Bavendamm^ expressed the opinion 
that the organism used two reactions as sources of energy, both a photosynthetic one and 
the oxidation of the sulphide. Since the cells failed to develop in the absence of either light 
or hydrogen sulphide, but could exist in the absence of oxygen, it seems more likely that the 
two processes are more closely related. It may be that the light serves as an agent to reduce 
carbon dioxide to at least intermediary synthetic substances and furnishes oxygen for the 
oxidation of the sulphide. This reaction is used as a further source of energy for metabolic 
processes. The following reaction was suggested by Baas-Becking and Parks :3 

XII. 6CO.+ i2H.S = C6H„06-F6H.O+i2S 

AF298 = + 2 1 2,000 calories 

Under these conditions the deficiency of energy is supplied by the photosynthetic reaction. 
BACTERIA WHICH OXIDIZE FERROUS OR MANGANOUS COMPOUNDS 

Precipitation of large amounts of ferric hydrate has been repeatedly observed from 
many natural waters such as mineral springs, mines, and flows from other subter- 
ranean streams. Associated with such deposits there generally occur cells of fila- 
mentous and other bacteria which are incased in sheaths of ferric hydrate. Some are 
multicellular organisms and others small spherical, oval, or bent cells incased in ir- 
regular masses of ferric hydrate or carrying ribbon-like streamers of the substance 
attached to the cells. In many cases these incrustations have been considered to be 
sufficient evidence to indicate that these organisms are autotrophic. Pure culture 
studies'" have shown that some of them are obligate autotrophs {Leptothrix ochraceae, 
L. trichogenes, Gallionella fcrruginca, G. minor) and others facultative {Leptothrix 
crassa, Crenothrix polys pora). Manganese compounds may be substituted for ferrous 
compounds with some of these bacteria. The reaction by which they may obtain 
energy may be represented as follows: 

XIII. 4FeC03+0.+6H.O = 4Fe(OH)3-F4CO. 

AF298 = — 8 1 ,000 (approximate) 

Since chemical oxidation of ferric hydrate may be so common in natural waters, 
the relative importance of the iron bacteria as factors in iron deposition is not known. ■'^ 

' Engelmann, T. W.: Pfliigcrs Arch. f. d. gcs. Physiol., 30, 95-124. 1SS3; 42, 183-86. 1888; 
Botan. Zeit., 46, 66 r. 1888. 

' Loc. cit. 3 Loc. cil. 

^ Winogradsky, S.: Bolan. Zcil., 46, 261-70. 1888; Centralhl.f. BaktcrioL, Abt. II, 57, 1-21. 1922; 
Lieske,R.: Jahr.f. Wiss. Botan., 4g,gi~i2j. zgii;Cenlra!bl.f. BaklcrioL, .\hl. II, 49, 4.1:^-2$. 1919; 
Molisch, H.: Die Eisenbakterien. 83 pp. Jena: Gustav Fischer, 1910; Cholodii}^, N.: Die Eiscn- 
baktericti. 162 pp. Jena: Gustav Fischer, 1926. 

5 Harder, E. C: U.S. Gcol Survey, Prof. Paper i ij. 89 pp. 1919; Gruner, J. W.: Econ.Gcol., 17, 
407-60. 1922; Halvorson, H. O., and Starkey, K. L.: /. Pliys. Chew., 31, 626-31. 1927; Starkey, 
R. L., and Halvorson, H. O.: Soil Sci., 24, 381-402. 1927. 



ROBERT L. STARKEY 329 

Even the existence of autotrophic iron bacteria has been questioned on theoretical 
grounds.' The precipitation of iron irrespective of its mechanism is of considerable 
economic importance. This is suggested by the extensive deposits of iron ore of 
aquatic origin, deposition with rusting of iron pipes, the fouling of drinking waters, 
and the formation of iron hardpans in soils, 

BACTERIA WHICH OXIDIZE HYDROGEN 

Hydrogen arises naturally in considerable abundance as a result of decomposition 
processes under anaerobic conditions. This is particularly noticeable in stagnant 
waters. It may also arise from mines and numerous non-biological sources. Bio- 
logical oxidation may be active in preventing any great accumulation of this gas. It 
may be oxidized under aerobic conditions by a large number of simple bacteria all 
of which appear to be facultative autotrophs.^ Some of those isolated by Grohmann 
have endospores. The reaction proceeds to water by the following reaction: 

XIV. H.+§0.=H.O 

AF298 = — 56,000 calories 

The quotient of H^iOj in pure-culture experiments is close to 2. No consistent ratio 
of C02(assimilated):H2(oxidized) was observed by Ruhland, but from observations 
on the physiology of other autotrophic bacteria one might infer that in the biological 
transformation such a relationship exists. 

These organisms occur widely in soils and may live upon many different organic 
compounds as well as hydrogen. 

It has been suggested that other organisms have autotrophic habits and utilize such 
compounds as selenium,^ carbon monoxide,'' arsenites,s and methane.* As concerns the first 
three compounds the evidence appears too limited at present to be more than suggestive. 
Although the bacterial oxidation of methane has been quite definitely established it seems 
undesirable to classify this process as autotrophic since methane may more logically be con- 
sidered as an organic compound. Although classification is naturally arbitrary and its dis- 
tinction is not as sharp in nature as seems to be suggested, it should eliminate confusion 
and lead toward simplicity and clearness. These advantages do not seem to be gained by 
including such organisms as methane-oxidizing bacteria among the autotrophic forms. 

' Baas-Becking and Parks, loc. cit. 

2 Kaserer, H.: CentralU.f. Bakleriol., Abt. II, 15, 573-76; 16, 681-96. 1906; Beijerinck, M. W. 
and Van Delden, A.: ibid., 10, 33-47. 1903; Nikitinsky, J.: ibid., ig, 495-99. 1907; Nabokitch, A. 
J., and Lebedeff, A. F.: ibid., 17, 350-55. 1907; Niklewski, B.: ibid., 20, 469-73. 1908; Jahrb.f. 
Wiss. Botan., 48, 113-42. 1910; Lebedev, A. J.: Bcr. d. Dent. Bot. GeselL, 27, 598-602. 1910; Groh- 
mann, G.: CentralU.f. Bakieriol., Abt. II, 61, 256-71. 1924; Ruhland, W.: Jahrb.f. Wiss. Botan., 
63, 321-89. 1924. 

3 Lipman, J. G., and Waksman, S. A.: Science (N.S.), 57, 60. 1923. 

■* Beijerinck, M. W., and Van Delden, A.: loc. cit.; Kaserer, H.: CentralU.f. Bakteriol., Abt. II, 
16, 681-96, 769-75. 1906; Lantzsch, K.: ibid., 57, 309. 1922. 

s Green, H. H.: Fifth and Si.xth Rpls. Vet. Res. Dept. of Agr. of the Union of So. Africa, pp. 
595-610. 1918. 

^Sohngen, N. L.: CentralU.f .Bakteriol., Abt. II, 15, 513-17. i9o6;Munz, E.: Zur Physiologic 
der Methanbakterien (Inaug. Diss., Halle). 63 pp. 1915; Aiyer, P. A. S.: Mem. Dept. Agr. India 
(Pusa), Chem. Ser., 5, 173-94. 1920. 



330 AUTOTROPHIC BACTERIA 

Although biological oxidation of phosphorus compounds has not been investi- 
gated, it is reasonable to believe that autotrophic processes may be concerned in such 
transformations. 

INFLUENCE OP ORGANIC MATERIALS ON AUTOTROPHIC BACTERIA 

The influence which organic compounds exert upon autotrophic bacteria is still 
a disputed point. These organisms fail to develop where organic materials furnish the 
sole source of energy in the medium. Further, no organic materials replace carbon 
dioxide as a source of carbon. However, even in the presence of the specific inorganic 
sources of energy, organic materials may exert pronounced inhibitory effects upon the 
development of autotrophic bacteria. 

Of the iron bacteria, L. ochracea appeared indifferent to considerable concentra- 
tions of organic matter, but G. ferriiginea was quite sensitive to such substances as 
peptone, sucrose, and asparagine.' For the higher sulphur bacteria moderate amounts 
of organic materials do not appear to be injurious.^ 

Oxidation by Th. thiooxidans was not appreciably affected by even 5 per cent 
glucose.^ Considerable citric acid (i per cent) exerted no effects on growth, but lower 
concentrations were tolerated than of sulphuric acid. It was early noted that some 
organic materials were toxic to nitrifying bacteria." Glucose injured growth of nitrate 
formers at 0.045 PS'" cent and completely inhibited development at 0.27 per cent. The 
nitrate formers were less susceptible to such injury than the nitrite formers. Cole- 
man5 noted no injury to growth of nitrate formers at 0.02-0.05 per cent glucose, but 
larger amounts were distinctly toxic. No nitrite formation took place at 0.2 per cent 
glucose, and even 0,02 per cent was injurious. Toxicity is very different with different 
compounds.* 

It has been noted with Th. thiooxidans'' as well as the nitrifying organisms'^ that, 
although glucose alone does not support growth, it disappears if introduced into 
cultures containing the specific inorganic energy source. 

Although under optimum conditions higher plants may utilize close to 80 per cent 
of the energy furnished by the light,' more commonly from i to 3 per cent is used. 
Between 5 and 10 per cent of the energy liberated in the oxidation of the inorganic 
substances is utilized by the autotrophic bacteria to reduce carbon dioxide to organic 

' Cholodny, N. : loc. cil. 

^ Winogradsky, S.: loc. cil.; Bavendamm, W.: loc. cit. 

3 Waksman, S. A., and Starkey, R. L.: loc. cil.; Starkey, R. L.: loc. cit. 

■I Winogradsky, S., and Omeliansky W.: Centralbl.J. Baktcriol., Abt. II, s, 329-43, 377-87, 429- 
40. 1899. 

5 Coleman, L. C: Centralbl. f. Baktcriol., Abt. II, 20, 401-20, 484-513. 190S. 

•^Meyerhof, O.: loc. cit.; see also Beijerinck, M. W.: Folia Mikrobiol. (3d year), 2, 91-113. 
1914; Winogradsky, S.: Compt. rend. Acad. Sci., 175, 301-4. 1922; Murray, T. J.: Proc. Soc. Exper. 
Biol. b° Med., 20, 301-3. 1923; Sach, J.: loc. cit.; Fred, E. B., and Davenport, A.: Soil Sci., 11, 389- 
404. 1921. 

7 Starkey, R. L.: loc. cit. * Coleman, L. C: loc. cit. 

"Warburg, O.: Naturmssenschaften, g, 354-58. 1921; 13, 985-93. 1925; Ztschr. phys. Chemic, 
106, 191-218. 1923; Warburg, O., and Negelein, E.: ibid., 102, 235-66. 1922; 108, 101-2. 1924; 
Naturwissenschaftcn, 10, 647-53. 1922. 



ROBERT L. STARKEY 



33^ 



compounds. The energy values for some of the autotrophic bacteria have been 
calculated by Baas-Becking and Parks as free energy efficiency/ 



Reaction 



Author 



N03-+|0. = N03- 



-+H.O+2H+ Meyerhof* 

Meyerhoff 



S+i|0.+H,0 = H.SO,. 



WinogradskyJ 
(Beggiatoa) 
1 Waksman and Starkey§ 
[ {Th. thiooxidans) 



6KN03-t-sS-|-2CaC03 = 3K.S04 

+ 2CaS04+2CO.+ 2N. 
5Na.SA+8KN03+2NaHC03 = 6Na.SOj L' k IT 



Beijerinck|| 



+4K.SO4+4N.+ 2CO.+H.O 
H,+|02 = H.O Ruhland** 

* Loc. cit. t Loc cit. % Loc. cil. § Loc. cit. 1 1 Loc. cii. f Loc. cil. ** Loc. cit. 



Free Energy 
Efficiency 
Percentage 

7-9 
5-9 
8.3 

8.3 

S-o 

9.0 
26.4 



The mechanism of assimilation of carbon dioxide by autotrophic bacteria shows 
some striking similarities to the photosynthetic reaction in higher plants. Klein and 
Svolba' have distinguished between assimilation, respiration, and oxidation in 
studies of two autotrophic bacteria, one an obligate and the other a facultative auto- 
troph. It was observed that in the assimilation reaction carbon dioxide was reduced, 
and that in the course of its use for the synthesis of organic compounds formaldehyde 
was formed.- Respiration, or the disintegration of synthesized organic compounds, was 
accompanied by the formation of acetaldehyde. The energy for these synthetic re- 
actions was derived from the process of oxidation of the specific inorganic compound. 

' Loc. cit. 

^ Klein, G., and Svolba, F.: Ztschr. f. Botan., 19, 65-100. 1926; see also Loew, O.: Biochem. 
Zlschr., 140, 324-25. 1923; Kluyver, A. L., and Donker, H. J. L.: Chemie d. Zelle u. Geivebe, 13, 134- 
90. 1926. 



CHAPTER XXV 
THE ROOT-NODULE BACTERIA OF LEGUMINOUS PLANTS 

EDWIN BROUN FRED 
University of Wisconsin 

The growing of leguminous crops for soil improvement is probably one of the old- 
est applications of bacteriology. More than two thousand years ago the Romans, and, 
at an earlier date the Chinese, were aware of the fertilizing effect of leguminous plants. 
Although the Roman farmers made use of these plants to increase the fertility of the 
soil, they did not know how the beneficial effect was produced. It was not until the 
early eighties of the last century that investigators demonstrated clearly that this 
beneficial effect of leguminous plants was due to the fixation of nitrogen within the 
root nodules. 

Outstanding among the early investigators were HellriegeP and his associate 
Wilfarth in Germany, Atwater^ in America, and Lawes and Gilbert^ in England. 
As a result of their work, intensive studies were carried out in various countries 
and the conclusion reached that nodule formation was caused by the associated 
growth of the leguminous plant and some lower organism, and also that nitrogen fixa- 
tion takes place in the root nodules. For the first time in 1888, Beijerinck^ in Holland 
obtained pure cultures of nodule bacteria and demonstrated their abihty to produce 
root nodules. 

RELATION or BACTERIA TO HOST PLANT 

The life of the root-nodule organism outside of the plant and the agencies which 
lead to its entry into the tissues are not well known. It has been suggested that the 
bacteria are attracted to the roots by the secretion of a substance of unknown com- 
position. At best, the movement of the bacteria in the soil is slow, perhaps i inch in 
twenty-four hours.^ 

The bacteria get into the parenchyma of the root through the root hairs or 
through other epidermal cells. These infected root hairs show a characteristic bend- 
ing at or near the root tip. Once within the tissues the bacteria multiply rapidly, 
forming threadlike filaments with many branches throughout the hair and into the 
parenchyma of the root. Here in the innermost cells of the root cortex, just outside 
of the endodermis, the bacteria bring about conditions favorable for a rapid multipli- 
cation of the surrounding cells and thus the formation of the younj^; nodule begins. As 
the cells multiply the young nodule soon pushes out the overlying cortical paren- 

' Hellriegel, H.: Lmidw. Vers. Stat., 33, 464. 1886. 
' Atwater, W. O.: Am. Chem. J., 8, 398. 1886. 
3 Lawes, J., and Gilbert, J. : Proc. Roy. Soc, London, 47, 85. 1890. 
^Beijerinck, M. W.: Bot. Ztg., 46, 725. 1888. 

s Thornton, H. G., and Gangulee, N.: Proc. Roy. Soc, London, B, 99, 427. 1926. 

332 



EDWIN BROUN FRED 



333 



chyma and epidermis and thus forms a swelling on the side of the root/ In general, 
nodules consist of a mass of thin-walled parenchymatous cells, rich in protein, and 




Fig. I. — Young pea plant with nodules 




Fig. 2. — Soy bean plants with nodules 

usually almost filled with bacteria. These thin-walled swollen cells occupy the greater 
part of the tissue of the nodule. A layer of cork and branches of a vascular system are 

' Brenchley, W. E., and Thornton, H. G.: ibid., 98, 373. 1925. 



334 ROOT-NODULE BACTERIA 

also present. By means of this system of vascular tubes the plant supplies the bac- 
teria with sugars and other food substances, and in turn takes away the nitrogenous 
compounds prepared by the bacteria. 

The shape, size, and position of the nodules vary with the dififerent leguminous 
plants, e.g., alfalfa nodules are small, finger-shaped swellings, single or in bunches or 
clusters; soy-bean nodules are larger, usually spherical, and rarely in clusters. Fig- 
ures I and 2 show the nodules of pea and soy bean. 

Of the ten thousand or more species of leguminous plants described by botanists, 
all except a very few, like Kentucky coffee tree and wild senna, show root nodules. 
The bacteria, however, from these various plants are not all alike. In general, plants 
closely related harbor the same kind of bacteria.' Some idea of the relationship among 
the bacteria and higher plants is shown in the groups of the more commonly cultivated 
plants. Below is a list of common leguminous plants divided into groups on the basis 
of cross-inoculation. The plants of each group have their own specific organism which 
can inoculate any member of the group but will not interchange with the plants of 
another group. 

CROSS-INOCULATION GROUPS 

1. Alfalfa, white sweet clover, yellow sweet clover, Hubam, bur clover, yellow trefoil, and 
fenugreek 

2. Red, mammoth, alsike, crimson, Egyptian, and white Dutch clovers 

3. Garden, canning, and field peas; hairy, spring, and wild vetches; broad bean; lentil; 
, sweet pea; and perennial pea 

4. Cowpea, peanut, Japan clover, velvet bean, lima bean, partridge pea, wild indigo, and 
tick trefoil 

5. Garden, field, navy, kidney, wax, and scarlet runner beans 

6. Lupines and serradella 

7. Soy beans 

8. Wood's clover (Dalea) 

9. Sanfoin 
10. Locust 

The explanation for the grouping of the plants into the so-called "cross-inocula- 
tion" groups has not been determined. Probably some phase of the physiological 
complex of the plant is responsible. Support for this assumption may be seen in the 
results of recent precipitin tests with the seed proteins of leguminous plants.^ These 
tests show that the seed proteins of those leguminous plants belonging to any one 
cross-inoculation group are closely related. 

There is some evidence that the bacteria within a given cross-inoculation group 
are not all alike. The organisms from pea and vetch are interchangeable, and yet the 
best results are usually secured from pea bacteria on peas and vetch on vetches. All 
in all, there exists a high degree of specificity. Why this difference should exist is at 
the present time unknown. 

' Burrill, T. J., and Hansen, R.: Illinois Agr. Exper. Sla. Bull. 202. 1917; Fred, E. B., Whiting, 
A. L., and Hastings, E. G.: Wisconsin Agr. Exper. Sla. Research Bull. 72. 1926. 
^ Baldwin, I. L., Fred, E. B., and Hastings, E. G.: BoL Gaz., 83, 217. 1927. 



EDWIN BROUN FRED 



335 



NITROGEN NUTRITION 

The process of nitrogen fixation is carried out by the bacteria within the nodule. 
The plant furnishes the carbohydrate and the bacteria the combined nitrogen. Just 



TABLE I 





Total Nitrogen 




Dry Basis 


Stage of Growth 


Roots 


Nodules 




Percentage 


Percentage 


Blossoms forming 


1.6 

1.8 
1-4 


5- 2 


Seed beginning to form 


2.6 




I- 7 







what causes the rapid autolysis of the bacterial cells within the nodule is not definitely 
known. It is reported' that the presence of a bacteriophage brings about the rapid 





Not inoculated Inoculated 

Fig. 3. — Effect of root-nodule bacteria on the growth of clover 

dissolution of the bacteria within the nodule, and thus the proper equilibrium is main- 
tained between the higher plant and the bacteria. 

The intimate relationship between the nodule and nitrogen content of the plants 
is shown by the results of chemical analyses. Young nodules of plants are much richer 
in nitrogen than the rest of the root, while at a later period the nodules are much 
lower in nitrogen. Analyses of lupine nodules and roots at various stages during 
the growth of the plant illustrate this point (see Table I).' 

Concerning the nature of the nitrogenous compounds of nodules, it has been 
found that about 50 per cent are soluble in water. ^ Of the protein-free soluble nitrogen 

' Gerretsen, F. C, Grijns, A., Sach, J., and Sohngen, N.L.: Ccntralbl.f. Baktcriol., Abt. II, 60, 
311. 1923; Grijns, A.: Doctor's thesis, p. 104. Wageningen, Holland: Veenman & Zonen, 1926. 
^ Stoklasa, J.: Landw. Jahrb., 24, 827. 1895. 
3 Strowd, W. H.: Soil Sc, 11, 123. 1921. 



336 



ROOT-NODULE BACTERIA 



about i6 per cent exists as primary amino nitrogen and 20 per cent as amid nitrogen. 
The nitrogen of the nodule is rapidly transferred from the underground nodule to the 

TABLE II 
Effect of Inoculation on the Nitrogen in Soy Beans 





Nitrogen in Pounds per Acre 




Tops 


Roots 


Nodules 


Total 


Bacteria 


S7-IO 

7.46 


2.40 
0.87 


S.86 


65-36 


Without bacteria 


8.33 






Gain due to bacteria 


49 • ^'4 


1-53 


5-86 


57.03 







UNINOCULATED 



INOCULATED 



seed and stems. Thus the activity of the bacteria results in the formation of a nitrogen 

supply for the plant. Figure 3 shows the effect of the bacteria on the growth of red 

clover on poor, sandy soil. 

Since nitrogen may be the 

element limiting plant 
growth, it is to be expected 
that the addition of the 
proper bacteria to certain 
leguminous plants will make 
possible a profuse growth 
and larger amounts of pro- 
tein. Some examples of the 
effect of the nodule bacteria 
on the host plant are pre- 



Nitrogen 

TAKEN FROM 

Air 



None. 



Total 
Nitrogen 

Crops 



\ 



Nitrogen 
taken from 
Soil 





\ 



WOA 



sentedinTable II.' Figure 4 
is prepared from the results 
of field tests of soy beans 
grown with and without bac- 
teria. Field tests with new 
crops show that inoculation 
of the seed with the nodule 
bacteria has a double ad- 
vantage to the farmer. There 
is an increase in the protein 
and often an increase in the 
yield. Numerous analyses 
have been made of alfalfa, 
clover, peas, and soy beans 
grown with and without 
nodule bacteria, some of 
which are given in Table III. The results are in accord with the general facts 
brought out by field tests. Aside from the favorable effects of inoculation on nitro- 
' Fred, E. B.: ibid., p. 469. 1921. 



Fig. 4. — Source of nitrogen for inoculated and uninocu- 
lated soy beans. Areas in black represent total nitrogen; shaded 
areas represent nitrogen from air and from soil in pounds 
per acre. 



EDWIN BROUN FRED 



337 



gen content and yield, in the case of canning peas the presence of the nodule bac- 
teria often results in more uniform growth and hence an improvement in the 
quality of the peas. 

FACTORS THAT INFLUENCE NODULE FORMATION 

When leguminous plants are grown in soil abundantly supplied with nitrate ni- 
trogen, the plants may develop luxuriantly and yet fail to show nodules, although the 
soil and seed have been richly inoculated with the proper bacteria. That this lack of 
nodules is not due to a toxic effect on the bacteria is well established. From all of the 
tests carried out it has been found that the suppression of nodule formation is not a 
direct effect of the nitrates but rather a case of change in the metabolism of the higher 
plants.' The addition of certain salts, such as phosphates and sulphates, and also car- 
bon compounds, such as sugars and organic acids, have been found beneficial to 

nodule production.^ 

TABLE III 





Weight 
Per Acre 


Nitrogen 
Per Acre 




Lb. 


Lb. 


Dried alfalfa with bacteria 


2,866 

1,715 

1,717 

874 


87 
46 

43 
27 


Dried alfalfa without bacteria 


Green peas (seed) with bacteria 

Green peas (seed) without bacteria 



Nodule formation and consequently nitrogen fixation are also greatly influenced 
by soil reaction, temperature, moisture, and oxygen relations. 



CHEMISTRY OF NITROGEN FIXATION 

If the average amount of nitrogen fixed per acre per year is taken as 100 lb., then 
the energy required by the bacteria would be that from about 10,000 lb. of car- 
bohydrate. This estimate is based on the results of pure-culture studies assuming 
that the nodule bacteria fix 1-2 mgm. of nitrogen for 100 mgm. of sugar consumed. 
Obviously this requirement of carbohydrate is far in excess of the amount supplied to 
the root nodules by the plant. It is highly probable, therefore, that the organisms in 
the plant nodule function much more efficiently than outside of the host. Christian- 
sen- Weniger'' suggests that the process is exothermic. Just how this fixation takes 
place is not known. It is probably a reduction process which results in the formation 
of ammonia. 

PURE-CULTURE STUDIES 

If the roots of a normal leguminous plant bearing nodules are thoroughly washed 
and the contents of a young nodule pressed out on a glass slide, fixed, and stained, 
great masses of variously shaped bacterial cells will be seen. 

From these active young nodules, it is compartively easy to isolate pure cultures 
of the nodule-forming bacteria. The ordinary culture media, nutrient or plain agar, 

'Fred, E. B., and Graul, E. J.: /. Af?i. Soc. Agron., 8, 316. 1916 

^Wilson, J. K.: Cornell Agr. Exper. Sla. Bull. j,S6. 1917. 

3 Christiansen-Weniger, F.: Centralbl.f. Bakteriol., Abt. II, 58, 41. 1923. 



338 ROOT-NODULE BACTERIA 

is not very satisfactory for the growth of these organisms. Much better results are 
obtained if special sugar media almost free of combined nitrogen or if sugar media 
plus extracts of plants are used. On mannitol phosphate agar at 2o°-25° C. well- 
developed colonies are obtained within five to ten days. 

The colonies of bacteria vary in appearance depending on the kind of leguminous 
plant from which they were obtained. From alfalfa, clover, and pea nodules the col- 
onies are usually large, raised, opaque, and sticky, while those from the nodules of 
soy bean are usually small, slightly raised, producing an almost transparent film which 
slowly spreads over the surface of the agar medium.' 

Morphology. — When cultures from a synthetic nitrogen-free agar medium are ex- 
amined the cells usually appear as short rods. In addition to the rods, cocci and large 

unevenly stained rods are found. These 
banded rod- forms may in turn become 
greatly swollen, and are then known as 
"bacteroids." The coccus forms which 
arise from the banded mother-cells are 
at first non-motile, but later become 
actively motile. As these cocci become 
older they elongate and return to the rod 
form, and are often motile. Still later 
they appear as the banded non-motile 
rods. According to Lohnis and Smith,^ 
Bewley and Hutchinson,-^ and more 
recently Thornton and Gangulee,^ the 
various forms through which the organ- 
isms naturally pass make up the so- 

„ -n . • r . J 1 r called "life-cycle." Figure =; is a photo- 

FiG. 5. — Bacteria from root nodule of pea -^ . 

plant showing large vacuolated forms. Xisoo. micrograph of SOme of the typical bac- 

teroid forms. 

The bacteroid form may not appear on culture media, but in the nodule this greatly 
enlarged and banded or highly vacuolated form is of common occurrence and some- 
what characteristic of the plant species. Small amounts of caffeine, cumarine, and 
other vegetable alkaloids added to the culture medium promote the formation of 
bacteroids.s The bacteroids produced in this way are not so numerous as those found 
in the nodule. Many of the small coccus forms may develop from one of the branched 
swollen rods. As the nodules disintegrate the root-nodule bacteria pass back into the 
soil as these small cocci. 

Flagellation. — According to the number and arrangement of the flagella these bac- 
teria may be divided into two great groups.** Those of the alfalfa, clover, pea, and 

' Burrill, T. J., and Hansen, R.: loc. cit.; Fred, E. B., Whiting, A. L., and Hastings, E. G.: loc.cit. 

= Lohnis, F., and Smith, N.R.: J. A gr. Research, 6, 6^$. 1916. 

3 Bewley, W. F., and Hutchinson, H. B.: J. Agr. Sc, 10, 144. 1920. 

''Thornton, H. G., and Gangulee, N.: loc. cit. 

5 Barthcl, C: Ann. deVInst. Pasteur, 35, 634. 1921. 

•^ Shunk, I. v.: J. Bact., 6, 239. 1921; ibid., 5, 181. 1920. 




EDWIN BROUN FRED 339 

bean groups possess numerous peritrichous flagella ; while those of the cowpea and 
soy-bean groups possess a single flagellum attached at a corner of the cell (mono- 
trichous) . 

Acidity relations. — The two general groups of nodule bacteria established by 
flagellation may be subdivided according to their behavior toward acidity. The acid- 
ity or critical pH for bacteria differs with the several cross-inoculation groups. In gen- 
eral, the limiting acidity is that known to be injurious to the higher plant. Below is 
given the critical pH for the bacteria of several of the more common leguminous 
plants/ 

Critical or Limiting pH 



Alfalfa and sweet clover 4 

Peas and vetches 4 

Clovers and beans 4 

Soy beans 4 

Lupine 3 



Carbohydrate fermentation. — Differences between the cross-inoculation groups as 
well as the strains within a given group are easily recognized by differences in the fer- 
mentation of carbohydrates.^ Since the fermentation of carbohydrates is weak, a 
medium of low-buffer capacity and a long period of incubation must be used if changes 
in pH value are to be noted. 

In general, the members of the peritrichous group are much more vigorous fer- 
menters than are those of the monotrichous group. Each of the cross-inoculation 
groups may be recognized by the use of appropriate carbon compounds, and in several 
of the cross-inoculation groups subgroups are established. For example, strains of the 
alfalfa organism produce much acid from mannitol, while others do not. 

Serological reactions. — Because of the high specificity of the agglutination test, 
it has been found of value in separating strains within a single cross-inoculation group. 
The sera usually show a high titre, agglutinating homologous antigen in dilutions of 
1-20,000, 1-30,000, or even higher. Repeated tests indicate that these differences in 
serological properties remain constant after long cultivation under varying condi- 
tions.^ 

Gum production. — One of the most noticeable characters of the nodule bacteria is 
their pronounced gum formation, especially when grown on plant extracts plus sugar. 
If pea or clover bacteria are grown on the extract of young pea plants, the formation 
of gum may be so profuse that the entire culture becomes viscous, like a weak gelatin. 
The addition of acetone or alcohol to these well-developed cultures precipitates this 
gum. Great variation in the amount of gum production is observed between the cul- 
tures of the different cross-inoculation groups and between strains within each group. 

Chemical tests show this gum to be a nitrogen-free carbon compound. It does 
not reduce Fehling's solution in the natural state but is readily hydrolyzed by acid 
or heat with the production of a reducing sugar. 

' Fred, E. B., and Davenport, Audrey: /. Agr. Research, 14, 317. 1918. 

= Baldwin, I. L., and Fred, E. B.: Soil Sc, 24, 219. 1927. 

3 Stevens, J. W.: ibid., 20, 45. 1925; Wright, W. H.: ibid., 20, 95. 1925. 



340 ROOT-NODULE BACTERIA 

Nitrogen fixation. — In spite of numerous investigations on the assimilation of ni- 
trogen in the absence of the host plant, this phase of the root-nodule problem has 
proved most baffling. The nodule bacteria under proper conditions will combine small 
amounts of atmospheric nitrogen, 2-3 mg. of nitrogen for each 100 cc. of culture. 
Compared with the amount fixed in the plant, this is insignificant. A satisfactory 
explanation for this difference in behavior in the absence of the host plant has not 
been found. 

Strain variations. — Only recently has the importance of the variation between 
strains within a cross-inoculation group been realized. The relatively small differ- 
ences between strains in cultural characteristics, carbohydrate fermentation, gum 
production, and serological properties have been shown to be correlated. The practi- 
cal importance of this lies in the fact that certain strains in symbiosis with plants have 
been more efficient in fixing nitrogen, and this activity of the strain has been shown to 
be related to the behavior of the strain in laboratory tests. 

CULTURES OF BACTERIA FOR THE INOCULATION OF LEGUMES 

Since 1887' it has been known that the transfer of soil from a well-inoculated field 
may exert a beneficial effect on the growth of leguminous plants in a new location. The 
bacteria carried in the soil cause nodule development and thus furnish nitrogen to the 
plants. More recently much easier and quicker methods have been found for intro- 
ducing nodule bacteria. Pure cultures of the bacteria are grown in liquid or solid 
media, and a water suspension of the bacteria is applied directly to the seed. In this 
way the bacteria are brought in close contact with the young plant. To insure nitro- 
gen fixation the use of bacteria to inoculate the seed has become a well-established 
practice, especially when a leguminous plant is seeded for the first time. 

The property of leguminous plants to accumulate atmospheric nitrogen in large 
quantities gives them an enviable role in economic agriculture. 

' Kellerman, K. F.: Centralbl.f. Bakleriol., Abt. II, 34, 42. 191 2. 



CHAPTER XXVI 
MICRO-ORGANISMS IN RELATION TO SOIL FERTILITY 

JACOB G. LIPMAN 

New Jersey Agricultural Experiment Station, New Brunswick, N.J. 

The average cultivated and improved soil is a good culture medium for higher 
plants and for micro-organisms. A soil is said to be fertile when it manifests ability to 
produce large crops throughout a series of years. By the same token it is infertile 
when its crop-producing power is below the average. But a soil fertile or infertile for 
higher plants is usually also fertile or infertile for micro-organisms. This is borne out 
by widespread experiments and observations which provide ample proof that an inti- 
mate relation exists between the macrofiora of soils, on the one hand, and their micro- 
flora and fauna, on the other. 

THE NATURE OF SOIL FERTILITY 
INTERNAL FACTORS 

A soil consisting, for the most part, of minute fragments is said to be "fine- 
textured soil," and when made up of particles of relatively large size, it is designated 
as a "coarse-textured" soil. The relation of the fineness of soil material to texture may 
be well illustrated by the figures given by Lyon, Fippin, and Buckman, who note that 
for the average gram of material there will be found, in coarse sand, 1,670 particles; 
in fine sand, 131,900 particles; in very fine sand, 1,676,500 particles; in silt, 35,934,000 
particles; and in clay, 45,632,000,000 particles. Thus soil texture must necessarily 
affect the rate of movement of water and air, the temperature, microbiological activi- 
ties, and the concentration and composition of the soil solution. Obviously, then, the 
different factors just noted exert a reciprocal influence on one another. 

Wide differences exist in the chemical composition of soils. Their origin, their 
age, and the environment under which they were formed all serve to determine their 
value as a culture medium. Sandstones, limestones, feldspars, granites, and other 
rocks when exposed to weathering will give rise to soils of different physical and 
chemical quality. In humid climates the soil is exposed to constant leaching. Basic 
ions such as calcium, magnesium, potassium, sodium, and iron are gradually made 
soluble and carried away. Given a time long enough, everything except the silica 
skeleton of the soil may be more or less thoroughly removed. In the earlier stages of 
the process the loss of basic material may not be as clearly reflected in the vegetation, 
but sooner or later the increased hydrogen-ion concentration is certain to affect the 
crops, as weU as the soil micro-organisms. On the other hand, in arid and semi-arid 
regions the leaching of the soil is less thorough, an acid reaction does not develop so 
readily, if at all, the soil tends to become basic in character, and not infrequently 
an undue accumulation of soluble salts (known as alkali) takes place. Hence the 
composition of the parent-rock, the length of the weathering period, and environ- 
mental factors of temperature, rainfall, glaciation, and aridity have always played a 

341 



342 SOIL FERTILITY 

prominent role in determining the growth and distribution of plants and, in conse- 
quence, also of micro-organisms. The fact that the latter carry on their activities 
largely in the moisture films surrounding the soil particles, that these films represent 
solutions of varying composition and concentration, that the organic and inorganic 
colloidal material in the soil is the seat of many chemical reactions, and that the 
intensity of biological changes is directly reflected in the intensity of chemical changes, 
is evidence that chemical and biological soil factors are intimately related. 

Organic matter is the direct source of food and energy of the heterotrophic soil 
organisms. It is the indirect source of energy for most of the autotrophic organisms. 
For this reason too much importance cannot be attached to organic matter as a link 
in the chain of soil fertility. Aside from the organic matter of recent origin found on 
the surface of the soil and within its tilled portion, there are older residues in the sur- 
face and subsoil of plants, soil-infesting insects, and micro-organisms. The quantity 
of organic matter will ordinarily vary from less than i per cent in light sandy soils 
to more than 5 per cent in heavy silt or clay loams. In muck and peat soils the organic 
matter content may range up to 85 per cent. An acre of surface soil 6| inches deep 
is usually assumed to weigh 2,000,000 pounds; hence, at 2 per cent there would be 
present about 20 tons of organic matter in this quantity of soil. In the next layer of 
the same depth the quantity of organic matter is ordinarily only half as great. It 
becomes relatively still less in the following layers. There is also a marked difference 
in the quality of the organic matter at different depths, particularly as to the carbon- 
nitrogen ratio. Everything being equal, the greater the depth from which the organic 
matter is taken, the narrower its carbon-nitrogen ratio. This is due primarily to 
variations in the proportions of carbohydrates and proteins brought about by micro- 
biological activities. As would be expected, the older residues in the subsoil represent 
material that had been used over and over again for the building of microbial cells. 

Excessive concentrations of soluble salts injure higher plants and depress the 
activities of soil micro-organisms. In extremely acid soils soluble salts of aluminium 
and iron often produce toxic effects. In some abnormal soils compounds of boron, 
copper, nickel, arsenic, and manganese may be present in unduly large amounts and 
exert toxic action. Certain organic compounds, toxic to higher plants, have been 
isolated from soils. Some of these compounds may be injurious also to micro-organ- 
isms. In modern fertilizer practice large amounts of chemical fertilizer are often ap- 
plied in the row or hill. When such treatment increases beyond a certain point, the 
concentration of the soil solution, seed germination, plant growth, and microbio- 
logical activities may be interfered with. Altogether, therefore, toxic or injurious 
substances may arise in the soil either as the result of the weathering of the rock 
material or the chemical changes brought about by cropping and the use of com- 
mercial fertilizers. 

Both positive and negative influences are exerted by micro-organisms in the soil. 
The transformation by them of various organic substances involves the production of 
large quantities of carbon dioxide, the formation of ammonia and nitrates, and the 
mineralization of organic matter. As the end products of decay and fermentation, 
carbonates, phosphates, sulphates, and other simple salts are again made available for 
the use of plants. Nitrogen fixing bacteria, both symbiotic and non-symbiotic, may 



JACOB G. LIPMAN 343 

add more or less substantial amounts of combined nitrogen to the reserves of this 
constituent. Under some conditions nitrates may be reduced and thus withheld from 
the crop. In the presence of sufficiently large amounts of cellulose, starch, sugar, etc., 
bacteria and fungi may use up available nitrogen compounds fast enough to offer 
serious competition to higher plants. The kinds, numbers, and distribution of the 
micro-organisms in the soil are affected by the food supply and the nature of the soil 
environment. In their turn the micro-organisms modify the soil environment of crops 
by increasing or decreasing the food supply of the latter, and it is the task of the skil- 
ful farmer to treat his soil in such manner as to reduce to a minimum the negative 
effects and raise to a maximum the positive effects of soil microbiological activities, 

EXTERNAL FACTORS 

The amount and distribution of rainfall, the conservation of soil moisture, and 
the use of irrigation water are related in an important way to the growth of plants. 
Optimum moisture conditions for plants are established when the pore space in the 
soil, that is, the space not occupied by the soil particles, is filled with water to the 
extent of about 60 per cent. When there is too much or too little water in the soil, 
reduction and oxidation processes and the diffusion of gases may be shifted in the 
direction unfavorable to normal growth. Micro-organisms, like higher plants, are 
directly affected by the thickness of the moisture films surrounding the soil particles, 
the movement of capillary water, the concentration of the soil solution, and the diffu- 
sion of soil gases. Fine-textured soils may at times contain an excess of water, while 
coarse-textured soils may frequently contain an excess of air. Hence in the former 
the mineralization of organic matter is retarded, and in the latter it is hastened often 
to the detriment of the farmer. This will account for such agricultural practices as 
drainage, subsoiling, tillage, liming, green-manuring, etc., intended to speed up the 
circulation of water and air in fine-textured soils. It will account, also, for marling, 
claying, liming, green-manuring, and other practices as a means of retarding the 
movement of water and air in the more open soils. By controlling the water and air 
supply the farmer can influence the microbiological activities in the direction most 
favorable for crop growth. 

Temperature, like water and air, is a major soil-environment factor for both higher 
plants and micro-organisms. There is a direct relation between the range of soil 
temperature and the amount of chemical change produced by the soil microflora. 
Similarly, there is a direct relation between the intensity of microbiological activity 
and the growth and yield of crops. In tropical and subtropical regions microbio- 
logical activities are never dormant wherever the moisture supply is sufficiently 
abundant. This accounts for the rapid decomposition of plant residues as an offset 
to more luxuriant vegetation and the more rapid accumulation of organic matter 
both in the soil and on its surface. In such environments the circulation of carbon 
and nitrogen between soil and atmosphere is manifestly more active than it is in 
regions farther north. On the other hand, in temperate and subarctic regions the rate 
of both accumulation and dissipation of organic matter is less intense. There must be, 
therefore, a certain relation between temperature, the microbiological activities in the 
soil, the supply of carbon dioxide to vegetation, and the rate of growth of the latter. 



344 SOIL FERTILITY 

Both soil moisture and temperature have a more or less direct relation to soil 
aeration. As has already been pointed out, the proper circulation of air and water in 
the soil is essential for the normal activities of both plants and micro-organisms. 
Temperature affects this circulation by influencing the rate at which inorganic and 
organic substances become soluble. In its turn, concentration of the soil solution will 
affect surface tension and the movement of capillary water. Temperature affects 
also the diffusion of gases and influences the oxygen pressure in the soil solution. 
Hence we recognize that moisture and temperature both help to determine the 
prevalence and activities of obligate and facultative aerobes and anaerobes, and the 
extent and intensity of oxidation and reduction processes. In localities where the 
winters are long and the spring and summer temperatures relatively low, evaporation 
is retarded, and the activities of aerobic bacteria are slowed down. Conditions become 
favorable then for the accumulation of peat as it is found in North America, Siberia, 
Northern Europe, and elsewhere. 

Topography, like moisture and temperature, must receive consideration as a factor 
capable of influencing soil fertility. Weathered material is constantly being moved to 
lower levels. The steeper the slope, the more rapid is the downward movement of 
such material. Hence, topography comes into play as a factor in determining the 
depth of the soil, not only on the hillsides, but also in the valleys. So