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^2 ^^ 



JOURNAL 



OF 



BACTERIOLOGY 



VOLUME I 



BALTIMORE, MD. 
1916 



COMPOSED AND PRINTED AT THE 

WAVERLY PRESS 

By the Williams & Wilkins Company 

Baltimore, Md., U. S. A. 



CONTENTS 

Number 1, January, 1916 

Foreword — The Genesis of a New Science — Bacteriology. W. T. Sedgwick. . 

The Pedagogics of Bacteriology. D. H. Bergey. Presidential Address, Ur- 
bana meeting 5 

Further Studies on Bacterial Nutrition: The Utilization of Proteid and 
Non-Proteid Nitrogen. L. F. Rettger, N. Barman, and W. S. Sturges... 15 

Studies on Soil Protozoa and their Relation to the Bacterial Flora. I. 
James M. Sherman 35 

A Culture Medium for Maintaining Stock Cultures of the Meningococcus. 
C. G. A. Roos 67 

Bile Compared with Lactose Bouillon for Determining the Presence of B. 

coli in Water. M. M. Obst 73 

Society of American Bacteriologists. Abstracts of Papers Presented at Sev- 
enteenth Annual Meeting, Urbana, III. December 28-30, 1915 81 

Abstracts of American Bacteriological Literature: 

Bacteriology of Food 123 

Bacteriology of Soils ; 123 

Bacteriology of Water and Sewage 125 

Dairy Bacteriology 125 

Disinfection 126 

Immunology 126 

Laboratory Technique 129 

Medical Bacteriology 130 

Physiology of Bacteria 131 

Plant Pathology 132 

Number 2, March, 1916 

Preliminary Report on Synthetic Media. C. J. T. Doryland 135 

On the Significance of the Voges-Proskauer Reaction. Max Levine 153 

Studies on Soil Protozoa and Their Relation to the Bacterial Flora. II. 

James M. Sherman 165 

Are Spore-forming Bacteria of any Significance in Soil under Normal Condi- 
tions? H. J. Conn 187 

A Possible Function of Actinomycetes in Soil. H. J. Conn 197 

Practical Observations on the T-'tration and Adjustment of Culture Media. 

Bertha van Houten Anthony and Clarence V. Ekroth 209 

A Species of Alcohol-forming Bacteria Isolated from the Interior of Stalks 
of Sugar Cane Infested with the Cane-borer Diatraea saccharalis. W. 

L. Owen 235 

iii 



IV CONTENTS 

Abstracts of American Bacteriological Literature: 

Animal Pathology 249 

Bacteriology of Water and Sewage 250 

Immunology 251 

Laboratory Technique 256 

Medical Bacteriology 257 

Protozoa and Other Animal Parasites 266 

Number 3, May, 1916 

Frontispiece: Portrait of Professor T. J. Burrill 

In Memoriam, Thomas J. Burrill. Erwin F. Smith 269 

Resolutions adopted at the Urbana Meeting of the American Bacteriolo- 
gists in regard to the work of Professor Burrill 271 

Studies on Aerobic Spore-bearing Non-pathogenic Bacteria. Part I. Intro- 
duction. W. W. Ford 273 

Spore-bearing Bacteria in Milk. J. S. Lawrence and W. W. Ford 273 

The Number of Colonies Allowable on Satisfactory Agar Plates. Robert S. 

Breed and W. D. Dotterrer 321 

A Modification of the Hygienic Laboratory Method for the Production of 
Tetanus Toxin. Harriet Leslie Wilcox 333 

A Method of Anaerobic Plating Permitting Observation of Growth. Horry 

M. Jones 339 

Testicular Infusion Agar. A Sterilizable Culture Medium for the Gono- 
coccus. Ivan C. Hall 343 

Book Review. Der Erreger der Maul- und Klauenseuche. By Heinrich 
Stauffacher. Gary N. Calkins 353 

Abstracts of American Bacteriological Literature : 

Animal Pathology 357 

Bacteriology of Air and Dust 360 

Bacteriology of Food 361 

Bacteriology of Soils 361 

Bacteriology of the Mouth 362 

Bacteriology of Water and Sewage 363 

Classification of Bacteria 364 

Disinfection 364 

Immunology 364 

Industrial Bacteriology 372 

Laboratory Technique 373 

Medical Bacteriology 376 

Paleontology 384 

Plant Pathology 384 

Number 4, July, 1916 

Biological Variations of Bacteria. I. M. R. Smirnow 385 

A New Culture Medium for the Tubercle Bacillus. W. Whitridge Williams 
and Ward Burdick 411 



CONTENTS V 

Bacillus Abortus (Bang) as an Etiological Factor in Infectious Abortion in 

Swine. Edward S. Good and Wallace V. Smith 415 

The Relation of Protozoa to Certain Groups of Soil Bacteria. T. L. Hills. . 423 
A Study of the Boas-Oppler Bacillus. P. G. Heinemann and E. E. Ecker. . . 435 

A Contribution to the Bacteriology of Silage. J. M. Sherman 445 

Book Review. Laboratory Manual in General Microbiology. Ward Giltner 453 
Abstracts of American Bacteriological Literature : 

Bacteriology of Food 455 

Bacteriology of the Mouth 455 

Bacteriology of Soil 456 

Bacteriology of Water and Sewage 457 

Disinfection 459 

Immunology 461 

Industrial Bacteriology. 463 

Medical Bacteriology 464 

Number 5, September, 1916 

The Bacteriology of the Bubble Fountain. Dorothy F. Pettibone, Franklin 
P. Bogart and Paul F. Clark 471 

The Advantages of a Carbohydrate Medium in the Routine Bacterial Exam- 
ination of Milk. James M. Sherman 481 

On a Species of Treponema Found in Rabbits. Hans Zinsser and J. G. 

Hopkins 489 

Studies on Spore-Bearing Non-Pathogenic Bacteria. Part II. W. W. Ford 

and others 493 

Spore-Bearing Baateria in Dust. C. A. Laubach 493 

Spore-Bearing Bacteria in Water. C. A. Laubach 505 

Spore-Bearing Bacteria in Soil. C. A. Laubach and J. L. Rice 513 

Miscellaneous Cultures. W. W. Ford 518 

Classification. W. W. Ford 527 

A Rapid and Simple Indol Test. Paul R. Cannon 535 

Bacterial Nutrition, a Brief Note on the Production of Erepsin by Bacteria. 
Nathan Berman and Leo F. Rettger 537 

A Practical Method for the Identification of Guinea-Pigs under Treatment. 
A. Parker Hitchens 541 

A Note on the Preparation of Agar Agar Culture Media. C. L. Williams and 
H. P. Letton 547 

Book Reviews. McFarland's Pathogenic Bacteria and Protozoa; D. Green- 
burg. Mallory's Principles of Pathologic Histology; F. P. Gay 549 

Abstracts of American Bacteriological Literature: 

Animal Pathology 553 

Bacteriology of Soils 553 

Bacteriology of the Mouth 563 

Bacteriology of Water and Sewage 564 

Classification of Bacteria 565 

Dairy Bacteriology 566 

Disinfection 567 



VI CONTENTS 

Abstracts of American Bacteriological Literature — Continued 

Immunology 568 

Laboratory Technique 576 

Plant Pathology 577 

Public Health Bacteriology 578 

Medical Bacteriology 579 

Number 6, November, 1916 

Studies in the Nomenclature and Classification of Bacteria. The Problem 

of Bacterial Nomenclature. R. E. Buchanan 591 

The Oxygen Requirements of Biological Soil Processes. T. J. Murray 597 

The Preparation of Culture Media from Whole Blood. Raymond A. Kelser. 615 
Preliminary Note on the Classification of Some Lactose Fermenting Bac- 
teria. Max Levine 619 

A New Ice Sampler. Myrtle Greenfield 623 

Apparent Recovery of a Hen Infected with Bacillary White Diarrhea. (As 

Determined by the Macroscopic Agglutination Test.) George D. Horton 625 
Observations sur I'lnfiuence Chimique des Milieux de Culture sur le D6ve- 
loppement et la Production de I'Indol par les Coli-Bacilles et par les 

Bacilles Typhiques. Edgard Zunz and Paul Gyorgy 627 

Some Regulating Factors in Bacterial Metabolism. I. J. Kligler 663 

Book Review. Practical Textbook of Infection, Immunity and Specific 

Therapy. By John A. Kolmer, M.D., Dr. P.H. Hans Zinsser 673 

American Bacteriological Literature: 

Bacteriology of Soils 675 

Bacteriology of Water and Sewage 680 

Classification of Bacteria 681 

Immunology 682 

Laboratory Technique 693 

Medical Bacteriology 694 

Physiology of Bacteria 703 

Plant Pathology 705 

Public Health Bacteriology 706 

Index 709 



VOLUME I 



NUMBER 1 



JOURNAL 



OF 



BACTERIOLOGY 



OFFICIAL ORGAN OF THE SOCIETY OF AMERICAN 
BACTERIOLOGISTS 



JANUARY, 1916 




// is cliarncteristic of Science and Progress that they contirtuallij 
open new fields to our n'sion . — Pasteur 



PUBLISHED BI-MONTHLY 

WILLIAMS & WILKINS COMPANY 

BALTIMORE, U. S. A. 

THE CAMBRIDGE UNIVERSITY PRESS 

FETTER LANE. LONDON; E. C. 

Application has been made for entry as aecond-claas matter at the Post Oflice at H.iltiinorf. Mil. 
under the ant of Maroh 3, 187ft 



Bacteriological Pepton 



Fairchild Building 

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FAIRCHILD BROS, k FOSTER 



JOURNAL OF BACTERIOLOGY 

OFFICIAL ORGAN OF THE SOCIETY OF AMERICAN BACTERIOLOGISTS 



DEVOTED TO THE ADVANCEMENT AND DIS- 
SEMINATION OF KNOWLEDGE IN REGARD TO 
THE BACTERIA AND OTHER MICRO-ORGANISMS 



Editor-in-Chief 
C.-E. A. WINSLOW 

Yale Medical School, New Haven, Conn. 



Managing Editor 
A. P. KITCHENS 

Glenolden, Pa. 



C. C. Bass 
R. E. Buchanan 
P. F. Clark 
H. W. Conn 
F. P. Gay 

F. P. GORHAM 

F. C. Harrison 



Advisory Editors 

H. W. Hill 
E. O. Jordan 
A. I. Kendall 
C. B. Lipman 
C. E. Marshall 
V. A. Moore 
M. E. Pennington 
E. B. Phelps 



L. F. Rettger 
L. A. Rogers 
M. J. Rosenau 
W. T. Sedgwick 
F. L. Stevens 

A. W. V\'lLLIAMS 

H. Zinsser 



S. H. Ayers 
F. Bachmann 
D. H. Bergey 
O. Berghausen 
C. P. Brown 
P. E. Brown 

V. BlRCKNER 

H. J. Conn 
M. W. Cook 
J. T, Emerson 
L. W. Famulener 



Abstract Editors 

D. Greenberg 
P. B. Hadley 
C. M. Hilliard 
I. J. Kligler 
j. a. kolmer 
H. L. Lang 

H. W. Lyall 
W. J. MacNeal 

E. C. L. Miller 
E. H. Nollau 
L. Pearse 



E. B. Phelps 
G. H. Robinson 
W. Sadler 

G. H. Smith 

F. L. Stevens 
F. W. Tanner 
R. M. Taylor 
E. B. Vedder 

A. R. Ward 

B. White 



ANNOUNCEMENT 

Although there are numerous journals in the United 
States that deal with various special phases of bac- 
teriology (as applied to Medicine, Sanitary Science, Agri- 
culture and the like), there has been no journal in the Eng- 
lish language to represent the science as a whole. 

The Society of American Bacteriologists has estab- 
lished the Journal of Bacteriology as its official organ 
and as a medium for the discussion of the more general 
problems of the science — the structure and physiology 
of the microbes, the inter-relationships of microbic types, 
the effects of physical and chemical agents upon microbic 
life, the mutual interactions of microbes growing together 
in various media, the nutritional needs and products of 
metabolic activity of various microbes, and new methods 
of laboratory technique— and similar advances in knowl- 
edge which are so fundamental as to be of vital interest 
to workers in all parts of this great field. 

The Journal of Bacteriology will publish abstracts 
of all of the papers read at the meetings of the Society 
and will print the more important of them in full, but 
its columns will be open for the publication of suitable 
communications by other persons whether members of 
the Society or not. It will include in its scope not only 
the bacteria but other related micro-organisms, yeasts, 
molds, protozoa, etc. While it is planned to make the 
Journal in particular an organ for the more fundamental 
and general aspects of bacteriology, it will necessarily 
include many papers whose interest is mainly technical, 
particularly in those fields of bacteriology which have 
now no satisfactory organ of publication at their disposal. 

The Journal will include not only original papers but 
also abstracts of bacteriological literature published else- 
where. The abstracts will at first be limited to papers 
published in the United States and Canada, and it is 
hoped will cover this field with reasonable completeness 
beginning with papers pubhshed since January 1, 1916. 
Later on the abstract department will probably be broad- 
ened to include the foreign literature. 



CONTENTS 

W. T. Sedgwick: Foreword — The Genesis of a New Science — Bacteriology. . 1 

D. H. Bergey: The Pedagogics of Bacteriology, Presidential Address, 

Urbana Meeting 5 

L. F. Rettger, N. Berman, and W. S. Sturges: Further Studies on Bac- 
terial Nutrition: The Utilization of Proteid and Non-proteid Nitrogen. 15 

J. M. Sherman: Studies on Soil Protozoa and Their Relation to the Bacterial 
Flora. 1 35 

C. G. A. Roos: A Culture Medium for Maintaining Stock Cultures of the 
Meningococcus 67 

M. M. Obst: Bile Compared with Lactose Bouillon for Determining the 
Presence of B. coli in Water 73 

Society of American Bacteriologists. Abstracts of Papers Presented at 

Seventeenth Annual Meeting, Urbana, 111. December 28-30, 1915 81 

Abstracts of American Bacteriological Literature: 

Bacteriology of Pood 123 

Bacteriology of Soils 123 

Bacteriology of Water and Sewage 125 

Dairy Bacteriology 125 

Disinfection 126 

Immunology 126 

Laboratory Technique 129 

Medical Bacteriology 130 

Physiology of Bacteria 131 

Plant Pathology 132 

The Journal of Bacteriology is issued bimonthly. Each volume will con- 
tain approximately 600 pages. Subscriptions are taken only by the volume and 
not by the year. 

The price of the Journal is $5.00 a volume for all points within the United 
States and Canada; foreign subscriptions $5.50 (23s), 

No claims for copies lost in the mails can be allowed unless such claims are 
received within 30 days of the date of issue. Claimants must directly state 
that the publication was not delivered at their recorded address, "Missing from 
files" is no proof of nonreceipt. The publishers will not be responsible for loss 
due to change of address unless notification is received at least one week in advance 
of issue. 

Fifty reprints of articles will be furnished to contributors free of cost when 
ordered in advance. A table showing cost of reprints, with an order slip, is sent 
with proof. 

Manuscripts should be sent to Prof. C.-E. A. Winslow, Yale Medical School, 
New Haven, Conn. 

All other communications pertaining to editorial work should be addressed 
to A, P. Hitchens, Glenolden, Pa. 

SUBSCRIPTIONS AND ADVERTISEMENTS 

United States and Canada. 

Subscriptions and correspondence concerning business matters should be 
addressed to the Williams & Wilkins Company, 2419-21 Greenmount Avenue, 
Baltimore, Md. 

Great Britain and British Dominions with the Exception of Canada 

Subscriptions from Great Britain and British dominions, with the exception 
of Canada, and correspondence pertaining thereto, should be addressed to Mr. 
C. F, Clay, Manager, Cambridge University Press, Fetter Lane, London, E. C. 



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FOREWORD 
THE GENESIS OF A NEW SCIENCE —BACTERIOLOGY 

W. T. SEDGWICK, Sc.D. 

First President, Society of American Bacteriologists 

"Die Bakteriologie ist ein Kind der jiingsten Zeit." — Fraenkel, 1886. 

Sciences are not made but born, and lineage often sheds light 
upon development. It was an acute observation of the late 
C. S. Peirce that some of the most fruitful of modem sciences 
have been bred by the crossing of older ones. Mathematical 
astronomy, physical chemistry, physiological psychology, astro- 
physics are examples, and the same thing is true of the applied 
sciences, as witness electrical, chemical and sanitary engineering; 
bio-chemistry; electro-chemistry. 

Bacteriology is the offspring of microscopical science hybridized 
with the art of bacterial cultivation,— in other words, of micro- 
scopy and bacterial horticulture. The compound microscope 
was invented and bacteria and other micro-organisms were 
observed in the seventeenth century but no great progress was 
made in our knowledge of microbes— with the single exception 
of yeast— until methods for their selective cultivation or breed- 
ing similar to those long practised in agriculture and horti- 
culture were discovered and introduced by Pasteur, Lister and 
Koch. In his studies in zymology and his long and arduous 
battle against spontaneous generation, Pasteur became pro- 
ficient in ''sterihzing" nutrient liquid soils or "substrata" 
which he afterwards planted or "inoculated" with "traces", of 
micro-organisms. These traces after incubation and multi- 
pUcation produced overgrowths if not pure cultures of partic- 
ular kinds of micro-organisms, in manageable quantities suffi- 
cient for reasonably thorough examination. In this way, Pasteur 

1 



2 W. T. SEDGWICK 

was able to magnify microscopic into macroscopic characteristics, 
and for the first time made it practicable to differentiate and 
classify bacteria with some accuracy. It should also be remem- 
bered that Pasteur's disciple and follower, Joseph Lister, 
made improvements in the method of pure culture by ''dilution" 
during his studies upon milk and the lactic fermentation, 

Pasteur, very early in his work, had insisted upon the indis- 
pensabiUty of the microscope in all investigations of yeasts and 
other microbes, as well as in fermentations, putrefactions and 
diseases (of wine and beer), which they produce, and only those 
who have taken the trouble to read the preposterous paper in 
which Liebig, the most eminent chemist and fermentation expert 
of his day, ridicules the use of the microscope — a paper which 
Huxley has rightly pronounced the most surprising that ever 
appeared in a sober scientific journal — can appreciate the 
immense service done by Pasteur in developing the microscope 
as an instrument of research. It was his insistence upon the 
use of the microscope superadded to a rigid and refined technique 
all along the line which enabled him to win one of the hardest 
fought and most important scientific battles of the nineteenth 
century, namely, that on behalf of biogenesis. 

Pasteur is thus at once the pioneer and the founder of that 
wonderful science of which the present new Journal is to stand 
as an American exponent. 

But it is very doubtful if bacteriology would ever have at- 
tained even a tithe of its present development and importance 
if the methods of Pasteur and Lister had not been supplemented 
and largely displaced by those of Robert Koch, who is at once the 
protagonist of the new science and the architect of that imposing 
superstructure now known as bacteriology, built chiefly since 
1881 by Koch himself and his pupils upon the foundations laid 
by Pasteur. 

In his earUer work, Koch employed substantially the methods 
of liquid culture of Pasteur and Lister, but before long he vastly 
improved upon these cultures by thickening them with gelatine 
or agar — a step to which he was led through the use of potato 
an opaque medium for which it was obviously desirable to sub- 



FOREWORD 3 

stitute something transparent. Brefeld and other botanists had 
ah-eady used gelatine, for "gelatine was first employed by 
Vittadini in 1852 in the culture of microscopic Fungi and has 
been frequently used since that time, especially by Brefeld. 
Klebs more recently, in 1873, recommends it especially for the 
cultivation of bacteria." (DeBary, 1886). 

It was in 1881 that the gelatine-tube culture method and in 
1883 that the gelatine-plate culture method were introduced by 
Koch — his first great work, namely, that upon anthrax, and 
also that on typhoid fever, having been done before this time, and 
his well known "rules," "postulates" or ''laws" having also 
been laid down before his perfection, if not before his invention, 
of the sohd-culture method. 

The word "bacteriology" had appeared before 1886 but the 
subject had no existence anywhere much before that time and 
very little for a year or two later. In the United States, bac- 
teriology was hardly heard of before 1885 but by 1890 it had 
become well known. Unfortunately, neither Great Britain nor 
America can make any claim to the earliest work. The terms 
"sterilization" and "cultivation," in our modern sense, and the 
word "microbe," were introduced by Pasteur and his school. 
The terms "pure culture," "colony," "gelatine," "agar," 
the use of the oil immersion objective, and the art of dyeing 
microscopic specimens of bacteria, come from Koch and the 
German school. Bacteriology is now, however, very widely 
cultivated both in Great Britain and the United States, and it 
is fitting that a Journal of Bacteriology should be pubUshed 
in the English language in honor of the thirty or more years of 
service which one of the broadest and most fundamental of the 
biological sciences may now claim. 

Because of the intensely practical bearings of bacteriology 
upon medicine, and especially because of the marvellous surgical 
and pathological discoveries which were the first, and must ever 
remain the greatest, fruits of bacteriology, its botanical, agri- 
cultural, sanitary, industrial, household and economic impor- 
tance were at first obscured and neglected. But of recent years 
these have rapidly become clear and even conspicuous, and today 



4 W. T. SEDGWICK 

hardly any field of the vast domain of the parent science, biol- 
ogy, is more esteemed or more cultivated than is bacteriology. 
Nor is this strange for, thanks to the microscope and the methods 
of bacteriology, the microscopic world, of which almost nothing 
was known when Pastern* began his researches, stands before 
us today in a revelation of extent, variety, magnitude and inter- 
est second only in importance and impressiveness to that other 
and distant world which has been revealed by the telescope and 
the methods of astronomy. The region of the "infinitely httle" 
seems, however, even more directly and intimately connected 
with our everyday life than that remoter world. The micro- 
scopic world, indeed, lacks the majesty and grandeur of that 
telescopic world which lies so far beyond our reach and over- 
awes us with sheer distance, heat, light and immensity. But 
the revelations of the microscope and the lessons of bacteriology 
have so direct, so intimate, and so fateful association with 
almost every aspect of the conduct of our daily and personal 
life — with food and drink, with health and disease, with life 
and death even — ^that they gain in intimacy what they lose in 
grandeur. 

Bacteriology must henceforward be recognized as a broad and 
fundamental branch of science, coordinate with, rather than sub- 
ordinate to, the other grand divisions of biology such as medicine, 
agriculture, zoology and botany. It is today of immense theoreti- 
cal and practical importance, and it bids fair to become vastly 
greater and more important tomorrow. Hence the obvious 
desirability of a Journal which shall cover the whole field and 
be devoted to the subject in its broadest aspects. 

The time has forever gone by when bacteriology can be regarded 
merely, or even chiefly, as the handmaid of medicine or pathology. 
It is no less the servant of agriculture, of industry, of sanitation 
and of household life. It is already important in sanitary engi- 
neering, and indispensable in the arts of food production and 
food conservation. In its further' differentiation and develop- 
ment the present Journal should be a powerful factor. May the 
event justify both our hope and our expectation. 

Mcissachusetts Institute of Technology 
Boston, February, 1916 



THE PEDAGOGICS OF BACTERIOLOGY^ 

DAVID H. BERGEY 

Assistant Professor of Bacteriology, University of Pennsylvania 

I propose to consider the subject of teaching bacteriology from 
several standpoints, and especially the place of bacteriology in 
scientific education and in medical education. 

The students who, in the past, have demanded a knowledge 
of bacteriology as a part of their instruction in general biology 
have been much fewer than the number that should be seeking 
this knowledge; in fact the demand has been really insignificant 
in comparison with the importance of the study. An expla- 
nation for the neglect of bacteriology as a part of the general 
training of students of the biological sciences is difficult to find, 
but it is evident that the teachers have been to blame, chiefly 
because they have failed to emphasize the importance of the 
study from an educational standpoint. They have been inter- 
ested, more in the practical apphcation of a knowledge of bac- 
teriology, than in the development of the educational importance 
of the subject. 

The aim in modern education is to train the individual for 
usefuhiess. With the present crowded curricula in schools and 
colleges it is essential that the material presented for the train- 
ing of students be selected with the greatest care. Each study 
should be carefully weighed in order to determine its educational 
value. It is necessary not only to select the subjects to be taught, 
but also to arrange the order in which they may be presented so 
as to obtain the greatest benefit from each. 

In the modern organization of society the interests of different 
calhngs are so diverse as to call for general as well as special 
trammg. This fact is now recognized in the preliminary education 

1 Presidential address, Seventeenth Annual Meeting, Society of American Bac- 
teriologists, Urbana, 111., December 28, 1915. 

5 



6 DAVID H. BERGEY 

demanded of those who expect to enter the various professions. 
There is a special need for broad general education in science 
for all persons who wish to be equipped for the most efficient 
service to mankind. The aim of education should be, not merely 
to give information, but to indicate how that information should 
be used, and only in so far as education aids in the promotion 
of the general welfare, does it meet the ideal. The extent of 
the training of each individual must depend upon his ability 
to receive and apply the knowledge which is being disseminated 
in educational institutions. 

A particular science may be studied from two principal as- 
pects, namely, the practical application of the knowledge gained 
to the solution of problems in a special field, and the educational 
value of a knowledge of the science in broadening one's concept 
of the various forces and agencies in nature. 

The science of bacteriology has extended its ramifications in 
so many directions that its study has become of interest and 
direct value to the student in many fields. A knowledge of 
bacteriology enters in a prominent way into most of the activities 
of mankind, and for this reason it should receive much wider 
recognition as a subject for general educational training than it 
is receiving today. The educational value of the study of bac- 
teriology has received recognition slowly and for a study of such 
immense practical importance it has been taken up, for its edu- 
cational value, by a comparatively small number of students 
in our colleges and universities. Yet there are few subjects 
taught that touch upon so many phases of man's activities or 
so many of the conditions influencing his environment as does 
a course in bacteriology, and, it is safe to say, few other subjects 
can have greater educational value. No one can fully appreciate 
the relation of bacteria to many vital problems without having 
studied the subject at first hand. It is only by seeing the activ- 
ities of the bacteria in the test tube, under diverse conditions, 
that one can gain an insight into their prominent place in many 
biological processes. 

The relation of the bacteria to the nitrogen cycle in nature is 
most illuminating to the student. The function of the bacteria 



PEDAGOGICS OF BACTERIOLOGY 7 

in the decomposition of organic matter as they work over the use- 
less constituents of dead plants and animals into forms in which 
they may be utihzed as food by the higher plants is of the greatest 
importance in nature. The control and purposeful utihzation of 
this same function of the bacteria in the preparation and pres- 
ervation of food materials for man and animals, and the relation 
of the bacteria to water and sewage purification, are examples of 
the regulation of bacterial action for the economic and hygienic 
advantage of the human race. Of equal significance are the utiU- 
zations of the functions of the bacteria in agriculture, in domestic 
science and the industries; and of even greater importance are 
the methods of controlling the action of the bacteria in their re- 
lations to sanitary science and clinical medicine. 

The earhest practical application of bacteriology was to the 
fermentation industries through the investigations of Louis 
Pasteur. This was soon followed by his pioneer work in dis- 
eases of animals, especially chicken cholera and anthrax. In 
this latter field Pasteur laid the foundations for our later work 
in immunology and protective inoculations while the studies of 
Robert Koch paved the way for the application of bacteriology 
to the solution of problems in the etiology of disease and in 
sanitary science. 

The earliest demand for a knowledge of bacteriology came from 
the medical profession, concerning the activities of the patho- 
genic bacteria, and the first courses were given to graduates in 
medicine. These were followed later by courses for under- 
graduate students of medicine, of dentistry, and of veterinary- 
medicine. The extension of our knowledge of the activities of 
bacteria in nature, in fields other than disease production, soon 
led to the development of courses for the sanitarian, the student 
of dairying, and the student of agriculture. In all these courses 
chief stress was laid upon the practical application of the knowl- 
edge gained to the solution of problems arising in these difi'erent 
fields. The concentration of endeavor and interest along such 
lines has yielded a great fund of knowledge which is now being 
utilized in enhancing the welfare of man. 

In recent years there has been a slowly growing demand for a 



8 DAVID H. BERGEY 

knowledge of bacteriology on the part of students in biology, 
especially by seniors in arts and sciences, and by graduate stu- 
dents. The chief interest of this group of students is to gain a 
broader insight into the relations of the bacteria to many of the 
important problems of life. Some of these students are pre- 
paring to teach biology, and others are already teaching in 
high schools and colleges. 

While in the earlier courses, offered to graduates and under- 
graduates in medicine, the subject matter presented was intended 
largely to facilitate the application of the knowledge gained to 
practical questions in medicine and sanitary science, the courses 
for students in the arts and sciences have taken on a somewhat 
different aspect. For these students it has been deemed pref- 
erable to lay greater stress upon the broad fundamental bio- 
logical principles involved and much less emphasis upon the 
practical application of the knowledge. The students of chemis- 
try and biology in the graduate school, and in the senior class of 
the course in arts and sciences, have greater interest in the general 
information obtainable from the study of bacteriology, than in 
the more intricate problems of infection and immunity, which are 
of primary interest to the medical student. For this reason the 
general course in bacteriology for science students should be 
developed so as to acquaint them with the relations of the bac- 
teria to problems of food production and conservation and to 
problems in domestic and sanitary science. 

Bacteriology can be studied with greatest profit by students 
in their junior and senior years in college, or by graduate stu- 
dents, after they have had a broad training in biology, chemistry, 
physics and the languages. The student of bacteriology should 
have had instruction in general botany and zoology, in plant 
and animal physiology, in general inorganic and organic chemis- 
try as well as in elementary physics. With a knowledge of 
these subjects he will be in a position to understand something 
of the biological relation of the bacteria to the welfare of man and 
especially to the problems of sanitary science. The broadening 
of the prehminary education of the medical student so as to in- 
clude chemistry, biology, physics and the modern languages has 



PEDAGOGICS OF BACTERIOLOGY 9 

made it possible to place the teaching of bacteriology to these 
students on a much higher plane than was formerly attainable. 
Their understanding of the far-reaching activities of the bacteria 
has thereby been greatly increased and their application of the 
knowledge gained, to the solution of the problems which con- 
front them as practitioners of medicine, is already showing 
abundant fruit in the more intelhgent attitude which medical 
men are assuming toward questions relating to the public health. 

The student who takes up the study of bacteriology as a part 
of his education in the biological sciences should possess a pre- 
liminary training equal to that required of medical students. 
With this broader foundation it becomes possible for the teacher 
to present the subject in a more philosophical way, and the 
general training which the student receives is correspondingly 
made more beneficial. 

The amount of instruction in bacteriology offered to science 
students must vary with the time available for the study and 
with the general and professional training which the individual 
student is seeking. The minimum course should be one of 
twelve hours a week for one semester and should be devoted to 
general bacteriology. After the student has acquired some 
knowledge of bacteriological technique and of the general char- 
acters of the bacteria, attention should be directed to the activities 
of the bacteria in decomposition, in fermentation, in water and 
sewage purification, in the dairy industries and in food production 
and preservation. For students who desire more profound 
knowledge along the various hues of general and apphed bacte- 
riology, more detailed courses should be arranged to meet their 
special needs, the course to be given depending in part on the 
application which the students desire to make of the knowledge 
they are seeking. 

The best course of study in bacteriology for the student in 
biology or general science has not as yet been developed. For 
students beginning the study a combined lecture, laboratory, 
and seminar course seems to give satisfactory results. The 
lecture should be largely a part of the laboratory exercises and 
should consist in explanatory remarks preceding each new phase 



10 DAVID H. BERGEY 

of the subject that is taken up. It is desirable to give the stu- 
dent a brief explanation as to what he is expected to do or see 
and how he is to proceed in conducting the laboratory exercises. 
When the student has carried out a series of laboratory exercises 
the subject may be developed on a broader plane by a lecture 
emphasizing the importance of the observations made and their 
relation to other aspects of the study. 

The almost infinite number of ways in which the bacteria and 
their activities react upon human life, especially in their relation to 
the production of disease in plants and animals, and their relation 
to the various industrial activities, particularly in food production 
and food preservation, give us inexhaustible material for study in 
the classroom. The knowledge which the student of bacteriology 
gains is of such great personal interest and importance that he is 
easily carried along, step by step, from simple observations to 
the most complex and vital phenomena of hfe. 

The study of bacteriology serves unusually well for training the 
powers of observation and judgment. Every lesson is per se an 
object lesson and one in which the student is not only the ob- 
server, but, the demonstrator as well. Moreover the remarkable 
susceptibiUty of the bacteria to environmental influences will 
permit of each demonstration being modified in a variety of 
ways. This possibility of varying the demonstrations precludes 
the probability of a loss of interest on the part of the student. 

It will be profitable to begin a course in general bacteriology 
with exercises in staining various types of bacteria. The stu- 
dent should record his results briefly and amplify the record with 
line drawings of the organisms and cultures studied. In this 
way he acquires some knowledge of the relative size, grouping, 
staining reactions, and rapidity of growth of the bacteria. The 
next step may be the isolation of bacteria in pure culture from 
mixtures and the cultivation of several species of these pure 
strains upon the common media employed for this purpose. 
In the systematic study of a culture the student may follow 
the general plan of description as contained in the Society card. 
This will acquaint him with the vocabulary generally employed 
in this work and will help him to recognize some of the ac- 



PEDAGOGICS OF BACTERIOLOGY 11 

tivities of the bacteria. After he has become familiar with 
bacteriological technique and the methods of studying indi- 
vidual cultures, he should be given pure cultures of the com- 
mon types of bacteria. It is desirable that he should be able 
to recognize all the ordinary bacteria that may be encountered 
later in his work as contaminations so as to be able to avoid 
confusing them with other bacteria that may be of importance 
in the study that he is conducting. With the foregoing exercises 
as a foundation the student is prepared to take up the study of 
the bacteria in water, soil, air, milk, butter, cheese; in the differ- 
ent orifices of the body; and in the excretions from the body. 
In these studies it will be possible to observe merely a few of the 
more common bacteria encountered, but each phase of the sub- 
ject can be amplified by lectures, assigned readings, and dis- 
cussions in the seminar. In the foregoing studies special exer- 
cises may be arranged to enable the student to comprehend 
the relation of bacteria to decomposition, putrefaction, fer- 
mentation, nitrification, denitrification and nitrogen fixation, or 
these phenomena may be independently attacked after the more 
common bacterial flora in nature have been studied. If the latter 
course is pursued the relation of the bacteria to these processes 
should be taken up briefly as phases of the phenomena present 
themselves, while the detailed study of the phenomena is carried 
out later. 

A general course in bacteriology is not complete unless the 
student is given at least a brief introduction to the relation of 
bacteria to the diseases of plants and animals. This study 
should include the methods of recognizing the causative agents 
of disease, the manner in which they produce disease, and the 
ways in which recovery from infection occurs. The student 
should also have an introduction to the bacteriological side of 
important problems in preventive medicine, especially the 
eflSciency of disinfection by the use of chemicals, heat and fight. 
The relation of bacteria to the purification of water and sewage, 
and to the preservation of milk, eggs, meat and vegetables should 
be developed by lectures, assigned readings and exercises in the 
laboratory. 



12 DAVID H. BERGEY 

The position of bacteriology in the curriculum in some medical 
schools is unsatisfactory, especially where bacteriology is taught 
to first year students. It is largely a waste of time to attempt 
to teach clinical bacteriology to a student who knows nothing 
about disease in general and is not expected to take up the study 
of clinical medicine until one or two years later. The difficulty 
can be overcome in large part by requiring that the student 
receive a course of instruction in general bacteriology as a part 
of his premedical training and then receive instruction in clinical 
bacteriology during the second semester of the second year, or 
the first semester of the third year of his medical course. In the 
present arrangement of the curriculum of the medical school, if 
bacteriology is taught entirely in the first year, the student has 
usually not completed the study of physiology nor has he, as a 
rule, begun to study pathology and clinical medicine. The 
anomalous position of bacteriology in the medical curriculum is 
probably due to the fact that those responsible for the condition 
fail to appreciate the broad biological relations of the science of 
bacteriology. 

The student of clinical bacteriology who lacks a knowledge 
of physiology, of pathology, and of clinical medicine, suffers a 
serious handicap in appreciating the principles that underlie the 
pathogenic action of the bacteria and the reactions of the body 
to infection. The problems of infection and immunity have 
the most important relations to normal and abnormal conditions 
of the body and these relations cannot be fully comprehended 
without a knowledge of physiology and of pathology. 

Many of the colleges that prepare students for the medical 
course could be equipped, without great expense, if not already 
prepared to do so, to give a course in elementary bacteriology in 
their biological departments through teachers of those depart- 
ments who would develop the subject on a broad biological basis. 
With such a prehminary training in general bacteriology the 
medical student could then take up clinical bacteriology with 
much greater profit in the second or third year of his medical 
course because he could appreciate the relation of the subjects 
to their clinical application. 



PEDAGOGICS OF BACTERIOLOGY 13 

If general bacteriology is placed in the premedical course it 
will be necessary to lengthen that course to three years, 
at least. This would involve no hardship for those students 
taking a combined science and medical course in seven years,, 
nor for the students entering a medical school that requires a. 
college degree as a requisite for entrance to the medical course. 
This plan would also afford opportunity to extend the premedical 
course so as to include organic chemistry and a broader training 
in biology and the modern languages, as many students enter 
the medical school with insufficient preparation in these three: 
subjects. 

In the second half of the second year or the beginning of the 
third year of the medical course, the student should have com- 
pleted his studies in physiology and have had a course in general 
pathology. He would then pursue his studies in clinical bacteri- 
ology with much greater intelligence and profit. 

The course in bacteriology adapted to the needs of medical 
students should consist, at present, of prehminary work in the 
acquirement of technique, the ability to isolate and recognize 
individual species of bacteria, the study of the conunon sapro- 
phytic bacteria and their important functions in nature, espe- 
cially their relation to decomposition, putrefaction and fermen- 
tation, and the utilization of the functions of the bacteria in the 
purification of water and sewage. As persons with a broad 
scientific training, graduates in medicine should have as deep an 
insight into all of the foregoing activities of the bacteria as it is 
possible to give them. 

With this fundamental knowledge the medical student is in a 
position to comprehend more fully the relation of the bacteria to 
disease, and the various measures which are employed by sani- 
tarians to combat and eradicate disease. 

The more practical side of the training of medical students 
will deal with the recognition of the pathogenic bacteria, a knowl- 
edge of the effects which they produce in the body in causing 
disease, and the reactions of the body in overcoming the disease. 

The extent to which the medical student should be trained 
in the various phases of clinical bacteriology cannot be stated 



14 DAVID H. BERGEY 

categorically, but it may be emphasized that the more detailed 
the laboratory studies in infection and immunity, the greater 
the assistance to the student in obtaining a grasp of the subject 
and hence the more intelligent the application which he will make 
of the knowledge obtained, to the problems in clinical medicine 
and therapeutics. 

If the medical student were to receive instruction in general 
bacteriology in his premedical course, it would be possible to 
devote more time to clinical bacteriology and its application to the 
diagnosis, treatment and prevention of disease during his medi- 
cal course. Colleges and universities should therefore be equipped 
to give courses in general and special bacteriology to students in 
the premedical, the arts and sciences, and the sanitary engineer- 
ing courses, as well as to science students in the graduate school. 
Besides courses in general bacteriology, more advanced courses 
should be offered, especially in the bacteriology of water and 
sewage, in dairy bacteriology, in agricultural bacteriology, in do- 
mestic science bacteriology and in sanitary science bacteriology. 

It is evident that if the knowledge to be gained through a 
course in general bacteriology were more widely diffused amongst 
persons in all walks of life, there would be far less credence given 
to the extravagant and false claims of the horde of quacks and 
faddists who are now preying upon an ignorant and credulous 
public. The light of truth alone can reheve us of the depredations 
of those who claim to practice those "isms" that have been 
raised up because of the general ignorance of mankind. 

In order to further the development of bacteriology and to 
extend the teaching of the subject to students of the biological 
sciences, it would be desirable for this Society to organize a 
teaching section for the discussion of problems in the teaching 
of bacteriology at each annual session. Through the interchange 
of views and the discussion of the principles of teaching the sub- 
ject, the science of bacteriology, as well as education in general, 
would reap great benefit. 



FURTHER STUDIES ON BACTERIAL NUTRITION: 

THE UTILIZATION OF PROTEID AND 

NON-PROTEID NITROGEN 

LEO F. RETTGER, NATHAN BERMAN and WILLIAM S. STURGES 
From the Sheffield Laboratory of Bacteriology and Hygiene, Yale University 

The highly interesting observation of Bainbridge (1911) that 
certain aerobic and facultative anaerobic bacteria of the gelatin- 
liquefying and non-hquefying types are of themselves unable to 
initiate decomposition of purified native proteins has been fully 
corroborated by Sperry and Rettger (1915). The last-named 
authors have shown further that the putrefactive anaerobes B. 
putrificus, B. oedematis (B. oedematis-rnaligni, Zopf) and B. 
Feseri {B. anthracis-symptomatici, Kruse) are likewise devoid of 
this property; and that the vegetable protein edestin, like egg 
and serum albumin, does not undergo disintegration by direct 
bacterial action. It was but natural to assume, therefore, that 
the protein nitrogen cannot be utilized by bacteria unless it is 
first simplified and made available for cell nutrition through the 
action of a proteolytic enzyme, strong acid or alkali, or some other 
cleavage-producing agent. 

Solutions of purified proteins were prepared by the methods 
now used in all biochemical laboratories and involving the crystal- 
lization of the proteins. The test media were usually the same 
as those employed by Bainbridge, and contained the following 
ingredients, besides the protein; sodium chloride 0.5 per cent, 
sodium sulphate 0.2 per cent, calcium chloride 0.1 per cent and 
acid potassium phosphate 0.1 per cent. The only possible source 
of nitrogen was the protein, except in certain check tests in 
which small amounts of peptone were employed. The solutions 
containing the purified proteins were steriUzed by filtration 
through the laboratory Berkefeld. 

The test media were inoculated from 24 hour slant agar cul- 
tures of the various organisms, with the special precaution of 

15 



16 L. F. RETTGER, N. BERMAN AND W. S. STURGES 

introducing only a small number of the bacteria and as little 
extraneous matter as possible. The fate of the bacteria and of 
the proteins was determined in three ways; first, by the plate 
method of determining the numbers of cultivable bacteria at the 
beginning of the experiments and after varying intervals or 
periods of incubation ; second, by noting any change in the appear- 
ance of the media; and finally by determining the amount of 
coagulable protein at different times, during the course of the 
experiments. 

The results obtained by Sperry and Rettger (1915) were so 
definite and consistent as to leave no doubt as to their signifi- 
cance. It was assumed that the purified proteins resisted decom- 
position by direct bacterial action because of their original or 
imchanged condition as native proteins; hence, sterilization by 
heat was to be avoided, as heating at coagulation temperature 
undoubtedly causes changes in the protein molecule. 

The present investigation is in part a continuation of the work 
of Sperry and Rettger on the action of bacteria on purified 
proteins. Instead, however, of studying the behavior of bacteria 
toward unchanged (unheated) proteins, the experiments were 
conducted on test media containing coagulated egg albumin as 
the only possible source of nitrogen. The investigation also in- 
cluded a study of the behavior of bacteria toward proteoses 
and peptones, and of so-called "bacterial autolysis." 

I. THE BEHAVIOR OF BACTERIA TOWARD PURIFIED COAGULATED 

EGG ALBUMIN 

The egg albumin was prepared by the method of Hopkins and 
Pinkus (1899). The test medium containing the albumin and 
inorganic salts was the same as that used by Bainbridge and by 
Sperry and Rettger, with the exception that the medium was 
sterilized by heat and hence contained coagulated albumin. The 
methods of inoculation, incubation and determination of results 
were the same as those described in the earher paper from this 
laboratory, (Sperry and Rettger, 1915). Special attention was 
given to the enumeration of bacteria by the usual plate method 



UTILIZATION OF PROTEID AND NON-PROTEID NITROGEN 17 



immediately after inoculation and after definite periods of incu- 
bation at 30°C. The results are given in the accompanying 
Tables I and II. 

With very few exceptions, the results show that there was 
httle multiplication of the bacteria with which the medium was 
inoculated. An increase of 100 per cent, or even 1000 per cent, 

TABLE I 

Enumeration of bacteria in inoculated test medium containing heated {coagulated) 
egg albumin. Counts per cubic centimeter of medium* 



ORGANISMS 



Prot. vulgaris I . . . . 

Prot. vulgaris II 

Prot. vulgaris III. . . 
Prot. vulgaris IV . . . 
Prot. vulgaris V . . . . 

Prot. mirabilis 

B. subtilis 

B. prodigiosus 

Staph, aureus II. . . . 

Staph, aureus II 

Staph, aureus III. . . 
Staph, aureus IV . . . 
Staph, aureus V . . . . 
Staph, aureus VI . . . 

B. coli I 

B. coli II 

B. coli III 

B. typhi I 

B. typhi II 



IMMEDI- 


24 


48 


72 


4-7 


2 


3 


ATELY 


HOURS 


HOTJRS 


HODHS 


DATS 


WEEKS 


WEEKS 


2,600 


3,752 


2,350 






4,800 




1,150 


4,450 


5,700 






XX 




146 


3,900 






2,800 






358 


1,650 




4,032 


1,070 


3,520 


3,840 


800 


3,680 


3,136 






4,990 


3,700 


480 






1,980 




3,000 


544 


1 




1,470 






XX 




640 




XX 






XX 




1,400 


2,500 


2,150 










3,800 


4,800 


7,450 










50 


2,100 






1,700 






365 


864 




990 


860 


134 


. 75 


204 


800 


865 






84 


620 


150 








640 


160 


89 


17 








2,000 


1,200 


600 


352 






2,300 




2,000 


260 


1 






3,800 




4,700 


6,100 


288 




705 






480 




165 






191 




768 


832 



4 WEEKS 
AFTER 

INOCULA- 
TION 



1,775 



1,180 

XX 

XX 



406 



53 

675 

350 

7,200 

162 



Note: XX indicates too many colonies on the agar plates to count. 
* Dilutions of 1 : 10,000 were employed in these tests. 

in the numbers of organisms would not be unexpected even in 
what may be termed a nitrogen-free medium which is constantly 
exposed to the atmosphere. In the tests with B. prodigiosus, B. 
subtilis and one of the Proteus vulgaris strains the numbers of 
colonies on the agar plates became too numerous to count. 
Furthermore, there were visible indications that the protein was 
undergoing disintegration. These results are, therefore, in strik- 



18 



L. F. RETTGER, N. BERMAN AND W. S. STURGES 



ing contrast to the rest. The most plausible explanation of the 
proteolytic action in these tubes is that these organisms pro- 
duced a very active proteolytic enzyme early in the course of 
their growth on the slant agar, so that sufficient enzyme was 
introduced into the test medium along with the bacteria to bring 
about cleavage of the albumin and thus prepare it for nitrogen 
assimilation by the bacteria. These tests are being repeated. 

In all of the experiments except the three just commented 
upon there was no visible indication of bacterial disintegration 



TABLE II 



Control experiments. Enumeration of bacteria in inoculated medium containing 
heated (coagulated) egg albumin and 1 per cent of peptone 



ORGANISMS 


IMMEDI- 
ATELY 


24 HOURS 


48 

HOURS 


72 

HOURS 


4-7 

DATS 


2 

WEEKS 


3 

WEEKS 


4 WEEKS 

AFTER 
INOCULA- 
TION 


Prot. vulgaris I . . . . 

Prot. vulgaris II 

Prot. vulgaris III. . . 
Prot. vulgaris IV . . . 

B. subtilis 

Staph, aureus I 

Staph, aureus II. . . . 
Staph, aureus III. . . 
B. coli 


11 

1,472 

46 

1 

2 

680 
94 

1 
1 


17,300 
XX 
21,000+ 

XX 

13,000 


1,900 
XX 


XX 
XX 


XX 
XX 

XX 
XX 


XX 




XX 


B. typhi 


256 









Note: XX indicates too many plate colonies to count. 

In all of the experiments recorded in tables I and II the test medium con- 
tained the following inorganic salts: Sodium chloride 0.5 per cent, sodium sul- 
phate 0.2 per cent, calcium chloride 0.1 per cent, and acid potassium phosphate 
0.1 per cent. 

of the egg albumin. In fact, the liquid portion of the medium 
remained clear and colorless, and the medium could not be dis- 
tinguished from the uninoculated tubes, either by its appear- 
ance to the naked eye or by the odor. Control tubes contain- 
ing the same ingredients plus 1 per cent peptone rapidly under- 
went marked change. The protein became involved and, in the 
tubes containing gelatin-liquefying organisms, was gradually di- 
gested. In every instance the liquid part of the medium soon 
became turbid, and frequently more or less colored (see Table II). 



UTILIZATION OF PROTEID AND NON-PROTEID NITROGEN 19 

Tests were also conducted with anaerobes of the type of B. 
putrificus and B. oedematis, which are characterized by their 
strong proteolytic and putrefactive properties. No quantitative 
bacterial determinations were attempted with these anaerobes, 
however, and the observations were confined to a study of the 
physical characters of the medium. No change whatever could 
be noted in the medium; the clear liquid and the coagulated 
albumin remaining apparently unaffected even after three to 
four weeks of incubation. Inoculation of egg-meat medium from 
these tubes with the aid of a platinum loop clearly demon- 
strated the presence of putrefactive anaerobes by the rapid and 
characteristic decomposition which took place in the standard 
egg-meat medium. 

II. THE BEHAVIOR OF BACTERIA TOWARD PROTEOSES AND 

PEPTONES 

In text books and other bacteriological publications the assump- 
tion is made that proteoses and peptones are readily attacked by 
all known bacteria which are easily cultivated on artificial media. 
So firmly has "peptone" established itself as an important in- 
gredient of the common and standard bacteriological laboratory 
media that its value as the source of nitrogen supply in the cell 
metabolism of bacteria is taken as a matter of course. It is 
true that meat extract which is practically protein-free is also 
looked upon as practically indispensable, but not because it fur- 
nishes food as such to the organisms. By many at least it is 
regarded as a stimulator of cell metabohsm, due to the various 
extractives present. 

It is one of the objects of this paper to show that proteoses and 
peptones follow essentially the same law of resistance to direct 
bacterial action as do the native proteins, egg albumin, serum 
albumin and edestin. While the scope of the investigation has 
as yet been somewhat limited, sufficient data appear to us to have 
been acquired to warrant their publication at this time. 

It is a well-known fact that the proteoses and peptones result- 
ing from the action of proteolytic agents hke pepsin and trypsin 
upon native proteins, and indeed all proteoses and peptones, have 



20 L. F. RETTGER, N. BERMAN AND W. S. STURGES 

thus far resisted all attempts to isolate or purify them. Hence, 
it has been impossible to employ all the methods of investiga- 
tion in a study of their bacteriological-chemical relations which 
are applicable in connection with certain albumins, as for instance 
egg albumin. Peptones are now regarded as amino acid com- 
binations of varying complexities, rather than proteins. Witte's 
peptone, which is essentially a mixture of albumoses and pep- 
tones, is far from being made up purely of these nitrogen com- 
plexes, although it has long been regarded as the standard for 
bacteriological purposes. The various American brands are 
undoubtedly even less pure than the Witte product. It does not 
follow, however, that they are of correspondingly less value as 
food for bacteria. 

In our study of the behavior of various types of bacteria 
towards proteoses and peptones the Biuret test for proteins has 
been employed to great advantage. The method which has 
been advocated and used by Vernon (1904) for the quantitative 
estimation of peptone has, with slight modifications been em- 
ployed by us in the present investigation and in the experiments 
on bacterial autolysis. A brief description of this method is 
given here. 

The tests were made in Nessler tubes. One cubic centimeter 
of the inoculated culture fluid was added to 20 cc. of a 4 per cent 
solution of sodium hydroxide and 2 cc. of a centinormal solution 
of copper sulphate. To the same mixture of alkali and copper 
sulphate in a second tube a standard solution (0.25 per cent) of 
Witte's peptone was added until the same degree of color was 
produced as in the test medium. The quantity of peptone 
required in matching the colors was taken as a measure of the 
amount of peptone present in the inoculated and incubated cul- 
ture fluid. For example, if 1 cc. of standard peptone solution 
was required the value of the biuret test was recorded as 1.0, 
since both hquids gave the same color in the same concentration. 
Test fluids and controls contained the same amount of Witte's 
peptone at the outset as the standards, namely 0.25 per cent. 

Peptone solutions containing from 0.2 to 2.0 per cent of Witte's 
peptone were at first employed as culture media for the different 



UTILIZATION OF PROTEID AND NON-PROTEID NITROGEN 21 

organisms, but it was soon found that the amount of peptone 
present should not exceed 0.25 per cent. When the peptone was 
used in higher concentration sHght reductions in the amount of 
the proteins could not be detected, or at least could not be 
determined accurately. In the lower dilutions, however, the 
various degrees of decomposition were easily observed. 

For the autolysis experiments standard peptone solutions were 
frequently employed for color comparison, while the work on the 
relation of the growth of different bacteria to proteoses and pep- 
tones involved the employment of the standard solution only as 
a check or control for the inoculated flasks. The results are not 
given in per cent, but are represented in the tables by 0, X, XX, 
XXX and XXXX. The first of these symbols, 0, indicates no 
reduction of the proteoses and peptones, as compared with the 
controls, X a slight decomposition, XX fair, XXX strong, and 
XXXX complete reduction of these soluble proteins. Besides 
the ''peptone" the test media often contained other agents, as 
will be seen in the tables, namely ammonium sulphate, beef 
extract and glucose. Furthermore, all of the fluids contained 
0.5 per cent of sodium chloride. 

The results require but little comment. With few exceptions, 
no disappearance of albumoses and peptones could be noted in 
flasks which were inoculated with members of the colon-typhoid 
group of organisms, even after four weeks of incubation. In 
the flasks showing a reduction of the biuret reaction the appar- 
ent loss of the soluble proteins was slight, and may be accounted 
for by other factors than an actual decomposition by the bacteria 
with which they were inoculated. In all of these experiments 
the bacterial growths were fairly luxuriant, particularly when 
the test medium contained beef extract or ammonium sulphate. 
Even in those instances in which slight reduction of the soluble 
proteins was recorded, at least two weeks, and as a rule three weeks 
or more, were required to show the apparent reduction. 

The above experiments are being repeated. Similar tests are 
also being made with media containing peptone and the ingredi- 
ents of the Uschinsky medium, with soluble purified casein, or 
nutrose and with dialyzed proteoses. Thus far results similar 



TABLE III 

Showing the behavior oj certain gelatin-non-liquefying bacteria towards Witte' 

peptone 







DECOMPOSITION OF 


PROTEOSES AND PEPTONES 




At 37° C. 




At 20° C. 




ORGANISMS 


MEDIA 




















% 




1 


1 






1 
1 


1 








(M 


CO 


■* 


^ 


M 


M 


-* 




2 per cent peptone 

























B. coli ■ 


0.5 per cent peptone. . . 




























0.25 per cent peptone . . 


























' 


0.25 per cent peptone. . 




























0.25 per cent peptone 




















0.5 per cent beef ex- > 








X 


X 




X? 









tract 










xo 












B. coli (H). ■ 


0.25 per cent peptone 1 







1.0 per cent glucose . . ^ 




















0.25 per cent peptone 




















0.25 per cent ammo- ' 















X 


X 


X 




nium sulphate 




















0.25 per cent peptone . . 








X 


xo 
















0.25 per cent peptone ] 




















0.5 per cent beef ex- ■ 







X 


? 







X 


•? 




tract 























B. coli (U). - 


0.25 per cent peptone 1 







1.0 per cent glucose . . J 




















0.25 per cent peptone. 




















0.25 per cent ammo- - 










xo 










xo 




nium sulphate , 




















0.25 per cent peptone 




















0.5 per cent beef ex- > 







X 









X 






tract 


















B. coli (A). ^ 


0.25 per cent peptone 1 
1.0 per cent glucose . . J 
0.25 per cent peptone 










X 










X 




0.25 per cent ammo- • 



















X 


x 




nium sulphate 




















0.25 per cent peptone . . 
















X 










0.25 per cent peptone 




















0.5 per cent beef ex- • 







X 


X 








X 


X 




tract 

















X 


XXX 




B. typhi 

(Y.M. S.) " 


0.25 per cent peptone. 1 
1.0 per cent glucose. . j 


XX 




0.25 per cent peptone 




















0.25 per cent ammo- • 


























nium sulphate 


















B para- 1 
typhi B. . J 


0.25 per cent peptone . 


























B. pullorum. 


0.25 per cent peptone . 










X 










X 


B. aerogenes 


0.25 per cent peptone . 

























22 



UTILIZATION OF PROTEID AND NON-PROTEID NITROGEN 23 

to those already recorded have been obtained. These will con- 
stitute part of a future publication from this laboratory. 

The results given in Table IV are in marked contrast to the 
preceding. The pronounced and rapid decomposition of the 
soluble proteins left no doubt as to the ability of gelatin-Hquefying 
bacteria to convert them into products which no longer give the 
biuret reaction. Sp. cholerae and Staphylococcus aureus were, 
however, much less active than the others. 

It may be of interest to note the sparing action of glucose on 
the proteoses and peptones in the flasks which were incubated 
at 37°C. At room temperature (20°C.) the protein-sparing action 
did not last beyond the first two weeks. 

III. BACTERIAL AUTOLYSIS 

The term ''autolysis" has been used somewhat indiscriminately 
by bacteriologists. Whether it is to denote actual decomposition 
of the intracellular proteins by the action of the bacteria them- 
selves, or of certain enzymes, or whether it is meant to signify 
merely a liberation of intracellular substances without change in 
their chemical structure, is often left undetermined. The word 
has for many years had definite significance, however, in bio- 
chemical literature, carrying with it the idea of self-digestion, as 
the term implies. This can, of course, be its only true meaning. 

It has frequently been shown that real bacterial autolysis is 
a common phenomenon in organisms of the Bacillus prodigiosus 
and Bacillus pyocyaneus type which elaborate strong proteolytic 
enzymes (Rettger, 1904; Levy and Phersdorff, 1902), especially 
under conditions of food deprivation and relatively high tempera- 
tures. It is to be questioned, however, whether the so-called 
"autolysis" of cultures of B. coli and B. typhi during long incu- 
bation, and the liberation of endotoxin, as claimed by some 
investigators (Conradi, 1903) is a process of real self-digestion. 

The present study of autolysis was carried on with certain 
well-known proteolytic organisms, namely B. prodigiosus, Pro- 
teus vulgaris, Ps. fluorescens (B. fluorescens liquefaciens, Fliigge), 
B. subtilis and B. ramosus, and with B. typhi and several differ- 
ent strains of B. coli representing the gelatin-non-liquefying class 



24 



L. F. RETTGER, N. BERMAN AND W. S. STURGES 









J§ 


X! , >< X 




XXX 

xxxx 


XXX 

xxxx 
xxxx 






X 










°>^y^^^ 








>^ 








s 




(N- 




X! 




IS 

o 


d 

o 


■>»< 


M XX! 








X! 




CO 


O |i< 




X X 

X X 
X X 


n 1^ 

X >^M 


XI 
X 




x: 

X! 
X! 


o 


i 


X >^ 




X 
X 
X 

X 


d>^ 
° ^>^ 


o 




X! 

X 
XJ 


a 

Si? 




•^ 


















CO 






o o X o o X X 




o 


o o 


o 








O 




















^ 


H 
O 








































o 








X. X 




X 


X 






X 


.e 


b 




„ t^ X X X 




r-. X 


X 






XI 


s^ 


O 




1 




y 


° X 


o 




X 


o 


2 




■* 


X X 




X 






X 


e 


H 






<a 






<0 








rO 




CJ 






o 








Cn 


S 




m 


a 




X 


XI a 






X 


5 


cu 
o 










° X 


3 J? 
° X.S 


o 




X 
X 







d 


CO 


^ 
^ 




X 


X s 






X 


O^ 




CO 










•+J 








•<^ 




a 






a 








^ 




< 


1 
1 


X x-^ 

oXiM o X'o 

?S 1^ o 




° X 


X-9 

° xi 


o 




X 
X 
X 








e^ 


X -g Xu 




X 


XO 






X 






X 1X1 














to 






IB 


o o^ o &r^xi 

X aX 




o 


n 


o 






"o- 






*"* 














O 


































a 


a 






a 














3 


3 






3 










q; 0^ ^ 0^ a* q; c 
Cl 13 fl fl fl CI fi 




M »-i >, 


Oi n, 0) •-; 01 4) 

« ^ 9 s as 


0) 


0) 

d 


'S 


•w 








o o o o o o o 


o 


o o o 


O o o o O O 


o 


o 


O 


^ 








^j -i-i -(J -i_> -u -fj -t^ 


-u 


" "S a 


a § a a a a 


o 


+i 


a 










a, ex a a a a o, 


a 


3 


a 










o; 0) a* 0^ o c:' 0^ 


<u 


Oi ^ O C O 0( 




o 


s 




< 




a a a a a a a 


a 


do a c3 


a tc a rt a a 


"3d 


a 


3 






3 




+J -4^ -^ -*^ +-^ -i-J -^^ 


,^ 


-tJ -U -1^ 


^J -U •+J -fcj -u -»^ 


+J 


-i-i 


,i^ 


§ 




a 




a d ;3 a c E3 CI 


a 


CI a CI 


a a a a c a 


Cl 


a 


a 


o 




s 




<D G) O <D "15 Q^ <U 


o 


01 a; <u (u 


01 01 01 Ol m 01 O 


dJ 


01 


01 aj 


^ 








O C) o O O O O 


o 


o o o ^ 


a 1^ <^ ii ^ ^ '^ 


o 


o 


o ^ 








t4 ^ t^ &-( fH t-t ^ 


^H 


tn i< fcH ^ 


(h t- t< t< 5 t- (H 


;-< 


!h 


f- s 










O 0^ C» <D O OJ Q^ 


(D 


OJ 01 O -^ 


(D 01 0> Ol 43 Ol Ol 


Ol 


Ol 


« J3 










a a a a a a a 


a 


a a a_a 


a a a a_a a a 


a 


a 


a^ 










lO lO lO >0 'O 


»o 


lO lO 3 


lO lO iC 3 lO lO 




lO 


iC 3 










lO (M (M <M (M C^ >0 


c^ 


O (N (M 00 


(M O <M <M CO M 04 


o 


(M 


(M CO 




d d d d d d d 


d 


^ d ci 


d .-i d d d d 


'-' 


d 


d 






00 

s 




i 2 § « : : 

O =^ 3 o3 m • 




3 --A 


^ V '*^ 

1 

"5 '-^ 




.2 








00 

s 










> fa 

OQ 


QQ 


3 








o 




5 " -2 a • • 




3 2 


s 2 


3 


c4 

















01 fc^ 
O M 


01 

o 














02 CO M CQ DQ 




Pw 


Oh 


Ol 







UTILIZATION OF PROTEID AND NON-PROTEID NITROGEN 25 





S 






X 

X 










X 


X 


X 


M 




X 


o 






X 


X 


X 


X 




X 








X 


X 
X 
X 
X 


X 
X 
X 
X 


X 




X 
X 
X 
X 


o 


M X 








X 


X 






X 




X.. 






X 




X 
X 


o 




X 
X 


o 


X X 








o 










o 


XX 








X 


X 






X 




X 






X 


X 


X 






X 




X o 






X 


X 


X 


o 




X 




x° 








X 


X 






X 




X 






X 


X 


X 


o 




^x 

XX 


XI 






X 


X 


X 






X 




X 






X 


X 


X 
X 






>^ 


x^^ 






X 
X 


X 


X 


o 




xxx'-' 






X 


X 


X 






^ 




X 






X 


X 




















X 
X 




o 






o 


X ^ 

x° 








X 






















■k3 




















o 
o3 






a 








a 






u 






_3 








3 




9i 


Oi -s 


0^ ,1-, 


a; 




« 


lu <» rtl 


OJ 


•^ 




a 


« ?, 


a § 


fl 


a 


C3 


CI O M 


fl 


C 




O 


o iJ 


O o 


o 


o 


o 


O O o 


o 


C 




■*^ 


-•-^ t-> 


•*^ o 


-♦J 


g 


-tJ 


■^ -tf o 


+J 


a 




a 


CU <D 


a 3 


a 


a 


a 


a a a 


a 


a 




<u 


<D <U 


« j:i 


a> 


OJ 


(D <» .^ 


<D 




&, 


a J2 


a bo 


a 


e3 


a 


a a bc 


a 


ci 




-*J 


■*j -ti 


-u -i-= 


,^o 


-kJ 


-*i 


-kJ -t:s += 


+j 


->J 




a 


a a 


a a 


a 


C 


fl 


a c a 


a 


a 




V 


<D <D 


(U 0) 


<u 


a> CD 


o 


a> 4) <u 


0) 


<D 


o 


« 


O O 


o o 


o 


w ^ 


o 


o o u 


o 


O 


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;h 


%^ >H 


V> kH 


tH 


^- 2 


kl 


iH «-C f- 


;_ 


(h 


o3 


<u 


<B (D 


O lU 


<B 


<u ^ 


a> 


<u (D a> 


0) 


(D 


^ 


cu 


a a 


a a 


a 


a^ 


a 


a a a 


a 


a 


_a 


lo 


"3 


lO 


lO 


>o 3 




"^ -^ 


o 


lO 


3 


(N 


IM U5 


C-l o 


C^) 


C^ to 


o 


to IM O 


M 


(N 


m 


d 


d d 


d ^ 


d 


d 


(M 


d> d ^ 


d 


d 




/ 1 














1 

o 












0} 








• ^H 




















bO 












'•*i 








^ 












XI 








o 












a 








(« OS 












<n 








a 3 

X 












m 








m 











3 


O 


Xi 


to 


to 


a 


^ 










o 


>> 


a 


X! 


c3 


-c 


(V 


HI 


J-l 


rt 




o 


c 


T! 


'^ 


c 


fl 




O 


I/l 




M 










3 


c3 


T3 


-n 


o 


C) 


o 


a 


a 


43 


tn 


-i-j 




a 

o 

S-i 


-n 


t*-l 


3 


to 
3 






o 


<u 



-3 -3 «« 



" 


3 


"^ 


cS 


n1 


Vh 




el 


a 


<U 


— ( 


a 




a 




o! 


H 


to 


H 


T! 


O 





26 



L. F. RETTGER, N. BERMAN AND W. S. STURGES 



of bacteria. The experiments fall into two distinct groups; in 
the first, tests were made as to the ability of bacteria to digest or 
destroy their own proteins under highly favorable conditions of 
temperature and environment. Representative gelatin-hquefy- 
ing and non-liquefying organisms were employed. The second 
set of experiments had as its object a study of the fate of purified 
egg albumin, Witte's peptone, and dialyzed proteoses when added 
in small amounts to autolyzed bacterial suspensions or to sus- 
pensions which had the necessary conditions for proteolysis pro- 
viding the organisms were capable of digesting themselves. 

The different organisms were grown on slant agar and, in a 
few instances, on potato. The surface growths were washed off 
with distilled water and transferred to sterile bottles. These 
suspensions which were of the consistency of a thin paste were 
incubated at 37 °C. with 5 per cent toluol. Definite amounts of 
this material were tested from time to time by the quantitative 
biuret method, a 0.25 per cent solution of peptone being em- 
ployed as a standard for color comparison. The exact plan of 
the experiments and the results are given in the following tables. 

TABLE V 
Autolysis experiments with gelatin-liquefying bacteria 





BIURET REACTIONS AND COLOR COMPARISONS WITH STANDARD 


§2^ 






PEPTONE SOLUTION (0.25 PER CENT) 


^ 


OBQANISMS 


















^H" 




Ist 
day 


2nd 
day 


3rd 
day 


4th 
day 


5th 
day 


6th 
day 


7th 
day 


8th 
day 


15th 
day 


Z o a 

^ H Cl. 


[- 


0.05 












0.0 








B. subtilis < 


0.25 
0.07 




Faint 






0.0 








1.0 


B. prodigiosus . . . < 

f 


5.0 
2.0 
2.0 


4.5 


1.0 
1.5 


1.0 


Faint 
1.0 


0.05 


0.0 
0.5 




Faint 




Prot. vulgaris . . . I 


1.0 
1.0 




Faint 
Faint 








0.0 
0.5 








B. ramosus 


0.15 


0.10 


0.03 

















Note: The above figures are based on the relative strengths of color obtained 
in the tests, each being compared with the degree of color given by 1.0 cc. of the 
standard (0.25 per cent) solution of Witte's peptone which is taken as 1.0. In 
all of the tests 1.0 cc. of the autolysis material was employed. 



UTILIZATION OF PROTEID AND NON-PROTEID NITROGEN 27 

TABLE VI 

Autolysis experiments with different strains of Bacillus coli 





BIUBET REACTIONS AND COLOR COMPARISONS WITH 


STANDARD PEPTONE 






SOLUTION (0.25 


PER CENT) 









P3 




































>> 


1^ 


T3-T3 


03 


>> 


to 




>> 

CO 


1' 




00 






5l 

g 




B. coli I 


2.0 


2.0 


2.0 




? 




1.0* 














B. coli II ... . 


1.0 




1.0 




1.0 






1.0 


1.0 














B. coli III. .. 


1.0 




1.0 






1.0 


1.0 


1.0 


0.75t 












1.0 


B. coli IV.... 


2.5 






2.5 
















2.5t 








B. coli V 


1.0 






1.0 
















1.0 








B. coli VI.... 


1.0 


1.0 


1.0 


















1.0 








B. coli VII... 


1.0 
















1.0 














B. coli VIII.. 


0.1 
















0.1 




0.1 




0.1 


0.1 





*Contamination with Subtilis-like organism. 

t Developed a condition which rendered the test fluid cloudy, and hence made 
quantitative study impossible. 

TABLE VII 
Combined autolysis experiments with gelatin-liquefying and non-liquefying bacteria 





BIURET REACTIONS AND COLOR COMPARISONS WITH STANDARD 
PEPTONE SOLUTION (0.25 PER CENT) 







OJ 


1"° 




>> 


>> 

03 

CO 




>> 

J3-13 

00 


>> 

1^ 




>> 


>> 


1-^ 


5^ 

OT3 


-^ 03 


go 


B. subtilis I 

B. subtilis II.... 

B. subtilis 

Ps. fluorescens. . 

B. coli (H) 

B. immobile 
(fluorescens 
non-liquefa- 
ciens) 


1.0 
0.2 

0.2 
0.5 
0.4 

0.6 

0.8 

0.5 


0.4 


0.0 
0.0 

0.8 
0.5 


0.3 
0.4 

0.6 


0.8 


0.0 
0.5 


0.4 


0.1 

0.6 
0.8* 


0.4 

0.0* 
0.5 


0.0 
0.4 

0.6 


0.4 


0.4 


0.4 


0.4 


1.0 


B. typhi (Ho)... 

M. luteus (ce- 

reus flavus) . . . 





* At the end of ten days of incubation of the autolysis bottle (B. typhi) 2 cc. 
of completely autolyzed B. subtilis material was added. After eight days the 
contents of the bottle failed to give a biuret reaction. 



28 



L. F. RETTGER, N. BERMAN AND W. S. STURGES 



In the following experiments 1 cubic centimeter of autolyzed 
material or of bacterial suspensions which had been incubated 
long enough to allow of self-digestion, was added to 5 cc. of 
purified egg albumin, to Witte's peptone and to purified proteose. 
Biuret tests were made after definite periods of incubation at 
37°C. In each experiment the original biuret reaction is the 
same for all of the flasks including the control. 



TABLE VIII 



Proteolytic action of autolysis material on purified egg albumin, 
pension added to 5.0 cc. of the albumin solution 



1.0 cc. of 8US- 



ORGAJ^ISMS 


BIUHET BEACTION8 AND COLOR COMPARISONS WITH 
EGG ALBUMIN CONTROL 




Ist day 


3rd day 


5th day 


12th day 


20th day 


B. prodigiosus 


1.0 
1.0 

1.0 


1.0 
1.0 


0.5 
1.0 
1.0 


Faint 
1.0 
1.0 


0.0 


B. coli I 


1.0 


Control 


1.0 







TABLE IX 
Action of autolysis material {1.0 cc.) on solution of purified proteoses {5 cc.) 



ORGANISMS 


BIUREI 


REACTIONS AND COLOR 
PROTEOSE SOLUTION 


COMPARISON 
CONTROL 


WITH 




Ist day 


2nd day 


3rd day 


6th day 


10th day 


B. prodigiosus I 


1.0 
1.0 
1.0 
1.0 
1.0 
1.0 
1.0 
1.0 
1.0 


0.5 

1.0 
1.0 
1.0 
1.0 

1.0 


Faint 
Faint 



1.0 
1.0 
1.0 
1.0 
1.0 


Faint 
0.5 
1.0 
1.0 
1.0 
1.0 
1.0 




B. prodigiosus II 


0.0 


Prot. vulgaris I 




Prot. vulgaris II 




B. coli I 


1.0 


B. coli II 


1.0 


B. coli III 


1.0 


B. coli IV 


1.0 


Control 


1.0 







UTILIZATION OF PROTEID AND NON-PROTEID NITROGEN 29 



TABLE X 



Action of autolysis material (1.0 cc.) on solution of Witle's peptone {5 cc. of a o per 
cent solution). Gelatin liquefiers 



B. prodigiosus 

B. prodigiosus II ... . 

Prot. vulgaris 

Control 



BIURET REACTIONS AND COLOR COMPARISONS WITH 
PEPTONE CONTROL 



1st day 2nd day 4th day 6th day 12th day 20th day 



5.0 
5.0 
5.0 
5.0 



4.0 
4.0 
4.0 



2.0 
2.5 
3.0 
5.0 



1.0 
2.0 



0.5 
1.5 
5.0 



Faint 
1.0 
5.0 



Note: The controls were prepared by adding 1 cc. of distilled water to 4 cc. 
of the given peptone solutions. 



TABLE XI 



Action of autohjsis material (1 cc.) on solution of Witte's peptone {5 cc. of a 0.25 per 
cent solution). B. subtilis and B. coli comparison 



ORGANISMS 


BIURET REACTION AND COLOR COMPARISONS WITH 
PEPTONE CONTROL 




1st day 


2nd day 


4th day 


8th day 


12th day 


B. subtilis I 


0.25 
0.25 
0.25 
0.25 


0.13 
0.20 
0.25 
0.25 


0.08 


0.0 
0.12 
0.25 
0.25 




B. subtilis II 

B. coli I 


0.25 


Control 


0.25 



Note: This experiment was repeated with larger amounts of peptone. The 
results were practically the same as in this table. 



The above tables show clearly the ability of bacteria to 
digest themselves. This property appears to be confined, how- 
ever, to organisms which are known to elaborate a proteolytic 
enzyme — the gelatin-liquefying group. Not only do the organ- 
isms of this group rapidly destroy their own protein under 
favorable conditions of autolysis, but they readily attack and 
decompose egg albumin, peptones and partially purified pro- 
teoses when the autolyzing materials are brought in contact 
with these foreign proteins. 

On the other hand, the gelatin-non-liquefying organisms em- 
ployed in these experiments were unable to effect any change in 
the protein content of the respective suspensions, at least in so 
far as may be judged by the biuret tests. Furthermore, other 



30 L. F. RETTGER, N. BERMAN AND W. S. STURGES 

proteins, when added to the bacterial suspensions after periods 
of preUminary incubation, remained unaffected. In every in- 
stance where the test was satisfactorily carried out the quality 
and degree of color obtained in the biuret test remained un- 
changed, as is readily seen by comparisons with the controls 
or with the standard peptone solution. 

Autolysis of the bacterial cells was always accompanied by a 
change in the staining properties of the individual organisms. 
In many cases, as for example in the complete autolysis of B. 
subtilis material, the bacilli took on only a faint color; and the 
presence of numerous fine granules presented a picture far from 
the normal. A difference in staining properties was also occa- 
sionally observed in the organisms of the B. coli type, but this 
was never marked, and was not due to actual destruction of the 
cell protein, as was shown always by the biuret test. The change 
was due to some process other than autolysis, as for instance 
"washing" or "laking" of the bacterial cells. 

GENERAL DISCUSSION AND CONCLUSIONS 

The results of the present investigation strongly indicate that 
bacteria are unable to attack and bring about the decomposition 
of proteins without the aid of enzymes or other proteolytic agents. 
This applies not only to the more complex proteins like egg 
albumin, but in all probability to albumoses and peptones as 
well. Coagulated albumin shows the same resistance to the 
direct action of bacteria of both the gelatin-liquefying and non- 
liquefying types as the unheated and unchanged native proteins. 

By means of the quantitative biuret test of Vernon the dis- 
appearance of proteoses and peptones from solutions serving as 
test or culture media may be readily demonstrated. This method 
has been of much value to us in the present investigation. It is 
being employed for the determination of other proteins also, as 
for instance casein in the form of nutrose. 

Even during prolonged incubation of flasks containing the 
necessary inorganic salts for bacterial metabolism, together with 
proteoses or Witte's peptone, little if any loss of these soluble 



UTILIZATION OF PROTEID AND NON-PROTEID NITROGEN 31 

proteins could be observed if the flasks had been inoculated with 
members of the colon-typhoid group or with other gelatin-non- 
liquefying bacteria. On the other hand, organisms which are 
known to elaborate proteolytic enzymes, as for example B. 
prodigiosus and B. subtilis, rapidly brought about destruction of 
the proteins. Te^t media containing purified coagulated egg 
albumin or dialyzed proteoses as the only possible source of 
available nitrogen were, with few exceptions, not attacked, how- 
ever, even by the gelatin-liquefyers, if the inoculations were 
made with but comparatively few organisms and from a culture 
less than twenty-four hours old. 

The slight reduction in the amount of "peptone" which was 
observed in a few instances may have been due to agents other 
than enzymes or bacterial cells, as for instance acids and am- 
monia. It is significant that such reductions did not become 
apparent until at least two to three weeks after the time of 
inoculation. These slight losses in the soluble proteins, if they 
were losses, usually occurred in flasks containing luxurious 
growths, and may possibly be due to adsorption by the bacteria 
and other suspended matter and by the w^alls of the flasks which 
were more or less coated. The possibility of the occurrence of 
small amounts of a proteolytic enzyme having the properties of 
erepsin (Cohnheim, 1901, Vernon, 1904) cannot be ignored. 
However, if such an enzyme is produced by organisms of the 
B. coll and B. typhi type it is of little importance, as no indi- 
cations of any proteolytic action whatever were apparent during 
the first two weeks, and since only very minute quantities can 
be produced even under the most favorable cultural conditions. 

The statement that purified albumin and dialyzed proteoses 
were not attacked even by gelatin-liquefying bacteria if the test 
fluids were inoculated with few organisms taken from very young 
cultures may appear at first paradoxical. The results, which 
are in harmony with those of Bainbridge and the earlier investi- 
gations in this laboratory on purified albumins, readily admit of 
an explanation. When the test medium contains no other possi- 
ble source of nitrogen for cell metabohsm besides the purified 
protein it is not attacked by any bacteria unless a sufficient 



32 L. F. RETTGER, N. BERMAN AND W. S. STURGES 

amount of the inoculating material is introduced to carry with 
it the necessary enzyme to bring about cleavage of the protein. 
In a medium containing nitrogen which is directly available, 
bacterial multiplication will take place, though the number of 
bacteria introduced is small. If such a medium also contains 
protein, and if the organism is one which under favorable con- 
ditions elaborates a proteolytic enzyme, the protein undergoes 
cleavage as the result of the enzyme action. These points have 
been demonstrated repeatedly. 

What are some of the important sources of nitrogen avail- 
able for bacterial metabolism without the aid of an enzyme? 
Our attention will naturally be directed to aixiino acids which 
in animal physiology are now known to play such an important 
part in nutrition. Witte's peptone contains amino acids which 
may be demonstrated readily by any of the well-known tests, 
particularly the Sorensen method (Sorensen, 1908). The amount 
of amino acids present in the American brands of peptone is con- 
siderably greater than in the Witte product. This undoubtedly ex- 
plains why we have consistently obtained more luxuriant, though 
not necessarily more characteristic, bacterial growths in media 
which contained the American products than in the standard 
Witte. 

It appears at this time indeed probable that so-called "peptone 
media" largely owe their value as culture media to the amino 
acids and perhaps other nitrogenous substances which readily 
give up their nitrogen as the result of direct bacterial action, 
and unless bacteria are present which elaborate proteolytic en- 
zymes, little if any of the proteoses and peptones in the medium 
is utihzed. Indeed it may be necessary for us to go even further 
than this, and to adopt the view that the bacterial cell can not 
utilize any protein until after it has been broken up by some other 
agent and the nitrogenous portion converted into simple form. 
If this view should obtain it will be necessary for us to alter 
materially our conception of the value of peptone, nutrose, and 
other soluble as well as insoluble proteins as culture media, 
especially in so far as the group of gelatin-non-liquefying bac- 
teria is concerned. 



UTILIZATION OF PROTEID AND NON-PROTEID NITROGEN 33 

Such a view as is tentatively presented here is certainly in 
harmony with the results of Loewi (1902) Abderhalden and others 
who in elaborate investigations have shown that animals like the 
dog may be maintained in nitrogenous equilibrium for long periods 
of time when fed on a diet in which all protein material had been 
replaced by the products of prolonged digestion of proteins. 
These experiments imply that the cleavage products of the pro- 
teins are resynthesized in the animal body. According to Abder- 
halden no cells can directly assimilate and utilize foreign food 
material. Complex nitrogenous food material must be prepared 
for the cell through enzyme action. This breaking down and the 
reconstruction of food are just as necessary as it is to reduce a 
church to the very bricks which constitute it before it can be 
converted into a school-house. This may perhaps apply to the 
bacterial cell as well as in the field of animal cell nutrition. 

REFERENCES 

Abderhalden, et al. Protein synthesis in the animal body. Zeit. f. physiol. 

Chem., vols. 42, 44, 47, 52, 57, 59, 77 and 78. 
Bainbridge, F. a. 1911 The action of certain bacteria on proteins. Jour. 

Hyg., xi, 341-55. 
CoHNHEiM, O. (1901) Die Umwandlung des Eiweiss durch die Darmwand. 

Zeit. f. physiol. Chem., xxxiii, 451-65. 
CoNRADi, H. (1903) Ueber losliche durch aseptische Autolyse erhaltene Gift- 

stoffe von Ruhr und Typhusbazillen. Deut. med. Woch., xxix, 26-28. 
Hopkins, F. G., and Pinkus, S. N. (1899) Observations on the crystallization 

of native proteins. Jour. Physiol., xxiii, 130-36. 
Levy, E., and Phersdorff, F. (1902) Ueber die Gewinnung der schwer 

zuganglichen in der Leibensubstanz enthaltenen Stoffwechselproducte 

der Bakterien. Deut. med. Woch., xxviii, 879-80. 
Loewi, O. (1902) Ueber Eiweisssynthese im Thierkorper. Arch. f. exp. Path. 

u. Pharm., xlviii, 303-30. 
Rettger, L. F. (1904) On the autolysis of yeasts and bacteria. Jour. Med. 

Res., xiii, 79-92. 
SoRBNSEN, S. p. L. (1908) Enzymstudien. Biochem. Zeit., vii, 45-101. 
Sperry, J. A. AND Rettger, L. F. 1915 The behaviour of bacteria towards 

purified animal and vegetable proteins. Jour. Biol. Chem., xx, 445-59. 
Vernon, H. M. (1904) The peptone-splitting ferments of the pancreas and 

intestine. Jour. Physiol., xxx, 330-69. 



STUDIES ON SOIL PROTOZOA AND THEIR RELATION 
TO THE BACTERIAL FLORA. I^ 

JAMES M. SHERMAN 

From V4te Bacteriological Laboratories of the Wisconsin Agricultural Experiment 

Station, University of Wisconsi7i 

I. INTEODUCTION 

The occurrence of protozoa in soil 

The knowledge that protozoa occur in soil dates back nearly 
as far as does the science of microbiology, but it is only recently 
that specific studies have been directed at the micro-fauna of 
the soil. Miiller (1887) reported studies concerning some soil 
protozoa which he thought played a part in the destruction 
of organic tissue, and thus were to be considered as important 
agents in the formation of humus. Celli and Fiocca (1894) 
studied the protozoa of the soil and described several forms of 
amoebae obtained from this source. Beijerinck (1896) described 
an amoeba which occurred in his cultures of nitrifying bacteria;, 
and later (1901) called attention to a variety of amoebae, monads 
and infusoria which appeared in cultures with Azotobacter. 
Frosch (1909) isolated a number of saprophytic amoebae from 
garden soil similar to those found in the intestinal tracts of ani- 
mals. Tsujitani (1908), likewise described some amoebae which 
occur in soil. Hiltner (1907) noted many types of protozoa 
which appeared in cultures made from soil, and stated that 
these organisms certainly do not play an unimportant role. He 
noted the presence of various ciliates, flagellates, and amoebae, 
some of which he said were often present in unusually large 
numbers. Stormer (1907) also studied the protozoan fauna, and 
demonstrated that the soil contains a considerable number of 
these organisms, especially amoebae. 

1 Presented at Seventeenth Annual Meeting of the Society of American Bac- 
teriologists, Urbana, 111., December 29, 1915. 

35 



36 JAMES M. SHERMAN 

Within the past few years more attention has been directed 
toward the soil protozoa with the result that they have been 
demonstrated to be of general occurrence in the soils of those 
parts of the world which have been studied. In England, Russell 
and Hutchinson (1909) (1913), Russell and Golding (1912), 
Goodey (1911), Martin (1912), and Martin and Lewin (1914) 
have noted the constant presence of protozoa in soil. Similar 
observations have been made by Wolff (1909) (1912), France 
(1911), Killer (1913), Emmerrich, Leiningen and Loew (1912), 
and Cunningham and Lohnis (1914) in Germany; by Lodge and 
Smith (1912), Gainey (1912), Rahn (1913) and Sherman (1914) 
in the United States; by Cauda and Sangiori (1914) in Italy, 
Peck (1910) in Hawaii, and Greig-Smith (1912) in Australia; 
while Loew (1911) (1913) has observed them in the Alps, in Porto 
Rico, the Island of Borkum, and in Japan. Important contribu- 
tions to our knowledge of the types of protozoa which occur in 
soil have been made by Wolff (1909), France (1911), Goodey 
(1911) (1914). IVIartin (1912), and Martin and Lewin (1914). 

The relation of protozoa to bacteria 

It is well known that bacteria constitute the chief food for 
many types of protozoa. Many of the ciliates in particular are 
especially destructive to bacteria, although this property is pos- 
sessed by the other classes as well. Indeed, Calkins (1901) has 
said that probably all protozoa ingest bacteria with the excep- 
tion of the parasitic forms and those which live on other protozoa. 
This view, however, has been modified considerably in recent 
years and it is now known, as is stated by Minchin (1912), that 
a number of protozoa are saprozoic in nature and obtain their 
food by absorption. A considerable portion of the non-pai-asitic 
flagellates belong to this class. 

In many places in nature bacterial development is limited by 
the action of predatory protozoa. Huntemtiller (1905) and 
Korshun (1907) have proven that the micro-fauna plays an 
important part in the purification of water. The possibility 
that protozoa are inimical to the soil bacteria has only very 



STUDIES ON SOIL PROTOZOA 37 

recently received serious consideration, probably because of the 
lack of evidence that these organisms exist in an active, free- 
living condition in the soil. 

The phagocytic theory of soil fertility 

Interest in the soil protozoa was given a great stimulus in 
1909 when Russell and Hutchinson (1909) of the Rothamsted 
Experimental Station announced their theory which involves the 
protozoa as a factor detrimental to the soil bacteria, and, there- 
fore, to soil fertility. This theory, commonly known as the 
phagocytic theory of soil fertility, was proposed in an effort to 
explain the phenomena associated with the partial sterilization 
of soil with heat or with volatile antiseptics. The increased 
yields of crops obtained after partial sterilization is explained, 
by the sponsers of this theory, on the view that the soil protozoa 
prey upon the bacteria and thus act as a limiting factor on the 
microflora of the soil. The process of partial sterilization is 
thought to destroy the protozoa while the bacteria are greatly 
reduced, but not exterminated. When the protozoa are sup- 
pressed, the bacteria which remain are allowed to multiply un- 
hindered and so attain numbers greatly in excess of those found 
in normal soils. A greater number of bacteria results in the 
elaboration of a greater amount of plant food, hence larger crops 
are produced. 

No direct proof has been produced in support of this theory, 
but Russell and Hutchinson and their associates (1909), (1912), 
(1913) have presented much evidence of an indirect nature which 
indicates strongly that some biological factor, detrimental to 
bacteria, does exist in the soil. 

Other views on the partial sterilization of soil 

In opposition to the protozoan theory of Russell and Hutchin- 
son, several other explanations have been advanced to account 
for the beneficial effects of volatile antiseptics upon the higher 
and lower forms of plant life in the soil. Koch (1899) claims 
that the antiseptic acts as a stimulant directly upon the bac- 



38 JAMES M. SHERMAN 

terial flora of the soil, and likewise upon the higher plants. In 
support of this stimulation theory some very convincing data 
have been furnished by Koch (1899) (1911), Nobbe and Richter 
(1904), Egorov (1908), Fred (1911), Gainey (1912) and others. 

The selective theory of Hiltner and Stormer (1908) holds that 
volatile antiseptics exert a selective action on the bacterial flora 
of the soil. It is thought that the soil is so changed that the 
subsequent development of the beneficial types of bacteria is 
enhanced, while the harmful forms are suppressed. These in- 
vestigators believed the increased crop yields obtained to be due 
to the increase in the amount of plant food elaborated by the 
beneficial bacteria. 

Bolley (1910) (1913 a) 1913 b) claims that the improvement of 
soil by partial sterilization is in many cases due to the destruc- 
tion of certain parasite fungi which attack the plants and thus 
hinder their growth and development. Another function per- 
formed by volatile antiseptics according to Grieg-Smith (1911) 
is the liberation of plant and bacterial food through the solution 
of the "agricere" or soil wax. 

Other points have been noted in the works of various investi- 
gators which partially account for the action of certain anti- 
septics in soil exclusive of their effect upon the protozoan fauna. 
Buddin (1914) has shown that the treatment of soil with sulphur 
dioxide increases the number of bacteria very appreciably with- 
out exterminating the protozoa, while certain other compounds 
such as pyridine cause an increase in the number of bacteria due 
to the fact that their decomposition products furnish an excellent 
source of food for the soil micro-organisms. Hutchinson and 
MacLennan (1914) have shown that the partial sterilization of 
soil with caustic lime leads to a chemical breaking down of some 
of the organic matter of the soil and thus stimulates the subse- 
quent activities of the bacteria. Fred (1915) in his work on the 
action of carbon bisulphide in soil has demonstrated that all of 
this compound does not evaporate when added to the soil, but 
that some of it remains and is changed to sulphates. 

Some workers believe that the value of partial sterilization by 
heat is due to the destruction of soil toxins which limit the 



STUDIES ON SOIL PROTOZOA 39 

activities of the micro-organisms and plants. Whether toxins 
occur in soil is a disputed question, but Whitney and Cameron 
(1904) (1910), Schreiner and Reed (1907 a) (1907 b), and Picker- 
ing (1910 b) have demonstrated quite satisfactorily that plant 
toxins do exist; while Bottomley (1911) and Greig-Smith (1911) 
have submitted data which point to the existence of bacterio- 
toxins in the soil. 

Others explain the beneficial action of heat by the changes 
it produces in the soil compounds. Frank (1888), Pickering 
(1910 a), Lyon and Bizzell (1910), Stone (1912) and others have 
demonstrated an increase in the amount of soluble plant and 
bacterial food in partially sterilized soil. Pfeffer and Franke 
(1896) and Kriiger and Schneidewind (1899) have shown an 
increased assimulation by plants in heated soils. Pickering 
(1910 b), Wilson (1914) and others have further proven that 
heat may also produce a toxic compound in the soil, the toxicity 
increasing with an increase in the temperature used, so that soils 
heated to very high temperatures have a detrimental effect 
upon plant growth. 

The effect of protozoa on the bacterial flora of the soil 

There has been only a limited amount of work done upon the 
action of the protozoa in soil aside from the indirect evidence 
which has been acquired in the study of partial sterilization. 
Russell and Hutchinson (1913) have failed in their attempts to 
reduce the number of bacteria in partially sterilized soil by the 
addition of mass cultures of protozoa; Lipman, Blair, Owen and 
McLean (1910) in their work on ammonification in soil were 
unable to detect any influence upon this process due to the pro- 
tozoa; while Grieg-Smith (1912) obtained entirely negative re- 
sults in his efforts to show that protozoa are detrimental to the 
bacterial flora of the soil. Cunningham (1914), on the other 
hand, claims to have demonstrated that protozoa do limit the 
number of bacteria in soil. 



40 JAMES M. SHERMAN 

Outline of work undertaken 

Preliminary to any work on the part played by the soil pro- 
tozoa, two essential points should be established: (1) whether 
protozoa occur in soil in numbers sufficient to be a factor in soil 
fertility; and (2) whether protozoa lead a trophic life in the 
soil. Unless these two points can be settled in the affirmative, 
it would appear that discussions concerning the role played 
by the micro-fauna of the soil must be considered more or less 
futile. The first part of this work was therefore directed toward 
getting more definite information as to the number of protozoa in 
soil and the nature of their existence therein. 

A study was made of the effect of protozoa upon the bac- 
terial flora of the soil by the isolation of animal-pure cultures 
of some representative soil protozoa and inoculation into 
protozoa-free cultures of soil bacteria, in solutions and in soil, 
and also by the comparison of the activities of bacteria in 
sterilized soil reinoculated with normal soil and with ''protozoa- 
free soil." 

The last part of the work was devoted to a study of the action 
of volatile antiseptics in soil in an effort to throw some light on 
the part played by the protozoa. 

II. THE NUMBER OF PROTOZOA IN SOIL 

Present status 

Although the soil protozoa have attracted considerable atten- 
tion in recent years, few data are at hand showing the number of 
such organisms actually found in normal soils. Stormer (1907) 
showed that fertile soils sometimes contain several thousand 
amoebae per gram as determined by the agar plate method of 
enumeration. Hiltner (1907) reported the finding of large num- 
bers of protozoa in soil, and said that flagellates and amoebae 
had been found in numbers reaching millions per gram. He did 
not report the specific data upon which this statement was based, 
nor did he give the details of the method by which the numbers 
of protozoa were determined. Lodge and Smith (1912), on the 



STUDIES ON SOIL PROTOZOA 41 

other hand, investigated field soils of Massachusetts and claimed 
that the number of protozoa present would have to be increased 
many fold in order to be considered a factor in the limitation of 
bacterial numbers. Gainey (1912) studied the protozoa in 
Missouri soils and likewise concluded that the number was not 
sufficient to be a factor in soil fertiHty. Rahn (1913) by use of 
the dilution method determined the number of protozoa in 
Michigan soils. From the limited data submitted by him it 
would appear that the soil contains about one thousand protozoa 
per gram. Killer (1913) tested various methods for the deter- 
mination of the soil protozoa but concluded that all of the 
methods now known are of limited and doubtful value. Recently 
Cunningham (1914) has reported studies of German soils which 
he has demonstrated to contain quite large numbers of protozoa. 

Methods 

The dilution method, which has been employed to some 
extent for estimating the number of protozoa in soil, has been 
used in this work. It is obviously impossible to devise one 
medium which will favor equally the development of all of the 
various forms of unicellular animal organisms found in the soil. 
In the preliminary work undertaken, various media were used 
with the idea of finding which forms of protozoa are most abun- 
dant in soil, and which media are best adapted to those par- 
ticular forms. 

From observations on several media it was early observed that 
the flagellates make up the greater portion of the protozoan 
population of the soils which were studied. The survey of media 
has not been very extensive, but a dilute soil extract has given 
the most satisfactory results for determining the number of 
flagellates. This medium is also well suited for the growth of 
ciliates and amoebae. The use of soil extract seems appropriate 
as it would appear that the orgam'sms favored by this medium 
would be the ones most likely to be leading a trophic life in the 
soil. One per cent hay extract, which has been used by most 
investigators in the study of the soil protozoa, has not given as 



42 



JAMES M. SHERMAN 



satisfactory results as has soil extract, in the comparison made 
at this laboratory. 

Soil extract prepared by boiling one part of soil in three parts 
of distilled water, filtering clear, and adding a small excess of 
CaCOs, has been used. This has been modified by using only 
one part of soil in nine parts of water and one part of a one 
per cent hay extract, plus CaCOs. The presence of this small 
amount of hay extract does not appear to exert any inhibitory 
effect upon the flagellates, while the ciliates seem to be benefited. 
In sampling soil not less than ten grams have been taken, and 
duplicate dilutions have always been made. The cultures have 
been incubated at 20°-25°C. and examined every few days for a 
period of about ten days. 

Results 

The data obtained on twelve Wisconsin soils, representing 
various types under different treatments, are tabulated in Table 
I. 

TABLE I 
Approximate number of protozoa in various Wisconsin soils 





TYPES OF SOU, 


TREATMKNT OP SOIL 


DILUTIONS 


NO. 


1/1,000 gram 


1/10,000 gram 




A 


B 


A 


B 


1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 


Clay loam 
Clay loam 
Rich loam 
Sandy loam 
Peat soil 
Loam soil 
Clay loam 
Loam soil 
Clay loam 
Sandy loam 
Sandy loam 
Sandy loam 


Apple orchard 

Blackberry patch 

Garden 

Tobacco field 

Uncultivated 

Pasture 

Vineyard 

Alfalfa field 

Not planted 

Clover 

Castor bean bed 

Corn field 


+ 

+ 

+ 

+ 

+t 

+ 

+ * 

+ 

+ * 

+ 

+ 

+ 


+ 
+ 

+ 
+ 
+ * 
+ 
+ * 
+ 
+ 
+ 


+ 

+ 
+ 
+ 

+ 
+ 


-1- 
+ 
+ 

+ 
+ 

+ 
+ 



A and B represent the duplicate samples of each soil. 

+ = presence of protozoa. 

— = no protozoa present. 

* Colpoda found in addition to flagellates. 

t Amoebae found in addition to flagellates. 



STUDIES ON SOIL PROTOZOA 



43 



That the high protozoan content noted above is not pecuhar 
to Wisconsin soils is showTi by the data obtained from three 
Virgiaia soils and one Tennessee soil given in Table II. These 
samples were from representative soils in a good state of culti- 
vation. 

The figures of the foregoing tables show that, in the sixteen 
soils recorded, in every soil protozoa were found in both samples 
of the 1/1,000 of a gram dilution, while of the total of 32 dilu- 
tions representing 1/10,000 of a gram each, 18 revealed the pres- 
ence of protozoa. When we consider that, (1) it is extremely 
improbable that every individual protozoan grows when taken 

TABLE II 
The approximate number of protozoa in Virginia and Tennessee soils 





SOURCE 


TYPE OF SOU. 


TREATMENT OP 
SOIL 


DILUTIONS 




1/1,000 gram 


1/10,000 gram 


o 


A 


B 


A 


B 


1 

2 
3 
4 


Virginia 
Virginia 
Virginia 
Tennessee 


Clay 

Clay loam 

Loam 

Compact red clay 


Garden 
Wheat field 
Grass field 
Corn field 


+ 
+ 
+ 
+ 


+ 

+ 

+ 


+ 
+ 


+ 
+ 

+ 



A and B represent the duplicate samples of each soil. 
+ = presence of protozoa. 

— = no protozoa present. 

* Balantiophorus elongalus found in addition to flagellates. 

from the soil and introduced into a hquid medium; (2) the 
liquid medium used is probably not adapted for the growth of 
all protozoan inhabitants of the soil ; and (3) some of the samples 
representing only 1/10,000 of a gram contained three distinct 
types of animal organisms, the statement that the average fer- 
tile soil contains about 10,000 protozoa per gram seems con- 
servative.- 

In cultures inoculated with 1/1,000 of a gram of soil, a variety 
of flagellates are usually to be observed. As seen from the above 

- it should be borne in mind that these data are not intended to give an accu- 
rate estimate of the numbers of protozoa in soil, but only to show whether they 
occur in numbers sufficient to be regarded as a possible factor in soil fertility. 



44 JAMES M. SHERMAN 

data, the ciliates do not ordinarily occur in numbers approxi- 
mating 1,000 per gram. Colpoda cucullus which has been widely 
noted as an inhabitant of soil, appears to be the most generall}^ 
distributed ciliate. This organism may be very often, but not 
constantly, found in 1/100 of a gram of soil. In one Virginia 
soil (Table II), Balantiophorus elongatus was found in the 1/1,000 
of a gram dilution. With the methods employed, it would not 
be safe to draw any conclusions regarding the amoebae other 
than that they do not occur in numbers nearly as great as do the 
flagellates in ordinary soils. Although the culture media and 
period of observation used are not adequate for work with the 
amoebae, it is worthy of note that in the peat soil, containing a 
high humus and water content (in which we should expect to 
find numerous amoebae) the method was of sufficient service 
to show their presence in 1/1,000 of a gram in one of the two 
samples taken. In spite of the limitations of the methods used, 
amoebae would probably have been revealed had they been of 
general occurrence in large numbers. Using the same medium 
and inoculating with a large amount of soil, amoebae have been 
observed as early as the third day. 

In the dilutions of 1/10,000 of a gram, with one exception, 
only four general types of protozoa have as yet been noted: 
Monas sp. Dimorpha radiata (?) and two other flagellates which 
have not been identified. 

III. THE GROWTH OF PROTOZOA IN SOIL 

Previous work 

In order to correlate the protozoa with any of the vital func- 
tions of the soil, it is first necessary to demonstrate that they ai-e 
active in soils of normal moisture content. Goodey (1911) made 
a very careful study of the ciliates of the soil and concluded thjit 
these organisms exist in soils of normal moisture content onh- 
in the encysted condition. When free water was to be found 
on the soil active protozoa could be demonstrated, but no active 
ciliates could be detected in ordinary soils. Russell and Golding 
(1912) working with water-logged, "sewage sick" soil, demon- 



STUDIES ON SOIL PROTOZOA 45 

strated the presence of active protozoa. Under these con- 
ditions, as was pointed out by Goodey, many protozoa may 
become active. When we consider the minute size of these 
organisms it would appear obvious that they may become active 
whenever the soil is in a saturated condition. Martin (1913) 
by means of a special method which he has developed has been 
able to prove definitely that soil contains active protozoa. Cun- 
ningham (1914) has collected data which indicate that some of 
the protozoa exist in soil in the active state. 

Experimental 

In order to see if any of the soil protozoa are active, the 
following experiment was performed. Four pots each contain- 
ing two kilograms of sterilized soil with an optimum moisture 
content of about 17 per cent were inoculated with two grams each 
of a normal soil, which showed 10,000 protozoa per gram by the 
dilution method. These pots were then brought up to varying 
moisture contents by the addition of sterile water, as follows: 

per cent 
H2O 

Pot No. 1 11 

Pot No. 2 12 

Pot No. 3 15 

Pot No. 4 22 

Since one gram of normal soil, with a protozoal count of 10,000 
per gram, was used to inoculate 1,000 grams of sterile soil, the 
resulting mixture should contain, approximately, only 10 of these 
organisms per gram. After fifteen days incubation at 20 to 25°C., 
these pots were sampled, and in every case flagellates were found 
in the 1/10,000 dilutions. This could not be explained except on 
the assumption that these animal organisms had undergone 
rapid multiplication. The forms found in greatest numbers in 
these pots were the same forms which were noted as the pre- 
dominating types in normal soils. Monas sp. did not occur 
in the 1/10,000 dilution cultures from the soils containing only 
11 and 12 per cent moisture. In the case of pot 4, with a mois- 
ture content of 22 per cent, Colpoda cucullus was found in the 



46 



JAMES M. SHERMAN 



1/100 dilution, showing that this organism was probably active 
in the soil containing this high amount of water. 

To obtain more definite data on this point, with known organ- 
isms, an experiment was performed in which pots of sterihzed 
soil were inoculated with animal-pure cultures of three of the 
types of flagellates mentioned in the first part of this paper. 
The pots were inoculated with 0.1 gm. each of soil cultures of 
the respective organisms per 1,000 grams of sterile soil. The 
water content was then held at 15 per cent (a little below normal 
for a soil of this type). Determinations for protozoa were made 
immediately after inoculation, and then at intervals for fifteen 
days. The results obtained are given in Table III. 

TABLE ni 
The multiplication of three types of protozoa in soil. (H^O content 15 per cent.) 



ORGANISM 



Monas (sp). 
D. radiata. . 
Flagellate A 



NUMBER PER ORAM 



Start 



1 

1 

10 



days 9 days 12 days 15 days 



10 
100 
100 



10 

100 

1,000 



1,000 

1,000 

10,000 



10,000 
100,000 
100,000 



In similar tests using Colpoda cucullus, Balantiophorus elongatus 
and Oxytricha sp. no multiplication could be detected during 
a period of thirty days. These results appear to substantiate 
the data of Goodey on the ciliates. 

A point which probably deserves mention in this connection, 
is that when sterilized soil is inoculated with normal soil, the 
protozoan fauna rises in numbers above that of normal soil, 
just as does the bacterial flora. In other words, it is probable 
that the micro-organic balance remains about the same. In 
Table IV are given the results obtained on five pots of sterilized 
soil which were inoculated with normal soil. 

Higher dilutions have not been tried, but it is not unlikely 
that under such conditions the number of protozoa may reach 
1,000,000 per gram. This fact is not of great significance, but it 
has a practical application in indicating that the subsequent 
work on the relation of the soil protozoa to the bacterial flora 



STUDIES ON SOIL PROTOZOA 



may be carried out in a sterilized soil medium without causing an 
apparent disturbance in the balance between these two classes 
of organisms. 

TABLE IV 
Approximale number of protozoa in reinoculated sterile soil 





DILUTIONS 


POT NO. 


1/10.000 gram 


1/100,000 gram 




A 


B 


A 


B 


1 


+ 


+ 


+ 




+ 


2 


4- 


+ 


+ 




- 


3 


+ 


+ 


+ 




+ 


4 


+ 


+ 


- 




+ 


5 


+ 


_L 


+ 




+ 



A and B represent the duplicate samples of each pot. 
+ = presence of protozoa. 
— no protozoa present. 

IV. THE BEHAVIOR OF BACTERIA IN SOILS CONTAINING PROTOZOA 
AND FREE FROM PROTOZOA 

Methods 

In an effort to show the influence of the soil protozoa upon the 
bacterial flora the following method was employed. Pots of 
soil covered with, a layer of non-absorbent cotton between layers 
of cheese cloth, to prevent reinfection, were sterilized in the 
autoclave under fifteen pounds steam pressure for one hour. 
Some of the pots were then inoculated with unsteriHzed soil in 
order to introduce all of the biological factors peculiar to normal 
soils, while the others were inoculated with a special soil con- 
taining a varied mixture of soil bacteria but free of protozoa. 

This "protozoa-free soil" was made up of a mixed flora ob- 
tained from several different soils by the isolation of as many 
kinds of bacteria as could be obtained. For this purpose 
several different soils were plated out on beef extract, casein, 
Heyden and Ashby's agars and all of the colonies which devel- 
oped transferred to sterile soil. Other portions of soil which had 
been partially sterilized by heat sufficiently to kill all protozoa 



48 JAMES M. SHERMAN 

were also added to this soil culture. The flora was made more 
complex by the addition of dilutions of various soils which repre- 
sented 1/1,000,000 of a gram, and which contained no protozoa. 
It would seem that the addition of these dilutions would seed 
the soil culture with those types of bacteria which predominate 
in normal soils. The protozoa-free soil so prepared was pro- 
tected from contamination and its freedom from protozoa was 
verified at frequent intervals. 

In all of the experiments in this part of the work at least one 
kilogram of soil was used in each pot. For most of the trials 
larger amounts were used according to the size of the pots. 
The moisture content of the soils was maintained at about two- 
thirds of their water holding capacities. 

For the determination of the number of bacteria the soils were 
plated on Heyden agar and the counts made after ten days 
incubation at laboratoiy temperature. 

The number of bacteria in soils containing protozoa and free of 

protozoa 

Many determinations have been made of the number of bac- 
teria in sterilized soils which were reinoculated with normal soil 
and with the special protozoa-free soil previously described. 
Before the results from these tests are discussed, it should be 
recognized that the method is open to severe criticism. A com- 
parison is made of two soils containing quite different flora. It 
is not to be expected that the flora of the artificial soil used 
approaches in complexity the bacterial flora of the normal soil. 
The number of bacteria found in these soils would probably be 
different even though neither be influenced by any detrimental 
factor. It is logical to believe that the greater number of bac- 
teria would be found in the soil containing the more complex 
flora, since it would seem, the greater the variety of bacteria the 
greater would be the efficiency of the flora in the utilization and 
destruction of its own by-products. 

If this view is correct, the greater number of bacteria should 
be found in the soil inoculated with normal soil unless the micro- 



STUDIES ON SOIL PROTOZOA 



49 



flora of this soil is limited by some harmful factor. In all of the 
tests which have been made the soils free of protozoa contained 
greater numbers of bacteria than the corresponding soils which were 
inoculated with normal soil. The results obtained in a few repre- 
sentative trials are given in Table V. 

TABLE V 

The number of bacteria in soils free of protozoa and containing protozoa 

Test 1* — Incubation period two months 



POT 


INOCULUM PROTOZOA FREE 


INOCULUM NORMAL SOIL 




Bacteria per gram 


Average 


Bacteria per gram 


Average 


1 

2 
3 
4 
5 


390,000,000 

290,000,000 
274,000,000 
250,000,000 
270,000,000 


294,800,000 


206,000,000 
110,000,000 
232,000,000 
164,000,000 
166,000,000 


175,600,000 



Test 2 — Incubation period one month 



340,000,000 
275,000,000 



307,500,000 



106,000,000 
114,000,000 



110,000,000 



Test 3 — Incubation period three months 



230,000,000 
180,000,000 



205,000,000 



142,000,000 
140,000,000 



141,000,000 



* Each of these tests represents a distinct experiment and not a re-count on 
the same soil. 

An experiment was also performed to see if the above phe- 
nomena would occur on different tyipes of soil. For this pur- 
pose a rich muck soil, a clay loam and a poor sandy soil were 
used. The results obtained (Table VI) showed the characteristic 
difference in each case. 

The results obtained in the foregoing experiments can not be 
considered as proof that the soil protozoa are inimical to the 
bacteria because of the differences in the two soils under con- 
sideration. However, these data, together with the previous 
observations that the soil contains an adequate supply of protozoa 
and that some of these organisms are active, certainly appear to 
add weight to the theory of Russell and Hutchinson that protozoa 
serve as a hmiting factor upon the bacteria in the soil. 



50 



JAMES M. SHERMAN 



TABLE VI 



The number of bacteria in different types of soil containing protozoa and free of 

protozoa 

After fifteen days incubation 



INOCULUM 


NUMBER OP BACTERIA PER GRAM 




Muck soil 


Loam soil 


Sandy soil 


Protozoa-free 


1,030,000,000 
307,000,000 


617,000,000 
214,000,000 


24,200,000 


Normal soil 


11,900,000 



After sixty days incubation 



Protozoa-free . 
Normal soil.. . 



210,000,000 
108,000,000 



157,000,000 
70,000,000 



21,800,000 
6,700,000 



The effect of the complexity of the bacterial flora upon the apparent 
number of bacteria in soil 

As was previously pointed out the weakness in the tests in 
which the number of bacteria in soils containing protozoa and 
free of protozoa are compared lies in the fact that the flora of the 
two soils are different. Whether the complexity of the bacterial 
flora affects the apparent number of bacteria, as revealed by the 
agar plate count, is very important in this connection. An 
experiment was conducted in order to test this point. 

Twelve pots of sterilized soil were divided into four groups of 
three pots each. The soils in group A were then inoculated 
with all of the bacteria which developed on six Heyden agar 
plates from two different soils. Group B was inoculated with A 
plus a mixture of all the bacteria that developed on beef extract, 
nutrose, and casein agar plates taken from several different 
types of soil and from different depths of soil. Group C was 
inoculated with A and B plus the ''protozoa-free soil" used in 
the previous experiments. The pots in group D were inocu- 
lated with normal soil. After incubation periods of fifteen and 
thirty days at laboratoiy temperature, samples were taken and 
bacterial counts made using Heyden agar. The results obtained 
are given in Table VII. 

The results in Table VII show very clearly that the bacteria 



STUDIES ON SOIL PROTOZOA 



51 



in soil, as determined by the plate culture method, are diminished 
with an increase in the complexity of the flora. The soils used 
were all free of protozoa with the exception of those in Group D, 
yet there is a continual decrease in the number of bacteria found 
in each group as the number of kinds of bacteria is increased. It 
will be noted that there was a greater difference obtained in the 
bacterial counts between Groups B and C, neither of which 
contained protozoa, than there was between C and D, one of 
which contained protozoa while the other was free of these organ- 
It is very probable that this decrease in the number of 



isms. 



TABLE VII 
Effect of complexity of flora upon the apparent number of bacteria in soil 

Fifteen days 



NO. 




NUMBER 


PER GRAM 






Group A 


Group B 


Group C 


Group D 


1 

2 
3 


500,000,000 
600,000,000 
580,000,000 


460,000,000 
420,000,000 
460,000,000 


220,000,000 
240,000,000 
270,000,000 


190,000,000 
250,000,000 
270,000,000 


Average 


560,000,000 


447,000,000 


243,000,000 


237,000,000 


Thirty days 



Average . 



700,000,000 
580,000,000 
700,000,000 



660,000,000 



420,000,000 
420,000,000 
400,000,000 



413,000,000 



280,000,000 
250,000,000 
250,000,000 



260,000,000 



230,000,000 
200,000,000 
210,000,000 



231,000,000 



bacteria, due to an increase in the complexity, is only an apparent 
one and that the actual number of bacteria is just as great as in 
the soils containing a less complex flora. This view might be 
explained on the ground that in the more complex flora there 
was a larger percentage of bacteria which were not able to grow 
on agar plates. If in two soils each of which actually contained 
300,000,000 bacteria per gram but in one 90 per cent of the 
organisms were able to develop colonies on agar plates while in 
the other only 50 per cent had this property, the counts obtained 
would be 270,000,000 and 150,000,000 respectively. Since growth 



52 JAMES M. SHERMAN 

on agar was the chief means employed to obtain bacteria free 
of protozoa, there can be no doubt but that the protozoa-free 
soil used in these experiments contained a higher percentage of 
bacteria capable of development on agar plates than did the 
normal soil. 

The relation of the number of kinds of bacteria in soil to the 
total number is in itself a large problem. Whatever the explana- 
tion for the variation in the numbers of bacteria, as determined 
by the plate culture method, with the complexity of the flora 
may be, it appears very clear that the differences in the numbers 
of bacteria in the soils with and without protozoa obtained in 
the foregoing experiments were due in large part, at least, to the 
complexity of the bacterial flora itself. This casts doubt upon 
the belief that protozoa act as a limiting factor, but it is possible 
that the reduction in bacterial numbers in group D was in part 
due to these organisms. 

The effect of temperature upon the development of bacteria in soils 
with and without protozoa 

If the soil protozoa act as a limiting factor upon the bacteria 
it should be possible to demonstrate that fact by the subjection 
of soils with and without protozoa to conditions that would 
inhibit the growth of the protozoa but not that of bacteria. 
Russell and Hutchinson explain the fact that the soil contains 
more bacteria during the winter and early spring months than 
in the summer on the view that the protozoa are not active at 
such low temperatures. It seems, therefore, that observations 
on the development of bacteria in soils which contain protozoa 
and others free of protozoa when exposed to winter weather 
should throw some light on the subject. 

Four pots of sterilized soil were inoculated, two with bacteria 
alone and two with ordinary soil. The soils were kept at labo- 
ratory temperature for two months after inoculation and then 
placed out of doors during the months of December, January, 
February and March. The soils remained frozen throughout the 
greater part of this period. Bacterial counts were made just 



STUDIES ON SOIL PROTOZOA 



53 



before the pots were put out of doors and at the end of 45 days 
and 105 days periods outside. The data obtained are presented 
in Table VIII. 

If the soil protozoa have a detrimental effect upon the bac- 
teria we should expect the number of bacteria in the pots inocu- 
lated with ordinary soil to rise very markedly, while in the soils 
free of protozoa there should not be such an increase. There 
was, apparently, no difference in the behavior of the bacteria 
in the different soils. 

TABLE VIII 

Effect of low temperature upon the number of bacteria in soils containing protozoa 

arid free of protozoa 
Before being placed out doors 



POT 


WITHOUT PROTOZOA 


WITH PROTOZOA 




Bacteria per gram 


' Average 


Bacteria per gram 


Average 


1 

2 


300,000,000 
165,000,000 


232,000,000 


77,000,000 
74,000,000 


75,500,000 



Forty-five days after being placed out doors 



250,000,000 
270,000,000 



260,000,000 



130,000,000 
60,000,000 



95,000,000 



One hundred and five days after being placed out doors 



280,000,000 
336,000,000 



308,000,000 



100,000,000 
120,000,000 



110,000,000 



Another experiment was performed on the relation of tempera- 
ture to the bacterial flora in the presence and absence of pro- 
tozoa. Three pots of sterile soil were inoculated with normal 
soil while three other pots were inoculated with the protozoa- 
free soil. One pot of the soil from each lot was then incubated 
at each of three temperatures, 10°C., 22°C., and 37°C. for a 
period of 30 days. In this case also there should be a difference 
in the development of the bacteria in the two soils at the various 
temperatures if soil is possessed of a detrimental biological factor. 
The results (Table IX), as in the foregoing experiment, give no 
evidence of a phagocytic agent. 



54 JAMES M. SHERMAN 

TABLE IX 
Number of bacteria in soils kept at different temperatures 



INOCULUM 


NUMBER OF BACTERIA PER GRAM 




10°C. 1 22''C. 


37°C. 


Without protozoa 

With protozoa. ... 


460,000,000 
110,000,000 


300,000,000 
220,000,000 


210,000,000 
150,000,000 







In the soils kept at 37°C. there was the same difference between 
the numbers of bacteria in the soils containing and free of pro- 
tozoa as in the soils incubated at room temperature. The great- 
est difference was found in the soils incubated at 10°C., which 
fact is opposed to the protozoan theory, unless it be assumed 
that the protozoa act better at low temperatures. Such an 
assumption is not in accord with the known facts concerning 
them. 



Effect of moisture content upon bacteria in soils with and without 

protozoa 

It is generally acknowledged that protozoa require a larger 
amount of moisture than bacteria. An experiment was made 
with soils of a very low moisture content in order to eliminate 
the action of the ''detrimental factor," if such exists in the soil. 
Soil with an optimum moisture content of 18 per cent was dried 
so as to contain only 8 per cent water. This soil was steriliz'ed 

TABLE X 

Number of bacteria in soils of low moisture content 



por 


INOCULUM 


NUMBER OF BACTERIA PER GRAM 




30 days 


45 days 


60 days 


1 
2 


Without protozoa 
Without protozoa 


590,000,000 
770,000,000 


660,000,000 
654,000,000 


484,000,000 
390,000,000 


Average 


Without protozoa 


680,000,000 


657,000,000 


437,000,000 


3 
4 


With protozoa 
With protozoa 


270,000,000 
470,000,000 


170,000,000 
150,000,000 


70,000,000 
120,000,000 


Average 


With protozoa 


370,000,000 


160,000,000 


95,000 000 



STUDIES ON SOIL PROTOZOA 55 

and then reinoculated, one set without protozoa and the other 
set with protozoa. The moisture content was held at 8 per cent 
and samples were taken for bacterial analysis at the end of 30, 
45 and 60 days. The results are given in Table X. These show 
that the relation between the number of bacteria in the presence 
and absence of protozoa is the same in soils of low water content 
as when more moisture is present. This fact argues strongly 
against the protozoan theory. 

The development of bacteria in soils containing protozoa and free of 

protozoa 

As is well known, protozoa do not multiply as rapidly as 
bacteria. It is also the contention of Russell and Hutchinson 
that the biological factor, which they beUeve to exist in soils, 
requires much more time to develop in soil than is necessary for 
the bacterial flora. If such a factor exists in the soil there should 
be a difference in the development of bacteria in soils containing 
protozoa and soils free of protozoa. We should expect the bacteria 
to multiply very rapidly in each soil and to attain about the same 
maximum numbers. At a later period, in soil containing protozoa, 
the number of bacteria should diminish very appreciably, due to 
the development of the ''detrimental factor," while the number 
of bacteria in the protozoa-free soil should remain much nearer 
its maximum. It has been observed on a number of occasions 
that the difference in the number of bacteria which develop in 
the soils inoculated with normal soil and Avith protozoa-free 
soil is apparent fifteen days after inoculation, and also that the 
difference in the two flora apparently remains the same from 
the fifteenth day through the third month. (Tables V, VI, VII 
and VIII.) The harmful factor, if such exists, must develop 
within fifteen days after the soil is inoculated. 

Two pots of a rich garden soil, two of a field soil (loam) and 
two of a poor sandy soil were steriHzed. One pot of each was 
then inoculated with normal soil and the other pot from each 
set inoculated with protozoa-free soil. Bacterial counts were 
made every day for sixteen days. The data obtained are tabu- 
lated in Table XI. 



56 



JAMES M. SHERMAN 



A study of the data fails to give any evidence that the proto- 
zoa act as a limiting factor upon the soil bacteria. In general 
it will be seen that curves representing the numbers of bacteria 
throughout the period would run nearly parallel in the different 
soils. The difference in the numbers of bacteria in soils with 
and without protozoa was apparent from the start, which fact 
indicates that the phenomena is due to a difference in the bac- 

TABLE XI 
The development of bacteria in soils containing protozoa and in soils free of protozoa 





NUMBER OF BACTERIA IN MILLIONS PEB GRAM 


<« 


Garden soil 


Field soil 


Sandy soil 


Q 


A 


B 


A 


B 


A 


B 


1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 


150 

200 

400 

430 

1,400 

1,300 

1,420 

1,650 

950 

830 

620 

770 

1,050 

1,030 

845 


40 
30 
50 

275 
420 
620 
760 
925 
770 
430 
440 
360 
490 
307 
230 
257 


350 
315 
400 
800 
600 
600 
780 
900 
910 
450 
530 
640 
730 
617 
385 
367 


250 
115 
250 
200 
400 
410 
500 
450 
425 
220 
330 
275 
215 
214 
175 
204 


12 

12.8 

40 

38 

55 

65 

50 

48 

36 

28 

40 

32 

23 

24.2 

31.2 

19.4 


15 

8 
12 
12 
25 
20 
27 
21 
28 
12 
14 
12 

9.8 
11.9 
11.2 

7.8 



A = soil without protozoa. 
B = soil with protozoa. 

terial flora and not to the protozoa. There was no evidence 
that a detrimental factor developed during the latter part of 
the period. 

Discussion 

An examination of the data presented in the foregoing experi- 
ments reveals the fact that in all of the tests the soils which 
contained protozoa gave lower bacterial counts as determined 



STUDIES ON SOIL PROTOZOA 57 

by the plate culture method. This observation supports the 
theory of Russell and Hutchinson. It must be remembered, 
however, that the bacterial flora contained in the soils with and 
without protozoa were quite different; the flora of the protozoa- 
free soil was not so complex as that of ordinary soil. It was 
shown that, under the conditions of these experiments, the apparent 
number of bacteria was afl'ected appreciably by the number of 
kinds of bacteria present. That the difference in the two flora 
was an important factor in the disparity between the apparent 
number of bacteria in the soils with and without protozoa can 
hardly be doubted; the assumption that it was the only factor, 
however, would not be justified. 

If the bacterial flora in these tests was limited by the protozoa, 
these organisms must be able to grow actively at low tempera- 
tures (0°C.), at high temperatures (37°C.), and in soils of very 
low moisture content (Tables VIII, IX, and X). It would also 
have to be acknowledged that the harmful protozoa are capable 
of multiplying as rapidly in soil as the bacteria (Table XI). 
Such characteristics would be different from those of any protozoa 
now known. Admitting these possibilities, it must be concluded 
that the weight of the evidence is opposed to the view that 
the soil protozoa are harmful to the bacteria. 

An objection that may be raised to this work is that sterihzed 
soil was used as a substratum. It may be claimed that the 
particular kinds of protozoa which are believed, by some workers, 
to act unfavorably in the soil are not capable of development in 
sterihzed soil. Such a possibility must be recognized, though 
there is no reason for this belief, while the evidence we have on 
this subject (see Part III) is opposed to such a view. 

A very essential part of this work is the study of the bacterial 
processess in soils with and without protozoa. This phase of 
the work was undertaken by another worker in this laboratory 
the results of whose work will probably be ready for pubhcation 
in the near future. It may be stated now, however, that the 
data obtained are in perfect accord with the foregoing observa- 
tions. 



58 JAMES M. SHERMAN 

V. THE EFFECT OF PURE CULTURES OF PROTOZOA UPON THE SOIL 

BACTERIA 

Isolation of protozoa 

The term ''pure culture" as it is here used in connection with 
protozoa means only an ''animal-pure culture," since the cul- 
tures of protozoa were not obtained free from bacteria. The 
soil amoebae, as has been shown by Beijerinck (1896), (1897), 
Celli (1896), Tsujitani (1908), Frosch (1909), and others, may be 
cultivated on solid culture media and so can be isolated in the 
pure state more readily than the other types of protozoa. 
With the ciliates and flagellates the problem of obtaining cul- 
tures free of bacteria is more difficult. Berliner (1909) and 
Nagler (1909) have shown that certain of the flagellates and cili- 
ates will grow upon agar plates but this method has not given 
general satisfaction. Many methods have been tried, some of 
which have been partially successful, but none have proved very 
practical. 

It was found in this work that animal-pure cultures could be 
conveniently obtained by the dilution method. As was noted 
earlier in this paper in high dilutions of soil (1/10,000 gm.) the 
protozoa present are usually restricted to a few types. By 
inoculation of a large number of high dilutions of soil (1/10,000 — 
1/50,000 gm.) into soil extract a few cultures which contain only 
one type of protozoa will usually be obtained. In order to isolate 
types which do not occur in soil in very large numbers it is 
necessary to utilize an enrichment medium which favors the 
growth of the desired organism. When the desired type has 
obtained the ascendency, it may be readily isolated by the dilu- 
tion method. Hay extract, Ashby's solution, Giltay's solution, 
dilute peptone broth, and a weak beef extract solution have been 
used successfully for enrichment cultures. 

After the protozoa were obtained in animal-pure cultures it 
was attempted to simplify the bacterial flora contained in them. 
This was accomplished by passage through several successive 
subcultures in Ashby's solution. The types of bacteria capable 
of development in this medium are somewhat limited. 



STUDIES ON SOIL PROTOZOA 



59 



Methods 

The effect of the various protozoa isolated upon soil bacteria 
was tested in soil extract and then in soil. The soil tests were 
made by the addition of the protozoan cultures to sterihzed soils 
reinoculated with protozoa-free soil. Tumblers each containing 
200 grams of soil were used for these experiments, and the soils 
were incubated 30 days at laboratory temperature, which ranged 
from 20° to 25°C. The moisture contents of the soils were held 
as closely as possible to the optimum for the growth of plants 
and micro-organisms. (The optimum moisture content is gen- 
erally considered to be very close to the moisture equivalent of 
the soil.) Inoculations of the soil and soil extract cultures were 
made by the addition of one cubic centimeter of an active soil 
extract culture of the protozoan under investigation. 

The experiments reported here include the tests which have 
been made with six types of protozoa, the ciliates, Colpoda 
cucullus, and Balantiophorus elongatus, and the four flagellates 
which have been previously mentioned (Part II) as the most 
abundant types in soil: — Monas sp., Dimorpha radiata (?) and 
two others designated for convenience as flagellates A and B. 

Results with Colpoda cucullus 

In soil extract, as would be expected, the number of bacteria 
is greatly reduced in the presence of Colpoda cucullus. In soil, 
on the other hand, no such limiting action could be detected, 
which further substantiates the view that this organism is not 
active under ordinary soil conditions. 



TABLE XII 
Effect of Colpoda cucullus upon the number of bacteria in soil extract 





NTTMBER OF 


BACTERIA PER CUBIC 


CENTIMETER 




2 days 


5 days 


8 days 


Control 


100,000,000 
128,000,000 
102,000,000 
110,000,000 


140,000,000 

142,000,000 

9,400,000 

9,500,000 


144,000,000 


Control 

Colpoda 

Colpoda 


100,000,000 
5,000,000 
4,000,000 



60 



JAMES M. SHERMAN 



TABLE XIII 
Effect of Colpoda cucullus upon the number of bacteria in soil 

Test 1 



POT 


CONTROL 


AVERAGE 


C. CUCULLUS 


AVERAGK 


1 

2 


470,000,000 
510,000,000 


490,000,000 


405,000,000 
500,000,000 


452,500,000 



Test 2 



1,640,000,000 
2,160,000,000 



1,900,000,000 



2,640,000,000 
2,500,000,000 



2,570,000,000 



Test 3 



620,000,000 
670,000,000 
820,000,000 
780,000,000 



722,500,000 



770,000,000 
730,000,000 
810,000,000 
700,000,000 



752,500,000 



Results with Balantiophorus elongatus 

The results obtained with Balantiophorus elongatus are similar 
to those with Colpoda cucullus; in liquid cultures the bacteria 
are definitely suppressed while in soil no such action is evident. 
The slight difference noted in Table XV is easily within the limits 
of experimental error. Only one test of B. elongatus in soil is 
here reported; since this organism is not active in soil (Part III) 
it is obvious that it could not function as a bacterial limiting 
factor. 



TABLE XIV 
Effect of Balantiophorus elongatus upon the number of bacteria in soil extract 





NUMBER OP 


BACTERIA PER CUBIC 


CENTIMETER 




3 days 


5 days 


8 days 


Control . . . 


114,000,000 
136,000,000 


141,000,000 
60,000,000 


122,000,000 


B. elongatus 


9,000,000 



STUDIES ON SOIL PROTOZOA 



61 



TABLE XV 
Effect of Balaniiophorus elongatus upon the number of bacteria in soil 



POT 


CONTROL 


NUMBER OF BACTERI.V PER GRAM 




Average 


B. elongatus 


Average 


1 

2 
3 

4 


620,000,000 
670,000,000 
820,000,000 
780,000,000 


722,500,000 


700,000,000 
580,000,000 
520,000,000 
650,000,000 


012,500,000 



Results with Dimorpha radiata 

Dimorpha radiata is an active inhabitant of soil and should 
therefore have some function therein. From the data obtained 
that function does not appear to be the destruction of bacteria. 
Neither in soil extract nor in soil did there appear to be any 
limiting action upon the bacteria. 

TABLE XVI 
Effect of Dimorpha radiata upon the number of bacteria in soil extract 





NUMBER OF 


BACTERIA PER CUBIC 


CENTIMETER 




2 days 


5 days 


8 days 


Control 

Control 

D. radiata 

D. radiata, 


100,000,000 

128,000,000 
146,000,000 
190,000,000 


140,000,000 

142,000,000 

120,000,000 

90,000,000 


144,000,000 
100,000,000 
100,000,000 
116,000,000 





TABLE XVII 
Effect of Dimorpha radiata upon the number of bacteria in soil 

Test 1 



POT 


NUMBER OF BACTERIA PER GRAM 




Control 


Average 


D. radiata 


Average 


1 

2 


1,360,000,000 
1,220,000,000 


1,290,000,000 


2,050,000,000 
1,610,000,000 


1,830,000,000 


Text 2 


1 

2 


470,000,000 
510,000,000 


490,000,000 


310,000,000 
585,000,000 


447,500,000 



62 



JAMES M. SHERMAN 



Results with Monas sp. 

In the case of Monas sp. very interesting results were ob- 
tained. This organism, as has been shown, is active in soil. 
The findings reveal a very strikingly harmful effect upon bac- 
teria in soil extract while in soil this action did not appear to take 
place. The reason for this is not clear, but it has been noted 
within recent years that the behavior of micro-organisms appar- 
ently differs, in many cases, in soil and in solution. 



TABLE XVIII 
Effect of Monas (sp.?) upon the number of bacteria in soil extract 



NUMBER OF BACTERIA PER CUBIC CKNTIMBTEB 



2 days 



5 days 



8 days 



Control 

Control 

Monas (sp.?) 
Monas (sp.?) 



100,000,000 

128,000,000 

200,000,000 

46,000,000 



140,000,000 

142,000,000 

6,500,000 

6,800,000 



144,000,000 

100,000,000 

5,500,000 

6,000,000 



TABLE XIX 
Effect of Monas (sp.?) tipon the number of bacteria in soil 
Test 1 





NUMBER OP BACTERIA PER GRAM 




Control 


Average 


Monas 


Average 


1 

2 
3 


155,000,000 
105,000,000 
150,000,000 


133,300,000 


105,000,000 
290,000,000 
100,000,000 


165,000,000 



Test 2 



440,000,000 
360,000,000 
460,000,000 



420,000,000 



380,000,000 
240,000,000 
260,000,000 



293,300,000 



Tests 



240,000,000 
170,000,000 
160,000,000 
245,000,000 



204,000,000 



240,000,000 
165,000,000 
220,000,000 
260,000,000 



221,000,000 



STUDIES ON SOIL PROTOZOA 



TABLE XX 



63 



Effect of seven different strains of Monas (sp.?) upon the numbers of bacteria in 

soil 



POT 


NUMBBB OP BACTBEIA PER GRAM 




Control 


Average 


Monas 


Average 


1 

2 
3 
4 
5 

6 

7 


840,000,000 
930,000,000 
660,000,000 
800,000,000 
700,000,000 


786,000,000 


780,000,000 
660,000,000 
760,000,000 
930,000,000 
940,000,000 
710,000,000 
740,000,000 


788,600,000 



To verify the results of Table XIX, another test was made 
in which seven strains of the organism, isolated from as many 
different soils, were employed. A comparison was then made 
with five control pots which were free of protozoa. The results 
from this test (Table XX) agree entirely with the data given in 
the preceding tables. 



Results with Flagellate A 

This organism, Hke Dimorpha radiata, does not appear to be 
antagonistic to the soil bacteria as is shown by the tests both in 
soil and in soil extract. 

TABLE XXI 
Effect of Flagellate A upon the number of bacteria in soil extract 





NDMBBR OP BACTERIA PER CDBIC 
CENTIMETER 




3 days 


5 days 




57,000,000 
77,000,000 
95,000,000 
58,000,000 


31,000,000 




35,000,000 


Flagellate A 


31,000,000 


Flanpllatp A 


50,000,000 







64 



JAMES M. SHERMAN 



TABLE XXII 

Effect of Flagellate A upon the number of bacteria in soil 

Test 1 



POT 


NUMBER OF BACTERIA PER GRAM 




Control 


Average 


Flagellate A 


Average 


1 

2 


1,530,000,000 
1,510,000,000 


1,520,000,000 


2,660,000,000 
2,230,000,000 


2,445,000,000 



Test 2 



825,000,000 
900,000,000 



862,500,000 



725,000,000 
645,000,000 



685,000,000 



Test 3 



136,000,000 
122,000,000 



129,000,000 



136,000,000 
146,000,000 



141,000,000 



Results with Flagellate B 

The organism used in this test, designated as Flagellate B, is 
an active inhabitant of the soil, in which it occurs in numbers 
nearly as great as the three preceding flagellates. This organism 
does not appear to affect the number of bacteria, as determined 
by the plate culture method, either in soil nor in soil extract 
cultures. 

TABLE XXIII 

Effect of Flagellate B upon the number of bacteria in soil extract 
Period of incubation five days 



CUL- 


NUMBER OF BACTERIA PER CUBIC CENTIMETER 




Control 


Average 


Flagellate B 


Average 


1 

2 


23,000,000 
43,000,000 


33,000,000 


39,000,000 
37,000,000 


38,000,000 



STUDIES ON SOIL PROTOZOA 



65 



TABLE XXIV 
Effect of Flagellate B upon the number of bacteria in soil 



POT 


NUMBER OP BACTERIA PER GRAM 




Control 


Average 


Flagellate B 


Average 


1 

2 
3 
4 


240,000,000 
170,000,000 
160,000,000 
245,000,000 


203,800,000 


195,000,000 
165,000,000 
210,000,000 
170,000,000 


185,000,000 



Mixture of Protozoa 

It was thought that, although none of the individual protozoa 
which were tried in pure culture were able to decrease the number 
of bacteria in soil to a measurable extent, this might be accom- 
plished through the combined action of all of them. Two experi- 
ments were performed in which a comparison was made of pro- 
tozoa-free soil and of soil containing the six types of protozoa 
used in the preceding tests. For these tests the soils were 
allowed to incubate for two months as it might be contended 
that one month was not sufficient time to allow the "detrimental 
factor" to develop. These trials, in keeping with all of the tests 
which have been made with the individual organisms, gave 
wholly negative results. 

TABLE XXV 
Effect of mixture of protozoa upon the number of bacteria in soil 

Test 1 



POT 


CONTROL 


AVERAGE 


PROTOZOA 


AVERAGE 


1 

2 
3 
4 
5 


250,000,000 
390,000,000 
270,000,000 
290,000,000 
274,000,000 


294,800,000 


444,000,000 
372,000,000 
384,000,000 
336,000,000 
250,000,000 


357,200,000 


Test 2 


1 
2 
3 


180,000,000 
240,000,000 
160,000,000 


193,300,000 


180,000,000 
176,000,000 
172,000,000 


176,000,000 



66 JAMES M. SHERMAN 

The conclusion of this paper with bibhography will appear in 
the next number of the Journal of Bacteriology. 

Acknowledgment is made to Professors E. G. Hastings, A. 
S. Pearse and E. B. Fred of the University of Wisconsin from 
whom criticisms and suggestions have been obtained during the 
progress of this work. 



A CULTURE MEDIUM FOR MAINTAINING STOCK 
CULTURES OF THE MENINGOCOCCUS^ 

C. G. A. ROOS 
From the Mulford Biological Laboratories, Glenolden, Pa. 

The maintenance of certain pathogenic bacteria upon artificial 
culture media is sometimes attended with great difficulty. 
Among these organisms the meningococcus may be said to occupy 
the first place. Its peculiar biology — particularly its intra-cellu- 
lar ferment which is so potent a factor in its destruction — makes 
its viability at all times precarious. Furthermore its highly 
parasitic nature requires highly complex substances such as those 
upon which it grows in the human body. 

While strains of the meningococcus that have been accustomed 
to artificial cultivation may be maintained upon plain nutrient 
agar, this medium is not favorable to its continued cultivation; 
the addition of glycerine offers no advantage; glucose results 
in more rapid growth and consequently more rapid degeneration. 
In their early work Councilman, Mallory and Wright used Loef- 
fler's blood serum for both isolation and maintenance. Flexner 
used plain agar to which sheep serum was added. Some authors 
have used the serum of other animals — horse, goat, calf. Human 
serum and human ascitic fluid are conceded to be superior for 
isolation and for obtaining massive growth. The addition of 
the whole blood is possibly better than serum alone. Kutscher 
recommends a medium prepared with human placenta to which 
is added calf serum and glucose. For isolation Conradi used 
the centrifugaHzed spinal fluid, adding one part of the super- 
natant liquid to three parts of shghtly alkaline nutrient agar; 
upon this solidified medium he planted the sediment. 

1 Presented at seventeenth annual meeting of the Society of American Bac- 
teriologists, Urbana, 111., December 28, 1915. 

67 



68 C. G. A. ROOS 

Fluid media offer no advantage over solid media and they 
are of course not adaptable for isolation. Gelatin is not suit- 
able because the meningococcus does not grow at low tempera- 
tures. 

While many of these media, offer satisfactory conditions for 
growth during a few generations, a fair proportion of strains 
kept upon them suddenly fail to develop and in spite of persistent 
effort cannot be resuscitated. Furthermore on all the above 
mentioned media the cultures under ordinaiy conditions must 
be transplanted at short intervals — not longer than two or three 
days — ^and kept constantly in the incubator at 37.5°C. (Ex- 
ceptional strains are found which are unusually hardy and seem 
to require little more care than the common saprophytic bacteria.) 

With all the above culture media, those favoring the rapid 
growth of the meningococcus at the same time result in rapid 
ferment production and consequently rapid death of the culture. 

After innumerable trials we have found a medium which 
permits of relatively slow growth of the meningococcus with 
apparent suppression of ferment formation, thus resulting in 
greater viability. This medium has been in use for about two 
years and its superiority over the other media mentioned in the 
literature for the maintenance of stock cultures of the menin- 
gococcus seems to warrant its publication. This culture medium 
is a modification of the potato-blood-agar used by Bordet and 
Gengou for the isolation of B. pertussis. 

Preparation of medium 

1. Prepare potato extract as follows: 

a. Potato peeled, cut in small pieces and washed in running water 
for about two hours, 100 grams. 

h. Water containing 4 per cent double distilled glycerine free from 
acid, 200 cc. 

c. Mix and autoclave for forty minutes. 

d. Allow to stand over night and strain through cheese cloth. 

2, Make potato-extract-agar as follows : 

a. Mix in an Erlenmeyer flask: Potato extract, 50 cc; NaCl solution 
0.65 per cent, 150 cc; agar 5 grams. 



STOCK CULTURES OF THE MENINGOCOCCUS 69 

6. Heat in Arnold sterilizer until agar is melted, requiring from thirty 
minutes to one hour. 

3. Tube without filtering, and sterilize, in autoclave for about 
forty minutes. 

4. When wanted for use, melt the agar, cool to about 45°C. and 
add the desired amount of sterile defibrinated horse blood. 

The amount of blood to be added depends upon whether or 
not the meningococcus has become accustomed to the medium. 

In transplanting from another medium to this potato blood 
agar, a little difficulty may be experienced in getting the cultures 
started upon the new substratum. For this reason a large 
amount of the growth (not over twenty-four hours old) should 
be transferred to the medium containing about 20 per cent of 
defibrinated blood. In making the inoculation the culture 
should be rubbed slightly into the surface. This is incubated 
at 37.5°C. for about 2 days and then transplanted again to 
the potato-extract-blood-agar containing just sufficient blood 
to permit growth — that is, about 5 per cent. Subsequent 
transplants need not be made more often than every thirteen 
to fifteen days or longer, when kept at 37°C., provided that the 
cultures do not become too dry. In the case of cultures paraf- 
fined or sealed to prevent drying, a fair growth may be obtained 
after six weeks. 

The meningococcus grows well at 37.5°C. At lower tem- 
peratures it will remain alive for a considerable length of time, 
although no growth occurs; viability may be retained at room 
temperature apparently as long as, if not longer than, at incu- 
bation temperature. Besides, maintenance at this temperature 
renders paraffining or sealing less imperative for the prevention 
of drying. Freshly transplanted cultures that were incubated for 
twenty-four hours at 37.5°C., and then kept at room temperature, 
showed fair growth after 4 weeks. Ice box temperature will 
kill most strains of the meningococcus in a comparatively short 
time. Cultures grown at 37.5°C. for twenty-four hours, and then 
transferred to the ice box, grew well after five days, but after 
ten days about twenty-five per cent failed to show any growth 
at all, 50 per cent showed scanty growth, and only 25 per cent 
a fair growth. 



70 C. G. A. ROOS 

The meningococcus is an aerobic organism but like many 
other aerobes when first grown aerobically and then transferred 
to an atmosphere of hydrogen, it can be kept ahve longer than 
when oxygen is present. Cultures of meningococci were grown 
aerobically for twenty-four hours at 37.5°C. and then trans- 
ferred to a Novy jar, the air of which was replaced with hydrogen 
by means of a Kipp apparatus and a Schutte vacuum pump; 
the jar contained pjrrogaUic acid and sodium hydroxide which 
were permitted to mix after the air had been replaced several 
times by hydrogen. These cultures were then kept in the incu- 
bator; after ten weeks good growths were obtained on the first 
transplant. 

The appearance of the meningococcus growth on potato 
blood agar is not very characteristic. After twenty-four hours 
growth at 37.5°C. the individual colony has reached the size 
of a small pinhead. It is gray in color, smooth and rather moist 
looking, of an amorphous consistency, the surface elevation varj'^- 
ing from convex to pulvinate with border entire. With age, the 
color of the colony changes to dull gray, the consistency becomes 
tenacious, and the surface elevation more of the raised type. 

Although the production of pigment by some organisms is 
facilitated on potato blood agar, the area of discoloration char- 
acteristic of numerous strains of streptococci, — notably the 
Streptococcus viridans and pneumococcus on blood agar, and 
some Gram negative cocci on glucose agar as described by 
Elser and Huntoon — has never been observed by us with any 
strain. 

All of our thirty-eight strains have invariably remained Gram 
negative, regardless of culture medium used or age of culture. 
Occasionally, more frequently in old cultures, a few organisms 
may be seen that do resist for a .time the action of the decoloriz- 
ing agent and thus appear to be Gram positive. However, this 
is usually an indication of faulty technique or of contamination. 

Arrangement in pairs is most conmion, although single cocci 
and groupings in tetrads are numerous, with certain strains 
especially. True chain formation has never been observed. 
'\'ariations in the size of organisms in the different strains are 



STOCK CULTURES OF THE MENINGOCOCCUS 71 

negligible, those of individual cocci seeming to be determined 
more by the age of the culture, than by the culture medium. De- 
generation forms occur with all strains in quite young cultures. 
The potato-blood-agar furthermore is of value for differentia- 
ting between the meningococcus and the gonococcus; on this 
medium the gonococcus grows only when the medium has a high 
blood and diminished salt content — the growth is always very 
scanty and the characteristic differences are immediately apparent. 



BILE COMPARED WITH LACTOSE BOUILLON FOR 

DETERMINING THE PRESENCE OF B. COLI 

IN WATER 

MAUD MASON OBSTi 

Irregular results have often been obtained in the past in the 
routine bacteriological examinations of water. The presumptive 
test for B. coli made with lactose peptone ox-bile upon a sample 
of water would give no indication of gas in 10 cc. quantities on 
one day, while another sample from the same source would show 
gas in 1 cc. or in 0.1 cc. quantities on the following day. It was 
observed that when bile which had been stored for six weeks 
at 1°C. was used for four samples of water from a polluted spring, 
and bile which had been freshly collected was used for six 
samples from the same spring, the former produced no gas, 
while the latter produced gas in 0.1 cc. quantities. Similar 
results were obtained with the use of bile which had apparently 
been carelessly collected. 

In this laboratory it had been found necessary to obtain bile 
from an abattoir in a nearby city in 5-gallon lots, and to depend 
upon the workers in the abbatoir for its collection. When 
received at the laboratory, the bile was sterilized and stored at 
1°C. until used, which was frequently for two months or longer. 
The above results showed that it must be collected more fre- 
quently and by one who would use proper precautions. This 
required one-half day's time every week of a reliable helper, and 
an expenditure of fifty (50) cents for three gallons. Even then, 
the helper met with serious difficulties in gaining access to that 
part of the abattoir in which he could secure the unbroken gall 
bladder. 

1 This work was carried on under the auspices of the Microbiological La))ora- 
tory, Bureau of Chemistry, Washington, D. C. The author desires to acknowl- 
edge the valuable assistance rendered by Dr. Charles Thorn in the preparation of 
this paper. 

73 



74 MAUD MASON OBST 

These experiences led to communications with other bacteri- 
ologists,^ all of whom expressed dissatisfaction with bile media. 
Prof. Edwin 0. Jordan, University of Chicago, has stated, ''Bile 
from different animals varies in composition, and this is probably- 
one reason for the variable results obtained when this substance 
is added to culture media. Dried bile and bile salts have been 
used by various observers. In my own experience bile salts, 
like fresh bile, inhibited B. coli to some extent." Previous to 
the time when he made this statement, Jordan determined the 
''degree of inhibition" of B. coli by bile and reported that from 
one-third to one-half of the vital cells of B. coli were thus in- 
hibited (Jordan, 1913). He stated also that there was no 
evidence that these cells were more attenuated or in any way 
less vigorous biologically than the others. 

A resume of the literature was then made, with regard to the 
origin of the use of bile as a medium. It was found that Jack- 
son (1906) had experienced the difficulty of having B. coli 
inhibited by other organisms when he used glucose or lactose 
bouillon as recommended by Theobald Smith (1893, 1895). 
Jackson, therefore, experimented with MacConkey's bile salt 
agar, "Platner's Crystallized Bile," which consisted of a mix- 
ture of the two bile salts, and finally with his own medium which 
he made by adding 1 per cent lactose to fresh ox-bile, and which 
gave satisfactory results in his work. 

Sawin (1907) corroborated Jackson in his recoromendation of 
the use of lactose bile and regarded it as a satisfactory and deli- 
cate indicator of minor sewage pollutions of springs and wells. 

When the necessity of finding a substitute for bile was rec- 
ognized, dried bile was considered. This substance, being 
obtained from liquid bile, varies in composition approximately 
in the same manner as the original material. Biochemical 
laboratories overcome these variations to some extent by drying 
bile from large quantities of mixed liquid biles, but this pre- 
caution does not produce an entirely satisfactory product. 

» Dr. F. L. Rector, Great Bear Spring Co., New York, N. Y. ; Dr. W. W. Browne, 
College of the City of New York; Prof. S. C. Prescott, Massachusetts Institute of 
Technology; Prof. Earle B. Phelps, Hygienic Laboratory, Treasury Departmeot. 



BILE COMPARED WITH LACTOSE BOUILLON 75 

The expense of obtaining bile salts discouraged their use, and 
it therefore seemed necessary to find a more uniform substance 
which could be easily obtained. In regard to glucose bouillon 
Weston and Tarbett (1907) have reported experiments upon 
the comparison of lactose bile and glucose bouillon in the 
examination of sixty-three samples of water, showing that 
although glucose permitted the production of gas from a larger 
number of samples than bile, yet B. coli could be confirmed from 
the glucose fermentation tubes in only 13 per cent of these 
samples. This result was confirmed by a small number of 
experiments conducted in this laboratory in 1911-1912. 

Members of the Hygienic Laboratory of the U. S. Pubhc 
Health Service have suggested the use of lactose bouillon and 
referred to satisfactory results which they have obtained from 
its use during the past few j^ears. Their suggestion was strength- 
ened by the report given by Theobald Smith (1895) that 
Chantemesse and Widal looked upon gas-production in lactose 
bouillon as conclusive evidence of the presence of B. coli, and 
by the work of Hall and Nicholls (1914) which showed that if 
the percentage of lactose added to bile be increased to 15 per 
cent the productioxi of gas would be more rapid. Organisms of 
the B. coli group are the only aerobes^ commonly found in water 
which will ferment lactose with the production of gas. The few 
anaerobes which might be found to produce gas may be elimin- 
ated by subsequent transfers to Endo's medium. It was, there- 
fore, thought practical to substitute this medium for bile. 

When a medium is used in large quantities, as bile usually 
is, comparative costs become important. Lactose and pep- 
tone are used in equal quantities in both media. The differ- 
ence in cost of the two media depends upon the cost of the raw 
bile in one and the meat extract used in the other. Meat ex- 
tract at contract price costs approximately three cents for the 
quantity required for making one hter of nutrient broth. The 

' The Committee on the Standard Methods of Water Analysis, in their report 
read before the meeting of the American Public Health Association, held at 
Rochester, N. Y., in September, 1915, defined the B. coli group as including all 
aerobic bacteria which produce gas in lactose broth. 



76 MAUD MASON OBST 

ox-bile requires one-half day each week of the time of the helper 
and fifty cents for three gallons for its collection, thereby cost- 
ing approximately ten cents per liter of medium. Lactose 
broth can be made as desired from ingredients which do not 
materially change during storage. 

In the experiments here reported check analyses were made 
upon a series of samples of water, using lactose peptone ox-bile 
and lactose broth. The bile was never used later than a week 
after collection. It was sterilized upon receipt and stored during 
this period at a temperature of 1°C. It was enriched with 1 per 
cent lactose and 1 per cent peptone, and tubed m Dunham 
tubes. The lactose bouillon was made from neutral nutrient 
broth prepared with 0.5 per cent Liebig's meat extract, 1 
per cent peptone and 1 per cent lactose. This medium was 
compared with lactose peptone ox-bile in the examination of 191 
samples of water with the following results: 

No gas-producing organisms in 10 cc. quantities in either lactose 
or bile in 68 samples. 

Gas-producing organisms in the same dilutions in lactose and in 
bile in 59 samples. 

Gas-producing organisms in higher dilutions in bile than in lactose 
in 3 samples. 

Gas-producing organisms in higher dilutions in lactose than in bile 
in 61 samples. 

Lactose showed gas-producing organisms in one-half as much water 
as were shown by bile in 12 samples. 

Lactose showed gas-producing organisms in one-fifth as much water 
as were shown by bile in 12 samples. 

Lactose showed gas-producing organisms in one-tenth as much water 
as were shown by bile in 19 samples. 

Lactose showed gas-producing organisms in one-hundredth as much 
water as were shown by bile in 5 samples. 

From every sample B. coli was isolated from the highest 
dilution tube showing gas. This shows the ratio of inhibition 
of bile on B. coli when compared with lactose bouillon to be 
about 2:1. 



BILE COMPARED WITH LACTOSE BOUILLON 77 

In some instances only a small bubble of gas appeared in the 
inner tube in the bile fermentation tubes, while the production 
of gas in lactose filled two-thirds of the inner tube. In one 
instance a culture of B. paratyphosus B., which showed char- 
acteristic agglutination in 1 : 500, was obtained from a lactose 
fermentation tube. 

In order to obtain a direct comparison by total counts of the 
growth of the B. coli group on lactose and on bile collected at 
different times and stored for varying intervals, firm substrata 
were prepared by adding 1.5 per cent agar to each medium. It 
was found necessary to exert great care in filtering the bile agar 
in order that the final product should be free from a precipitate 
which would render the counting of the colonies diflficult. The 
bile was collected each week, sterilized, and either made into 
agar at once or stored at 1°C. until used. For the tests, fifteen 
strains of B. coli, with characteristics described in the accom- 
panying table, were grown for three days at 37°C., with daily 
transfers in 10 cc. nutrient broth. 

In recording the results, it was found that the inhibition by 
the bile could be shown more clearly by taking the number of 
colonies which developed upon the bile agar as one, and con- 
sidering the multiple of this number, which expressed the count 
on lactose agar from the same dilutious of the original culture 
plated and incubated at the same time, as the ''ratio of inhibition" 
of the bile. 

The bile which was collected during the month of June (see 
table) was tested with the cultures two days after receipt and 
again some weeks later. With a few exceptions the inhibition 
exerted by the fresh bile was less, in amount, and more regular 
for the different strains than that of the same bile after storage. 
When the actions of the various organisms upon the individual 
samples of bile are considered, it is seen that the variations in 
the degree of inhibition are greater for the bile that was held in 
storage before being tested; and, in general, this variation in- 
creased with the time of storage. One sample of bile (see table, 
(e) ) was of a pronounced red color and contained a heavy red- 
brown precipitate. This sample showed an inhibitive action, 



78 MAUD MASON OBST 

which was markedly irregular and which permitted no compari- 
son with any other sample. 

The contents of three individual gall bladders, collected on 
different dates, were also tested. The ratios of inhibition (see 
table, (f)) for any one organism on the three agars are nearly 
uniform and on the individual bile agar the different strains 
showed only a slight variation. 

A comparison of the average inhibitive action of each sample 
of bile upon the different strains of organisms shows a vari- 
ation from 2.4 to 3.8, and for the freshly collected bile a ratio of 
approximately 2.4 for all strains. 

CONCLUSIONS 

In these experiments lactose bouillon used as a substitute for 
lactose peptone ox-bile permitted the development of about twice 
as many B. coli from water as the bile medium. 

Lactose bouillon costs less in money and labor. The difficulty 
of obtaining pure, fresh bile puts it almost out of the reach of 
many workers. The stored bile is proved to show progressive 
deterioration. Lactose bouillon can be prepared when desired 
and can be made more uniform. It need contain no precipitate 
to clog the inner tube or to affect the activity of the organism. 

REFERENCES 

Hall, I. W. and Nicholls, F. (1914) Earlier indications of gas formation 

by coliform organisms; with description of a modified fermentation 

tube. Centr. f. Bakt., Abth. I, Orig. 75, 140. 
Jackson, D. D. (1906) A presumptive test for B. coli. Biological Studies 

by the Pupils of W. T. Sedgwick. U. of C. Press., 292. 
Jordan, E. O. (1913) The inhibitive action of Bile upon B. coli. Jour. Infect. 

Dis., 12, 326. 
Sawin, L. R. (1907) Experience with lactose bile medium for the detection 

of B. coli in water. Jour. Infect. Dis., Supp. No. 3, 33. 
Smith, T. (1893) The fermentation tube, with special reference to Anaerobiosis 

and gas production among bacteria. The Wilder Quarter-Century 

Book. Ithaca, N. Y. 187. 
Smith, T. (1895) B. coli communis. Amer. Jour. Med. Sci., N. S. 110, 283. 
Weston, R. S. andTarbett, R. E. (1907.) Comparative results obtained by 

the use of lactose bile and dextrose broth media for the detection of 

B. coli in water. Jour. Infect. Dis., Supp. No. 3, 39. 



BILE COMPARED WITH LACTOSE BOUILLON 



79 





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SOCIETY OF AMERICAN BACTERIOLOGISTS 

Abstracts of Papers 

Presented at Seventeenth Annual Meeting, Urhana, Illinois, 
December S8 to SO, 1915 

SYSTEMATIC BACTERIOLOGY 
Under Supervision op S. H. Ayers 

Studies on the Classification of the Colon-Typhoid Group. C.-E. A. 

WiNSLOW AND I. J. KlIGLER. 

The committee on the classification of the colon-typhoid group, 
appointed at Philadelphia, has adopted a standard series of tests for 
titrable acidity, hydrogen ion concentration, milk reactions, indol 
production, gelatin liqefaction and chromogenesis. This preliminary 
report is based on the application of certain of these tests to 150 strains 
from the American Museum of Natural History collection; each strain 
being tested independently in New Haven and in New York. 

Our results so far indicate that there are at least three major groups 
in the colon-typhoid scries, the B. coli group clotting milk and producing 
a final high hydrogen ion concentration in glucose broth and forming 
indol; the B. aerogenes group, clotting milk but producing a final low 
hydrogen ion concentration in glucose broth and failing to form indol; 
and the B. typhi group giving a final alkalin reaction in milk but yield- 
ing a high hydrogen ion concentration in glucose broth and failing 
to form indol. The B. coli group includes at least three types, B. com- 
munis (indol positive, sucrose negative) — B. communior (indol posi- 
tive, sucrose positive) and B. acidi-lactici (indol negative, sucrose 
variable). The B. aerogenes group is generally indol negative and 
sucrose positive and includes the gelatin liquefying B. cloacae as well 
as the typical B. aerogenes. The B. typhi group is indol negative 
and includes at least three types — B. dysenteriae (reaction in milk 
varying back and forth about the neutral point), B. typhi (initial 
acidity followed by alkalinity), and B. paratyphi (alkaline throughout). 

The indol reaction as determined by the use of a tryptophane medium 
with 71 strains, gave results identical with those obtained by the 
use of peptone on the same strains two years ago. A positive Voges and 
Proskauer reaction is correlated with negative indol and a negative 
methyl red test for hydrogen ion concentration and clearly marks off 
B. aerogenes as a distinct group (the high gas ratio cultures of 
Rogers and Clark). 

81 



82 ABSTRACTS 

The Characteristics of Bacteria of the Colon Type Occurring in Human 

Feces. L. A, Rogers, Wm. Mansfield Clark, and H. A. Lubs. 

A total of 113 cultures were obtained from 17 individuals and classi- 
fied on the basis of carbon-dioxid hydrogen ratio, indol formation, 
gelatin liquefaction and the fermentation of certain carbohydrates 
and alcohols. All but 6 of the 113 cultures fermented glucose in the 
absence of oxygen with the production of almost exactly equal volumes 
of carbon dioxid and hydrogen. 

This ratio agrees with that given by 99 per cent of the cultures 
obtained from bovine feces and differs radically from that given by 
nearly all of the grain cultures. Further agreement with the bovine 
feces cultures is seen in the high percentage of indol formers, the absence 
of gelatin liquefiers, a low percentage of carbohydrate fermenters but 
a relatively high ability in fermenting the alcohols. 

The remaining six cultures produce nearly twice as much carbon dioxid 
as hydrogen and otherwise agree in a general way with the high ratio 
group which predominated in the grain cultures. 

While this type occurred in relatively small numbers the actual 
number may amount to several hundred thousand in each gram of 
material. It is possible that the more frequent occurrence outside 
of the animal body of the high ratio type may be because it is more 
resistant to unfavorable conditions and consequently survives after 
the low ratio type has disappeared. 

The Ttjpe of Colon Bacillus Occurring in Surface Waters. L. A. Rogers. 

A collection of 137 cultures of the colon type isolated from waters of 
greatly varying degrees of contamination were separated into two dis- 
tinct groups. One of these included about one-third of the cultures 
and was evidently identical with the type which has been found to 
include 95 to 99 per cent of the colon bacilli of bovine and human 
feces. This type was found occasionally in springs in which there was 
no evident source of contamination but was especially abundant in 
rivers and streams usually considered to be polluted with sewage. 

The second group which occurred in practically all waters examined 
was identified with a type which, while it responds to all of the usual 
tests for B. coli, occurs in feces in relatively small numbers. Cultures 
isolated from grains belonged almost exclusively to this type. The 
significance of this type in water cannot be determined but the charac- 
teristic fecal colon type can be demonstrated in polluted water with 
reasonable certainty. 

Some Problems in Bacterial Nomenclature. R. E. Buchanan. 

The following conclusions are reached: 

1. A standing committee of the society, either the committee on 
classification or a new committee should be asked to consider problems 
of bacterial nomenclature and make suitable recommendations to the 
societv. 



ABSTRACTS 83 

2. Such a committee could study the international codes of nomen- 
clature adopted by the botanists and zoologists and report such modifi- 
cations as might be necessary to adapt them to the needs of 
bacteriologists. 

3. They could recommend a suitable date of departure for bacterial 
nomenclature. 

4. They could report upon the historical validity of the names that 
are used for bacterial groups, particularly genera. 

5. They could prepare a list of recognized generic names. 

6. They could seek the active cooperation of societies having the 
same interests, in this and other countries. 

7. They could prepare resolutions to be presented at the next inter- 
national botanical congress expressing the wishes of the society. 

8. They could prepare a list of suitable names for the designation 
of chemical changes brought about by bacteria. 

The Hemolytic Streptococci Found in Milk. Their Significance and their 

Relation to Streptococci of Human Origin. David John Davis. 

The occurrence of epidemics of streptococcus sore throat having 
some relation to the milk supply has directed the attention of recent, 
workers to the pathogenic properties of the various streptococci found 
in milk. 

From supplies of milk obtained under various conditions including 
both pasteurized and certified milk a collection of strains was isolated 
and subjected to various tests. They were also compared with a col- 
lection of human hemolytic streptococci as regards their pathogenicity 
for animals. 

Especial attention was given to the study of the property of heat- 
resistance on account of its relation to pasteurization. 

Only those streptococci were selected whose colonies were surrounded 
by a distinct clear zone of hemolysis on human blood agar plates 
(Type B, Theobald Smith). 

The feebly hemolytic streptococci (type A) were often noted in the 
milk but were not collected and studied since the interest in sore throat 
epidemics has centered about the cocci with a clear wide zone. 

Three hundred and twenty-eight sample specimens of bottled milk 
were collected from different dairies in the city of Chicago. Excepting 
45 samples from one dairy which furnished certified milk all the speci- 
mens were pasteurized ; and with the exception of two dairies (17 samples) 
the holding process was used. 

The time of the year during which these examinations were made 
was from October 1914 to March 1915. 

Blood agar (human) was used in plating and the counts were made 
after incubation at 37°C. at the end of 48 hours. The colonies of 
hemoljdiic streptococci were carefully noted and counted on the plates 
and later their identity was confirmed by further tests. 

Eighty-five samples yielded on culture streptococci of the strongly 
hemolytic variety. The number in different samples varied consider- 



84 ABSTRACTS 

ably ranging from a few hundred to several thousand per cubic centi- 
meter. In the certified milk they were about in the same proportion as 
in the pasteurized samples. From one dairy in 16 specimens of milk no 
hemolysing streptococci were found. In all others some were found. 

A study of these 85 strains of hemolysing streptococci was made 
as regards their morphology, cultural characteristics and certain other 
properties. They vary considerably among themselves. They are 
more resistant to heat than human strains of hemolytic streptococci. 
They possess little or no virulence for rabbits ; therefore in all probability 
not for man. They rapidly acidify and coagulate milk and grow well 
at 20°C. They may form short or long chains but as seen in milk they 
often appear in pairs or a chain of few elements. While they are all 
definitely hemolytic (type B, Theobald Smith) the characteristics of 
the hemolytic zone on plates may vary in certain respects. 

These milk strains are different from certain strains of hemolytic 
streptococci found at times in diseased udders in cows. These latter 
resemble the strains of hemolytic streptococci from human sources 
and are virulent for rabbits. 

There is no reason to consider that these milk strains have any 
sanitary significance. The importance however of certain strains of 
hemolytic streptococci in relation to epidemics of sore throat makes 
it necessary to study carefully all such organisms in milk. 

By itself the hemolytic property has no more value for identification 
purposes than many other characteristics and perhaps less than some. 
But it is of great importance on account of the practical value of the 
blood agar plate method as a means of initial separation of hemolytic 
strains from the many strains of non-hemolytic and feebly hemolytic 
streptococci found in milk. 

Hydrogen-ion Concentrations in Cultures of Streptococci. S. Henry 

Ayers. 

Hydrogen-ion concentrations were determined in cultures of strepto- 
cocci from the following sources: 34 from various human infections, 18 
from the human mouth, 89 from milk and 60 from the udder, feces and 
mouth of the cow, making a total of 201 cultures. 

The cultures were grown in a broth containing 1 per cent cerevisine 
(a dry-yeast preparation), 1 per cent peptone, 1 per cent of the test 
substance, and distilled water. All the streptococci grew well in this 
medium and it does not contain a fermentable sugar. Many of the 
cultures would not grow in extract broth or in infusion broth when the 
muscle sugar was removed by fermentation with B. coli. 

Since the acidity may be defined in terms of hydrogen-ion concen- 
tration this was determined instead of the titrable acidity. The 
test substances used were glucose, lactose, and cane sugar, raffinose, 
mannite and inulin, and the hydrogen-ion concentrations were de- 
termined by a colorimetric method. 

The hydrogen-ion concentration can be represented by the symbol 
P+H and on this basis the neutral point of absolutely pure water is 



ABSTRACTS 



85 



P+H = 6.8 at 30°C. When P+H is less than 6.8, a solution is acid 
and when greater it is alkaline to pure water. 

A study of a large number of cultures of streptococci seems to indi- 
cate that two limiting zones of hydrogen-ion concentration are reached, 
as may be seen in the table. 





HYDROGEN-ION CONCENTRATION 




" 4.6-4.8 


5 5-6.0 


34 from human infections 


8 
18 

79 
54 


26 


18 from human mouth 





89 from milk 


10 


60 from udder, feces and mouth of cow 


6 







These hydrogen-ion concentrations have been found to be fairly 
constant and would probably be more definite if the values were de- 
termined electrometrically. 

It seems evident from these results that streptococci reach more or less 
definite hydrogen-ion concentrations, which fact may serve to help in 
their classification. 

The difference in the limiting hydrogen-ion concentration has not 
been correlated thus far with any other reactions, but it is rather strik- 
ing that such a large percentage of the streptococci from cases of human 
infection reach only the lower zone of limiting hydrogen-ion concentration. 

This difference among the streptococci, brought out by the hydrogen- 
ion concentration, can not be shown accurately by titration methods, 
since the titrable acidity depends upon the composition of the medium. 

The Value of Lactose Bile for the B. Coli Presumptive Test. John W. 

M. Bunker. 

The use of lactose bile media for the B. coli presumptive test has 
met with criticism because of the difficulty of obtaining fresh whole 
bile whenever and wherever needed. It has been mainta,ined by some 
investigators that the use of lactose broth without bile is as efficient 
as the bile medium. Others have advanced the view that the inhibitive 
action of bile is so great that with its use weak forms of B. coli are lost. 

The use of dried bile in the form known as Bacto Oxgall in 10 per 
cent solution has given results always as satisfactory as those obtained 
when preparing the medium from new whole bile, in tests upon oysters, 
milk, cream, ice cream, polluted water, and sewage. 

When used in a 5 per cent solution dried bile has provided a medium 
which detects B. coli by fermentation in higher dilutions occasionally 
than when 10 per cent solution or Standard Lactose Bile is employed. 
The 5 per cent solution has never failed to show fermentation in equally 
high dilutions as the 10 per cent solution or the Standard medium. 

A water to which was added a small quantity of B. coli culture when 
stored for a long time and tested daily with whole bile medium, ten 



86 ABSTRACTS 

per cent dried bile, 5 per cent dried bile, 2 per cent dried bile, and lactose 
broth, showed the presence of B. colt at first in all media, later only in 
media containing 5 per cent or less of dried bile, and finally in lactose 
broth alone. 

A water heavily polluted with feces tested in the same manner showed 
gas in one higher dilution in the 10 per cent dried bile and in whole bile 
than in the other media containing less amounts of ox gall. This 
water was so bad in appearance that a sanitary examination would 
have been unnecessary and therefore represents an exaggerated case. 

For these somewhat contradictory results the following explanation is 
offered. It is believed that they are really not contradictory but are 
typical of the results which, being reported by different workers dealing 
with different material for test purposes, have confused the issue for 
some time. 

The intestine is not filled with bile but bathed with it and the environ- 
ment of intestinal organisms may be regarded as one of diluted bile. 
It seems reasonable then to suppose that a test tube of diluted bile 
offers a more favorable environment for these organisms than a test 
tube of whole bile. Organisms which develop in the standard medium 
do so in spite of the concentration of bile, and the weaker ones succumb. 

The view is offered that in testing highly polluted material where the 
presence of organisms which will develop in the presence of lactose is 
so great that there is danger of overgrowth of B. coli and the shutting 
off of fermentation, the inhibiting property of bile toward non intestinal 
organisms is desirable. 

Where the danger of overgrowth of B. coli is not great as in mildly 
polluted samples, lactose broth may be as efficient and will probably 
be more delicate. 

The danger in using lactose broth is not that too many organisms 
will be included by the test, but that too many may be lost by over- 
growth of others. 

The question which ought to engage the attention of sanitary analysts 
is not "Shall we use bile or shall we use lactose?" but "How much bile 
plus how much lactose?" 

The results of experimentation upon which this paper is based 
indicate that a medium composed of 1 per cent peptone, 1 per cent 
lactose, and from 2 to 5 per cent dried bile is much more delicate in de- 
tecting intestinal organisms by the fermentation test than is our present 
Standard medium, and is in the majority of cases more reliable than 
lactose broth alone. 

A Chromogenic Bacillus. Frank L. Rector. 

This organism was isolated from water. It is a small rod with 
rounded ends, measuring from two to four microns long by seven- 
tenths of a micron wide. Occurs singly. Is motile. Stains evenly 
and easily; is Gram negative. Forms no nitrites, and indol production 
is doubtful. Coagulates milk in twenty-four hours. Forms gas in 
glucose, saccharose and glycerin; no gas in lactose. Thermal death 
point is 66°F. for ten minutes. 



ABSTRACTS 87 

Produces a red pigment at room temperature. Rapidly liquefies 
gelatin and produces ammonia. Is a facultative anaerobe. Group 
number is 221.1012701. 

On the Correlation of the Voges-Proskauer and the Methyl Red Reaction. 

Max Levine. 

The methyl red reaction of Clark and Lubs was suggested as a rel- 
atively quick and simple routine test for the differentiation of the colon- 
aerogenes group of bacteria. This test correlates strikingly with the 
accurately determined gas ratio. It is desirable that the methyl red 
reaction should also be correlated if possible with some previously 
employed test in order that the valuable work of the Department 
of Agriculture may be adequately compared with the numerous pre- 
vious investigations. 

From a study of 187 coli-like bacteria, 31 of which were obtained 
from other investigators and 156 isolated from raw and septic sewage 
and from the feces of the cow, pig, horse, sheep, and man, it was found 
that only those organisms which gave the Voges-Proskauer reaction 
were alkaline to methyl red. One hundred and fifty-nine of the organisms 
were negative to the Voges-Proskauer test; these were all acid to 
methyl red, while all of the 28 which reacted positively to the Voges- 
Proskauer test were alkaline to methyl red. It was also observed that 
although 23 per cent of the organisms isolated from sewage gave these 
two reactions (methyl red alkaline, V.P. + ), no such organisms were 
isolated from the feces of the horse, cow, pig, sheep, and man. 

The scarcity of organisms giving the Voges-Proskauer reaction in 
human feces is dwelt upon by MacConkey and also pointed out by 
Clemesha. Coli-like bacteria which give this reaction are character- 
istically of non-fecal origin. They resemble, in many other characters 
the Bacillus aerogenes (Escherich) and probably represent soil forms. 

TECHNIQUE 
Under Supervision of Jean Broadhurst 

Acidity of Media. H. A. Notes. 

Media for bacteriological purposes are standardized to definite de- 
grees of acidity or alkalinity. There are several factors which may 
affect the accuracy with which the reaction of the media is determined. 
The paper, as presented, took up several of these factors and gave 
results of experimental work carried out in the Horticultural Research 
Chemistry and Bacteriology Laboratories of the Purdue Agricultural 
Experunent Station. The following conclusions were reached: 

1. Agar-agar and gelatine should be the best obtainable. A grade 
of each, such that unfiltered solutions contain no visible solid matter, 
is obtainable. 

2. Only necessary chemicals of the highest purity should be used. 

3. The resulting products of reactions that will occur when a number 
of compounds are put together should be understood before one at- 
tempts to make combinations of them in a medium. 



88 ABSTRACTS 

4. No indicator, used for the titration of media is accurate in all 
cases (even phenolphthalein which is very sensitive to acids will not 
indicate phosphoric acid accurately when it is in mixtures). 

5. Enough media should be used for titration so that inaccuracies 
in measuring are minimized and errors in the actual titration will not 
be magnified in subsequent calculations. 

6. The acid equivalent of media varies with the temperature of the 
media and the amount of variation is dependent upon the compounds 
present in the media. 

7. Media should be added to carbon dioxide free distilled water and 
titrated at the temperature at which they are to be held when organisms 
are growing. 

8. Distilled or double distilled water does not mean carbon dioxide 
free water. The carbon dioxide content of distilled water will not be 
a constant from day to day at a given laboratory. 

9. Carbon dioxide in distilled water makes it possible to have a 
medium titrate, with phenolphthalein, 1.0 per cent acid^ when it is alka- 
line or neutral. 

10. The acidity of media that has been properly made does not 
increase appreciably when the length of time of sterilization is increased 
or when repeated sterilizations are made in the autoclave. 

A Culture Medium for Maintaining Stock Cultures of the Meningococcus. 

C. G. A. Rocs. 

This medium has been in use for about two years and experience has 
demonstrated its superiority over the other media mentioned in the 
literature for the maintenance of stock cultures of the meningococcus. 
This culture medium is a modification of the potato blood agar used 
by Bordet and Gengou for the isolation of B. pertussis. 

Preparation of Medium. 

1. Prepare potato extract as follows: 

a. Potato peeled, cut in small pieces and washed in running 

water for about 2 hours 100 gm. 

b. Water containing 4 per cent doubled distilled glycerine free 

from acid 200 c.c. 

c. Mix and autoclave for 40 minutes 

d. Allow to stand over night and strain through cheese cloth. 

2. Make Potato-Extract-Agar as follows: 
a. Mix in an Erlenmeyer flask — 

Potato extract 50 c.c. 

NaCl Sol. 0.65 per cent 150 c.c. 

Agar 5 gm. 

6. Heat in Arnold sterilizer until agar is melted, requiring from 30 min. 
to 1 hour. 

3. Tube and sterilize without filtering in autoclave for about 40 minutes. 

4. When wanted for use, melt the agar, cool back to about 45°C. and add 

the desired amount of defibrinated horse blood. 

' Requires 10 cc. of normal alkali per liter to make it neutral. 



ABSTRACTS 89 

The amount of blood to be added depends upon whether or not the 
meningococcus has become accustomed to the medium. In trans- 
planting from another medium to this potato blood agar, a little difficulty 
may be experienced in getting the cultures started upon the new sub- 
stratum. For this reason a large amount of the growth (not over twenty- 
four hours old) should be transferred and about 20 per cent of blood be 
added to the medium. In making the inoculation the culture should 
be rubbed slightly into the surface. This is incubated at 37.5°C. for 
about two days and then transplanted again to the potato extract 
agar containing just sufficient blood to permit growth — that is, about 
5 per cent. Subsequent transplantation need not be made more often 
than every thirteen to fifteen days or longer, when kept at 37°C., 
provided that the cultures do not become too dry. In the case of 
cultures paraffined or sealed to prevent drying, a fair growth may be 
obtained after six weeks. 

Pasteurization Applied to Mold Spores. Charles Thom and S. H. 

Ayers. 

A series of experiments was devised to test the effects of temperatures 
commonly used in pasteurization upon the spores of pure cultures of 
a series of species of Penicillium, Aspergillus, Mucors, one form of Fus- 
arium, and Oidium ladis. Results are summarized as follows: 

1. The holder process of pasteurization in which milk was heated 
to 145°F. (62.8°C.), and maintained at that temperature for 30 minutes 
killed the conidia of every species investigated, except those of Asper- 
gillus repens, A. flavus, and A. fumigatus. The molds which survive 
are only found occasionally in milk. 

2. The flash process of pasteurization, where milk was heated to 
165°F. (73.9°C.), for a period of 30 seconds, destroyed the spores of 
all the molds tested with the exception of many spores of one form and 
occasional spores of three more forms. At 175°F. (79.5°C.), only 
occasional spores of two forms developed. 

3. When the heating process was performed in dry air for a period 
of 30 minutes at 200°F. (93.3°C.), 31 out of 42 forms of Penicillium and 
7 out of 24 forms of Aspergillus were destroyed, but none of the cultures 
of Mucors. A temperature of 250°F. (121. 1°C.) over a period of 30 
minutes killed all the forms of Penicillium tried, but left an occasional 
living spore in one species of Aspergillus and 3 out of 6 Mucors. 

The use of 0.01 Cubic Centimeter Pipettes in Bacterial Milk Analysis. 

James D. Brew. 

There are two common ways of measuring milk for counting bacteria 
with a microscope; one, by loops and the other by capillary pipettes. 
Prof. H. W. Conn, in his report on an investigation recently conducted 
in New York City, concludes that the wire loop as used by one of the 
laboratories making the counts appears to yield results as accurate as 
those secured by using 0.01 cc. pipettes. This conclusion is based 
upon comparative counts; but owing to possible wide variations in 



90 ABSTRACTS 

microscopical counts, due to other factors than possible inaccuracy 
in measuring the quantity of milk under examination, a better means 
of testing is by comparing the weights of the amount of milk delivered. 
Twenty samples, weighed as discharged by one pipette, had a variation 
of 3 per cent, while under the same conditions the weights of the milk 
discharged from a loop varied 51 per cent. 

Poorly formed tips and faulty calibration cause difficulty in securing 
the amount of milk desired. The tip should be a truncated cone with 
the flattened end a circle of about 2 mm. in diameter. Pipettes should 
be calibrated so as to deliver (not contain) 0.01 cc. of milk (about 0.0103 
grams). This may be done by weighing the amount of milk dis- 
charged into the hollow of a clean, hollow ground microscope slide, 
covered to prevent evaporation. A pipette should deliver 4 to 5 per 
cent in excess of 0.01 cc. of mercury if this is used in calibrating, as 
mercury does not adhere to the walls of the pipette as do water and 
milk. 

Pipettes should be clean, but sterilization is unnecessary. Sterilizing 
does not remove bacteria, and growth is stopped in quickly dried smears. 
Smears from ten samples of milk made by individual, sterile pipettes 
averaged 6150 bacteria per cubic centimeter. Smears made from the 
same samples by individual, clean pipettes averaged 4200 per cubic 
centimeter while those made from the same samples by one pipette 
which was recontaminated between each delivery in a high count milk 
and cleaned by rinsing in clean water, averaged 4600 per cubic centi- 
meter. Smears made from the same samples by one pipette which was 
recontaminated between each delivery in a high count milk and rinsed in 
the sample of milk from which the next smear was to be made, averaged 
338,900 per cubic centimeter while when smears were made in the 
same way but with the pipette unrinsed the average was 17,723,000 
per cubic centimeter. These results indicate that for quantitative 
milk work, individual, sterile pipettes have no measurable advantage 
over individual, clean pipettes and that neither have any advantage 
over the use of one pipette for all samples, provided it is cleaned by 
rinsing in clean water. Dirty pipettes or pipettes handled carelessly, 
cause measurable errors. 

A Simple Apparatus for Isolating Anaerobes. Zae Northrup. 

A simple apparatus for isolating anaerobes consists in a 25 cc. burette 
complete with rubber tubing, glass tip and pinchcock. This is filled 
with any desired liquid nutrient medium, plugged with cotton, the de- 
livery tube protected from contamination by inserting it in a small 
test tube and the whole apparatus set up on a ring stand and sterilized. 

This tube may be inoculated with any material containing anaerobes. 
In a comparatively short incubation period the various classes of micro- 
organisms will adjust themselves to their optimum oxygen require- 
ments which will be noted sooner or later by a clear zone at the upper 
portion of the liquid varying in depth from a few millimeters to 8 or 
9 cc. in some instances. 



ABSTRACTS 91 

The organisms growing anaerobically may then be drawn off and 
sub-cultured further in these tubes until practically nothing but anaero- 
bic organisms will be present. These may be isolated by the anaerobic 
plate method. 

If successive sub-cultures are to be made from the same tube it is 
necessary to have the rubber tubing on the burette several centimeters 
in length so that a pinchcock may be placed about 3 cm. above the 
original one, the rubber tubing cut with sterile scissors and a sterile 
burette tip inserted. This is necessary as after one withdrawal of 
hquid, aerobic organisms may grow in the liquid remaining in the tip. 

Modification in Staining Technic. Zae Northrup. 

The following modification in the technic of Gram's stain is recom- 
mended for beginning students in bacteriological laboratory work. 
If a coverglass preparation of two morphologically different organisms, 
one gram positive, the other gram negative, is made on the same cover- 
glass and stained by Gram's method, the student is enabled to get the 
differentiation very clearly. B. coli and B. subtilis lend themselves 
to this differentiation very nicely. 

Amniotic Fluid as a Bacterial Culture Medium. AVard Giltner and 

L. C. LUDLUM. 

Amniotic fluid is a normal transudate from the blood of the pregnant 
mammalian female. It is a very dilute albuminous solution contain- 
ing various salts and also cellular elements in suspension. While its 
composition varies with the different species of animals and with the 
stage of pregnancy it was believed that its composition was sufficiently 
constant and quite probably, qualitatively and quantitatively, in 
conformity with the food requirements of many parasitic bacteria. 
In fact it was believed that on account of the ease of collection and the 
large quantities available, bovine amniotic fluid would serve as a sub- 
stitute for, or perhaps have advantages over ascitic fluid or blood serum 
media. At all events amniotic fluid need not be collected aseptically 
since it can be steriHzed either in the autoclave or by Tyndall's method. 

In our experiments amniotic fluid has been used in place of broth 
with no additions, and with agar and gelatin or with glycerine. 
With the colon-typhoid group, B. coli gave excellent growths, B. 
typhi grew scantily and B. cholerae-suis only fairly well. Staph, 
aureus grew best on glucose agar and on glycerine amniotic agar. None 
of the strains of Streptococcus pyogenes studied grew well except in 
bouillon. The growth was least on the solid amniotic agar which is 
a fact of considerable interest since, in the few tests conducted, B. 
diphtheriae from swabs could be isolated readily on the amniotic agar. 
The isolation of new strains of B. diphtheriae on amniotic agar was 
facilitated by the inhibiting action on streptococci. Involution forms 
of B. diphtheriae were not so abundant on amniotic agar as on Loeffler's 
blood serum medium. An old culture of B. tuberculosis grew with 



92 ABSTRACTS 

marked vigor. For a number of years we have been satisfied that 
plain amniotic agar was a very nearly ideal medium for B. abortus 
(Bang). 

Study of Effect of Dilution Water on Bacterial Suspensions. H. M. 

Weeter. 

A series of tests were made to determine the quantitative changes 
in bacterial suspensions held in dilution water. From suspensions 
held for two hours at room temperature triplicate plates on lactose agar 
were made at fifteen minute intervals. 

Of fourteen tests made with dilutions of one part milk in one hun- 
dred thousand parts water, five showed decreases ranging from ten to 
seventy per cent in two hours, seven showed no definite change, and 
two gave increases of thirty-two and thirty-nine per cent. 

Since colonies of the lactic acid type seemed to be the ones disappearing 
thirty-two tests were made with milk inoculated with these organisms 
in dilution water from five different sources. The same dilution 
was used as before. Thirty of these tests gave unmistakable reductions 
in numbers, amounting in some cases to fifty per cent in fifteen minutes. 

Additional tests of dilutions below one to one thousand made with 
milk containing a mixed flora did not show any decrease during two 
hours, but an increase was observed on longer standing. 

The nature of the organisms present and the amount of material 
added to the dilution water from the original bacterial medium are 
probable factors determining the effect of dilution water on the bacteria 
present. 

Variation in Plate Counts Under Research Conditions. M. J. Prucha. 

Results of seven experiments are presented in this preliminary report. 
In each experiment about one hundred lactose agar plates were pre- 
pared using the same dilution of milk. 

Further study is needed to give sufficient basis for drawing definite 
conclusions, but the results so far point to the conclusion that the 
average of three plates from the same dilution approaches reasonably 
closely to the average of a hundred plates made from the same dilution, 
when that average is between one and two hundred colonies per plate. 

Another Use of the Double-Plate Method. W. D. Frost and Freda M. 

Bt^chmann. 

This method was used by Frost in 1904 to study antagonism among 
bacteria. It was slightly modified, renamed and used by Churchman 
in 1912 to study the bactericidal action of aniline dyes. 

It is here modified to obviate the necessity of using either a glass or 
metal division by putting in a petri dish, before sterilization, a semi- 
disc of cheesecloth. The bottom of the dish is entirely flooded with the 
medium to be used and when hard the piece of cloth with the adherent 
agar is lifted out with sterile forceps. The clear portion of the dish 
is flooded with the medium containing the material to be studied. 
Streaks of the organism to be used are then made over each side of the 



ABSTRACTS 93 

dish. In this way the agar on both sides of the dish is in perfect con- 
tact which makes difTusion readily possible. 

These plates are being used to study the effect of spices and condi- 
ments in inhibiting yeasts, molds, and bacteria. This method serves 
as a satisfactory means of determining the preserving action of these 
substances. 

INDUSTRIAL BACTERIOLOGY 
Under Supervision of D. H. Jones 

A Possible Function of Actinomycetes in soil. H. Joel Conn. 

A comparison has been made at the New York Experiment Station 
between the number of Actinomycetes in sod and in cultivated soils. 
The samples have been taken in pairs, one from sod and one from 
cultivated soil, the two spots selected always being within a few yards 
of each other. Thirty-five pairs of samples have been taken; and a 
considerable variety of soil types have been sampled. The compara- 
tive counts have been made by means of gelatin plate cultures. 

Sod soil, almost invariably, has given a higher count of Actino- 
mycetes than cultivated soil; also the Actinomycetes have comprised 
a greater proportion of the total flora in sod soil than in cultivated 
soil. In the thirty-five samples of cultivated soil, Actinomycetes com- 
prised, on the average, 20.5 per cent of the total flora; in the samples 
of sod soil, 37.5 per cent, or almost twice as high a proportion. There 
were only three or four exceptions in the whole series, and in those the 
difference in favor of cultivated soil were so small as to be insignificant. 
The differences in favor of sod soil on the other hand have never been 
negligible, and sometimes have been extreme — as in one case where 
Actinomycetes comprised only 2 per cent of the flora in the cultivated 
sample but 16 per cent in the corresponding sod sample, or in another 
where they comprised 15 per cent of the flora in the cultivated sample, 
but actually as much as 60 per cent of the flora in the corresponding 
sod sample. 

A further series of tests comparing three spots in a single soil type, 
one fallow, one in new sod, and one in old sod, showed 10-15 per cent 
Actinomycetes in the fallow spot, 21 to 25 per cent in the new sod 
and 37 to 39 per cent in old sod. 

The most probable explanation of this difference seems to be that 
the Actinomycetes are active in the decomposition of grass roots, a 
point which is now being investigated at the New York Experiment 
Station. 

Media for Soil Bacteria. H. A. Notes. 

In the course of other investigations in the Horticultural Research 
Chemistry and Bacteriology Laboratories of the Purdue Agricultural 
Experiment Station it was necessary to select a medium for the plating 
of soil organisms. The media first chosen for comparison were the 
Lipman and Brown agar, H. J. Conn's sodium asparaginate agar, and 
soil extract agars. Agar-agar alone was also used in comparison with 
the above media. 



94 



ABSTRACTS 



In plating tests the Lipman and Brown agar proved to be the best 
of the media studied, not only from colony counts, but in the variety 
of organisms grown. This latter is judged from the macroscopic 
appearance of the plates and by the fact that some organisms growing 
on the Lipman and Brown agar would not live when transferred to 
other media. 

The colonies on the sodium asparaginate agar were large, but in 
only one of over fifty tests of organisms growing well on this media 
did the organisms fail to grow on agar alone. 

The two best media were so different in constitution and the organ- 
isms that grew best on them acted so differently when transferred from 
one medium to the other that those ingredients furnishing carbon and 
nitrogen were investigated further. These tests resulted in other 
tests where 15 gms. of (best obtainable) agar-agar was the basis of all 
the media. This was used both alone, and in combination with am- 
monium nitrate, starch, Witte's peptone, sodium asparaginate, Liebig's 
extract of beef; soil extracts, wheat straw extracts, and leaf extracts 
were also used. 

The results of this work and of later work are shown in the follow- 
ing table. The soil organisms reported on were chosen because of their 
macroscopic differences. The known pathogens and known non-patho- 
gens were the only known organisms studied. 

Media tests 
(15 grams agar-agar is basis of all media.) 



Lipman plus Brown 

0.05 gram Witte Peptone 

2 grams St. plus 0.05 Peptone . . 
H. J. Conn's media 

1 gram Na. asparaginate 

2 grams St. and 1 gram Na. asp 

Soil plus 1 gram NH4NO3 

Soil plus 1 gram Starch 

Soil plus 2 grams Starch 

1 gram NH4NO3 plus 1 gram 

Starch 

1 gram Starch 

2 grams Starch 

7 .5 grams Gelatine 

7.5 grams Gel. plus 1 gram Starch 
7.5 grams Gel. plus 2 grams Starch 
7.5 grams Gel. plus 2 grams Starch 

plus soil 

5 grams Liebi g' s Ext . plus 10 grams 

Peptone 

5 grams Liebig'sExt. plus 4 grams 

Starch 



24 son, 

ORGAN- 
ISMS 



7 KNOWN PATHOGENS AND 8 KNOWN 
NON-PATHOGENS 



Rank 3 days Rank 6 days Rank 8 days 



Patho- 
gens 



12,13 

17 

10,11 

6 

16 

8 

15 

14 

10,11 



9 

5 

12,13 

7 
4 

3 



Non- 
Pathogens 



4« 



7 

16 

13 
8, 9, 10 

14 
8,9,10 

17 

15 
8, 9, 10 



11 
6 

12 
5 

4 



Non- 
Patho- 
gens 



7 

16 

12 

8 

13,14 

9 

17 

15 

13,14 



11 
5 

10 
6 

4 



Patho- 
gens 



11 

16 

9 

6 

14 

7, 8 

17 

13 

12 



10 

5 

15 

7,8 
4 



Non- 
Patho- 
gens 



9 

16,17 

12, 13 

7 

14 

8 

16, 17 

15 

13 



10 
5 

11 
6 
4 



ABSTRACTS 95 

Are Spore-Forming Bacteria of any Significance in Soil Under Normal 

Conditions? H. Joel Conn. 

A series of tests has been made at the New York Experiment Station 
to determine whether B. mycoides, B. cereus and B. megatherium, 
the most common spore-forming bacteria in soil, occur under normal 
conditions as spores or as vegetative rods. Diluted soil infusion was 
heated at a temperature of 75 to 85°C., heated and unheated samples 
of the infusion plated, and the colonies of these three types on the two 
sets of plates counted. It was assumed that the colonies on the plates 
from heated infusion represented spores and that the difference in 
favor of the unheated infusion, if any, represented the vegetative forms. 

In a series of 22 tests the average count of all three spore-formers 
was: 775,000 per gram, unheated; 726,000 per gram, heated. They 
were higher 14 times in unheated infusions, 8 times in heated. The 
greatest difference in favor of the unheated infusion was 530,000 per 
gram, which was offset by a difference of 450,000 in favor of the heated 
infusion; a fact which suggests that both were within the limits of 
experimental error. Considering the organisms separately: 

B. mycoides was higher 12 times in unheated infusions, 8 times in 
heated. 

B. cereus was higher 10 times in unheated infusions, 8 times in 
heated. 

B. megatherium was higher 12 times in unheated infusions, 8 times 
in heated. 

The number of spores or the total number of spore-formers present 
did not increase even in a pot of soil mixed with a heavy application of 
fresh horse manure. 

These figures suggest that bacteria of this group normally occur in 
soil only as spores, in which form they cannot be active. This is 
surprising, as they are universally present, and have always been 
considered important. What their actual significance may be is a 
question. 

Ferrification in Soils. P. E. Brown and G. E. Corson. 

Preliminary studies of the oxidation of iron in the soil, or ferrifi- 
cation, have shown that the process is rather a complicated one. An 
attempt was made to ascertain whether soils have a ferrifying power, 
whether such a power is bacterial or chemical in nature or the result 
of a combined action of several groups of factors, whether different 
soils have varying ferrifying powers, whether ferrification can be 
measured in the laboratory and finally whether the process is of any 
significance from the soil fertility standpoint. 

Much difficulty was experienced in devising methods for the work, 
owing to unsatisfactory chemical methods and many series of tests 
were carried out merely to solve some of the chemical problems in- 
volved. A method has been devised which, although still rather crude, 
will permit of tentative conclusions. 

Soils from a wide variety of sources were tested and it appears that 
ferrification is a process of common occurrence in the soil, that different 



96 ABSTRACTS 

soils possess differing ferrifying powers, and that the process is in part 
chemical and in part bacterial in nature. A line of investigations is 
thus opened up which may prove of much interest. Ferrification in 
soils apparently should be studied further both from the technical and 
from the practical standpoints. 

Coll-like Organisms of the Soil. B. R. Johnson. 

Forty-two samples of soil were examined for the presence of coli- 
like bacteria. Eighteen samples were from manured and twenty- 
four from unmanured areas. It seems from this study that in both 
manured and unmanured soils, the incidence of coli-like organisms is 
considerably greater, if a crop is being raised than if the soil is fallow. 

In a preliminary study of 363 cultures as to their reaction with methyl 
red and the Voges Proskauer test, 261 were found to be alkahne. Of 
these over 84 per cent reacted positively to the Voges Proskauer test. 
Of the 219 cultures which gave the Voges and Proskauer reaction, over 

97 per cent were alkaline to methyl red. This striking correlation be- 
tween the two reactions has been previously pointed out by Levine, who 
observes that such forms are rarely found in feces, but relatively abun- 
dant in sewage. Rogers and his co-workers found methyl red alkaline 
coli to be the prevailing type on grains and seldom present in cow 
manure. The prevailing coli-like organisms in the soil are apparently 
of a non fecal type and they may be differentiated from fecal strains by 
the Voges Proskauer and methyl red reactions. 

The Influence of Soil Solution on the Longevity of Microorganisms Sub- 
jected to Desiccation. Ward Giltner and Virginia Langworthy. 
It is already well known that bacteria resist desiccation in soil for a 
much longer time than in a naked or unprotected condition such as 
might be offered by the surface of a solid culture medium. It is also 
known that any porous substance, not in itself antimicrobial, will offer 
protection to microbes against the deleterious effects of desiccation. 
There is a question as to just what factors are responsible for the pro- 
longed life of microbes in the soil. Soil solution, as used in these tests, 
was extracted by the paraffin oil displacement or pressure method. 
Tests were made with other solutions, viz., physiological salt, 0.1 per 
cent agar, gelatin, albumin, gum arable, soluble starch, also nutrient 
broth and milk using Ps. radicicola and drying in quartz sand after 
suspension in the different solutions. The results were that: (a) after 
suspension in normal salt, gum arable, starch or agar solutions drying 
in sand was rapidly fatal, few or no bacteria being alive after one 
month, (b) after suspension in gelatin or albumin solution drying was 
less rapidly fatal, (c) after suspension in milk or bouillon drying in 
sand was still less rapidly fatal. Suspension in soil solution followed 
by drying in sand gave in one case better results than with milk and 
all other solutions used except broth and in another case better than all 
other solutions except milk and broth. Tests were also made of the 
longevity of Ps. radicicola dried in quartz sand and in clay loam. 



ABSTRACTS 97 

Further tests were made to determine the changes in numbers and 
kinds of microorganisms naturally occurring in soil solution when it is 
dried in different types of soils, sand, sandy loam, clay, clay loam and 
muck, using soil solution from a rich garden loam. 
Conclusions from all the experiments were that : 

1. The survival of nonspore-bearing bacteria in air-dry soil is due, 
in part, to the retention by the soil, of moisture in the hygroscopic 
form. This, however, is not the only factor, for the longevity of bac- 
teria in a soil is not directly proportional to its grain-size and hygro- 
scopic moisture. 

2. Bacteria, at least those tested, resist desiccation longer in a rich 
clay loam than in sand under the conditions of our experiment. 

3. The solution extracted from a rich clay loam contains substances 
which have a protective influence upon bacteria subjected to desic- 
cation. 

Reaction of the Soil Solution as an Index of Biological Changes in the Soil. 

J. F. Morgan and O. M. Gruzit. 

One of the essential problems in the study of soil fertility is to adjust 
the reaction of the soil. This reaction essentially influences the chemi- 
cal, the physical conditions and the biological life of the soil. 

The junior author has found in his preliminary study of the soil 
solution adjusted to various degrees of reaction with n/100 mineral 
acid and n/100 alkali and mixed with pure sterile quartz sand, that 
this reaction had some effects upon the number and the type of bacteria. 

An acid reaction of n/1200 had a distinct toxic action on the growth 
of the bacteria and the most suitable reaction for the growth of the 
soil bacteria was in the neighborhood of n/1000 alkali. 

When changes occur from alkaline to neutral and to acid, the numbers 
of bacteria increase up to the point where the solution is barely alkaline 
and then, decrease after this point is passed. In cultures with an acid 
reaction, the lowering of this acidity causes the soil bacteria to increase 
rapidly. 

The Soil Solution as an Index of the Biological Changes in the Soil. 

J. Franklin Morgan. 

The soil solution is a homogeneous mixture of the soil water and the 
soluble soil constituents, both mineral and organic. 

The soil solution offers a good medium for the study of some of the 
biological changes in the soil. As it is the work shop of the micro- 
organisms, this solution will contain the products of their work. 

In some nitrogenous experiments with dried blood, tankage, and 
cotton seed meal, marked changes in the forms of nitrogen and physical 
conditions of the solution were noted at the different periods of ex- 
tractions. 

The longer periods showed a decrease in ammoniacal-N and an in- 
crease in nitrate-N. In all cases there was an increase in the total 
solids. This had its effect upon the physical conditions of the solution, 
e.g., specific gravity, specific conductance and similar phases. 



98 ABSTRACTS 

The Indirect Effect of Certain Soil Treatments Upon Bacterial Activity. 

P. L. Gainey. 

Different methods of preparing seed beds for winter wheat at the 
Kansas Station have given very large differences in the accumulation 
of nitrate nitrogen prior to seeding. Early (July 15) versus later, and 
deep (eight inches) versus more shallow plowing, have given higher 
nitrate contents. Efforts to trace observed differences to variations 
in bacterial flora have failed. Evidence was presented showing a cor- 
relation between moisture content of the surface soil and nitrate ac- 
cumulation. A summary of data accumulated indicates that the 
various treatments have had but slight effect upon the organisms con- 
cerned in nitrate formation, except in so far as this activity is controlled 
by other factors. 

Studies on Soil Protozoa and their Relation to the Bacterial Flora. J. M. 

Sherman. 

The occurrence and activity of protozoa in soil. The results obtained 
from sixteen fertile soils representing various soil types indicate that 
these soils contain about 10,000 protozoa per gram. The predominating 
protozoa in the soils studied were flagellates. Ciliates and amoebae 
were occasionally found in numbers approximating 1,000 per gram. 

It was demonstrated that certain types of flagellates are capable of 
multiplication in soil. The ciliates which were tested were not able 
to increase in soil when kept at its normal moisture content. 

The effect of protozoa upon the soil bacteria. Observations made on 
soils containing protozoa and free of protozoa, at various temperatures, 
with different moisture contents, and on various types of soil indicated 
that the protozoa in the soils studied did not have a limiting action upon 
the bacterial flora. 

The effect of specific types of protozoa, in animal-pure cultures, 
upon the soil bacteria was also studied. The ciliates limit the develop- 
ment of bacteria markedly in soil extract, but are not able to exert this 
effect in soil, since they do not lead a trophic existence under ordinary 
soil conditions. 

Of four types of active soil flagellates which were tested three had 
no apparent effect upon the number of bacteria, either in soil or in 
soil extract. The fourth organism, a species of Monas, had a marked 
limiting action upon the bacterial flora in soil extract but apparently 
had no effect in soil. 

The effect of volatile antiseptics upon the soil micro-organisms. The 
treatment of soil with volatile antiseptic does not free it of protozoa. 
The active soil protozoa again multiply and attain their normal num- 
bers within one month after treatment. 

The maximum numbers of bacteria in partially sterilized soils are 
not found while the protozoa are suppressed, but after these organisms 
have again reached their maximum numbers. 

The number of bacteria in treated soils cannot be decreased by rein- 
oculation with one per cent of untreated soil. 



ABSTRACTS 99 

Comparisons made of treated and untreated soils under various 
conditions failed entirely to give any evidence in support of the theory 
that there exists in soil a harmful biological factor which is destroyed 
by the action of volatile antiseptics. 

The Relation of Protozoa to Certain Groups of Soil Bacteria. T. L. Hills. 

In this work studies were made of the effect of protozoa on ammoni- 
fication, nitrification and free nitrogen fixation in soil. 

Ammonification. Three sets of the same sandy loam soil were used. 
One set was left untreated, another heated to 90°C. for one hour and 
the third heated as above and later inoculated with 1 per cent of the 
original untreated soil thus introducing the supposed harmful factor, 
the protozoa. The ammonia and nitrate determinations after 30 days 
revealed the following: in the untreated soil the ammonia content 
remained about the same while the nitrate increased slightly; in the 
heated soil the ammonia increased considerably but the nitrate re- 
mained the same, as the nitrifiers had been destroyed by heating the 
soil. In the heated and reinoculated soil the ammonia decreased 
slightly but there was a decided increase in nitrate formation. The 
protozoa introduced did not seem to have any detrimental effect on the 
production of ammonia and its subsequent oxidation to nitrate. 

Nitrification. Flasks containing soil were sterilized by heating at 
15 pounds pressure for two hours. Then one half of them were inocu- 
lated with a suspension of normal soil in sterile distilled water and the 
remaining half were inoculated with the same amount of protozoa free 
soil. This latter soil was obtained by sterilizing soil and inoculating 
it with as many different kinds of bacteria as could be isolated by the 
plate method. All flasks were then inoculated with protozoa free 
cultures of Nitrosomonas and Nitrobacter. Then a definite amount 
of ammonium sulphate was added. The ammonia and nitrate were 
determined after 28 days' incubation. There was but very little 
difference in the rate of conversion of ammonia to nitrate in the two 
different soil cultures. 

Free nitrogen fixation. Sterile soil cultures were inoculated one 
half with normal soil and the remainder with protozoa free soil. Then 
a suspension of Azotobacter (and I per cent mannite in sterile solution) 
was added and after incubation of 21 days at 25°C. the cultures did 
not show any appreciable difference as regards the amount of nitrogen 
fixed, the difference being quite well within the limit of experimental 
error. In liquid cultures sterile Ashby's solution was used, one set 
being inoculated with soil containing protozoa and the other set with 
soil free from those organisms. Suspensions of Azotobacter were also 
added. After 21 days' incubation at 25°C. the total nitrogen analyses 
revealed a noticeable difference in free nitrogen fixation; those cultures 
free from protozoa fixing 2.05 mgs. as an average per 100 cc. of solution 
in excess of those containing protozoa. It seems evident that the 
protozoa finding here a medium suitable for their development de- 
stroyed many of the Azotobacter cells. 



100 ABSTRACTS 

In conclusion it would seem from the experiments above cited that the 
protozoa do not have a detrimental effect on the processes of ammoni- 
fication, nitrification and free nitrogen fixation in the soil. 

A Study of the Nodule-Forming Bacteria. F. O. Ockerblad. 

This paper deals with the relative longevity of Ps. radicicola (twelve 
strains from the more common legumes) in sealed and unsealed culture 
bottles such as are used for distribution of nodule-forming bacteria. 
The media used are a liquid medium (1000 cc. ash leachings from 5 
grams wood ashes plus 1 per cent saccharose) and a solid medium (same 
as above plus 1 per cent agar). 

In preparation all bottles were inoculated and incubated at room 
temperature (20°-22°C.) for two weeks, then half the number of each 
of the solid and liquid cultures of the different strains were sealed by 
removing the cotton plug and inserting cork stoppers which had been 
soaked in mercuric chlorid 1: 1000 and flamed at time of insertion. At 
10-day intervals a culture of each strain on both the solid and liquid 
media is analyzed by plating in ash agar to enumerate the number of 
living cells and by making direct count with Thoma counting chamber 
for total numbers, dead and alive. 

The bacteria in the liquid cultures both the sealed and unsealed 
are dying quite rapidlj'-, with the greatest rapidity in the sealed cultures. 
On the solid medium the number of living cells in the sealed cultures is 
decreasing, approximately 25 per cent in 20 days; while the unsealed 
are showing little or no decrease. The total number of bacteria on the 
solid medium is greater than in the liquid culture media. 

If we may be permitted to draw conclusions from limited and in- 
complete data we should say that a liquid medium for the distribution 
of nodule-forming bacteria is unsuitable because of the small total 
number and of a high mortality; and that the deterioration of cul- 
tures on solid medium in sealed form should be recognized. 

Quantitative Media for the Estimation of Bacteria in Soils. R. C. Cook. 

Comparative tests of several different media upon twenty soils are 
reported. 

The length of incubation period and manner of sterilization were 
incidentally studied as affecting the comparative values of the respec- 
tive media with the indication that five days is of sufficient duration to 
secure satisfactory counts. 

Lipman and Brown's modified synthetic agar. Temple's peptone 
agar. Brown's albumen agar, and Conn's sodium asparaginate agar 
were compared with several other agar media having varied sources 
of nitrogen. 

Highest counts were obtained quite consistently with the sodium 
asparaginate agar during the first part of the work. Later a medium 
was developed in which ammonium nitrate and urea were employed; 
this gave results fully as good as any other in a limited number of tests. 
Albumen agar in which the albumen was dissolved in sodium hydroxide 



ABSTRACTS 101 

instead of water gave much more consistent counts, and in most cases 
followed closely the sodium asparaginate agar, surpassing it in one or 
two instances. Molds quite frequently affected the ease in counting 
in the albumen agar, but less so in the ammonium nitrate-urea agar. 
In the case of the asparaginate agar, however, there was no difficulty 
experienced from this cause. 

It was also observed that inasmuch as all soils do not behave in the 
same manner toward the different media it is essential to use several 
soils in the comparisons; otherwise misleading results may be obtained. 

Differences between the counts on the various media were not as 
large as might be expected and there seems to be no justification for the 
belief that any particular one will be most satisfactory in all cases. 

Bacteria, Actinomyces, and Fungi in Soils. Selman A. Waksman. 

This investigation has been undertaken with a view to demonstrate 
the relationship between these three groups of microorganisms in 
different soils and at different depths. Soils of different texture and 
structure were used, and samples taken at six different depths. A six- 
day incubation period has been used for the counts of the bacteria 
and fungi and a fourteen-day period for the actinomyces counts. The 
results indicate that soils rich in bacteria are also rich in fungi and 
actinomyces. The largest numbers of all the three groups occur within 
the upper eight inches of the surface soil. The bacteria decrease regu- 
larly with depth, in numbers and also in percentages relative to the 
total numbers of microorganisms. The numbers of fungi decrease also 
with depth and they almost disappear below eight to twelve inches. 
The actinomyces numbers decrease with depth, but below eight to 
twelve inches their numbers remain constant up to thirty inches, and 
their percentage relative to the total numbers of microorganisms in- 
creases regularly with depth, because the bacterial numbers decrease, 
and fungi almost disappear. At a depth of 1 inch the bacteria form 
81 to 86.5 per cent, fungi 6.2 to 7.1 per cent, and actinomyces 7.3 to 
12.1 per cent; at 30 inches the bacteria form 16.4 to 42.1 per cent, fungi 
to 5.6 per cent, and actinomyces 52.7 to 83.6 per cent of the total 
microorganic flora of the soil developing on agar plates. The actino- 
myces form a numerous group of soil microorganisms, especially in the 
ower soil depths; over 30 species of them have been isolated. The 
following groups of fungi occur in the soil in the largest numbers: 
Penicillia, Mucors, Aspergilli, Cladosporia, Trichodermae, Fusaria, 
and Alternaria. Many more fungus types have been isolated, but 
their numbers are limited. 

FOOD 
Under Supervision of Charles Thom 

Comparison of the Number of Water Bacteria Growing on agar at 37° C. 

and on Gelatin at 20°C. Fred W. Tanner. 

The recommendation of the Committee on Standard Methods for the 
Examination of Water and Sewage of the American Public Health 



102 



ABSTRACTS 



Association in their 1912 report, that the colony count on agar be 
adopted as the standard, has not met with the approval of many- 
bacteriologists. In order to secure more data on this subject the 
Illinois State Water Survey began a series of comparative tests. A 
large number of analyses was made of which 4379 are considered in 
this paper. 

In order to reach a more definite basis for comparison the analyses 
were arranged in the following classes according to their sources. The 
table indicates the ratios which were found. In each case the agar 
count was taken as unity. 



Deep wells 

Shallow wells 

Raw Lake Michigan water 

Raw river water 

Treated water 





K 






tt 






s^ 


o 


o 




o 






n 


fri 

< 
« 


a o 




•< 


o 


» 


a 


^ -i 


a 


m 






D 


^ ^ 


o 


6. 


«2 


-< 


s 


s§ 


■< 




^w 


K 


X 


J K 




O 






"i 


hE^ 


> 


E5 


< 


■< 


s 


o 


■< 


712 


54 


1.1-1 


25-1 


658 


1-1.4 


1648 


148 


1.6-1 


17-1 


1500 


1-1.4 


405 


3 


156-1 


466-1 


402 


1-16 


537 


56 


1.4-1 


4.1-1 


481 


1-9.0 


1077 


151 


1.9-1 


12-1 


926 


1.1-8 



s 



1-76 

1-68 

1-633 

1-250 

1-35 



The relation of agar colonies to gelatin colonies on those samples 
showing the larger number on agar does not exceed 10 to 1, with one 
exception and this exception results from the consideration of only three 
analyses. With those samples showing the greater number on gelatin, 
the ratio does not exceed 1 to 10, except in one case and this again 
is on raw Lake Michigan water. This would seem to indicate that 
in badly polluted waters we might expect a high ratio, but with pure 
waters the counts on the two media closely approach each other. 



Scientific Methods of Control in the Mineral Water Industry. Frank 

L. Rector. 

Methods of protecting the source and handling the product of the 
Great Bear Spring Company from the spring to the consumer are dis- 
cussed. This company owns 600 acres of land comprising the entire 
watershed of a group of springs, 11 in number, whose flow is about 
one-half million gallons daily. The springs are situated five miles 
south of Fulton, New York. 

Some 360,000 evergreen trees have been used to reforest this tract 
of land. Water from three of the springs is used. The springs are 
enclosed in enamel or glass-lined steel caissons with light-proof covers. 
They are perfectly protected from surface drainage. 

The water is shipped in large tanks, also glass and enamel-lined, of a 
capacity of from seven to ten thousand gallons. Cars are sterilized 
by steam at 10 pounds pressure for one hour before filling. After 



ABSTRACTS 103 

filling, each car is given a twenty-four hour test for gas production before 
being shipped. 

Upon arrival at destination the car is connected by a metal hose to 
the storage tanks in the building and the line sterilized with steam before 
the car is emptied. The storage tanks are glass lined and are sterilized 
when empty. 

Bottles are washed inside and out with hot water and soda solution, 
rinsed with hot sterile water, and sterilized at 104°C. for thirty minutes. 
When cool they are filled automatically, stoppered and sealed. Only 
sterilized glass stoppers are used. The piping system is sterilized daily 
with steam. 

The laboratory work consists in checking methods of operation by 
frequent sampling of various parts of the system. Also frequent 
inspections of the different bottling houses are made, and a score card 
record of the visit is kept. Recording thermometers check the tempera- 
ture of the sterilizers and these records are kept on file. 

Analytical results show a product unchanged in the course of handhng. 

Bacteria in Commerical, Bottled Waters. Maud Mason Obst. 

The official supervision of commercial, bottled waters has led to the 
accumulation of a large amount of data concerning their bacterial 
content. Waters from 167 sources, both foreign and American, have 
been examined. Many contained large numbers of organisms, includ- 
ing B. cloacae, paratyphi, mycoides, aerogenes, subtilis, aurantiacus, 
maritinum, ovale, prodigiosus, fluorescens (liquefaciens) , fluorescens, 
(non-liquefaciens), M. citreus, long-chain streptococci, and unidentified 
chromogens. Occasionally, common molds were found, and from one 
source a sporotrichum occurred in large numbers. A sample from one 
spring gave cultures of P. italicum, and from one import sample were 
obtained Actinomyces. B. coli were isolated from 57 per cent of the 
domestic samples and from 49 per cent of the import samples in 10 cc. 
quantities, from 44 per cent of the former and 28 per cent of the latter 
in 0.1 cc. quantities, and from 9 per cent and 3 per cent, respectively, 
in 0.001 cc. quantities. 

In certain cases, inspections of the springs have located the sources of 
pollutions in some controllable place, as in the bottles or bottling houses 
or in a less easily controlled place, as in the spring. When the source 
of pollution could not be removed, the bottled product was not con- 
sidered safe for human consumption. 

Communications from other bacteriologists have shown that nearly 
all expect to find bottled waters more nearly bacteriologically pure than 
municipal supplies, and many feel that bottled waters should at least 
contain no B. coli in more than one of 10 cc. portions. 

Comparison of Rapid Method of Counting Bacteria in Milk with Standard 

Method. W. D. Frost. 

The method consists of making small plate cultures, four square 
centimeters in area, on microscopical glass slides. One twentieth of a 



104 ABSTRACTS 

cc. of milk, or less, is mixed with an equal amount of nutrient agar. 
These "lilliputian" plates are incubated at 37°C. for from three to 
twelve hours, depending upon the character of the milk. The little 
plates are then air dried, fixed, treated with ten per cent acetic acid in 
alcohol, stained in Loeffler's methylene blue (1:4), slightly decolorized 
in alcohol, and dried. The colonies are stained a deep blue, while the 
background is a light blue. 

The number of colonies in twenty microscopic fields is counted and 
the number of colonies on the entire plate calculated. This number 
multiplied by the dilution factor gives the number of bacteria per cubic 
centimeter of milk. The magnification used should be from 100 to 200 
diameters. Results of thirty-seven comparative tests are given. The 
number of bacteria in these milks varied from 675 to 20,750,000 per 
cubic centimeter. 

The correspondence seems reasonably close. The difference between 
the two counts usually amounts to less than the differences which occur 
between duplicate plates or the counts obtained by means of different 
dilutions in the same analysis, or the counts obtained on the same milk 
by different analysts. 

The preparation of the plates requires less time than the preparation 
of the standard plates, the staining and counting a trifle more. No 
expensive apparatus is required. The amount of culture medium is 
very small. The time required to complete an analysis is never more 
than twelve hours and in many, if not most, cases can be reduced to 
four or five hours. 

Notes on Brine Pickle Fermentation. C. W. Brown. 

In salting cucumbers there may enter the tank many types of micro- 
organisms; yet only those that can tolerate 12 to 20 per cent salt are 
concerned in the normal fermentation. The acidity of new brine is 
practically zero and increases gradually during two to six weeks to 50 
per cent fo or above — a maximum of 75 to 100 per cent. The 
principal acids are lactic and acetic in ratio of approximately 2: 1 with 
traces of propionic, butyric, benzoic. During fermentation gases are 
evolved; the volume is equal to approximately one-half the volume of the 
tank and consists chiefly of carbon dioxid — 80 to 90 per cent; the samples 
contained no hydrogen, no oxygen, a trace of methane and a residual gas, 
presumably nitrogen. In the samples of brine analyzed alcohol was 
found in traces only. 

The acid bacteria are facultative anaerobes, short rods or cocci ar- 
ranged chiefly in chains of 2 to 5 members, they produce acid from glu- 
cose and lactose, litmus milk after a time is rendered acid but is not 
loppered. The ability to produce gas is questioned in that there is 
evidence of the strains isolated producing sufficient gas to saturate 
or nearly saturate the liquid medium. The gas formed during pickle fer- 
mentation is produced largely by yeasts, which can tolerate the high 
percentage of salt: However, motile short rods — colon type — may be 
isolated frequently from brine during the first stages of the fermen- 



ABSTRACTS 106 

tation and these bacteria produce hydrogen in no less quantity than one 
part to three parts carbon dioxid. 

The scum yeasts or torulae which begin to develop upon the surface 
of the brine during fermentation and later form a thick scum are 
acid consumers. In wide mouth bottles plugged with cotton the acidity 
of a 10-inch column of pickle brine was reduced from 74 per cent t^ to 17 
per cent in 45 days at room temperature and to alkaline 12 per cent in 
less than a year's time. Sterile pickle brine in test tubes inoculated 
with pure cultures of the scum yeasts was reduced in acidity from 35 
per cent w to neutral or alkaline within 30 days time at 20°C. The 
first acid to be consumed is the lactic, leaving the acetic until the last, 
that is, the ratio changes until the acetic is the predominant or only acid. 
Tubes of pickle brine agar or even tubes of litmus agar to which sterile 
commercial lactic acid is added — as much as 200 per cent t^ (1.8 per 
cent pure acid) — when inoculated with these scum yeasts are rendered 
neutral or alkaline adjacent to the growth of the yeast within a few 
days' time. Under similar conditions acetic acid is consumed with 
difficulty. 

The fermentation of brine pickles is an associative action of various 
microorganisms resulting in (1) the using up of those constituents of the 
cucumber which may be used readily as microbial food — protein made 
soluble, sugar changed to acid, etc. — ; and (2) in preservation of the 
depleted cucumbers (brine pickles) in the brine containing the by- 
products. When the acidity which is a potent factor in preservation 
is destroyed from the surface of the tank downward by the scum yeasts 
the brine pickles are liable to decomposition. 

Sampling Milk for Bacterial Analysis. Robert S. Breed. 

In the series of comparative studies on the plate and microscopical 
methods of counting bacteria in milk which are in progress at the New 
York Agricultural Experiment Station, some tests of methods of samp- 
ling have been carried out. In these tests, comparative counts have been 
made in order to discover whether samples of milk taken in clean test 
tubes containing preservatives (formaline or corrosive sublimate) were 
as satisfactory for use in making microscopical counts as iced samples 
taken in sterile tubes. The results secured are not sufficient to warrant 
a positive statement but indicate that samples taken with preservatives 
are as satisfactory as are the iced samples and much more convenient 
to handle. When an effort was made to keep the preservative samples 
for days or weeks, it was discovered that they became less satisfactory, 
the longer they stood. This was not because the organisms lost their 
staining power or because of any growth of organisms in the samples 
but because the bacteria floated to the top with the cream which 
became compact on standing. Some of them also fell to the bottom. 
Because of the fact that it was impossible to shake a sample so as to 
break up both the cream and the sediment perfectly, the counts secured 
from the samples after standing tended to be lower than they should 
have been. 



106 ABSTRACTS 

Counts made from the cream and sediment of both iced and preserva- 
tive samples showed that this concentration of the bacteria in the cream 
and in the sediment occurred in all of the samples. Where no cream 
was present as in skim milk, the bacteria did not rise to the surface but 
sedimented in large numbers showing that the reason for their con- 
centration in the cream was because they were buoyed up on the fat 
drops. In the samples studied, there was a strong tendency for the 
larger clumps of bacteria to concentrate in the cream, the bacterial 
groups which occurred in the sediment rarely consisting of more than two 
individuals. 

The Pasteurization of Dairy By-products. Robert S. Breed and 

W. D. DOTTERRER. 

In some work done for the New York State Commission for the 
Investigation of Bovine Tuberculosis during the summer and fall of 
1915 on the pasteurization of whey, it has been found that whey, 
heated between 140 and 180°F. and allowed to cool slowly in the whey 
tank, sours with an almost pure lactic acid fermentation due to lactic 
acid bacilli belonging to the Bacillus bulgancus group. Immediately 
after heating the numbers of bacteria in the raw whey are reduced from 
millions to tens of thousands per cubic centimeter. During the 18 to 
20 hour period which elapses before the whey is returned to the farmers 
and during which time it is cooling slowly, there is a rapid growth of the 
lactic acid bacilli which have survived the heating so that the whey 
contains from tens to hundreds of millions of these organisms per cubic 
centimeter as delivered to the farmers. The other types of bacteria 
present (largely spore forming bacilli) do not increase in number to 
any marked extent. The acidity of the whey as delivered to the far- 
mers was found to varj^ from 0.3 to 0.4 per cent calculated as lactic acid. 

On the other hand the unheated whey which was examined showed 
an acidity of 1.2 per cent and contained several million miscellaneous 
bacteria, one and a half billion lactic acid bacilli and about thirty 
million yeasts per cubic centimeter. Neither of the latter developed 
on the agar media and would not have been found if the micro- 
scopic method of counting had not been used. No heated whey was 
found which contained yeasts, a condition which suggested that the 
improvement in the quality of cheese frequently noted where pasteuri- 
zation has been adopted has arisen from the elimination of yeasts from 
the whey tank and so from the farmers' milk cans. 

On two successive days at one of the factories, the predominant 
lactic acid organism in the making vat was found to be a bacillus 
instead of the more common Streptococcus. 

The Effect of Air Pressure on Potable Waters During Stora^ge. W. D. 

Frost and Freda M. Bachmann. 

Steel pressure tanks are in common use for storing water. The 
question is raised whether or not the effect of the air under pressure 
in these tanks could be injurious to the contained bacteria, or, in other 



ABSTRACTS 107 

words, whether these plants could be depended upon to improve the 
water if it came from a contaminated well. 

Experiments conducted with small quantities of water (2 to 3 liters) 
held in steel chambers at varying degrees of pressure up to 100 pounds 
per square inch, and at the temperature of a warm room, showed that 
the contained bacteria increased very rapidly and to an enormous 
extent. B. coli was not affected by the pressure. 

When similar samples were held at the temperature of an ice box, 
but otherwise under the same conditions, the growth was marked but 
slow. When water held under similar conditions in the ice chest was 
partially renewed at intervals of 24 hours by pouring out half the water 
and putting in fresh, the number of bacteria appeared to remain nearly 
constant. 

An examination of several plants in actual operation showed that 
the water in these tanks remained practically constant so far as their 
bacterial content was concerned. 

The Bacterial Content of Market Oysters. Fred Berry. 

Eight samples of shell oysters and twenty-one samples of shucked 
oysters, collected from eighteen different retail markets in Chicago 
were examined according to the methods recommended by the Committee 
on Standard Methods of Shell-fish Examination. The first sample 
was collected October 13 and the last April 27. Additional tests were 
made to determine whether the use of- the combined shell liquor of 
fifteen oysters, as recommended by Smith, would necessitate a different 
interpretation from that based on the analysis of five individual oysters, 
as recommended by the Standard Methods Committee. A few samples 
of shucked oysters were re-tested after storing in the ice-box for forty- 
eight hours to determine the character of the bacteria which multi- 
plied most rapidly under such conditions. 

Of the eight samples of shell oysters, three contained an excessive 
number of B. coli. These were collected October 13, October 23, and 
March 2, and had a score of 41,140, and 120, respectively. The other 
five samples were collected in February and four of these had a score 
of 0, one a score of 23, and the other a score of 5. The lowest count 
on the shell oysters was 2600 on a sample collected February 8, and the 
highest was 7,740,000 bacteria per cubic centuneter on a sample col- 
lected March 2nd. 

Of the twenty-one samples of shucked oysters, none was free from 
B. coli. The minimum was 1 and the maximum 40,000 B. coli per 
cubic centimeter of oyster liquor. Fifteen of the twenty-one samples 
contained 100 or more B. coli per cubic centimeter. Eight of these 
fifteen contained 1000 or more B. coli per cubic centimeter. The 
count on shucked oysters varied from a minimum of 140,000 to a maxi- 
mum of 34,000,000 bacteria per cubic centimeter of oyster liquor. 

These results may be summarized as follows: 

1. The shell oysters purchased at Chicago during February contained 
fewer bacteria than those purchased in October and March. 



108 ABSTRACTS 

2. The season of the year apparently had Uttle influence on the 
character of the bacterial content of the bulk oysters, a majority of 
the samples containing a very large number of bacteria, many of which 
belonged to the B. coli group. 

3. No definite correlation existed between the total number of bac- 
teria and the number of gas formers found in the samples. 

4. The use of five or fifteen shell oysters for a sample did not materi- 
ally affect the interpretation as to the sanitary quality of the sample 
when judged by the U. S. Standards. 

5. On the basis of the bacteria developing on plain agar at 20°C., 
on Endo medium at 37°C., and the presumptive test for B. coli, the 
increase in bacteria in bulk oysters during 48 hours storage in the ice- 
box cannot be interpreted as being due mainly to an increase in intestinal 
bacteria. 

Normal Fermentation of Sauerkraut. Lester A. Round. 

The fermentation of sauerkraut was studied in two factories. In 
the first factory, microscopic and chemical examinations were made 
while in the second factory a bacteriological study was also made. 
The microscopic examination showed that bacteria alone are concerned 
with the proper fermentation. Wherever air came in contact with the 
kraut or brine, as at the top of the vat, yeasts grew ver}^ rapidly after 
the first week and produced a heavy foul-smelling scum which rapidh- 
destroyed the acid. Analysis of fresh juice from a vat just being 
filled showed the presence of five million bacteria, 80 per cent of which 
were glucose fermenters. The remaining 20 per cent were mainly, 
if not all, yeasts. The high count was due chiefly to the refilling of 
tanks which had just been emptied and the walls served as a means of 
inoculation with acid-producing organism. It was found that in the 
first 24 hours the plate count would go up to about 100,000,000. Dur- 
ing the first week it would go up gradually to 200,000,000 to 300,000,000. 

The rate of growth of bacteria and the rapidity of fermentation 
varied directly with the temperature and were much slower in cold 
weather than in warm. After reaching a maximum, the number of 
bacteria gradually decreased until at the end of five weeks there were 
present between four and ten million viable organisms. Lactose-bile 
fermenting organisms were found in small numbers at the start. These 
increased rapidly for the first few days and disappeared rapidly after 
the kraut showed an acidity of plus 7.0. These organisms probably 
came from the wagons, forks and shoes of the farmers who brought 
in the cabbage. Examination of the interior of the cabbage-head 
showed it to be sterile. 

Vats showing abnormal fermentation contained a different class of 
organisms. A study of such vats indicated that bad fermentations in 
a properly salted vat were due to the growth of unfavorable organisms 
during the first few days before the normal acid flora had been able to 
establish itself and produce sufficient acid to stop decomposition. In 
the course of normal fermentation there was found to be a slight in- 
crease in the temperature. 



ABSTRACTS 109 

A Study of the Effect of Spices on the Growth of Certain Organisms. 

Freda M. Bachmann. 

A study of the preservative effect of spices in foods was made in 
order to determine the relative efficiency of the different spices in 
inhibiting the growth of microorganisms. The molds used for inocu- 
lation were the common ones found on spoiled fruits and vegetables, 
species of Rhizopus, Penicillium, Aspergillus, and Alternaria. Of 
the bacteria, B. coli, B. subtilis, and B. prodigiosus were studied. A 
yeast isolated from Fleischman's compressed yeast was also used for 
inoculation. The molds and yeasts were grown on Thaxter's potato 
hard agar and the bacteria in the ordinary nutrient agar. 

A new method for obtaining a double plate was devised in which 
the agar without the spice covers one-half of a petri dish and that 
with spice the opposite half. In this way the organisms may be grown 
on two kinds of media in one plate. The organisms were grown in such 
double plates, also in spiced agar slants, and on steamed apples to 
which varying amounts of spice were added. Besides the study of the 
effect of ground spices, the alcoholic extracts, the active principles, 
and the oils were used. 

Cinnamic aldehyde is most effective in preventing growth of all the 
organisms studied. Eugenol and oil of allspice also have a considerable 
preservative effect. Nutmeg is of little value as a preservative and 
black pepper and ginger have practically no effect. It was found that 
there is considerable variation in the sensitiveness of different organisms. 
Molds were found to be more sensitive than the bacteria and yeast. 
There is a very considerable difference in the amount of spice necessary 
to prevent germination of mold spores and the amount necessary to 
inhibit a growth of the mycelium. The results of this study for the 
most part confirm those of Hoffman and Evans in their work on spices 
as preservatives. 

SANITARY BACTERIOLOGY 
Under Supervision of Henry Albert 

Influence of Conditions in the Barn Upon the Germ Content of Milk. 

M. J. Prucha and H. M. Weeter. 

The aim in this study was to measure the collective influence of the 
barn conditions and operations on the bacterial contamination of milk. 
Pails were steamed before each milking and the samples of milk for the 
analysis were taken from individual cows when the pail of milk was 
brought out from the barn into the adjacent milk room. 

The study was conducted from March to July in 1914 and 1915 in 
three different barns. Barn I was very clean, barn II was not as clean 
as barn I, and barn III was decidedly dirty. 1710 samples were taken 
in all. The results are summarized in the following table: 



110 



ABSTRACTS 





ATEBAGB NUMBEB OF BACTEBIA OF ALL SAMPLB3 




1914 


1915 


I 

II 
III 


2,288 
1,073 
6,604 


3,229 

873 

5,255 



Table showing the lowest and the highest counts and the average of fifteen samples 

from each cow, 1914 



BARN I 






BABN 


II 


BABN III 


No. 


Bacteria per 1 cc. 


No. 


Bacteria 


per 1 cc. 


No. 


Bacteria per 1 cc. 


of 








of 
cow 








of 
cow 








COW 


Average 


Low- 
est 


Highest 


Aver- 
age 


Low- 
est 


Highest 


Average 


Low- 
est 


Highest 


174 


183 


17 


532 


170 


222 


27 


582 


1,034 


2,667 


307 


20,365 


135 


325 


47 


1,215 


116 


265 


50 


815 


1,019 


2,748 


563 


7,285 


189 


387 


50 


2,502 


166 


295 


53 


890 


1,031 


3,150 


1,088 


9,725 


167 


444 


45 


1,780 


123 


329 


3 


1,078 


1,025 


4,320 


970 


22,146 


150 


506 


50 


1,457 


165 


356 


45 


1,087 


1,033 


4,598 


855 


18,520 


187 


585 


247 


1,932 


159 


373 


63 


920 


1,032 


4,603 


1,390 


9,275 


171 


602 


272 


1,480 


557 


603 


480 


820 


1,018 


5,324 


2,120 


13,735 


155 


613 


40 


2,560 


552 


635 


63 


4,025 


1,015 


6,414 


1,453 


12,108 


63 


657 


72 


3.257 


113 


636 


100 


1,307 


1,003 


13,120 


3,425 


29,800 


110 


665 


245 


1,300 


145 


657 


52 


1,860 


1,026 


19,092 


7,480 


63,835 


177 


758 


82 


1,783 


125 


698 


50 


4,373 










35 


723 


40 


5,705 


550 


735 


30 


4,862 










130 


751 


77 


2,760 


108 


770 


31 


2,247 










176 


826 


332 


1,323 


163 


813 


11 


2,530 










156 


763 


281 


2,117 


149 


966 


32 


5,075 










182 


a33 


282 


3,425 


553 


1,045 


108 


2,012 










179 


837 


103 


2,760 


556 


1,117 


92 


4,444 










26 


872 


140 


3,800 


117 


1,217 


70 


4,025 










192 


888 


242 


2,182 


551 


1,258 


44 


3,250 










73 


927 


192 


3,627 


554 


1,292 


107 


4,925 










74 


931 


97 


4,850 


137 


1,369 


433 


2,587 










178 


925 


412 


1,686 


183 


1,613 


225 


6,612 










186 


1,042 


347 


2,400 


131 


1,878 


232 


4,675 










152 


1,044 


90 


8,060 


175 


2,425 


1,362 


3,450 










190 


1,140 


337 


6,225 


118 


2,752 


11 


12,955 










134 


1,164 


167 


3,395 


555 


3,588 


29 


33,000 










154 


1,307 


192 


4,157 


















184 


1,391 


135 


6,275 


















112 


2,010 


342 


6,900 


















191 


2,213 


320 


10,135 


















180 


2,529 


132 


28,950 


















172 


3,874 


937 


8,505 


















188 


5,231 


67 


58,275 


















111 


6,835 


3,095 


15,812 


















55 


35,131 


2,255 


218,250 




















2,288 








1,073 








6,604 







Relation of Bacteriology to City Milk Standards. H. A. Harding. 

Standards presuppose something to be measured and measure- 
ments presuppose comparison. 

Satisfactory city milk standards should furnish a basis for accurately 
comparing the various milks which may be analyzed from three essen- 
tial standpoints: (1) food value, (2) freedom from disease germ.s. 
(3) cleanliness. 



ABSTRACTS 111 

Food value. Bacteriology bears no direct relation to food value. 
It might bear an indirect one if high term content was accompanied 
by a lowering of food value. In commercial milk this reduction is not 
appreciable, except as induced acidity interferes with certain uses of 
milk. 

Freedom from disease germs. Bacteriology has everything to do 
with this feature, but practically we have no method of determining 
the presence of such germs and protection must be sought through 
omnibus methods such as pasteurization. Pasteurization control is 
mainly through time and temperature. 

Cleanliness. Added uncleanliness is probably best measured by 
bacteriological counts if they are made at the time of infection. How- 
ever, as soon as the elements of time and temperature enter, such counts 
no longer indicate the character or extent of contamination. 

Conclusion. Quantitative bacterial standards of 1,000,000 or any 
similar number do not throw any hght upon two of the three elements 
which are important in judging a milk supply, and unless the age and 
temperature history of the milk is known they do not give any important 
information regarding the third element. 

Purification of Sewage by Aeration in the Presence of Activated Sludge. 

Edward Bartow. 

By blowing air into sewage then allowing the suspended matter to 
settle and decanting the supernatant Hquid, adding fresh sewage and 
repeating the operation, there is accumulated sludge which has the 
property of purifying sewage, in the presence of air in from four to 
five hours. The sludge obtained contains more nitrogen than sludge 
obtained by any other method of sewage purification. It has been 
shown by analyses and by experiments with growing plants that it is 
valuable as a fertilizer. By the process a bacterial reduction of 95 to 
99 per cent is effected. The cost of the process depends upon the cost 
of producing air. It has been estimated that it will be the most effec- 
tive and most economical method of sewage purification. This will 
be especially true if the sludge can be readily recovered and disposed 
of for use as a fertihzer. Plants of considerable size have been con- 
structed at Milwaukee, Cleveland and Champaign and the process 
will be given a thorough trial. 

Diphtheria Diagnosis by means of Blood Serum containing Potassium 

Tellurate. Will Shimer. 

The medium for diphtheria cultures devised by Conradi and Troch 
has not been generally adopted apparently for two reasons; first, the 
tellurite salt instead of the tellurate salts has been used by most workers, 
second, the Conradi medium was first recommended as a color differ- 
entiating medium as well as an inhibiting. The color differentiation 
medium is now believed to be of Httle help. 

The Bacteriological Laboratory of the Indiana State Board of 



112 ABSTRACTS 

Health has used three dilutions of potassium tellurate: e.g., 1.4, 1.5, 
and 1.6 cc. of a 1 per cent solution of freshly made up potassium tel- 
lurate for each 100 cc. ordinary Loeffler blood serum. 

Three hundred and eleven parallel diagnostic cultures on ordinary 
Loeffler's blood serum and the same number containing 1.6 cc. potassium 
tellurate per 100 cc. were made. This dilution of potassium tellurate 
medium gave 2 per cent less positives than the Loeffler's blood serum. 
Of 246 parallel diagnostic cultures on ordinary Loeffler's blood serum 
and the same number containing 1.4 cc. potassium tellurate per 100 
cc, the dilution of potassium tellurate medium gave 1.2 per cent less 
positives than did the Loeffler's blood serum. Of 890 parallel diagnostic 
cultures on ordinary Loeffler's blood serum and the same number con- 
taining 1.5 cc. potassium tellurate per 100 cc. the potassium tellurate 
dilution medium gave 2 per cent more positives than the Loeffler's blood 
serum. 

The increased number of positives obtained with the potassium 
tellurate medium is not by any means a measure of the complete 
advantage of this medium. Smears made from the potassium tellurate 
medium contain fewer bacteria, and their use decreases the time neces- 
sary to examine the microscopic slides almost half and lessens the work 
of getting pure cultures enormously. 

The Number of Bacteria in the Air of Cow Stables. G. L. A. Ruehle. 

In the course of an investigation of the air as a source of bacteria in 
milk which has been made at the New York Agricultural Experiment 
Station, it was necessary to make a large number of analyses of stable 
air under a variety of conditions. Altogether 1130 separate analyses 
of air samples were made but since many of them were duplicate 
analyses or were made under artificial conditions only 402 analyses are 
summarized in the work reported upon here. Of these 344 were made 
in the Station stable and 58 in commercial dairy stables. The aero- 
scope used in the majority of cases was a simple modification of the 
sand filter aeroscope recommended by the Committee on Standard 
Methods for the Examination of Air. The modification was of such 
a nature as to permit dry sterilization, at the same time eliminating 
some of the joints where leakage might occur. 

The average germ content of the air in 344 tests was 115 per liter. 
The lowest number of bacteria was found, as would naturally be ex- 
pected, when the barn was empty and everything was quiet. Sixt}' 
analyses made under these conditions showed an average of 41 per 
liter. The highest average numbers were found after milking was 
finished and silage was being fed. This caused the cattle to move 
about, stirring up an evident dust. Ten analyses taken under these 
conditions showed an average of 271 per liter. Individual tests among 
the 344 analyses gave results varying from to 825 per liter. The 
germ content of the air of thi-ee commercial stables was found to be 
similar to that of the Station stable except that four analyses taken 



ABSTEACTS 113 

under dusty conditions occasioned by feeding hay or corn stalks gave 
noticeably higher figures than any of those recorded above. The 
results of these four analyses were 1100, 2400, 3957 and 16,070 per 
liter respectively. 

From the foregoing results, it is evident that the air of dairy stables 
contains many more bacteria than have been found by Winslow and 
Browne {Monthly Weather Bureau, 42: 452^53, 1914) in country air, 
city street air, offices, factories and schools. This is not surprising as 
relatively dusty operations such as feeding dry hay, grain and the 
like must be carried out several times daily in every cow stable. In 
spite of this fact, it must not be concluded that air plays a great part, 
numerically, in the contamination of milk by bacteria. The studies 
made in order to discover the relative importance of this factor in 
milk contamination have shown that the air is a relatively unimportant 
source of bacteria in milk. The detailed results of the latter investi- 
gations are published in Bulletin 409 of the N. Y. Agr. Exp. Sta. which 
has just been issued. 

Validity of Presumptive Tests. W. F. Monfort. 

Each presumptive coli test proposed from time to time has been 
first applied locally. Its extension to other regions and other classes 
of waters has developed certain limitations. None has proved of 
universal application. It is therefore of first importance that the saving 
clause of Standard Methods, 1912, page 96, be given due consideration 
in evaluating any abridged test for the colon group before its adoption 
with respect to waters of a class or region new to the investigator. 
This discrimination is not always practiced. 

There follow some results of such an evaluation with respect to a 
surface water (Missouri River) from which a turbidity of 1000 to 12,000 
parts per million has been removed; the effluent is treated with bleach. 
Observations covering more than two years show this supply to con- 
tain usually not more than two organisms of the colon group per 100 
cubic centimeters. 

Neutral red bile-salt lactose broth gives positive results in all dilutions. 

Aesculin bile-salt broth, giving negative results with B. cloacae, 
yields a brown coloration with an organism of frequent occurrence 
belonging to the class B. fluorescens. 

Lactose-bile gives an error of over 73 per cent as compared with 
confirmation tests of lactose-fermenting, acid-forming, aerobic bacteria. 

In lactose broth 80 per cent of gas formers fail of confirmation. 

For a water of this class apparently nothing thus far proposed short 
of actual discriminatory tests, at least so far as outhned in the lately 
adopted "standard" for waters used on common carriers in interstate 
commerce, can be considered valid. 



114 ABSTRACTS 

INFECTION AND IMMUNITY 
Under Supervision of A. I. Kendall 

A Study of the Bacteria of Normal and Decayed Teeth. I. J. Kligler, 
Material collected from deposits on teeth of 40 individuals was 
studied with the object of determining the numbers and types of 
bacteria found in such deposits, normally and at various stages of 
decay. Twenty specimens were taken from healthy teeth in mouths 
of different states of cleanliness, and twenty from carious teeth in differ- 
ent degrees of decay. The complete results of this investigation were 
published in The Journal of the Allied Dental Societies, 1915, vol. x, 
pp. 141-166, 282-330 and 445-458. 

Bacterium pyogenes Associated with a Case of Multiple Arthritis in a 

Hog. Archibald R. Ward. 

The writer pointed out that polyarthritis of swine is a condition 
frequently encountered in postmortem inspection of meat. A case 
showing various stages of articular involvement from the early stages 
of synovitis to later stages showing erosion of articular cartilage, exos- 
tosis and anchylosis of the joints was subjected to bacteriological and 
pathological examination. Bacterium pyogenes was isolated in pure 
culture by the method suggested by Ktinnemann. This consists of 
employing agar to which has been added about 30 per cent of sterile 
raw cattle serum, just previous to pouring the plates. The organism 
was also isolated from three abscesses near a joint. The walls of two 
of these abscesses were in contact with the synovial membrane. The 
abscesses contained an odorous pus greenish yellow in color. 

The synovial membrane was highly reddened and was covered with 
vegetations in the form of minute vascularized tufts or tassels. Sec- 
tions of the membrane stained by the Gram method showed organisms 
similar to Bacterium pyogenes within certain cells. 

The organism in question has been found by European investigators 
to be very frequently encountered in chronic suppurative conditions 
in both cattle and swine, observations that have been confirmed by 
the present writer. 

Spirochaeta Hyos. — Its Antigenic Value in Complement Fixation Tests 
on Hog Cholera Sera. Studies on Hog Cholera. Walter E. King 
AND R. H. Drake. 

With antigen prepared from pure cultures of Spirochaeta hyos, 115 
complement fixation tests have been conducted up to the present time. 
Of these, 22 tests were with normal hog sera from 10 different animals, 
1 from an animal which exhibited a reaction only following inoculation 
with virus, 6 tests from 2 convalescent or naturally immune swine, 
84 tests with sera from 34 animals suffering from hog cholera (4 of 
which had been used as normals) and one test each with 2 different lots 
of hyperimmune serum. Negative readings occurred in all cases in 
which normal hog sera were subjected to complement fixation tests. 



ABSTRACTS 116 

sitive readings resulted in all tests with sera from cholera hogs with 
2 exceptions. 

Complement fixation is coincident with chnical symptoms and 
depends upon the virulence of the infecting material and the individual 
resistance of the animal. 

Tests of two convalescent hogs indicate that complement binding 
substances cease to exist in the blood of hogs when immunity against 
hog cholera becomes fully established. 

Control antigens made from cultures of B. choleras-uis, B. Voldagsen 
(Haendel), B. typhi-suis (Glaesser) fail to exhibit complement fixation 
with cholera sera. 

Antigen prepared from pure cultures of Spirochaeta hyos possesses 
no complement binding properties upon sera of hogs suffering from 
septicemia, B. cholera-suis infection, Anthrax, Ghon-Sachs infection, 
brine poisoning, or pneumonia from natural exposure. 

We believe that, by the observance of proper technique, the results 
recorded herein can be dupHcated without difficulty and that the 
method may be used to practical advantage as a reliable, accurate 
means of laboratory diagnosis of hog cholera. Furthermore, the 
results of these experiments support our former conclusions that Spiro- 
chaeta hyos merits serious consideration as an organism possessing 
specific pathogenic properties in relation to hog cholera. 

Antigenic Properties of Autolysed Bacteria. George H. Robinson. 

Meningococci were allowed to autolyse at different temperatures 
for varying periods of time. The filtrates and residues were tested for 
their complement fixing and complement absorbing properties. Only 
after 24 hours in distilled water at 56° is the fixing power of the filtrate 
greater than that of the residue. The fixing power of autolysed sus- 
pensions decreases in proportion to the extent of autolysis indicating 
a degradation of the protein. A small portion of the original antigenic 
substance is obtained in a filtrate after autolysis. A fresh, washed, 
bacterial suspension gives more satisfactory results as an antigen for 
complement fixation tests than an autolysate. 

The Effect on Horses of Feed Heavily Inoculated with B. coli Isolated 
from Oat Hay. Robert Graham and L. R. Himmblberger. 
The occurrence of B. coli or colon-like organisms on grains hap 
been demonstrated numerous times. Recently Rogers, Clark and 
Evans^ pubHshed a report of their studies of colon bacteria on grains. 
They isolated one hundred and sixty-six cultures, of which seventy- 
five were obtained from corn, six from barley, thirty from wheat and 
forty-one from oats. The grains used were secured from the grain 
inspection laboratory and should therefore represent average samples 
grown throughout the different sections of the United States. This 
widespread occiu-rence of colon-like organisms on grains, together with 
the fact that B. coli or colon like organisms appeared to be constantly 

^ Journal of Infectious Diseases, vol. 17, no. 1, 1915. 



116 ABSTRACTS 

present on oats which were proved to be the cause of a serious outbreak 
of a disease commonly known as "forage poisoning," suggested the 
possibihty of some pathogenic or virulent type occurring on grains, 
thus explaining some of the losses occurring to the live stock industry. 

To determine the effect produced by ingestion of strains of B. coli 
isolated at this laboratory, horses were fed a wholesome feed heavily 
inoculated with the isolated cultures of B. coli grown on broth and 
agar media. One horse received agar cultures on corn meal in addition 
to oats which had previously been inoculated. After four days this 
animal developed diarrhea, showed a sluggish attitude, and regardless 
of the amount of wholesome feed consumed lost in weight. A mule 
was fed 200 cc. of broth culture ranging from forty-eight to seventy- 
two hours old twice daily. In this animal loss of appetite occurred 
and the animal became weak and suffered from diarrhea. 

A third horse was fed for eighteen days on oats heavily inoculated 
with B. coli after being frozen for four days at 30°F. and allowed to 
thaw slowly. This animal evidenced an indifferent appetite, was 
greatly depressed and lost in body weight. Another horse was fed 
oats which had been previously inoculated with broth cultures of B. 
coli, with the result that the animal suffered loss in weight. 

In no case were we able to produce death by feeding, but the con- 
dition in the experimental horses was such as to suggest that feeds 
contaminated extensively with colon bacilli lower animal vitaUty and 
render the animal more susceptible to other injury. While most 
investigators consider colon contamination of grains the result of fer- 
tilizing soils with animal fecal matter, some believe that multiphcation 
actually takes place on the grain. For instance, Prescott (cited by 
Rogers, Clark and Evans) found B. coli on grains grown under con- 
ditions which gave no history of contamination with fecal material. 
If this be a tenable view it will account for the occurrence of B. coli 
in greater numbers than can be accounted for by the theory of con- 
tamination. Since the toxins of B. coli have been proven by Vaughn and 
Cooley^ to be intracellular, it follows that the effects observed by us 
must have been produced b,y disintegrated bacterial cells. In this 
connection we desire to mention the effect of daily intravenous injec- 
tions of dead colon bacilli washed from agar slants. Horses so treated 
evinced marked symptoms, shortly after treatment, consisting of profuse 
sweating, uneasiness, increased repiration and exhaustion. In one 
instance death resulted. In most cases, however, the symptoms sub- 
sided in from thirty minutes to four hours after injection, with a notice- 
able increase in tolerance from day to day. 

It is evident from these observations that the occurrence of B. coli 
as isolated from grain which was the causative factor of so-called 
"forage poisoning" bears no primary relation to the disease re- 
sulting from the feeding of the oats, but from a sanitary standpoint 
it seems advisable to protect animal feeds from B. coli contamination 
in so far as possible. 

* Journal American Medical Association, 1901. 



ABSTRACTS 117 

Further Studies of the Presence of and Significance of Agglutinins for 

Bact. abortus (Bang) in Cows' Milk. L. H. Cooledge. 

An application has been made of the complement fixation and ag- 
glutination tests using B. ahartus (Bang) as antigen and replacing the 
blood serum usually tested with milk. The two tests, when applied 
to milk from infected udders have checked closely, with the agglutina- 
tion test a trifle more delicate and reliable. For this reason only the 
agglutination test is reported in this work. 

In every instance where milk direct from the udder was found by 
animal inoculation or cultural methods to contain B. abortus it was 
also found to agglutinate B. abortus. Antibodies were apparently pro- 
duced locally due to a local B. abortus injection as in some instances 
the milk from only one quarter would be positive while in others 
all four might be positive with a negative blood reaction. In other 
instances milk from a quarter would agglutinate the organism when 
the bacterium could not be demonstrated in the milk by animal in- 
oculation. In these instances the agglutinins may have come from 
the blood but the indications are that they were produced locally by 
too shght an infection for the organism to be present in sufficient 
numbers to cause the disease with the 5 cc. of milk used for inoculation 
of guinea pigs. 

The antibodies usually considered as accompanying infection by 
this organism have recently been found in the blood of two men and 
one woman drinking milk from a herd containing infected animals. 
In two other instances these antibodies appeared in the blood of men 
drinking milk that was known to be naturally infected with this organ- 
ism. This method may prove to be another means of safeguarding 
certified and unpasteurized milk. 

This material has been submitted to the Journal of Agricultural 
Research. 

The Behavior of Streptococci of Human and Bovine Origin in the Cow's 

Udder.^ George Mathers. 

Bacteriological observations in many epidemics of acute tonsillitis 
indicate that the causative organism is a virulent hemolytic strepto- 
coccus and that the infection is milk-borne. In epidemics in which 
an infected milk supply is an important factor it becomes necessary 
to determine the source of the bacteria, and the method by which 
they gain entrance into the milk. In the instance of epidemic tonsil- 
litis the question naturally arises whether the udder of the suspected 
cow becomes infected with human streptococci, or whether the organisms 
causing the outbreak represent bovine streptococci that have suddenly 
acquired a heightened virulence for man. From a review of the litera- 
ture it seems probable that hemolytic streptococci derived from bovine 
sources are of httle sanitary significance, and the active factors in 

1 This work was made possible by means of a grant from the Winfield Peck 
Memorial Fund. 



118 ABSTRACTS 

epidemic sore throat are virulent streptococci of human origin. There 
is still some difference of opinion, however, as to the virulence of these 
human types of streptococci for the cow. Davis and Capps^ have 
reported experiments in which they were able to produce mastitis in 
cows by the injection of hemolytic streptococci of human origin into 
the udder, and they have demonstrated conclusively that mastitis 
may exist in a cow's udder without any physical signs being present 
other than the invading bacteria and an increased number of leukocytes 
in the milk. Smith and Brown^ are inclined to believe from their 
studies that the streptococci commonly associated with bovine mastitis 
are different from those found in epidemic sore throat and do not 
cause human throat infections. Moreover they infer that organisms 
of human origin do not cause bovine mastitis but may grow and multi- 
ply in the milk ducts, a condition which might explain outbreaks of 
tonsillitis. During the past year an experimental stud}^ has been made 
of the comparative virulence of human and bovine types of streptococci 
for the cow, along with observations as to the behavior of these organ- 
isms over long periods of time in the cow's udder and the following 
results have been obtained. 

In six instances mastitis has been produced in normal milch cows 
by the injections of small amounts of streptococcus cultures into the 
milk ducts. Streptococci derived both from human and bovine 
sources were used in these experiments. It was found that hemolytic 
streptococci with all the characteristics of the human type may be 
highly virulent for cows when injected into the milk ducts. They 
produce a severe mastitis which may result in an atrophy of the mam- 
mary gland. It was also observed that organisms of this type 
may grow and multiply in the milk ducts without causing any visible 
changes in the udder, but the milk in this instance contained an in- 
creased number of leukocytes and streptococci. Hemolytic strepto- 
cocci from milk and the Streptococcus lacticus may produce an acute 
inflammation of the milk ducts but this change in my experience was 
of a transitory nature and the mammary gland regained its normal 
function very rapidly. In these observations streptococci derived 
from human sources proved to be more virulent for the cow than the 
milk strains. 

In three instances of bovine mastitis all of which were due to hemo- 
lytic streptococci of the human type, there were no noteworthy changes 
in the morphology or cultural characteristics of the invading organisms 
observed in frequent examinations of the milk throughout the course 
of the infections. The distinguishing characters primarily noted for 
each organism are still present, and there are no modifications which 
might be considered as indicating a change from one type to another. 
These infections are still active 304, 272 and 234 days respectively 
after the udders were injected. Also there were no noteworthy changes 

' Jour. Infec. Dis., 1914, xv, 135. 
^ Jour. Med. Res., 1915, xxxi, 455. 
* Mathers, G., Jour. Inf. Dis., 191G. 



ABSTRACTS 119 

in the distinguishing characteristics of the streptococci of the bovine 
type during the course of the corresponding experimental udder in- 
fections. 

It is interesting to note that under experimental conditions the 
quarters of a cow's udder are apparently separate as regards infection, 
hence an examination of the milk from each quarter of the udder is 
necessary before a mastitis can be excluded in the case of a suspected 
cow. 

Bacterial Changes in Uniced Specimens of Water. Henry Albert, 

Jack J. Hinman, Jr., and Gharrett Jordan. 

It is well-known that bacteria tend to multiply rather rapidly in 
water allowed to remain at ordinary room temperature. The purpose 
of this investigation was to determine to what extent reliance may be 
placed on bacteriological examinations of water sent to a distant 
laboratory. 

Examinations of forty different specimens of water of various degrees 
of purity, were made immediately after collection and again at the end 
of 8, 24, 48, and 72 hours. The standard methods of the A. P. H. A. 
were followed. 

The following conclusions regarding the bacteriological findings are 
based on data obtained by a sanitary survey and by both chemical 
and bacteriological examinations: 

1. The usual limit of 100 per cubic centimeter as the total number of 
bacteria on standard agar plates at 20°C. may fau'ly apply to uniced 
samples of water if examined within 8 hours after collection.. 

2. When uniced opecimens of water are not examined until 24 hours 
after collection, the total number of bacteria at 20°C. which may be 
permitted in ''safe" water may be placed at 200 per cubic centimeter 
and if not examined until 48 to 72 hours after collection, at 500 per 
cubic centimeter. 

3. The presence of as many as 50 bacteria per cubic centimeter on 
standard Htmus lactose agar at 37°C. should throw suspicion on water 
examined within 8 hours after collection although a total of 100 may be 
permitted if not examined until 48 hours after collection. 

4. The presence of bacteria producing both acid colonies on standard 
litmus lactose plates and gas in standard broth throws suspicion on the 
water as polluted with sewage material, regardless of the length of time 
that the water has stood after collection. 

5. The total number of bacteria in specimens of water which were 
polluted with sewage material (or probably so) as determined by both 
a sanitary survey and a chemical analysis is so high that it is not safe 
to establish limits of bacterial counts. 

6. It is possible to depend on the results of bacteriological exami- 
nations of uniced specimens of water in a large proportion of cases 
provided the results are properly interpreted in the hght of the sanitary 
survey, the chemical findings and the bacterial changes that occur in 
such specimens of water. 



120 ABSTRACTS 

Further Studies on the Influence of a Lactose-Containing Diet upon the 

Intestinal Flora. Thomas G. Hull and Leo F. Rettger. 

An ordinary bread and lettuce diet to which is added a considerable 
amount of lactose will simplify the intestinal flora of the white rat to a 
single group of organisms — the aciduric group. Milk has the same 
effect but to a less degree. In rats that have been kept on a high pro- 
tein diet, B. Welchii and B. coli are prominent. When lactose is added 
to this diet the process is much the same as before but slower. All of 
the Welch bacilli and most of the colon bacilli disappear within five to 
ten days. The addition of meat to the lactose diet has very little 
effect if the aciduric flora has been previously established. Milk has but 
a slight effect upon the meat flora, probably due to the small amount of 
lactose present. 

Three to four hours after feeding a meal containing dry lactose, sugar 
can be found in suspension for the entire length of the intestine, as well 
as in the feces. If the lactose is in solution when it is fed, it can be 
found as far as the ileum. Thus it is seen how lactose, being slowly 
absorbed, favors the multiplication of the aciduric group. 

The reaction of the intestine apparently has little effect upon the 
flora, the acidity being no greater with the simplified flora than with 
the mixed flora. 

Feeding Experiments with Bacterium pullorum. The Toxicity of In- 
fected Eggs. Leo F. Rettger, Thomas G. Hull and Willl^m S. 
Sturges, Yale University. 

The problem of eradicating ovarian infection in the domestic fowl 
must needs assume still greater importance than heretofore, in the 
light of recently acquired data. Not only is it of the greatest signifi- 
cance to eliminate the permanent carriers of B. pullorum from all 
flocks of fowls from the standpoint of successful poultry breeding, but 
also because they constitute a possible source of danger to man. 

Eggs which harbor B. pullorum in the yolk in large numbers may 
produce abnormal conditions, when fed, not only in young chicks, but 
in adult fowls, young rabbits, guinea pigs and kittens. The "toxicity" 
for young rabbits is most pronounced, the infection usually resulting 
in the death of the animals. In kittens the most prominent symptoms 
are those of severe food poisoning with members of the para-typhoid 
group of bacteria. The possibility of infected eggs causing serious 
disturbances in young children and in the sick and convalescent of all 
ages must therefore receive due consideration. 

Ovarian infection of fowls is very common throughout this country. 
Hence a large porportion of the marketed eggs must be infected with 
B. pullorum. The latter conclusion is warranted by the fact that of 
more than 13,000 fowls which were tested by the agglutination method 
fully ten per cent were positive, and therefore gave unmistakable 
evidence of infection with the organism in question. When eggs 
which harbor B. pullorum are allowed to remain in nests under broody 
hens, or in warm storage places, for comparatively few hours, they con- 
tain large numbers of the organisms. 



ABSTRACTS 121 

Soft-boiling, coddling, and frying on one side only do not necessarily 
render the yolks free from viable bacteria; therefore, eggs which have 
gone through such processes may, like raw eggs, be the cause of most 
serious disturbances at least in persons who are particularly susceptible 
to such influence, and especially infants. 

Studies in Bacterial Nutrition. The Utilization of Proteid and Non- 

Proteid Nitrogen. Leo F. Rettger, William S. Sturges and 

Nathan Berman, Yale Universty. 

In a recent pubhcation by Sperry and Rettger it was shown that 
bacteria are unable to utihze protein nitrogen without the preHminary 
cleavage of the proteins by enzymes, etc., into their relatively simple 
products. Further investigations clearly demonstrate that not only 
unheated (unchanged) proteins resist direct bacterial action, but that 
purified albumin which has been heated to the point of complete coag- 
ulation and sterilization likewise remains unaffected. 

It also appears quite certain that albumoses and peptones are not 
attacked by bacteria, or at the most but feebly, without the aid of a 
proteolytic enzyme, strong acids, alkali, or extreme heat. Organisms 
like B. coli and B. typhi which do not elaborate proteolytic enzymes 
are unable, therefore, to make free use of albumose and peptone nitro- 
gen. This has been shown in culture tests with weak solutions of both 
the untreated and partially purified Witte's peptone. For the determi- 
nation of any possible loss of proteose and peptone, or of albumin, as 
the case may be, the quantitative biuret method as used and recommend- 
ed by Vernon has been employed with considerable satisfaction. 

What is often regarded as autolysis of B. coli and other gelatin- 
non-liquefying bacteria is not a process of digestion of the protein 
constituents of the bacterial cells, since there is no reduction in the 
amount of protein of the medium plus the suspension, and if the protein 
partially disappears from the cells it is due to agencies other than en- 
zymes, as for example small amounts of acid or alkali, and perhaps mere 
washing. 

A proteose or peptone-digesting enzyme, erepsin, has not been de- 
monstrated in any of the experiments. 

Yeasts, Probabhj Pathogenic, Recovered from Routine Throat Cultures. 

Arthur L. Grover. 

In the past various observers have noted the presence of yeast-like 
bodies in smears from the throat but no real attempt has been made 
to study these. 

The present investigation covers ninety-cultures showing yeast- 
like bodies. Fifty-six gave yeast cultures, 3 oidia, 2 leptothrix, 20 
gave molds, and 9 gave no fungus. It is interesting to note that the 
molds and yeasts have identical morphology in the primary smears. 

These 56 yeasts could be divided into 17 distinct varieties as shown 
by the following table: 



122 



ABSTRACTS 



d 


2; 

Q 


OS 

o 
o 

D 

o 


o 

o 

■<! 

O 


o 

% 


on 
O 

> 
.J 


to 
o 

1 


< 


DC 
O 

>:; 

M 


% 

< 

n 

o 


o 

Q 
'A 


S5 


S 
o 

o 
* 


-< 
» 

o 


APPEARANCE ON SLAMT 
AGAR 


1 

2 
3 

4 

5 

6 

7 

8 

9 

10 
11 

12 

13 

14 

15 

16 

17 


* 

gas* 
gas* 


gas* 

gas* 
gas* 

gas* 

gas* 

gas* 

gas* 


* 

gas* 
gas* 

gas* 

* 

* 


gas* 

gas* 


41 

gas* 

gas* 
gas* 

gas* 
gas* 


gas* 
gas* 

gas* 


* 

gas* 
gas* 


gas* 


gas* 
gas* 

gas* 

* 


gas* 

N* 


Yellowish green heaped up, 
confluent 

Thin whitsh film 

Yellowish green, flat, con- 
fluent 

Bright lemon yellow, heap- 
ed up, confluent 

At first white, later pink, 
confluent 

Like sheets of yellow peint 

Waxy white, confluent, 
raised up 

Creamy white, rather flat 
and dry 

Golden yellow, like sheets 
of paint 

Dirty gray, dry film 

White like mass of cream 
cheese 

Greenish yellow, heaped 

At first white, then yellow- 
ish, finally fawn colored, 
confluent 

Salmon pink, confluent 

White discrete colonies 
hirsute 

Gray, thin film. 

Pale yellow discrete colon- 
ies 



• = Acid or top yeast. 

N = Nitrites 

Subcutaneous injection into guinea pigs in eleven cases gave a general 
glandular enlargement. It was possible to recover the yeasts from 
the glands. Eight of these gave a false membrane in guinea pigs when 
rubbed on an abraded surface of the mucous membrane lining the cheek. 
In three cases this membrane extended down over the entire throat. 
The clinical history of the cases from which the yeasts were recovered 
in numerous cases showed membranous angina and the absence of the 
Bacillus diphtheriae. 



ABSTRACTS OF AMERICAN BACTERIOLOGICAL 
LITERATURE 

BACTERIOLOGY OF FOOD 

Effects of Refrigeration Upon the Larvae of Trichinella spiralis. B. H. 

Ransom. (J. Agr. Res., 1916, 5, 819-854). 

This work was planned to show whether the refrigeration of meat 
was a safeguard against the spread of trichinosis. Trichinous meat 
was kept for periods varying from a few minutes up to fifty-seven days 
at various temperatures below the freezing point of water, and then 
after gradual thawing was fed to test animals, generally rats. Re- 
frigeration at temperatures as low as 50°C. for twenty days or longer, 
although not always kilhng the larvae, so influenced them that the 
meat could no longer cause infection. A temperature of 41°C. generally 
killed them in ten days or less. The author concludes that a refriger- 
ation for twenty days at 41°C. may be regarded as always sufficient 
to render trichinous meat safe for consumption. — H. J. C. 

The Bacterial Examination of Sausages and Its Sanitary Significance. 

W. E. Cary. (Amer. Jour, of Pubhc Health, 1916, 6, 124-135). 

The author found that the bacterial content of sausages bears no 
relation to the sanitary conditions of the shop. The average count of 
16 samples taken from shops scored by the author as insanitary was 
24,000 per gram at 37°C. and 2,133,000 at 20°C., while the count of 
18 samples collected from sanitary shops was 241,000 per gram at 37°C. 
and 13,280,000 at 20°C. B. coli was found in 94 per cent of the samples. 
Organisms biologically related to, but not identical with, the enteritidis 
group were present in 25 per cent of the samples, and Proteus vulgaris 
was found in 33 per cent of them. Starch as an adulterant was detected 
in 56 per cent of the samples. Skins used as casings, if properly pre- 
pared, cannot be considered to increase the bacterial content. Cooking 
destroyed from 93.3 per cent to 100 per cent of the bacteria present. — 
D. G. 

BACTERIOLOGY OF SOILS 

Some Factors Influencing the Longevity of Soil Micro-organisms Sub- 
jected to Desiccation, with Special Reference to Soil Solution. Ward 
GiLTNBR and H. Virginia Langworthy. (J. Agr. Res., 1916, 5, 927- 
942.) 

It has been observed in the past that bacteria are able to resist drying 
for longer periods in soil than under other conditions. This has been 
thought to be due to the retention by the soil of moisture in hygroscopic 

123 



124 ABSTRACTS 

form. This cannot be the only factor, however, for the longevity of 
bacteria in various soils is not proportional to the grain-size and hygro- 
scopic moisture. 

Recently Van Suchtelen has succeeded in extracting the soil-solu- 
tion directly from soil. It was found in the course of the present experi- 
ments that if bacteria are suspended in the solution extracted by Van 
Suchtelen's method from a rich clay loam and are then mixed with 
sand and dried, they live longer than if suspended in physiological salt 
solution and then dried under similar conditions. This suggests that 
the reason why bacteria resist drying longer in a rich clay loam than 
in sand is not only because of the greater amount of hygroscopic mois- 
ture present but because there is something present in the soil-solution 
of the loam that has a protective influence upon the bacteria. The 
soil-solution was found by Van Suchtelen to contain a slimy material; 
and the writers suggest that this might be the substance protecting the 
bacteria when dried. — H. J. C. 

A Comparison of the Acid Production of the B. coli Group Isolated from 
Various Sources. W. W. Browne (Amer. Jour, of Public Health, 
1916, 6, 39-48). 

The author undertook this study to determine the amount of acid 
production in various carbohydrate solutions by members of the B. 
coli group, as a guide to the recentness or remoteness of pollution of 
oysters in Narragansett Bay. He found that members of this group 
isolated from either feces or oysters produced their maximum amount 
of acid in lactose and glucose when incubated at 37°C. for 24 hours; 
furthermore that the maximum amount of acid was produced by the 
end of 24 hours. One series of experiments showed that the largest 
amount of acid was produced in the monosaccharides and hexites 
(glucose, levulose, galactose, arabinose, xylose, isodulcite, mannite), 
less in the disaccharides (lactose, maltose) , and least in the trisaccharide 
(raffinose). That is to say the yield of acid varies inversely as the 
complexity of the sugar. The author concludes that the members of the 
B. coli group isolated from feces produce more acid in carbohydrate 
solutions than cultures isolated from oysters, the average differences 
being very slight, but apparently consistent in all the different fer- 
mentable media studied. — D. G. 

Relation Between Certain Bacterial Activities in Soils and Their Crop- 
Producing Power. Percy Edgar Browne. Journal of Agricultural 
Research 1916, 5, 855-869. 

These experiments as a whole represent a line of investigation in 
soil bacteriology which it is believed will ultimately place the subject 
on a more practical basis — a basis which will permit the direct appli- 
cation of the results obtained to the solution of soil-fertility problems. 
The relations between the bacterial activities studied and the actual 
crop yields on these plots have proved so striking and so consistent 
that it was felt that accidental coincidence had been practically elimi- 



ABSTRACTS 125 

nated and the results might be considerd to give a strong indication that 
certain bacterial activities in fields are very closely associated with 
crop yields. Furthermore, the tentative conclusion presents itself 
that tests of such bacterial activities in the laboratory may indicate 
quite accurately the crop-producing power of a soil, or, at least, the 
relative crop-producing power of several soils. If, further, more ex- 
haustive tests confirm these preliminary observations, it may be possible 
to secure advance information regarding the crop-producing power of 
soils by means of laboratory tests of bacterial action in those soils. — 
S. H. A. 

BACTERIOLOGY OF WATER AND SEWAGE 

Predicts Federal Control of Stream Pollution. Earle B. Phelps. 

Eng. Record, 1916, 73, 173^. 

Federal policy needed in the supervision of stream conditions and the 
necessary administrative body to put it into effect. — F. B. 

Air Diffusers Tested at Milwaukee, {Wis.) Sewage Plant. T. Chalkley 
Hatton. Eng. Record, 1916, 73, 255. 111. Sec. Amer. W. W. Assn. 
1916. 

Filtros plate, composed of quartz sand baked, of uniform porosity 
has given the most satisfactory results. The removal of 90 per cent of 
suspended matter, 95 per cent bacteria and an effluent stable for 5 days 
was secured at Milwaukee with the continuous flow tank by using 1.75 
cubic feet of air per gallon sewage with 4 hours aeration, 20 per cent 
activated sludge and from 10 to 15 minute sedimentation. The esti- 
mated cost is S4.38 per million gallon excluding engine room and plant 
attendance, and the cost of disposing of the sludge. — F. B. 

DAIRY BACTERIOLOGY 

Fermented Milks. L. A. Rogers. Bulletin 319, U. S. Dept. Agr. 

A brief resume of our present knowledge of this subject. The 
therapeutic and food value of fermented milk is discussed together 
with the method of preparation of buttermilk, kefir and yogurt. S. H. A. 

The Present Status of the Pasteurization of Milk. S. Henry Ayers. 

Bulletin 342. U. S. Dept. Agr. 

A summary of our present knowledge of the process of pasteurization. 
The subjects discussed are: Meaning of the term pasteurization; 
value of pasteurization; extent of pasteurization in the United States; 
methods of pasteurization; advantages of low temperature pasteuri- 
zation; temperatures and methods most suitable for pasteurization; 
supervision of the process of pasteurization; handling pasteurized 
milk; cost of pasteurizing milk; bacteria which survive pasteurization; 
modern theories of pastemization; and the necessity for pasteurization. 
— S. H. A. 



126 ABSTRACTS 

Agglutination Test as a Means of Studying the Presence of Bacterium 
abortus in Milk. L. H. Cooledge. (J. Agr. Res., 1916, 5, 871-875). 
In testing a large number of samples of milk to determine the pres- 
ence of the causal organism of contagious abortion, the only pre- 
viously proposed technique that proved available was animal inocu- 
lat'on — an unsatisfactory procedure because of the length of time re- 
quired. The writer, has therefore, worked out a method of employing 
the agglutination test, using 48-hour agar cultures of B. abortus as 
antigen. Negative results by this test always indicated absence of the 
organism in question; but positive results did not necessarily prove its 
presence. In making a long series of tests, however, it was found to 
reduce the number of suspicious cases sufficiently so that the use of 
animal inoculation was practical in those few cases in which some 
particular cow's milk did cause agglutination.— H. J. C. 

Study of Condensed and Evaporated Milks. Ida A. Bengston. Jour. 

Home Econ., 1916, 8, 29-33. 

The present extensive use of condensed and evaporated milk products 
makes the proper control of manufacture, and the establishment of 
standards of purity and food value imperative. Meager work has 
been done on the bacteriology of these milk products. The methods 
of preparation of evaporated milk may assure a sterile product. This 
is not true, however, of condensed milk. The bacteria found are chiefly 
those that survive pasteurization, and their number may be as high as 
1,000,000 per cc. The high sugar content of the condensed milk 
inhibits the multiplication of many forms. 

Streptococci, staphylococci, B. sporogenes, lactic acid producing 
bacilli, B. subtilis, B. mesentericus, B. coli, and yeasts have been found. 
— C. M. H. 

DISINFECTION 

Phenol Coefficient of Germicides. F. B. Kilmer, A. W. Clark and P. 

Hamiton. (Jour. Ind. and Eng. Chem., 1916, 8, 45. 

Study of reliability of Hygienic Laboratory method for testing dis- 
infectants. Tests made in two laboratories gave concordant results 
provided following medium was used: Liebig's extract, 3 grams; 
salt, 5 grams; Peptone (Witte), 10 grams; water, 1000 cc; Composition 
of medium important. — I. J. K. 

IMMUNOLOGY 

Anaphylatoxin and the Mechanism of Anaphylaxis. Richard Weil. 

Proc. Soc. Exp. Biol, and Med., 1915, 13, 37-39. 

Precipitin is identical with the antibody effective in passive sensiti- 
zation. Precipitating antibody heated at 72° for one-half hour lost 
its capacity to bind complement in the presence of antigen but still 
retained its sensitizing value as shown by injection into anmials. llie 



ABSTRACTS 127 

conclusion is drawn that complement plays no part in the anaphylactic 
reaction and therefore that anaphylatoxin plays no role in this phe- 
nomenon. — W. J. M. 

On the Mechanism of Anaphylaxis and Antianaphylaxis. J. Bronfen- 
Brenner. Proc. Soc. Exp. Biol, and Med., 1915, 13, 19-21. 
The author regards anaphylaxis as due to toxic split products of the 
normal serum proteins produced by the action of the normal tryptic 
ferment of the blood after the inhibitory influence of the colloids has 
been diminished by the specific interaction of antigen and antibody. 
Antianaphylaxis is explained as the result of antitryptic influence of 
split products of products of proteolysis. — W. J. M. 

Agglutination of Bacteria in vivo; Its Relation to the Destruction of Bac- 
teria Within the Infected Host and to Septicaemia. C. F. Bull. Proc. 
Soc. Biol., and Med., 1915, 13, 32-33. 

Intravenous injection of immune serum causes an abrupt clumping 
of bacteria in the circulating blood in bacteremia and their accumu- 
lation in the internal organs, where they are phagocyted. — W. J. M. 

The Utilization of '^Reactor" Milk in Tuberculo-medicine. C. B. Fitz- 
PATRicK. Proc. Soc. Biol, and Med., 1915, 13, 35-37. 
Cows in excellent physical condition, but reacting to tuberculin, 
were used. Seven patients with moderately advanced pulmonary 
tuberculosis were fed upon their milk and showed improvement as 
compared with control cases on normal milk. — W. J. M. 

Late Results in Active Immunization with Diphtheria Toxin- Antitoxin 

and with Toxin- Antitoxin Combined with Diphtheria Bacilli. W. H. 

Park and Abraham Zingher. Proc. N. Y. Path. Soc, 1915, N. S. 

16, 110-116. 

Individuals giving a negative Schick test before treatment are prob- 
ably immune for life. Those who give a positive Schick test and who 
have been exposed to diphtheria should receive either antitoxin alone or, 
for longer protection, both antitoxin and toxin-antitoxin injections. For 
general prophylaxis against diphtheria, not including immediate con- 
tacts, toxin-antitoxin alone, or with the addition of killed diphtheria 
bacilli, is recommended. The dose is 1 cc. of toxin-antitoxin (85 to 
90 per cent of the L+ dose of toxin to each unit of antitoxin) and 1,000,- 
000,000 bacteria, injected subcutaneously three times at intervals of 
six or seven days. The early and the late results should be controlled 
by the Schick test, early results within four weeks and late results from 
four months to two years after the immunizing injection. — W. J. M. 

Agglutination in Pertussis. O. R. Povitzky ai^d E. Worth. Arch. 

Int. Med. 1916, 17, 279-292. 

The Bordet-Gengou bacillus, grown upon coagulated horse blood 
veal agar, is readily agglutinated by immune serum. An agglutinating 



128 ABSTRACTS 

serum can generally be obtained from rabbits after ten or twelve intra- 
peritoneal injections of living bacilli at seven day intervals. The 
agglutination test demonstrates the unity of the pertussis group and 
differentiates this group from the hemoglobinophihc and pertussis- 
like organisms. In the diagnosis of pertussis a positive agglutination 
test at a dilution of 1 : 200 is necessary, in order to eliminate the pres- 
ence of natural agglutinins. — G. H. R. 

Treatment of Typhoid Fever by Intravenous Injections of Polyvalent 
Sensitized Typhoid Vaccine Sediment. Studies in Typhoid Immuni- 
zation VI. F. P. Gay and H. T. Chickering. Arch, Int. Med. 
1916, 17, 303-328. 

The report deals with the treatment of 53 cases of typhoid fever, the 
diagnosis being confirmed by laboratory examination, with the sensi- 
tized vaccine of Gay and Claypole. The treatment consists of one or 
more intravenous injections of 1/50 to 1/25 milligram of the vaccine, 
or, in some cases, an intravenous injection followed by three subcutane- 
ous injections of 1/10 milligram each. The symptoms following the 
intravenous injections are mild, and the results generally beneficial 
unless the dose is too large. Of these 53 cases, 66 per cent showed dis- 
tinct improvement and 34 per cent were relatively unaffected. The 
curative results are regarded as due to the hyperleukocytosis and the 
increased amount of antibodies induced by the vaccine. In a few 
patients having low antibody (agglutinin) titer the vaccine treatment 
was supplemented by the intravenous injection of considerable 
amounts of typhoid-immune goat serum. The superiority of sensitized 
over non-sensitized vaccine is due to the production of a specific 
hyperleukocj-^tosis. — G. H. R. 

The Mechanism of the Abderhalden Reaction with Bacterial Substrates. 

G. H. Smith and M. W. Cook. Jour. Infect. Diseases 1916, 18, 14-19. 

Bronfenbrenner, working with tissue substrates had arrived at the 
conclusion that the Abderhalden reaction can be resolved into two 
distinct sub-phases; (1) sensitization of the substrate by specific ele- 
ments of the immune serum, resulting in adsorption of antif erments ; 
(2) autodigestion of the serum; he also concluded that only the former 
of these reactions was specific. The present authors attempt to as- 
certain whether the same principles apply to the reaction when bacterial 
instead of tissue substrates are employed. Immune sera were obtained 
from rabbits immunized to (a) typhoid, (b) paratyphoid A, (c) 
Staphylococcus aureus; also serum from control rabbits. The serum 
of each rabbit was combined with its homologous substrate and also 
with the two non-specific substrates. After the serum-substrate 
contacts, the tubes were centrifuged and the sera dialyzed, and tested 
by the Ninhydrin method. The substrates were washed and each 
divided into four parts, to three of which fresh serum from the immunized 
rabbits was added; the fourth received normal serum. Contact in 
cold was allowed for 16 hours, after which the tubes were centrifuged, 



ABSTRACTS 129 

the serum dialyzed and tested. In the first place, each serum after 
having been combined with its specific bacterial substrate, reacted 
positively, the other combinations being negative. In the second place, 
each substrate that had already been so combined with its specific 
serum, upon being subsequently combined with the non-specific sera, 
acted on all of them so as to yield a positive reaction upon dialysis, 
thus demonstrating that this phase of the reaction is due to autodigestion 
of the serum and is non-specific. Whether the sensitization of the sub- 
strate corresponds with the usual antigen-antibody reaction is a point 
left for further study.— P. B. H. 

LABORATORY TECHNIQUE 

On a Colorimetric Method of Adjusting Bacteriological Culture Media to 

any Optimum Hydrogen ion Concentration. S. H. Hurwitz, K. F. 

Meyer and Z. Ostenberg. Proc. Soc. Exp. Biol, and Med., 1915, 

IS, 24-26. 

The indicator is phenolsulphonephthalein 0.01 per cent. The final 
adjustment is made after sterilization of the medium, with aseptic 
technic, the readings being made in a specially devised comparator 
against a standard color solution. — W. J. M. 

The Use of Brilliant Green for the Isolation of Typhoid and Paratyphoid 
Bacilli from Feces. Charles Krumwiede, Jr., Josephine S. Pratt 
AND Helen I. McWilliams. Jour. Infect. Diseases, 1916, 18, 1-13. 
The success of the authors and others in the use of brilliant green 
broth for the enrichment of typhoid and paratyphoid bacilli in feces 
led to the attempt to produce a dye agar. After many trials a 
medium of the following constitution was found to be satisfactory. 
Extract of beef (Liebig's) 3 gm., Witte's peptone 10 gm., salt 5 gm., 
agar 15 gm., water 1000 cc. Dissolve in autoclave; the final reaction is 
set to the Andrade indicator, adding 1 cc. to a 100 cc. bottle of agar; the 
reaction may be set at time of preparation or (preferably) when used. 
If the latter, after dissolving, render slightly alkaline to litmus, bottle in 
100 cc. amounts and autoclave. Just before use, adjust 0.6 to 0.7 per 
cent to phenolphthalein (hot titration) then add to each 100 cc. 1 per 
cent lactose and 0.1 of glucose (25 per cent sterile solutions) and finally 
the appropriate amount (0.2, 0.3 or 0.4 cc.) of a 0.1 per cent solution of 
brilliant green. Use about 16 cc. of agar for each plate, allowing them to 
stand open until agar has cooled. Inoculate as in Endo plates. The 
method of use is as follows: Rub up in extract broth a large sample of 
feces (1: 15 by volume.) Place one loop of suspension on a 0.2 cc. and 
on a 0.3 cc. plate; streak in order given and then on an Endo plate. 
Place two loops on each of a similar pair of green dye plates; streak in 
same order and then on Endo plate. Use a heavy platinum wire looped 
at end. For a direct agglutination test a macroscopic slide method is 
employed. For fishing, the Russell medium, with 1 per cent Andrade 
indicator substituted for litmus is employed. As an added precaution 



130 ABSTRACTS 

it is recommended that there be inoculated from the original fecal sus- 
pension 0.1 cc. into 1 per cent glucose extract broth containing 1 : 300,000 
of the brilliant green. If slight growth develops on the green plates, Endo 
agar is inoculated from the broth tubes after 18 hours. In tests made 
upon carrier and normal stools, and of convalescents prior to discharge it 
was found that many fecal types were restrained while the typhoid bacilli 
developed well. In one instance the positive results were increased 36 
per cent over Endo plates. The method also proved successful for the 
isolation of members of the paratyphoid-enteritidis group from feces. — 
P. B. H. 

MEDICAL BACTERIOLOGY 

The Effect of Continuous Electric Light in Experimental Arthritis. W. E. 

SiMMONDS AND J. L. MooRE. Arch. Int. Med. 1916, 17, 78-81. 

Exposure to continuous incandescent electric light prevented or ren- 
dered less severe experimental streptococcal arthritis in rabbits. When 
the light treatment was begun after the development of arthritis, treated 
animals improved, while control animals continued to develop new 
lesions. — G. H. R. 

Lesions Produced in Rabbits bij Repeated Intravenous Injections of Living 
Colon Bacilli. C. H. Bailey. Proc. Soc. Exp. Biol, and Med., 1915, 
13, 62-63. ... 

Colon bacilli were injected intravenously into rabbits at 3-4 day inter- 
vals over long periods. Animals surviving 88 to 142 days showed fibrous 
and hyaline changes in the kidneys, spleen and liver. In the spleen a 
material resembling amyloid was formed about the Malpighian bodies 
but the amyloid nature of this substance was not conclusively demon- 
strated.— W. J. M. 

Tuberculosis in Infancy. C. H. Dunn, Amer. Jour. Diseases of Children, 

1916, 11, 85-94. 

The author briefly reviews the various opinions that have been held 
concerning the portal of entry and the type of the organism in tuberculo- 
sis of children. The observations recorded consist of twenty-five 
autopsies upon infants under two years of age. The examinations were 
particularly directed toward the lungs and intestines, which were cut 
into small pieces and all suspicious portions sectioned and examined 
microscopically. In twenty-two of the twenty-five cases there was 
found what was regarded as the primary focus and portal of entry. The 
author therefore, disagrees with the opinion that the tubercle bacillus 
may in many cases enter the body and leave no local histological evidence. 
In twenty of the cases the supposed primary was located in the lung and 
in two it was found in the intestine. In only five cases were animal 
inoculations made and the type of organism studied. Four of these 
proved to be human and one bovine. The one bovine culture came 
irom one of the cases in which the primary lesion was located in the 
intestine.— R. M. T. 



ABSTRACTS 131 

The Bacterial Flora of Infected Gun Shot Wounds. — Louis A. LaGarde. 

The Military Surgeon— 1916, 38, 1-6. 

This article is written for the benefit of the military surgeon rather 
than for the bacteriologist, but reviews some of the bacteriological 
work that has been done on wounds in the present war. Thus Flem- 
ing examined 127 wounds and found that the B. Welchii was present 
in 103, Bacillus tetanus in 22, and streptococci in 102 during the first 
week. Gudgeon, Gardner, and Bawtree found that of 100 wounds all 
were infected, 99 with various combinations of aerobic and anaerobic 
bacteria, and one with a pure culture of B. Welchii. The article points 
out that with regard to the bacteriology of gun shot wounds, investigation 
during the present world war has so far resulted in no new bacteriologi- 
cal data.— E. B. V. 

Practical Points in the Prevention of Asiatic Cholera. Allan J. Mc- 
Laughlin, The Military Surgeon, 1916, 38, 22-29. 
McLaughlin quotes literature showing that presumably healthy 
individuals have been proven to harbor cholera vibrios in dejecta for 
periods ranging from 10 days to 69 days, and that Gaffky reported a 
case who was a carrier for 6 months. The carrier question has there- 
fore become one of the most important factors in any endeavor to stamp 
out cholera or prevent the entrance of the disease. These long time 
carriers make a farce of the ordinary 5 day quarantine detention with- 
out stool examination. Instead of this, the present method is to 
examine the stools of all contacts or suspects. In view of the fact 
that prompt diagnosis is essential, and to avoid time consuming ma- 
nipulations where large numbers of people are to be examined, the fol- 
lowing simple method is recommended: Plate on agar after primary 
inoculation in peptone enriching media, and test individual suspicious 
colonies by a macroscopic agglutination on a glass slide, using a very 
powerful cholera immune serum, which will agglutinate cholera in 
dilution of 1-4000. This serum may be used in dilution of 1-200 and 
in this strength will give prompt agglutination with cholera but not 
with other organisms. Goldberger's enriching solutions, an alkaline 
egg peptone and on alkaline meat infusion peptone are mentioned 
with the statement that laboratory tests indicate that they restrain 
the growth of ordinary faecal bacteria while promoting the growth of 
cholera vibrios, but that these media have not yet been tested in actual 
field work.— E. B. V. 

PHYSIOLOGY OF BACTERIA 

Effect of Natural Low Temperature on Certain Fungi and Bacteria. 
H. E. Bartram. (J. Agr. Res., 1916, 5, 651-655.) 
Dried cultures of certain molds, Actinomycetes, and bacteria proper 
were exposed to outdoor conditions at temperatures sometimes as 
low as — 30°C. More than half of the molds survived for four months 
under these conditions, but most of the bacteria died. Control cul- 



132 ABSTRACTS 

tures in the laboratory did not die. Writer does not state whether the 
cultures kept outdoors were exposed to sunlight as well as to cold. — 
H. J. C. 

Effect of Elemental Sulphur and of Calcium Sulphate on Certain of the 
Higher and Lower Forms of Plant Life. Walter Pitz. (J. Agr. 
Res., 1916, 5, 771-780.) 

These experiments were planned because there has been some dis- 
agreement in the past as to whether sulphur compounds increase or 
decrease plant growth. Tests were made to observe the effect of 
elemental sulphur and of calcium sulphate upon: (1) total number of 
bacteria in soil (determined by plate method), (2) growth of pure 
cultures of the organism causing red clover nodules, (3) accumulation 
of nitrates and ammonia in soil, (4) growth of clover in soil and in agar 
culture. The results indicate that elemental sulphur slightly stimu- 
lates the growth of red clover, but has a harmful effect upon all the 
other activities investigated; that calcium sulphate increases the 
growth of the legume organism and the growth of clover, but has no 
influence upon the general soil bacterial flora. — H. J. C. 

The Action of Schumann Rays on Living Organisms. W. T. Bovie. 

Bot. Gaz., 1916, 61, 1-29. 

The source of light was a hydrogen discharge tube, the top of which 
was closed by a transparent fluorite plate through which the Schumann 
rays were emitted. In general a small organism was killed more quickly 
than a large one. The organisms used were rotifers, amoebae, infu- 
soria, Spirogyra and fungus swarm spores. By a number of methods 
it was shown that the action of the light is on the organism directly 
and not indirectly by the formation of a toxic substance in the medium. 
The extreme destructive action of these rays is the result of strong 
absorption. Because of this absorption, the Schumann rays have a 
marked localized action, which gives them a peculiar value for investi- 
gations in the morphology and physiology of the cell. The change 
produced is often one which results in an alteration of the equihbrium 
of the water content of the protoplasm. In the Schumann region of 
the spectrum, as in the region of longer wave length, the destructive 
action of the Ught increases as the wave length decreases, and the light 
of the Schumann region is much more destructive than the light of the 
region of longer wave length. — J. T. E. 

PLANT PATHOLOGY 

A Serious Disease in Forest Nurseries Caused by Peridermium fila- 
mentosum. James R. Weir and Ernest E. Hubert. Jour. Agr. 
Res., 1916, 5, 781-785. 

Peridermium filamentosum Peck has been found to cause a serious 
disease of yellow pine seedhngs at the Savenac nursery located at 
Haugan, Mont. The fact that the same species of Peridermium at- 



ABSTRACTS 133 

tacks both the lodgepole pine and the yellow pine increases the diffi- 
culty of control of this fungus. — S. H. A. 

Sweet Potato Scurf. L. L. Harter. Jour, Agr. Res., 1916, 5, 787-793. 
The scurf disease of the sweet potato was first recognized in 1890 
by Halsted, who named the fungus " Monilochaetes infuscans," a 
new genus and species. He failed, however, to describe either the 
genus or species. The scurf has been found prevalent in nine States 
and sparingly in others, and on 16 varieties of sweet potatoes. The 
organism has been shown by inoculation experiments to be the true 
cause of the disease. A detailed discussion of the morphology of the 
organism is taken up, also its growth on different culture media at 
different temperatures. It was found that the organism on the host 
consisted merely of sporophores and conidia. In the culture, however, 
well-defined branched mycelia and spores developed. — S. H. A. 

Further Studies on Peanut Leaf spot. Frederick A. Wolf. Jour. 

Agr. Res., 1916, 5, 891-902. 

A continuation of work on the fungus diseases of peanuts, the ob- 
ject being to secure information regarding the agencies concerned in 
the distribution of leafspot, Cercospora personata (B. and C.) Ellis, 
and to correlate the destructiveness of the disease with the presence of 
certain climatic conditions. Crop rotation was not found effective 
under field conditions in eliminating leafspot; nor was the disease 
prevented by seed disinfection with copper sulphate or formaldehyde 
before planting. No correlations between the presence of certain con- 
ditions of temperature and moisture and the presence of leafspot 
exist because of the fact that air currents and certain insects are carriers 
of Cercospora personata. — S. H. A. 

Soil Stain, or Scurf of the Sweet Potato. L. J. Taubenhaus. Jour. 

Agr. Res., 1916, 5, 995-1001. 

The economic importance of the disease is discussed, also the occur- 
rence of soil stain, symptoms of soil stain, effect of the disease on the 
host, factors favorable to soil stain development, the cause of soil 
stain or scurf and the morphology and physiology of the fungus caus- 
ing the scurf. The fungus Monilochaetes infuscans was found to be 
difficult to culture because it is a very slow grower and is readily over- 
run by associated saprophytes. The conidiophores of M. infuscans 
are distinct from the mycehum, the older growth of which is also dark. 
The conidia are borne in chains which readily break up when moistened 
or disturbed. — S. H. A, 

Factors Involved in the Growth and the Pycnidium Formation of Pleno- 
domus fuscomaculans. George Herbert Coons. Jour. Agr. Res. 
1916, 5, 713-770. 
This paper gives the result of experiments performed with Pleno- 

domus fuscomaculans, a fungus pathogenic to the apple. The organ- 



134 ABSTRACTS 

ism was found to have a wider range of conditions suitable for growth 
than for reproduction. The quantity of food stuffs necessary for 
growth is extremely minute. Pycnidium production requires more 
food, but the meager amount present in distilled water is sufficient to 
allow the production of a few pycnidia. Magnesium sulphate and 
potassium dihydrogen phosphate in very dilute solutions furnish 
the necessary mineral elements for growth and reproduction. The 
carbon supply may be taken from a wide range of compounds of alco- 
holic structure. Carbohydrates furnish food material in the most 
available form, and of these xylose and maltose produce the best growth. 
The nitrogen assimulation is greatly influenced by the type of carbon 
nutrition. The influence of physical conditions on growth and re- 
production is also shown. The general problem of the paper was to 
study the effect of environmental factors upon Plenodomus fuscoma- 
culans especially as they influenced growth and reproduction. — S. H. A. 



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VOLUME I NUMBER 2 

JOURNAL 

OF 

BACTERIOLOGY 



OFFICIAL ORGAN OF THE SOCIETY OF AMERICAN 
BACTERIOLOGISTS 



MARCH, 1916 




II is characteristic of Science and Progress that they continually 
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PUBLISHED BI-MONTHLY 

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CONTENTS 

C J. T. Doryland: Preliminary Report on Synthetic Media 135 

Max Levine: On the Significance of the Voges-Proskauer Reaction 153 

James M. Sherman: Studies on Soil Protozoa and Their Relation to the 

Bacterial Flora. 11 165 

IH. J. Conn: Are Spore-forming Bacteria of any Significance in Soil under 

Normal Conditions ? 187 

tt. J. ConN: a Possible Function of Actinomycetes in Soil 197 

Bertha van Houten ANthoNy and Clarence V. Ekroth: Practical 

Observations on the Titration and Adjustment of Culture Media 209 

W. L. Owen: A Species of Alcohol-forming Bacteria isolated from the 
Interior of Stalks of Sugar Cane infested with the Cane-borer Diatraea 
saccharalis ^ 235 

Abstracts of American Bacteriological Literature: 

- Animal Pathology 249 

Bacteriology of Water and Sewage 250 

Immunology 251 

Laboratory Technique - • 256 

Medical Bacteriology 257 

Protozoa and other Animal Parasites 266 



The Journal op Bacteriology is issued bimonthly. Each volume will con- 
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PRELIMINARY REPORT ON SYNTHETIC MEDIA^ 

C. J. T. DORYLAND 
North Dakota Experiment Station, Fargo, North Dakota 

THEORETICAL DISCUSSION 

By a "Synthetic Medium" is meant a solution which con- 
tains only compounds of known composition and structure. 
Any medium which includes compounds of unknown composi- 
tion or structure is not a synthetic medium. This paper deals 
both with synthetic nutrient solutions, and, with solid synthetic 
media formed by the precipitation of an agglutinant from com- 
pounds of known composition and structure. In the latter 
case, the agglutinant formed by precipitation from known 
compounds should have a known structure and composition. 
We should properly exclude from the list of synthetic media 
all those which contain substances of unknown composition 
or structure, such as meat extracts, proteins of unknown struc- 
ture, agar and gelatine. In order, however, to illustrate the 
present conception of possible media, the scheme presented 
includes many compounds of unknown composition and struc- 
ture, such as the polysaccharids, tannins, glucosides and pro- 
teins. These compounds which have either an unknown com- 
position or unknown structure are placed in their logical posi- 
tion in the systematic arrangement, because their decomposi- 
tion products are more or less known and because in nature 
they, or their decomposition products, furnish the principal 
source of energy to saprophytic microorganisms, and because 
it may be necessary to fall back upon some of them in order to 
secure media for such microorganisms as cannot utilize media 
made of sunpler compounds. However, media which con- 
tain such compounds of unknown composition or structure 
cannot properly be classed as synthetic. 

' Presented at Seventeenth Annual meeting of the Society of American Bac- 
teriologists, Ilrbana, Illinois, December 28, 1915. 

135 

to 



136 C. J. T. DORYLAND 

It is clear from the results of numerous investigators that 
microorganisms can utilize various carbohydrates, alcohols and 
salts of fatty acids as sources of carbons, both for energy ma- 
terial and cell construction. It has, also, long been known that 
the optical isomers of a substance containing an asymmetrical 
carbon atom behave very differently toward biological agents, 
such as yeast, molds, and bacteria. The classical researches 
of Pasteur showed that Penicillium glaucum, assimilated only 
d-tartaric acid and not the 1-tartaric acid. Recent investi- 
gations have shown, however, that this organism will also de- 
compose the 1-tartaric but less rapidly than the d-tartaric form. 
Likewise there are other organisms, bacteria, yeast and molds, 
some of which prefer a d-form of an isomeric compound while 
others prefer the 1-form of the isomer. Investigations of the 
action of yeast on all the known hexoses has shown that only 
four are fermented, viz., the d-forms of glucose, mannose, galac- 
tose and fructose. Wlien the behavior of different species of 
yeast toward these hexoses is studied, it is found without a 
single exception that any species of yeast which ferments any 
one of the three hexoses, glucose, mannose or fructose, likewise 
ferments all three of them. We know, too, from the work 
of E. Fischer and his associates that certain enzymes which 
are able to decompose certain organic compounds cannot attack 
their opposite isomers. Thus, for example, a-methyl glucoside 
is hydrolyzed by the maltase (a-glucosidase) of yeast, while 
j8-methyl glucoside is hydrolyzed by emulsin (j8-glucosidase) . 
Emulsin does not act on the a-glucoside and maltase has no 
effect on the /3-glucoside. So fundamental is this action that 
the composition of an organic compound may often be deter- 
mined by its behavior in the presence of an enzyme. While 
lack of knowledge concerning the behavior of optically inactive 
compounds toward enzymes does not permit us to speak as 
definitely as we may concerning the optically active, neverthe- 
less it is probable that an enyzme is capable of attacking only 
a certain group, or formation. Such, for example, is the action 
of myrosin upon sinigrin and other sulphur containing glucosides, 
where the change is confined to the sulphur linkage. This 



PRELIMINARY REPORT ON SYNTHETIC MEDIA 137 

property of the enzyme no doubt regulates the abihty of the 
parent cell to utilize a compound, consequently, organisms can 
utilize as a source of energy and carbon for cell construction 
only those compounds whose configuration, or the configuration 
of a radical therein, matches the configuration of their enzymes. 
By commencing with the simplest carbon compounds capable 
of yielding energy we may, by admitting only one energy ma- 
terial at a time build up a series of synthetic media which are 
mutually inclusive and exclusive. That is, those organisms 
which have an enzyme in common can utilize a given compound, 
while those which do not possess the necessary enzyme will 
not develop, providing the compound is not too complex; other- 
wise, some of the decomposition products may have a suitable 
configuration. We find that carbon compounds having a cer- 
tain configuration or certain atomic groups can be utilized by 
all those microorganisms which develop the requisite enzyme. 
Therefore, as far as energy is concerned, it is possible to prepare 
a medium w^hich will allow the growth of only a given number of 
organisms. 

Numerous investigators have shown that bacteria as well 
as molds can utilize ammonia nitrogen. Gerlach and Vogel 
isolated and studied several forms of bacteria which were capable 
of utilizing nitrate nitrogen. It is a well known fact that a 
large number of molds also readily assimilate nitrate nitrogen. 
Renato Perrotti pointed out the fact that certain bacteria were 
capable of utilizing cyanide nitrogen. The assimilation of 
either ammonia, nitrate or cyanide nitrogen was dependent, 
always, upon the presence of suitable energy materials. 

Previous work indicates that the other nutrient elements 
necessary, for bacteria at least, are probably common to all, 
therefore, the development of synthetic media must follow 
two lines of cleavage — first, according to carbon compounds 
required for energy, and second, according to nitrogen compounds 
required for nutrition. 

In order to make the resume complete we need only men- 
tion those bacteria which require a source of carbon for energy 
and utihze free nitrogen, those which obtain their energy by the 



138 C. J. T. DORYLAND 

oxidation of ammonia or nitrite nitrogen, those bacteria which 
obtain their energy by the oxidation of sulphur and those which 
obtain their energy from the oxidation of iron carbonate. We 
are now in a position to summarize the food requirements of 
microorganisms. They need in general the common nutrient 
salts. Their nitrogen requirements differ, some being able to 
utilize ammonia, some nitrate, some cyanide and probably 
some nitrite nitrogen. They differ too in the compounds which 
they use as sources of energy. Among the prototrophic forms 
we find, respectively, those groups which can utilize iron, sul- 
phur, ammonia, nitrite or a non-nitrogenous organic compound 
as a source of energy, while among the saprophytic and para- 
sitic forms we find those which can utilize the non-nitrogenous 
organic compounds as sources of energy, as well as those which 
can utilize nitrogenous organic compounds. If we prepare 
nutrient synthetic solutions containing one of the simplest ni- 
trogen and one of the simplest carbon compounds there will 
grow in that medium only those organisms which can utilize 
both the nitrogen and carbon compounds present. For example, 
a medium which contains the necessary nutrient mineral salts 
and ammonia as a source of nitrogen . with a formate as a source 
of energy will allow the development of only those organisms 
which can assimilate ammonia nitrogen and utilize a formate 
as a source of energy, and for the carbon of its cell construc- 
tion. If an organism is present which can assimilate ammonia 
nitrogen but cannot utilize a formate as a source of energy and 
cell construction it cannot develop. Conversely, if an organism 
is present which can utilize the formate but not the ammonia 
nitrogen there will be no growth. Therefore, starting with 
the simplest nitrogen and carbon compounds we can arrange 
a series of media having a common source of nitrogen and a 
changing source of energy, or vice versa, having a common 
source of energy but a changing source of nitrogen. We have, 
therefore, the possibiHty of developing as many media as the 
product of the number of nitrogen sources multiplied by the 
number of nitrogen free energy compounds. To these may 
be added a large number of media which may be made from 



PRELIMINARY REPORT ON SYNTHETIC MEDIA 139 

compounds containing both energy and nitrogen. For such 
organisms as are not able to develop upon a medium contain- 
ing one of the simplest nitrogen salts, and a non-nitrogenous com- 
pound as a source of energy, we may develop a suitable medium 
by using a solution containing the essential mineral salts to 
different portions of which we add a different nitrogenous com- 
pound until we find a nitrogenous compound upon which it 
will develop. However, we must exercise caution here, particu- 
larly in the employment of complex compounds for mixed floras, 
because as a general rule the more complex the compound the 
greater the number of species which can utilize the compound 
or its degradation products. It is by simplicity of compounds 
that we may hope to control the growth of the great mass of 
saprophytes. 

Possible media may be grouped according to their energy 
requirements as follows: 

THE AMMONIA GROUP 

Ammonia as a source of energy, no other form of nitrogen. 

THE NITRITE GROUP 

Nitrite as a source of energy, no other form of nitrogen. 

THE SULPHUR GROUP 

Sulphur as a source of energy, + ammonia. 

THE IRON GROUP 

Iron as a source of energy, + ammonia. 

THE CYANIDE GROUP 

Cyanide as a source of energy (no other form of nitrogen). 
Cyanide as a source of energy, + ammonia nitrogen. 

NITROGEN FIXING GROUP (Oligonitrophilic) 
Compounds of carbon with hydrogen and oxygen as sources of energy, no 
nitrogen added. 

THE ALIPHATIC ACID GROUP 

The Aliphatic acids as a source of energy, + ammonia nitrogen. 

Monobasic Monobasic hydroxy Monobasic keionic 

Formic Glycolic Pyruvic 

Acetic d-lactic Acetoacetic 

Propionic 1-lactic Levulinic 

Butyric Hydracrylic 
Isobutyric 
Isovaleric 
Normal Valeric 



140 



C. J. T. DORYLAND 



Di-basic 
Oxalic 
Malonic 
Succinic 

Iso Succinic (Methyl 
malonic) 



Unsaturated di-basic 
Fumaric 
Maleic 
Mesaconic 
Citraconic 



Hydroxy di-basic 
Malic 

Tartaric (dextro) 
Tartaric (levo) 
Meso-Tartaric 

Tri-basic 
Citric 
Aconitic 
Tricarballylic 



THE AROMATIC ACID GROUP 

Aromatic acids as a source of energy, + ammonia nitrogen. 

Benzoic Salicylic 

Anisic Gallic 

Tannic Quininic 

Phthalic Amygdalic 

Phenylacetic Mandelic 
Cinnamic 

THE ALDEHYDE GROUP 

Aldehydes as a source of energy, + ammonia nitrogen. 

Formaldehyde Benzoic aldehyde 

Acetic aldehyde Cuminol 

Propyl aldehyde Cinnamic aldehyde 

Butyl aldehyde Salicylaldehj^de 

Butyl aldehyde, iso Vanillin 

Piperonal 

THE KETONE GROUP 

Ketones as a source of energy, + ammonia nitrogen. 

Aceton 
Butanon 
Pentanon 



THE ALCOHOL GROUP 

Alcohols as a source of energy, + ammonia nitrogen. 

Monohydric alcohols 
Methyl alcohol 
Ethyl alcohol 
Propyl alcohol, normal 
Propyl alcohol, iso 
Butyl alcohol, iso 
Butyl alcohol, primary normal 
Butyl alcohol, secondary normal 
Amyl alcohol, iso primary 
Amyl alcohol, active 



PRELIMINARY REPORT ON SYNTHETIC MEDIA 



141 



Polyhydric alcohols Aromatic alcohols 

Ethylene glycol Benzyl alcohol 

Glycerol 
Erythritol 
Arabitol 
Xylitol 
Mannitol 
Dulcitol 
Sorbitol 
Persitol 
Volemitol 
Adonitol 

THE CARBOHYDRATE GROUP 

Carbohydrates as a source of energy + ammonia nitrogen. 

Monosaccharids 
Trioses Tetroses Pentoses 

Glyceric aldehyde d and 1-Erythrose d and l-Arabinose 

Dioxyacetone d and 1-Threose d and 1-Xylose 

1-Ribose 
1-Lyxose 



Methyl Pentoses 
Rhamnose 
Fucose (Rhodeose) 
Chinovose 



Heptoses 
Mannoheptose 
Glucoheptose 
Galacto heptose 

Disaccharids 
Maltose 
Iso maltose 
Gentiobiose 
Cellobiose 
Lactose 
Iso lactose 
Melibiose 
Turanose 
Sucrose 
Trehalose 



Octoses 
Mannoctose 
Glucooctose 
Galactoose 



Hexoses 
Mannitol series 

d and 1-Glucose 
d and 1-Mannose 
Fructose 
Sorbose 

Dulcitol series 
d and 1-Galactose 
d and 1-Talose 
Tagatose 

Nonoses 
Mannononose 
Glucononose 



Trisaccharids 

Mannotriose 

Rhamninose 

Raffinose 

Gentianose 

Melicitose 



Tetrasaccharid 
Stachyose 



Polysaccharids 
Dextrins 
Soluble starches 
Starches, Inulin, 

etc. 
Gums 



142 C. J. T. D DRYLAND 

THE NON-NITROGENOUS GLTJCOSIDE GROUP 

Non-nitrogenous Glucosides as a source of energy, + ammonia nitrogen. 

Phenolic glucosides Oxycumarin glucosides 

Arbutin Aesculin 

Methylarbutin Daphnin 

Phlorhizin Fraxin 

Glycyphyllin Oxyanthraqunone glucosides 

Hesperidin Ruberythrinic acid 

Naringin Rubiadin glucoside 

Iridin Frangulin 

Baptisin Oxyflavone glucosides 

Alcoholic glucosides Apiin 

Salicin Fustin 

Populin Quercitrin 

Coniferin Sophorin 

Syringin Xanthorhamnin 

Aldehydic glucosides Miscellaneous glucosides 

Helicin Saponins 

Salinigrin Digitonin 

Acidic glucosides Digitalin 

Convolvulin Saponarin 

Jalapin Camatambin 
Strophantin 
Gaultherin 

THE ESTER GROUP 

Esters'" as a source of energy, +ammonia nitrogen. 

The entire series, after the first few types, may be repeated 
three times by substituting in place of the ammonia first, nitrite, 
-second, nitrate, -third, cyanide nitrogen. We would have then, 
nitrite-aliphatic-acid group; nitrite carbohydrate group; etc., 
or the nitrite series; and the nitrate aliphatic acid group and 
nitrate alcohol group, etc., or the nitrate series and cyanide- 
aliphatic-acid group and cyanide aldehyde group, etc., or the 
cyanide series. 

Should occasion arise we may exclude all forms of nitrogen 
and carbon from the solution and substitute a series of amino- 
acids, amides, amino compounds, ureides, proteins, nitrogenous, 
glucosides, or cyanogen compounds, and thus build up series 
of media with each respective group. 

* The utility of this group is doubtful, it being probable that if one of the 
constituents of the ester is available to an organism the ester may be also. 



PRELIMINARY REPORT ON SYNTHETIC MEDIA 143 

PREPARATION OP MEDIA 

Conditions 

All water used was double distilled from glass retorts and 
condensers. All chemicals used were the purest the market 
afforded. Each chemical was tested for impurities before use. 
All measures of growth were macroscopic. 

Tests with liquid solutions were made in test tubes. Tests 
with solid media were made in Petri dishes. The incubator 
temperature adopted was 28°C. All media used were made 
neutral to phenolphthalein. The influence of sterilization by- 
heat on composition and structure may be eliminated in most 
instances where an acid is used as a source of energy by pre- 
paring a medium in such a manner that it automatically steril- 
izes itself. This will be explained under preparation of media. 
Whenever this has not been possible the compound whose 
structure is liable to be influenced by high temperatures, has 
been sterilized by itself in neutral aqueous solution and added 
to the other sterile constituents, in correct proportion, by means 
of a sterile pipette. All transfers were made from cultures 
48 to 96 hours old grown on standard agar. 

Solid synthetic media 

It will be necessary to describe the solid media first in order 
to explain the reason for the concentration of the liquid media. 
After numerous trials with starch, cellulose, aluminum hydroxide, 
iron hydroxide and washed agar as agglutinants, it was finally 
demonstrated that silica jelly was the most suitable solid medium. 
The silica jelly was made by a modified "Stevens Temple Meth- 
od." (Centbl. Bakt., etc., II abt., vol. 21, 1908, p. 84.) 

The method consisted essentially of dissolving c. p. KgSiOs 
and c.p. Na2Si03 in water in sufficient amounts to give a con- 
centration of 34.2732 grams of H2Si03 per liter. One half this 
concentration of H2Si03 per liter gives a medium which will 
solidify in approximately five minutes, thus making a medium 
suitable for plating. The mixture of sodium and potassium sili- 



144 C. J. T. DORYLAND 

cate gives us sodium and potassium salts in the final medium 
instead of only sodium salts, thereby lessening the danger of too 
great a concentration of sodium salt. 

The detrimental influence of too great a concentration of 
the sodium and potassium salts can be still further lessened 
by using a mixture of acids; for example, we may use equiva- 
lent solutions of HCl, H2SO4 and H3PO4, thus giving in the 
finished medium chlorides, sulphates and phosphates of both 
sodium and potassium. Experiment demonstrated that the 
''Stevens Temple Method" might be still further modified by 
eliminating the MgCOs or Na2C03. 

The time of precipitation and gelatinization of H2Si03 de- 
pends largely on two factors, first, reaction and second, con- 
centration. Gelatinization is delayed or entirely prevented by 
either an excess of acid or an excess of the Na2Si03 or K2Si03. 
The most rapid gelatinization with any concentration takes 
place in a neutral solution. 

The solid synthetic media were prepared in the following 
manner: Solutions of HCl, H2SO4 and H3PO4 were each stand- 
ardized separately against the Na2Si03 and K2Si03 solution, 
so that 1 cc. of each acid would just neutralize 1 cc. of the sili- 
cate solution. ■ Whenever an organic acid such as formic, ace- 
tic, lactic or tannic, etc., was used as a source of energy it was 
made in sufficient concentration to just neutralize an equal 
volume of the silicate solution. These acids, that is the HCl, 
H2SO4, H3PO4, and the organic acid (let us say acetic) were 
then mixed together in such proportion that the resulting salts 
from the sodium and potassium silicate would be present in 
the final sihcate medium in quantities, approximately, inversely 
proportional to their osmotic action, thus giving a minimum 
osmotic pressure. Before standardizing the HCl there was 
added to it 0.5 gram MgS04, 0.01 gram CaCOj or CaO, 0.01 
gram of Fe2(S04)3 and 0.01 gram of Mn SO4, per hter. Am- 
monia nitrogen was added to the HCl as ammonium sulphate. 
Cyanide nitrogen was added to the HCl as potassium ferri- 
cyanide. Nitrite nitrogen was added to the neutral solution 
in the Petri dish, nitrate nitrogen was added to the acid mixture 



PRELIMINARY REPORT ON SYNTHETIC MEDIA 145 

as HNO3. All the sources of nitrogen^ were added in proportion 
to give one gram of their respective salts per liter. The mixture 
of acids was then placed in a sterile flask plugged with cotton 
and the flask connected with an automatic burette so that the 
burette would fill by siphon. The silicate solution was placed 
in another sterile cotton plugged flask and connected with another 
automatic burette, so that it too would fill by siphon. Each 
burette was allowed to fill and then stand several hours before 
use, so as to sterilize completely the flasks and burettes. The 
overflow cup of the burette was plugged with cotton, to prevent 
contamination from the air during titration. When sterile, 
5 cc. of the acid mixture was added to a sterile Petri dish, after 
which there was added 5 cc. of the silicate solution. The plate 
was then rotated to mix the two solutions thoroughly, and then 
inoculated. If a non-acid compound was used as a source of 
energy (say glucose) a sufficient amount of a sterile aqueous 
solution, to give 10 grams per liter, was added at this point. 
Numerous tests proved both the acid and silicate solutions to 
be sterile in less than one hour. The resulting sihca medium 
was neutral to phenolphthalein and set firmly in approximately 
five minutes. When a compound other than an acid was used 
as a source of energy it was added from a sterile aqueous solution 
to the medium in the Petri dish just before inoculation. The 
growth of organisms upon this sohd medium resulted generally 
in quite typical colonies, although with a few organisms some- 
what peculiar developments took place. 

The liquid media used had a concentration similar to the 
above with the exception that H2Si03 was absent. It is pos- 
sible that some organisms which failed to grow might have 
grown in a lesser concentration. This was not determined 
because the first consideration was to develop solid sj^nthetic 
media, and a more dilute silicate solution would not solidify 
soon enough; therefore the liquid media used as a check for 
the solid media must necessarily be of the same concentration. 
Parenthetically, it may be stated that the results of the liquid 

^ In future experiments, the availability of different salts of ammonia, nitrite, 
nitrate and cyanid must be tested. . , 



146 C. J. T. DORYLAND 

and solid media did not always agree. The acetic ammonium 
medium will give an approximate idea of the composition and 
concentration. 

Acetic-ammonia-silicate medium 

HsSiOs 17 . 1366 

NaCl 1.1620 

Na2S04 2.8209 

NaCsHsOz 1 .6343 

Na2H PO4 4. 2330 

K CI 3 . 2954 

K,S04 7.7024 

K C2H3O2 4.3357 

K2H PO4 11. 5500 

MgS04 0.5000 

CaO 0.0100 

Fe2(S04)3 0.0100 

MnSO 0.0100 

(NH4)2S04 1.0000 

Water double dist 1000.0000 

The acetic ammonia solution agrees with the above, except- 
ing that it does not contain the H2Si03, It may be made as 
follows: Make a solution of KOH and one of NaOH so that 1 cc. 
of each alkali will just neutralize 1 cc. of one of the acids which 
was standardized against the sihcate solution. The KOH 
and NaOH solutions are then mixed in such proportions that 
when the mixture is brought in contact with the acid mixture 
the resulting sodium and potassium chlorides, sulphates, phos- 
phates and acetates are present in quantities inversely propor- 
tional to their osmotic action. The acids may be prepared 
as previously described. The alkah mixture and the acid mix- 
ture may be placed in their respective bottles and connected 
by siphon with automatic burettes. Allow the burettes to fill 
as with the silicate medium and when sterile equal quantities 
of the alkali mixture and of the acid mixture may be run from 
the burettes in a suitable vessel. 

It must be borne in mind that when glucose or an alcohol, 
or any other non-acid compound is used as a source of energy 
the proportion of each of the remaining salts increases. Like- 
wise, the relative proportion changes when another acid, such 
as formic or lactic, is used in place of acetic acid. This fluctua- 



PRELIMINARY REPORT ON SYNTHETIC MEDIA 147 

tion of the chloride, sulphate and phosphate content of the 
medium might be eliminated in some instances^ by adding the 
organic acids in such quantities that the resulting organic salt 
of sodium and of potassium should always contain equivalent 
quantities of these bases. Such a procedure might, however, 
give greater fluctuations in the osmotic action than the former 
procedure. This question, with many others, must be left 
for future investigators. 

For convenience there is given below a concise method for 
making the ammonia-acetate-silicate medium and the ammonium 
acetate solution. These figures give quantities of salts which 
are only approximately inversely proportional to their osmotic 
action. Up to the present time the writer has been unable to 
secure all the necessary ionization constants. 

Weigh out: 8.40 grams of c.p. Na2Si03, 24.00 grams of c.p. 
K2Si03 and dissolve in 500 cc. of distilled water. Dilute HCl 
to a concentration so that 1 cc. of the silicate solutions does not 
quite neutralize 1 cc. of the HCl. Add to the HCl 0.5 gram 
of MgS04, 0.01 gram of CaO, 0.01 gram of Fe2S04 0.01 gram 
of MgS04 and 1 gram of (NH4)2 SO4, and standardize the re- 
sulting HCl solutions against the silicate, using methyl orange 
as indicator, so that 1 cc. is equivalent to 1 cc. of the silicate 
solution. 

Standardize a solution of H2SO4 in the same way, omitting 
the salts. 

Standardize H3PO4 and CH3COOH in a similar manner 
omitting the salts and using phenolphthalein as indicator. 

The acids may then be mixed in the following proportion: 

HCl 153^5 

CH3COOH 153.5 

H2SO4 77.0 

H3PO4 116.0 

One cubic centimeter of this acid mixture will just neutralize 
1 cc. of the silicate mixture, using phenolphthalein. 

* Acids whose solubility in water is so low that solutions, equivalent to the 
silicate solution, cannot be obtained will still further complicate the question. 



148 C. J. T. DORYLAND 

Ammonia-acetate solution 

Prepare a N/0.2578 solution of NaOH and a N/0.6205 solu- 
tion of KOH. Mix these solutions in equal proportions and 
substitute in the place of the silicate solution. The procedure 
from here on is the same as for the silicate medium. 

Results with pure cultures 

It has been the object in working with these media to test 
as many pure cultures of microorganisms upon each medium 
as could be obtained. In this way we shall be able eventually 
to determine all the compounds which each organism can use 
as a source of energy as well as those from which it can obtam 
its nitrogen. This will eventually enable us to group the known 
organisms (yeast and molds as well as bacteria) according to 
their ability to utihze energy material of a given chemical struc- 
ture and configuration. The same thing applies to their nitro- 
gen requirements. When this has been accomplished we may 
then eliminate all media suggested in the above groups that are 
common to a given group of organisms and retain for practical 
use the one which is the most serviceable. Thus eventuall}^ 
the above group of synthetic media will be reduced for general 
use to a few which are mutually inclusive and exclusive with 
occasional employment of others for special studies. 

Several of the media described above have been tested upon 
225 pure cultures of bacteria, 50 cultures of molds and 6 cultures 
of yeast. In the following table will be found the positive re- 
sults obtained from three glucose media and three acetate media ; 
only positive results are given because of lack of space. 

Out of 225 pure cultures of bacteria tested the following re- 
sults were obtained: On the glucose-ammonia medium there 
were 83 positive growths; on the glucose-nitrate medium 70 
positive growths; on the acetate-ammonia medium 25 positive 
growths; and on the acetate-nitrate medium 17 positive growths. 
With the glucose-cyanide medium there were 7 positive growths ; 
and with the acetate-cyanide medium there were 2 positive 
growths. 



PRELIMINARY REPORT ON SYNTHETIC MEDIA 



149 



Positive growths on glucose ammonia, glucose nitrate, glucose cyanide and 
acetate ammonia, acetate nitrate and acetate cyanide media 



NUMBER 


GLUCOSE 


ACETATE 


OF ORGANISM 


NHi 


NO, 


CN 


NH, 


NOs 


CN 


7 




X 










10 














15 




X 










17 




X 










18 


X 


X 










22 


X 


X 




X 






23 


X 


X 




X 






29 


X 


X 




X 


X 




30 


X 


X 






X 




37 


X 












40 




X 


X 








47 


X 


X 




X 


X 




52 


X 


X 




X 






56 














59 






X 


X 




X 


72 




X 










80 




X 










88 


X 


X 




X 






103 


X 


X 




X 






110 




X 










112 


X 


X 




X 






116 




X 




X 






121 




X 










122 




X 




X 






123 




X 




X 






124 


X 


X 










125 


X 


X 




X 






126 


X 


X 










130 


X 












135 


X 


X 




X 






136 


X 


X 










137 


X 


X 






X 




141 


X 












142 


X 








X 




143 




X 


X 








144 


X 












152 


X 


X 




X 


X 




161 


X 


X 




X 






170 


X 


X 




X 






175 


X 


X 










183 




X 




X 







150 



C. J. T. DORYLAND 







GLUCOSE 


ACETATE 


NUMBEB 








OF ORGANISM 


NH. 


NO: 


CN 


NHs 


NOa 


CN 


184 


X 




X 


X 






185 


X 


X 










187 


X 


X 




X 




X 


193 




X 










195 


X 


X 










197 




X 










224 




X 










229 


X 












230 




X 










232 


X 


X 










235 


X 












239 


X 


X 










240 


X 


X 










242 


X 


X 










244 




X 










246 


X 












257 


X 


X 










277 


X 












282 


X 












304 


X 


X 










306 


X 


X 










318 




X 










242 


X 












347 




X 










352 


X 












370 


X 


X 










371 


X 












373 


X 












374 


X 












378 




X 










380 




X 










383 


X 












385 




X 










405 




X 










424 


X 


X 










429 


X 


X 






X 




436 


X 


X 










439 




X 










468 


X 




X 








475 




X 










477 


X. 












478 


X 












487 


X 












491 




X 






1 





PRELIMINARY REPORT ON SYNTHETIC MEDIA 



151 



NUMBEH 


GLUCOSE 


ACETATE 


OF ORGANISM 


NH. 


NO. 


CN 


NH3 


NOj 


CN 


492 


X 












495 


X 












497 


X 












505 


X 












506 




X 










527 


X 


X 










528 


X 












539 


X 












540 


X 












541 


X 












544 


X 












549 


X 


X 










555 


X 


X 










572 


X 


X 










573 




X 










574 


X 












575 


X 












579 


X 












583 


X 


X 










589 


X 


X 










592 


X 












593 


X 


X 










694 














595 


X 












596 


X 












598 


X 












601 


X 












602 










X 




603 














610 














612 










X 




614 










X 




615 














617 




X 










618 










X 





Laboratory numbers have been used purposely because the 
writer is not positive concerning the identity of some of the 
organisms used in the test. It must be borne in mind that the 
above results would be even more striking if the negative growths 
were included. 

Space does not permit the presentation of more data, but the 



152 C. J. T. DORYLAND 

above table demonstrates clearly the possibilities of the pro- 
posed synthetic media. It is apparent at once that the pure 
species of bacteria included in these experiments may be grown 
on some of the above synthetic media and controlled, to a cer- 
tain extent, at least, by changing the energy or nitrogen source. 

SUMMARY 

The above scheme differs radically from the attempts to 
secure a universal medium, i.e., one upon which most bacteria 
of the saprophytic or parasitic groups will grow. It is on the 
contrary an attempt, by the use of definite sources of energy 
and definite sources of nitrogen, to exclude all species but those 
which can use the particular source of energy and nitrogen in- 
cluded in each case. If we are thus able to devise a series of 
mutually inclusive and exclusive media, we shall, after having 
tested the known species upon each, be able to state positively 
that any growth which appears upon any given medium is a 
member or members of a hmited number of types belonging 
to that group (designated by media). Furthermore, we shall 
be able to plate out a soil, a milk, a water, etc., on a series of 
media and know, not only the number of bacteria present but 
also the number of the different groups present. 

The schematic arrangement presented is tentative and is 
limited to water soluble compounds. Many of the groups sug- 
gested may have no energy value or the chemicals used may be 
too toxic or too expensive for practicable purposes. Other 
groups and compounds will no doubt be suggested by further 
study. Likewise, it is possible that subdivisions of the above 
groups may be made by using different sources of ammonia, 
nitrate or cyanide, etc., or by modifying other nutrient con- 
stituents or by changing the reaction. Up to the present time 
16 of the above media have been tested. One using oxalate as 
a source of energy was negative throughout, the others have 
given promising results. It is hoped, therefore, that these 
tentative statements will stimulate needed investigation along 
this line, and criticism is heartily invited. 



ON THE SIGNIFICANCE OF THE VOGES- 
PROSKAUER REACTION! 

MAX LEVINE 

From the Laboratories of the Engineering Experiment Station and the Department 
of Bacteriology of the Iowa State College, Ames, Iowa 

Theobald Smith (1895) first called attention to the ratio of 
the gases evolved in the decomposition of glucose by B. coli 
and its relatives. He pointed out that whereas B. coli produced 
twice as much hydrogen as carbon dioxide, equal volumes of 
these gases were formed by B. aerogenes.^ In consequence of 
the inaccuracies in the determination of the gases in the Smith 
tube, the gas ratio has been generally discarded as a differential 
criterion. However, the comparatively recent work of Harden 
in England, and particularly that of Rogers and his associates 
in the Dairy Division of the United States Department of Agri- 
culture, indicates that the gaseous and other decomposition 
products of glucose, if accurately determined, are of considerable 
importance in the differentiation of coli-like bacteria. 

In a careful quantitative study of glucose fermentation. Har- 
den and Walpole (1905), showed that B. coli evolved carbon 
dioxide and hydrogen in approximately equal volumes, and not, 
as had been observed by Smith, in the ratio of 1 to 2. On the 
other hand, the B. aerogenes formed twice as much carbon dioxide 
as hj^drogen instead of the equal volumes observed with the 
Smith tube. They point out that the difference between the 
gas ratio obtained with the Smith tube and their accurately 
determined ratio is due to the loss of carbon dioxide in the for- 
mer case owing to its solubility in the medium. 

1 Presented at the Seventeenth Annual Meeting of the Society of American 
Bacteriologists, Urbana, Illinois, December 28, 1915. 

* B . aerogenes as employed in this paper, is synonymous with B. lactis aerogenes, 
i.e., those organisms of MacConkey's type IV (sucrose + , dulcitol — ) which 
give a positive Voges-Proskauer reaction. 

153 



154 MAX LEVINE 

The real significance of the accurately determined gas ratio 
was not appreciated until 1914 when Rogers called attention 
to the striking correlation between this ratio and the source 
of the organisms. In three papers by Rogers, Clark and Davis, 
(1914) and Rogers, Clark and Evans (1914 and 1915), it is 
demonstrated quite conclusively that fecal strains of B. coli 
(at least those derived from bovine feces) break down glucose 
with the liberation of carbon dioxide and hydrogen in about 
equal volumes, while non-fecal (grain) strains form two or 
more times as much carbon dioxide as hydrogen. The sanitary 
significance of such a division is evident, but the accurately 
determined gas ratio is inapplicable to routine work. 

Clark and Lubs (1915) note that the gas ratio is correlated 
with the H+ ion concentration and that the difference in H+ ion 
concentration between the low and high ratio groups is such 
that it may be easily recognized by methyl red. When grown 
in appropriate glucose media the low ratio, (fecal) group is 
acid and the high ratio (non-fecal) group alkaline to this indicator. 

As no earlier investigators employed the methyl red reaction, 
the valuable work of these men cannot be compared directly 
or adequately with former investigations, unless some previously 
employed test is found which is well correlated with either the 
gas ratio or the methyl red test. The Voges-Proskauer reaction 
seems to serve this purpose. 

THE VOGES-PROSKAUER REACTION 

The chemistry of the Voges-Proskauer reaction has been 
worked out in detail by Harden and his associates in England. 
West refers to one of Harden's articles in which it is pointed 
out that the reaction is due to the production of acetyl-methyl- 
carbinol and urges that this test be studied further, as it is of 
considerable importance in recognizing B. aerogenes and B. 
cloacae. Among other investigators who have employed this 
reaction in studies on B. coli may be mentioned, Durham, Mac- 
Conkey, Rivas, Bergey and Deehan, Ferriera, Horta and Paredes, 
Copeland and Hoover, Clemesha, Archibald and more recently 
Khgler and Levine. 



THE VOGES-PROSKAUER REACTION 155 

The significance of this reaction has not been fully appre- 
ciated by bacteriologists, nor has it been generally realized that 
the test is due to a definite end product of glucose fermentation. 
It will therefore not be amiss to review somewhat in detail 
the nature and chemistry of the Voges-Proskauer reaction. 

The reaction takes its name from the fact that it was first 
observed by Voges and Proskauer in 1898, in their studies on 
the "Bacteria of Haemorrhagic Septicaemia." They describe 
the test as follows: 

On addition of caustic potash, we observed a new and interesting 
color reaction. If the tube be allowed to stand 24 hours and longer 
at room temperature, after the addition of the potash, a beautiful 
fluorescent color somewhat similar to that of a dilute alcoholic solu- 
tion of eosin forms in the culture fluid particularly at the open end 
of the tube exposed to the air. We have investigated a few of the 
properties of this coloring substance, which is not produced by the 
action of the alkali on the sugar, and have found that it is fairly resist- 
ant to the action of the external air. After a time however, it becomes 
paler, and finally gives place to a dirty greenish brown. 

It has been repeatedly observed in this laboratory, that, 
with some cultures, a distinct coloration which may be observed 
about five hours after addition of the potash fades or disappears 
entirely after twenty-four to forty-eight hours. 

In a study of the end products of the fermentation of glu- 
cose by B. coll, Harden and Walpole (1905-06) observed that 
the products ordinarily enumerated, (lactic, acetic, succinic 
and formic acids, ethyl alcohol and carbon dioxide) do not ac- 
count for all of the carbon in the sugar. Aside from these sub- 
stances, a crude glycol was also obtained. This crude glycol 
consists for the most part of 2:3 butyleneglycol (CH3-CHOH- 
CHOH-CH3). On oxidation it yields acetyl-methyl-carbinol 
(CH3, CHOH.CO.CH3), a volatile reducing substance, which, 
when mixed with potassium hydroxide in the presence of pep- 
tone, imparts an eosine-like coloration to the mixture on stand- 
ing. Butyleneglycol is oxidized to acetyl-methyl-carbinol by 
B. aerogenes, but not by B. coli. Harden (1905) ascribes the 
Voges-Proskauer reaction to the production of this carbinol. 



156 MAX LEVINE 

Walpole (1910) found that in the presence of oxygen B. aero- 
genes gave a larger yield of acetyl-methyl-carbinol from glucose 
and that fructose was decomposed in a similar manner. 

Neither acetyl-methyl-carbinol nor butyleneglycol, when mixed 
with potassium hydroxide give the eosin-like coloration. In 
the presence of peptone, however, the coloration develops on 
standing in the case of the carbinol, but not with the glycol. 
According to Harden (1905) the reaction is due to the further 
oxidation of the carbinol (CH3CO.CHOH.CH3) to diacetyl 
(CH3CO.CO.CH3) which reacts with some constituent of the 
peptone. In a later study Harden and Norris (1911) report 
that in the presence of strong potassium hydroxide solution 
diacetyl reacts with proteins to give a pink coloration together 
with a green fluorescence. With arginine, creatine, dicyanamide 
and guanidine acetic acid, the pink coloration is also obtained 
but the fluorescence is absent. The reaction depends on the 
presence of the group NH:C (NH2) N:HR. The exact signifi- 
cance of R. has not been determined. 

Among the organisms capable of forming acetyl-methyl- 
carbinol from carbohydrates may be mentioned, B. aerogenes 
Escherich, B. cloacae Jordan, B. subtilis Cohn, B. vulgatus Fliigge. 
Pere obtained volatile substances which reduced Fehling's solu- 
tion, by the aerobic fermentation of mannitol by B. subtilis 
and B. vulgatus and of glucose and glycerol by Tyrothrix tenuis. 

CORRELATION OF VOGES-PROSKAUER AND METHYL-RED REACTIONS 

A study of 167 coli-like bacteria obtained from various sources 
including raw and septic sewage, stock cultures from the Ameri- 
can Museum of Natural History, and feces of the horse, cow, 
sheep, hog and man, showed that only those which were alkaline 
to methyl red (in a medium made up of 0.5 per cent K2 H PO4, 
peptone and glucose) gave the Voges-Proskauer reaction. Of 
13 cultures which gave these reactions, 9 were from sewage 
and 4 from the museum collection. It should be noted that 
coli-like organisms giving these reactions were not obtained, 
even in a single instance, from the fecal samples. 



THE VOGES-PROSKAUER REACTION 157 

In order to test further, the correlation between the H+ ion 
concentration, and acetyl-methyl-carbinol production, 10 or- 
ganisms which Dr. Khgler had observed to be positive for the 
Voges-Proskauer reaction were obtained. Two of these failed 
to form gas from glucose and will not be considered further 
here. The remaining eight strains were alkaline to methyl 
red and gave a positive Voges-Proskauer test. 

Thirteen organisms were also obtained from L. A. Rogers. 
Five were acid to methyl red and gave a negative Voges.-Pros- 
kauer test. One failed to grow at 37°C. All of the others 
(7) were alkaline to methyl red and gave a positive reaction 
when tested for the formation of acetyl-methyl-carbinol in glu- 
cose-peptone solution. 

The high correlation between the Voges-Proskauer reaction 
and the indicator test of Clark and Lubs makes it possible to 
compare the work of Rogers and his associates with that of 
previous investigators. Such a comparison shows a striking 
unanimity of opinion as to the significance of these reactions, 
Rogers regards the high gas ratio and alkalinity to methyl red 
as characteristic of B. aerogenes-\ike bacteria. Practically all 
who have employed the Voges-Proskauer reaction have pointed 
out that this test is characteristic of B. aerogenes and B. cloacae. 
Of a large number of coli-like strains examined by MacConkey 
in 1905, only three B. aerogenes Escherich B. copsulatus Pfeiffer 
and B. cloacae Jordan gave the Voges-Proskauer reaction. Dur- 
ham in 1901 observed that this test was given only by those 
organisms which he regarded as belonging to the B. aerogenes 
group. 

THE DISTRIBUTION OF COLI-LIKE ORGANISMS WHICH GIVE A 
POSITIVE VOGES-PROSKAUER REACTION 

Coli-like organisms which form acetyl-methyl-carbinol in 
glucose peptone solution are rarely found in feces. A reasonably 
accurate and reliable idea as to the distribution of such bacteria 
in nature may be obtained from a study of the distribution of 
B. aerogenes and B. cloacae. 



158 MAX LEVINE 

MacConkey (1905) remarks on the scarcity of B. aerogenes 
in human feces. In the examination of 205 coli-like strains 
obtained from 22 samples of human stools, only 4 were B. aero- 
genes and of these 3 were isolated from a single sample. His 
observations on cow dung also indicated that this organism 
was extremely rare. 

Ferriera, Horta and Paredes in an examination of 117 lactose 
fermenting strains from human feces obtained a positive Pros- 
kauer (presumably Voges-Proskauer) reaction in only eight 
instances. Among 81 coli strains obtained from 46 species 
of animals (8 birds and 38 manunals) they found only two which 
gave a positive "Proskauer" test. 

The work of Clemesha is particularly significant because his 
conclusions are based on such large numbers of cultures. He 
examined 1207 organisms from human feces and 1029 from cow 
dung. In the latter B. aerogenes was found to be present in 
very small numbers and B. cloacae was sometimes common. In 
human stools, however, B. aerogenes and B. cloacae were very 
rare, nor was a sudden increase in the prevalence of these types 
ever observed. These findings are confirmed to a considerable 
extent by R. G. Archibald of the Wellcome Tropical Research 
Laboratories in an investigation of the water supply of Kliartoum. 

Of 117 cultures isolated in this laboratory from fecal sources 
(cow, horse, sheep, pig and man) not a single organism proved 
to be B. aerogenes, but of 39 organisms obtained from raw and 
septic sewage 9 (23 per cent) were of the B. aerogenes group 
(V.-P pos.). The relative abundance of these Voges-Proskauer 
positive organisms in sewage coupled with their extreme scarcity 
in human and other animal feces leads to the inference that they 
may perhaps represent soil forms. 

Clemesha found that in India B. aerogenes is more prevalent 
in rivers and lakes during the wet reason than during the dry 
period. He explains this phenomenon on the basis of a sup- 
posed multiplication of the organisms in water, but observes that 
all his experiments indicate that such multiplication does not take 
place, at least in artificial mixtures. Nevertheless he maintains 
that in large bodies of water, such as rivers and lakes, there is un- 



THE VOGES-PROSKAUER REACTION 159 

doubted multiplication of B. aerogenes. As to the prevalence 
of this organism he states, "In rivers, the period of time when 
rain is common is characterized by enormous increase in the 
number of lactis-aerogenes and yet we are perfectly certain that 
the organism is rare in feces." 

These observations may be easily explained on the assumption 
that B. aerogenes is a soil form. In a study of coli-like micro- 
organisms of the soil, now under way by B. R. Johnson and 
the author, preliminary tests have shown that a large propor- 
tion of cultures react positively for the Voges-Proskauer test, 
and are therefore of the aerogenes-cloacae group. 

ON THE FORMATION OF ACETYL-METHYL-CARBINOL FROM 
DIFFERENT CARBOHYDRATES AND ALCOHOLS 

Acetyl-methyl-carbinol, like carbon dioxid and various acids 
is a product of carbohydrate metabolism. The fermentation 
of carbohydrates with acid and gas formation is generally ac- 
cepted as a reliable basis for differentiation of B. coli. It is 
possible that a study of the production of acetyl-methyl-car- 
binol from various carbohydrates might also be of differential 
significance. 

Harden and Norris obtained acetyl-methyl-carbinol by grow- 
ing B. aerogenes or B, cloacae in peptone solutions containing 
glucose, fructose, mannose, galactose, arabinose, isodulcite, 
mannitol or adonitol, but this compound was not formed with 
glycerol ethyleneglycol, or acetaldehyde. 

Ferriera, Horta and Paredes, studied the Proskauer reaction 
(presumably the Voges-Proskauer reaction) with glucose, galac- 
tose, maltose, lactose, sucrose, dulcitol, mannitol, and inulin. 
The reaction was positive eight times out of 117 tests with 
glucose, 7 times out of 48 tests with galactose, and twice in 48 
tests with mannite. Practically all cultures gave traces with 
lactose and maltose while with dulcitol and inulin the reaction 
was invariably negative. These authors give an interesting table 
in which B. coli, B. cloacae and B. aerogenes are differentiated 
on the basis of the "Proskauer" reaction with different carbo- 



160 MAX LEVINE 

hydrates and alcohols. It appears that B. cloacae differs from 
B. aerogenes by the ability to give the reaction with mannite and 
galactose while the latter (5". aerogenes) may be distinguished 
from B. coll by its positive reaction in glucose and saccharose. 
The differentiation is suggestive and interesting but question- 
able, since the experimental evidence is inconclusive. 

The term "Voges-Proskauer Reaction" is generally under- 
stood to mean the production of an eosin-like coloration when 
a glucose broth culture is made alkaline with potassium hydroxide. 
To employ the same term when some other carbohydrate is 
substituted for glucose may lead to confusion. It is therefore, 
suggested that the term "Voges-Proskauer Reaction" be re- 
stricted to designate the formation of acetyl-methyl-carbinol 
from glucose, but when referring to its production from other 
carbohydrates or alcohols, the term Acetyl-methyl-carhinol 
Test or merely Carbinol Test be employed. The nature of the 
substance being tested for is thus indicated just as is the case 
-with the Indol Test. 

The following experiment was carried out as a preliminary 
study on the production of acetyl-methyl-carbinol from various 
substances by coli-like bacteria. Forty-six organisms were 
selected. Twenty were strains which previous studies had 
shown did not produce the carbinol from glucose. They repre- 
sent five sources, horse, sheep, cow, pig and man. From each 
source a culture representative of each of MacConkey's four 
groups was included. As far as possible no two cultures were 
from the same animal. The object of this selection was to 
obtain a group of Voges-Proskauer negative strains which would 
be likely to contain many different varieties. 

The other 26 cultures were strains which previous tests had 
shown could form the carbinol from glucose. These included 
9 strains isolated from sewage, and 17 strains obtained from 
Rogers and Kligler. 

The organisms were inoculated into a medium consisting of 
0.5 per cent di-potassium phosphate 0.5 per cent peptone and 
0.5 per cent of the test substance. The Digestive Ferments 
Company peptone was employed for the test with glucose. 



THE VOGES-PROSKAUER REACTION 



161 



With all other test substances Wittes peptone was used. In- 
cubation was at 37°C. for seventy-two hours. 

Of the 26 supposedly Voges-Proskauer positive organisms, 
4 failed to give the reaction in this experiment. Whether this 
phenomenon is due to the difference in peptone, variation in 
period of incubation, or loss of physiological function needs to be 
further investigated. With one of these organisms, it was observed, 
about eight months ago, that the test for acetyl-methyl-carbinol 
was negative until the seventh day of incubation. 

In the table below are summarized the results. The cultures 
are arranged in three groups. One group comprises those 
strains which, repeated tests have shown, do not form acetyl- 
methyl-carbinol from glucose, even on long incubation (7 days). 
Another group contains 22 strains which do form this carbinol 
from glucose. The third group includes the 4 cultures whose 
Voges-Proskauer reaction is questionable. 

Correlation between the formation of acetyl-methyl-carbinol in glucose peptone 
solution and in peptone solution containing other carbohydrates and alcohols 



TEST FOR ACETYL-METHYL CARBINOL 
IN GLUCOSE PEPTONE SOLUT ON 


positive 

(22 strains) 


NEGATIVE 
(20 STRAINS) 


QUEST- ONABLB 
(4 STRAINS) 


(T0GES-PR08KAUBR REACTION) 


No. 


Per 

cent 


No. 


Per 
cent 


No. 


Per 
cent 


Positive reactions with: 

Fructose 


22 

20 

21 

15 

21 

8 

13 



5 




100.0 
90.9 
95.5 
68.2 
95.5 
36.4 
59.1 
00.0 
22.0 
00.0 




17 
5 
1 







00.0 
00.0 
85.0 
25.0 
5.0 
00.0 
00.0 
00.0 
00.0 
00.0 



1 
2 

1 
1 






00.0 


Galactose 


25.0 


Maltose 


50.0 


Lactose 


00.0 


Sucrose 


25.0 


Raffinose 

Mannitol 


25.0 
00.0 


Glycerol 


00.0 


Salicin 


00.0 


Dextrin 


00.0 







Practically all strains gave a trace of acetyl-methyl-carbinol 
with maltose. A very striking fact indicated in the table is 
that coli-like organisms which do not form acetyl-methyl- 
carbinol from glucose are characterized by an inability to pro- 
duce this compound from the other substances tested. In only 



162 MAX LEVINE 

one instance (5 per cent) was the carbinol test positive with 
sucrose; in 5 cultures (25 per cent) traces were observed with 
lactose; but with fructose, galactose, raffinose, mannitol and 
saUcin the reaction was invariably negative. 

On the other hand, the carbinol test with the organisms which 
gave a positive reaction with glucose was almost always positive 
with levulose (100 per cent), galactose (90.9 per cent), and suc- 
rose (95.5 per cent), usually positive with lactose (68.2 per cent) 
and mannitol (59.1 per cent), and occasionally positive with 
raffinose (36.4 per cent) and salicin (22 per cent). 

These results show quite conclusively that the metabolism 
of certain carbohydrates by the fecal group of coli-like organism 
is fundamentally different from that of the non-fecal group. 

The carbinol test was usually most intense and distinct in 
sucrose peptone solution. 

CONCLUSIONS 

The Voges-Proskauer reaction and alkalinity to methyl red 
in 0.5 per cent di-potassium phosphate, glucose peptone solu- 
tion are correlated. These reactions were not given b}^ any of 
the 117 strains isolated from the feces of the horse, cow, sheep 
hog and man. 

A review of the literature on the distribution of B. aerogenes 
and B. cloacae (Voges-Proskauer positive organisms) corroborates 
the contention of Rogers and his associates that coli-like organ- 
isms which give a high CO2/H2 gas ratio, and an alkaline reaction 
to methyl red in 0.5 per cent peptone di-potassium phosphate 
glucose solution, are rare in feces. 

The natural habitat of coli-like bacteria which form acetyl- 
methyl-carbinol from glucose and other carbohydrates is prob- 
ably the soil. 

The production of acetyl-methyl-carbinol from different car- 
bohydrates and alcohols might serve as a differential index. 

Practically all strains gave a trace of the carbinol in maltose- 
peptone solution. 

Of the organisms which did not give the Voges-Proskauer 
reaction only 5 per cent formed acetyl-methyl-carbinol from 
sucrose, and 25 per cent gave a trace from lactose. With all 



THE VOGES-PKOSKAUER REACTION 163 

the other substances tested fructose, galactose, raffinose, manni- 
tol, glycerol, salicin, and dextrin the carbinol test was negative. 

Of the cultures which were positive for the Voges-Proskauer 
reaction, 100 per cent formed the carbinol from fructose, 90.9 
per cent from galactose, 68.2 per cent from lactose, 95.5 per cent 
from sucrose, 36.4 per cent from raffinose, 59.1 per cent from 
mannitol and 22 per cent from salicin. 

The Voges-Proskauer reaction is of considerable sanitary 
significance. It differentiates between fecal and non-fecal 
coli-hke organisms and may be an index of soil washings. 

BIBLIOGRAPHY 

Archibald, R. G. 1907. Lactose fermenting bacilli in surface waters, feces, 
etc. Wellcome Tropical Research Laboratories 4th Report, 319. 

*Bergey, D. H. and Deehan, S. J. 1908. The Colon-Aerogenes group of bac- 
teria. Journ. of Med. Res., 19. 175. 

Clark, Wm. M. and Lubs, H. A. 1915. The differentiation of bacteria of the 
Colon-Aerogenes family by the use of indicators. Journ. of Inf. Dis., 
17. 160. 

Clark, Wm. M. 1915. The final hydrogen ion concentrations of cultures of 
Bacillus coli. Journ. of Biol. Chem., ^2. 87. 

Clemesha, W. W. 1912. The bacteriology of surface waters in the tropics. 
Calcutta and London. 

Clemesha, W. W. 1912. A criticism of Dr. A. C. Houston's report on the 
biological characters of B. coli isolated from (1) raw, (2) stored river 
water, (3) stored and filtered water. Journ. of Hygiene., 12, 463. 

CoPELAND, W. R. AND HoovER, C. P. 1911. The interpretation of tests for 
B. coli communis Journ. of Inf. Dis., 9, 343. 

Durham, F. E. 1901. Some theoretical considerations upon the agglutinins, 
together with further observations upon Bacillus typhi abdominalis, 
Bacillus enteritidis, Bacillus coli communis, Bacillus lactis aerogenes, 
and some other bacilli of allied character. Journ. of Exp. Med., 
5, 353. 

Ferreira, a., Horta, A., and Paredes, C. 1908. Recherches sur le B. coli 
communis de I'intestin de I'homme. Archivos do Real Institute 
Bacteriologico Camara Pestana, Tome ii, Fasc. ii, 153. 

Ferreira, A. Horta, A., and Parades, C. 1908. Recherches sur le B. coli 
de I'intestin des Mammiferes et des Oiseaux. Archivos do Real Insti- 
tuto Bacteriologico Camara Pestana, Tome ii, Fasc. ii, 203. 

Harden, A. 1901. Action of B. communis on dextrose. Trans, of Chem. 
Soc. (1901), 604. 

Harden, A. 1905. Action on glucose of lactose-fermenting bacteria of feces. 
Journ. of Hyg. 5, 488. 

* Original article not available. 



164 MAX LEVINE 

Harden, A. 1905-06. On the Voges-Proskauer Reaction for certain bacteria. 

Proc. Roy. Soc, IT, 424. 
*Harden, a., and Norris, D. 1911-12. The bacterial production of acetyl- 

methyl-carbinol and 2:3 butylene-glycol from various substances. 

Proc. Roy. Soc. (B) %J^, 492. 
Harden, A., and Norris, D. 1912. The bacterial production of acetyl-methyl- 

carbinol and 2: 3 butylene-glycol from various substances. Proc. 

Roy. Soc. (B) 85, 73. 
Harden, A., and Norris, D. 1911. The diacetyl reaction for proteins. Jour. 

of Physiol., 47, 332. 
Harden, A., and Walpole, S. G. 1905-06. Chemical action of B. lactis aero- 
genes, (Escherich) on glucose. Production of 2:3 Butylene-glycol 

and acetyl-methyl-carbinol. Proc. Roy. Soc. (B), 77, 399. 
KiiiGLER, I. J. 1914. Studies on the classification of the colon group. Journ. 

Inf. Dis., 15, 187. 
Lemoigne, M. 1912. Fermentation du sucre par le Bacillus subtilis. Produc- 
tion du 2.3-butylene-gylcol. Compt. Rend. 155, 792. 
MacConkey, a. 1905. Lactose fermenting bacteria in faeces. Journ. of 

Hyg. 5, 333. 
MacConkey, A. 1909. Further observations on the differentiation of lactose- 
fermenting bacilli with special reference to those of intestinal origin. 

Journ. of Hyg., 9, 86. 
Per^ 1896. Cited by Harden and Norris. 1912. Ann. Inst. Pasteur, 

10, 417. 
RiVAS. 1908. Cited by West. 

Rogers, Clark and Davis. 1914. Journ. of Inf. Dis., 14, 411. 
Rogers, Clark and Evans. 1914. The characteristics of bacteria of the colon 

type found in bovine feces. Journ. of Inf. Dis., 15, 100. 
Rogers, Clark and Evans. 1915. The characteristics of bacteria of the colon 

type occurring on grains. Journ. of Inf. Dis., 17, 137. 
Smith, T. 1893. The fermentation tube with special reference to Anaerobiosis 

and gas production among bacteria. The Wilder Quarter-century 

Book, 197. 
*Smith, T. 1895. Ueber den Nachweis des Bacillus coli communis im Wasser. 

Centr. fur Bakt., 18, 494. 
*Thompson, J. 1911. The chemical action of B. cloacae (Jordan) on glucose 

and mannitol. Proc. Roy. Soc. (B), 84, 500. 
*Thomp80n, J. 1913. The chemical action of B. cloacae (Jordan) on citric and 

malic acids in the presence and absence of oxygen. Proc. Roy. Soc. 

(B), 86, 1. 
Voges and Proskauer. 1898. Beitrag zur Ernahrungs physiologic und zur 

differential diagnose der Bakterien der hamorrhagishen septicamie. 

Zeit. fiir Hyg., 28, 20. 
Walpole, S. G. 1910-11. The action of B. lactis aerogenes on glucose and 

mannitol. The effect of free oxygen on their production. The action 

of B. lactis aerogenes on Fructose. Proc. Roy. Soc. (B), 83, 272. 
West, F. D. 1909. Notes on the Voges-Proskauer reaction for Bacillus coli 

communis. Am. Journ. Pub. Hyg., 19, 227. 



STUDIES ON SOIL PROTOZOA AND THEIR RELA- 
TION TO THE BACTERIAL FLORA. 11^ 

JAMES M. SHERMAN 

From the Bacteriological Laboratories of the Wisconsin Agricultural Experiment 
Station, University of Wisconsin 

VI, THE EFFECT OF VOLATILE ANTISEPTICS UPON SOIL PROTOZOA 

Introduction 

It is claimed by Russell and Hutchinson and their co-workers 
that soils partially sterilized with volatile antiseptics are en- 
tirely freed from protozoa. Hutchinson (1913) further claims 
that the larger types of the soil protozoa are killed by the treat- 
ment of soil with caustic lime. On the other hand, the results 
reported by Gainey (1912) and b}^ Grieg-Smith (1911) indicate 
that the application of such amounts of volatile antiseptics 
as are used in practice does not exterminate the protozoa. Even 
if it be acknowledged that some types of the soil protozoa are 
able to resist the process of partial sterilization by antiseptics, 
we must still consider the contention of Russell and Hutchinson 
that the harmful factor is inactivated for a considerable period, 
when not exterminated. Further, the possibihty exists that 
the kinds of protozoa most detrimental to the bacterial flora 
are pecuharly susceptible to the antiseptics. Since the greater 
part of the protozoan fauna of the soil is inactive, the mere sur- 
vival of certain types is not necessarily important, but the effect 
of volatile antiseptics upon the active soil protozoa, on the other 
hand, would appear significant. 

Experiments 

Tests were made of partially sterilized soils to determine the 
number of protozoa and also the types. These tests were made 

^Continued from the Journal of Bacteriology, vol. i, no. 1, p. 35. 

165 



166 



JAMES M. SHEEMAN 



with pots each containing one kilogram of soil. The number 
of protozoa was determined by the dilution method, while the 
types were determined by the inoculation of 25 grams of soil 
into sterile hay infusion. 

The effect of volatile antiseptics upon the active protozoa 
was determined by the treatment of soils with carbon bisulphide 
and toluene and by determining the number of protozoa one 
day after treatment and again after two months. The results 
(Table XXVI) show that the active protozoa are not exter- 
minated and again multiply to numbers equivalent to those 
found in normal soils. Monas sp., Dimorpha radiata and Flagel- 
late A were all observed on the 1/10,000 dilutions after two 
months. 

TABLE XXVI 
Effect of toluene and CS2 on the soil protozoa 



POT NO. 


TREATMENT 


NUMBER OF PROTOZOA FEB GRAM 




1 day 


60 days 


1 


2 per cent toluene 


Less than 10 
Less than 10 
Less than 10 
Less than 10 


10,000 

10,000 
10,000 
10,000 


2 
3 


2 per cent toluene 

2 per cent CS- 


4 


2 per cent CS2 



In another experiment toluene, carbon bisulphide and chloro- 
form were used, and samples were taken at the end of one month 
to determine the number and types of protozoa present. In 
this test it was found that the protozoan fauna had not been 
simplified, as far as could be noted by microscopic examination, 
there being present a very complex mixture of ciliates, flagellates 
and amoebae. At the end of a month the active protozoa were 
again present in just as large numbers as are found in untreated 
soils. 

In the foregoing tests the antiseptics used were left in the 
soil. It was also thought desirable to treat some soils by the 
method followed by Russell and Hutchinson. These workers 
usually employed 1 per cent toluene and then after one day 
spread the soil out to allow the antiseptic to evaporate. Four 
pots of soil were treated after this manner and four other pots 



STUDIES ON SOIL PROTOZOA 



167 



TABLE XXVII 

(Effect of toluene, CS2 and CHCI3 upon the soil protozoa {one month after treatment) 



POT NO. 


TREATMENT 


PROTOZOA PER GRAM 


TYPES OF PROTOZOA 


1 


2 per cent toluene 


10,000 

10,000 
10,000 
10,000 
10,000 
10,000 
10,000 
10,000 
10,000 


C. F. A. 


2 


2 per cent toluene 


C. F. A. 


3 


2 per cent toluene 


C. F. A. 


4 


2 per cent CS2 


C. F. A. 


5 


2 per cent CS2 


C. F. A. 


6 


2 per cent CS^ 


C. F. A. 


7 


2 per cent CHCI3 


C. F. A. 


8 


2 per cent CHCI3 


C. F. A. 


9 


2 per cent CHCI3 


C. F. A. 









C = Ciliates; F = Flagellates; A = Amoebae. 



were treated with 1 per cent toluene but not evaporated. As 
is shown in Table XXVIII the results after one month were 
similar to those obtained in the other experiments. 

TABLE XXVIII 

Effect of toluene left in and evaporated upon the soil protozoa {one month after 

treatment) 



POT NO. 


TREATMENT 


PROTOZOA PER GRAM 


TYPES OP PROTOZOA 


1 


Left in 


10,000 

10,000 
10,000 
10,000 
10,000 
1,000 
10,000 
10,000 


C. F. A. 


2 


Left in 


C. F. A. 


3 
4 
5 

6 

7 


Left in 

Left in 

Evaporated 

Evaporated 

Evaporated 


C. F. A. 
C. F. A. 
C. F. A. 
C. F. A. 
C. F. A. 


8 


Evaporated 


C. F. A. 









Another test was made in which large amounts of volatile 
antiseptics were used in order to see if the protozoa could be 
entirely eliminated from soil. Soils were treated with 5 and 10 
per cent of toluene and carbon bisulphide and the antiseptics 
left in the soil. Even with such large arnounts of antiseptics 
the protozoa were not entirely eliminated, although the fauna 
was considerably simplified, especially with carbon bisulphide. 
As is shown in Table XXIX, ciliates, flagellates and amoebae 



168 JAMES M. SHERMAN 

were found in every case except in the one treated with 10 per 
cent carbon bisulphide in which no amoebae were observed. 
In all of these soils Monas sp., Dimorpha radiata and Flagellates 
A and B were present. 



Effect of large amounts 


TABLE XXIX 
of toluene and carbon bisulphi 


de upon the 


soil protozoa 


TREATMENT 


5 PER CENT 
TOLUENE 


10 PER CENT 
TOLUENE 


5 PER CENT 
CSi 


10 PER CENT 
CS2 


Types of protozoa 


C. F. A. 


C. F. A. 


C. F. A. 


C. F. 



Several attempts were made without success to demonstrate 
a stimulation of the protozoa, similar to that of the bacteria, 
subsequent to the application of volatile antiseptics to the soil. 
Moore (1912) in an address on the "Micro-organisms of the Soil" 
stated that results obtained in his laboratory indicated that the 
protozoa in soil not only withstood the action of antiseptics 
but that they might be increased by such treatment. Wood- 
ruff (1908) has shown that the multiplication of infusoria may 
be stimulated by small doses of alcohol. The dilution method 
for the determination of the number of protozoa is far too crude 
to measure small differences so the fact that it failed to demon- 
strate any increase in the number of protozoa following the 
apphcation of volatile antiseptics to soil cannot be considered 
of much importance. 

Discussion 

From the results herein reported it may be concluded that 
volatile antiseptics in the amounts used in practice do not free 
the soil from protozoa. The active soil protozoa not only sur- 
vive, but multiply rapidly and again attain their normal num- 
bers, usually within a month after treatment. It is difficult 
to explain the failure of Russell and Hutchinson to find protozoa 
in the soils which they treated. They noted the survival of 
certain flagellates which they do not, however, associate with 
the "detrimental factor." The failure of these workers to find 
ciliates and amoebae may be due to insufficient samples. The 
ciliates and amoebae are greatly reduced by the treatment of 



STUDIES ON SOIL PROTOZOA 169 

soil with volatile antiseptics; these organisms, being inactive 
in most soils, do not increase subsequently and so it is obviously- 
necessary to use a larger sample in order to demonstrate their 
presence. 

That the presence of protozoa in the partially sterihzed soils 
used in this work was not due to contamination was shown by 
holding ten pots of sterilized soil under identical conditions 
for one month and then taking samples for protozoa. Nine 
of these pots were found to be free from protozoa, while the 
tenth contained one small flagellate. 

These results argue strongly against the protozoan theory 
as an explanation of the phenomena of partial sterilization, but 
it cannot be said they positively disprove it, since, as was pointed 
out before, the particular kinds that are detrimental, if such 
exist, may be very sensitive to volatile antiseptics. 

VII. EXPERIMENTS RELATING TO THE POSSIBLE EXISTENCE IN 

SOIL OF A HARMFUL BIOLOGICAL FACTOR WHICH IS DESTROYED 

BY THE ACTION OF VOLATILE ANTISEPTICS 

Introduction 

The experiments made in his part of the work were planned 
in an effort to determine whether the beneficial action of vola- 
tile antiseptics upon the soil bacteria is due to the destruction 
of a detrimental factor which is antagonistic to them. This 
problem was attacked in much the same way as was the study 
of the soils containing protozoa and free of protozoa (Part IV). 
If normal soils contain a bacterial-limiting factor while partially 
sterilized soils do not, it would seem that that fact could be 
quite definitely established by a comparison of the numbers of 
bacteria found in these soils when subjected to various condi- 
tions. It should also be easy to demonstrate the presence of 
this harmful factor by the reinfection of the partially sterihzed 
soils with a small amount of untreated soil. 



170 



JAMES M. SHERMAN 



The effect of volatile antiseptics upon the subsequent development 
of bacteria and protozoa in soil 

It was thought that some hght might be thrown upon the 
protozoan theory by making bacterial and protozoal counts 
on soils subsequent to treatment with volatile antiseptics. If 
this theory is correct we would expect to find the greatest num- 
ber of bacteria in partially steriUzed soil at a time when the 

TABLE XXX 

Effect of volatile antiseptics upon the bacteria and protozoa in soil 

Fifteen days after treatment 



POT 


TREATMENT 


BACTERIA PER GRAM 


PROTOZOA PEB ORAM 


1 


Control 


15,000,000 
14,500,000 
14,000,000 
15,000,000 
13,000,000 


20,000 
20,000 


2 


Control 


3 


2 per cent toluene ' 


100 


4 


2 per cent toluene 


1,000 
100 


5 


2 per cent CS2 





Thirty days after treatment 




1 


Control 


20,800,000 
20,200,000 
48,000,000 
49,300,000 
44,400,000 


20,000 


2 
3 


Control 

2 per cent toluene 


20,000 
20,000 


4 
5 


2 per cent toluene 

2 per cent CS2 • 


20,000 
20,000 





Forty-five days after treatment 




1 


Control 


17,000,000 
21,000,000 
45,000,000 
46,000,000 
110,000,000 


20,000 


2 


Control 


20,000 


3 


2 per cent toluene 


20,000 


4 


2 per cent toluene 


20,000 


5 


2 per cent CS2 


20,000 



protozoa are depressed. This, however, does not appear to be 
the case as is shown by the following experiment. Determina- 
tions were made of the numbers of bacteria and protozoa in 
treated and untreated soils at intervals of fifteen days after 
treatment. The results of this test are given in Table XXX. 

This table shows that the maximum number of bacteria is 
not found while the protozoa are depressed, but rather that 



STUDIES ON SOIL PROTOZOA 



171 



the development of the two classes of micro-organisms subse- 
quent to treatment with volatile antiseptics runs parallel. 
This experiment was verified by another test in which normal 
and carbon bisulphide-treated soils were compared. In this 
test (Table XXXI) the number of bacteria in the treated soil 
rose above that of the control soil by the fifteenth day, but at 
this period the protozoa in the treated soil had also returned 
to their normal level. It is seen also that the number of bacteria 
continued to increase after the protozoa had again become as 
numerous as in untreated soil. 

TABLE XXXI 
Effect of CSi Upon the bacteria and protozoa in soil 



POT 


TREATMENT 


FIFTEEN DAYS 


THIRTY DAYS 




Bacteria 


Protozoa 


Bacteria 


Protozoa 


1 

2 


Control 

2 per cent CSj 


23,000,000 
94,000,000 


10,000 
20,000 


60,000,000 
240,000,000 


20,000 
20,000 



The reinoculation of partially sterilized soils 

In their work at the Rothamsted Station Russell and Hutchin- 
son (1913) claim to have demonstrated that the soil contains a 
detrimental factor since the bacterial content of partially steril- 
ized soil may be reduced by reinoculation with untreated soil. 
It is pointed out that when soil treated with a volatile antiseptic 
is reinoculated with 5 per cent of untreated soil the number of 
bacteria is reduced, while if only 0.5 per cent of normal soil is 
added no such reduction takes place. These observations are 
explained by the assumption that when onlj'- 0.5 per cent of 
untreated soil is added the harmful factor is not transmitted, 
but when 5 per cent is used for the inoculum the treated soil 
again becomes infected with the undesirable group of organisms. 
The soundness of this view may certainly be questioned, as 
it is difficult to understand why it should be necessary to use 
such a large amount of untreated soil in order to insure the pres- 
ence of a factor which is supposed to exist in amount sufficient 
to suppress the bacteria. A review of the work of Russell 



172 



JAMES M. SHERMAN 



and Hutchinson reveals the fact that in some of the tests the 
treated soils which were reinoculated with 5 per cent of untreated 
soil did not show an appreciable depression in the number of 
bacteria, and they qualify their conclusion on this point with the 
statement that, "the harmful factor is not invariably transmitted 
to the same extent from the untreated to the partially sterihzed 
soil and in a few cases indeed it is not transmitted at all." 

In the experiments which were carried out in this laboratory 
the partially sterilized soils were reinoculated with 1 per cent 
of untreated soil; since at least 1 kgm. of soil was used in each 
pot the inoculum never consisted of less than 10 grams of normal 
soil. It could hardly be doubted that this amount of soil would 
be sufficient to transplant the group of organisms, if such exist, 
which act as a limiting factor upon the bacterial flora. 

The work wliich has been done on the reinoculation of par- 
tially sterilized soils (Tables XXXII to XXXIV) fails to give 
any indication that a harmful factor is thus introduced. It 
would appear, on the other hand, that if reinfection of the treated 



TABLE XXXII 



Effect of reinoculation of treated soil with untreated soil {treatment of 2 -per cent 

toluene) 





TREATMENT 


NUMBER OF BACTERIA PER GRAM 




30 days 


60 days 


90 days 


1 


Control 


56,000,000 
66,000,000 
57,000,000 
62,000,000 


80,000,000 

75,000,000 

82,000,000 

100,000,000 


69,000,000 


2 


Control 


62,000,000 


3 
4 


Reinoculated 

Reinoculated 


79,000,000 
92,000,000 









TABLE XXXIII 



Effect of reinoculation of treated soil with untreated soil (treatment 1 'per cent toluene: 

evaporated) 



POT 


TREATMENT 


NUMBER OF BACTERIA PER GRAM 




15 days 


30 days 


1 


Control. ... 


149,000,000 
127,000,000 
152,000,000 
178,000,000 


95,000,000 


2 


Control 


81,000,000 


3 


Reinoculated 


130,000,000 


4 


Reinoculated 


92,000,000 









STUDIES ON SOIL PROTOZOA 



173 



TABLE XXXIV 
Effect of reinoculation of treated soil with untreated soil {treatment 2 per cent CS2) 



POT 


CONTROL 


AVERAGE 


REINOCULATED 


AVERAGE 


1 

2 
3 


273,000,000] 
218,000,000 [ 
285,000,000] 


255,300,000 


247,000,000] 
317,000,000 [ 
422,000,000] 


392,000,000 



Incubation period after reinoculation: 2 months. 

soil has any effect it is to increase the number of bacteria rather 
than to decrease it. However, the data on this point are doubt- 
less within the boundaries of experimental error. It is difficult 
to reconcile these findings with the theory of Russell and Hutch- 
inson. 



The number of bacteria in partially sterilized and normal soils 
at different temperatures 

One of the strongest points in the evidence produced by Rus- 
sell and Hutchinson to prove that the soil contains a harmful 
biological factor was the difference in the behavior of untreated 
and partially sterilized soils when incubated at different tem- 
peratures. Their results indicated that the maximum develop- 
ment of bacteria in the untreated soil was at low temperatures 
(5° to 12° C), while in treated soil the greatest number was 
found at 20°C., and at 30°C. there was a marked increase over 
that found at 12°C. — the maximum in the case of the untreated 
soil. This phenomenon they claim shows that the bacteria 
under normal conditions are limited by the detrimental factor 
and that their maximum development takes place under 
conditions unfavorable for the harmful factor. 

This point has been tested by the comparison of toluened 
and normal soils at 10°, 22°, and 37°C. The treated soil used 
had been treated with 2 per cent toluene three months previously. 
These soils were incubated for one month at their respective 
temperatures and then sampled and their bacterial counts de- 
termined. The results are given in Table XXXV. 



174 



JAMES M. SHERMAN 



TABLE XXXV 

The number of bacteria in treated and untreated soils at different temperatures 



TREATMENT 


NUMBEB OF BACTERIA PER ORAM 




10° C. 


22° C. 


37° C. 


Untreated 


21,000,000 
64,000,000 


23,000,000 
49,000,000 


22,000,000 


2 per cent toluene 


36,000,000 



These data are not sufficient to base any conclusions upon 
but it can not be said they indicate very much, either in favor 
of the protozoan theory or against it. It will be seen that the 
greatest difference in the numbers of bacteria in the treated 
and untreated soils was at 10°C., a point not in favor of the pro- 
tozoan theory. On the other hand, the least difference was found 
at 37 °C., which point may support the theory of Russell and 
Hutchinson. 

It was decided to carry out another experiment at 37°C. in 
order to throw more light on this point. Instead of using soils 
which had been previously treated, the soils were first placed 
at 37°C. and allowed to incubate at that temperature for one 
month. Half of them were then treated with 2 per cent carbon 
bisulphide. If the protozoan theory is correct the antiseptic 
should have very httle effect at this high temperature. One 
month after treatment bacterial counts were made. The results 
obtained are given in Table XXXVI. 





Effect of CS2 


TABLE XXXVI 

upon the number of bacteria in soil 


at 37°C. 




NUMBER OP BACTERIA PER GRAM 




Untreated 


Average 


2 per cent CS2 


Average 


1 

2 


21,000,000 
21,000,000 


21,000,000 


208,000,000 

248,000,000 


228,000,000 



The results are very striking; a difference of over ten fold in 
the number of bacteria in the treated and untreated soils being 
found. This observation indicates strongly that the beneficial 
.action of volatile antiseptics in soil is not to be explained by 



STUDIES ON SOIL PROTOZOA 



175 



its effect upon the protozoa. Soil extract and hay extract cul- 
tures made from untreated soil and incubated at 37°C. have 
failed entirely to reveal the presence of any of the active types 
of protozoa which have been mentioned as especially abundant 
in soil. In such cultures only a very few types of protozoa 
appear at all and these only slowly and in small numbers. 

The number of bacteria developing in sterilized soils reinoculated 
with untreated and with partially sterilized soils 

The preceding experiments appear to demonstrate quite 
conclusively that the beneficial effect of volatile antiseptics in 
soil is not due to the destruction of a biological factor, unless 
it be assumed that the treatment of soil so changes it that the 
harmful organisms are no longer able to develop in it, even though 
it is reinoculated with them. An experiment was planned in 
order to see if this explanation is a true one. Two pots of sterile 
soil were inoculated with 1 per cent of normal soil, while two 
other pots were inoculated with 1 per cent of a soil which had 
been treated with 2 per cent toluene. In case the antiseptic 
really destroys a harmful factor that fact should be indicated 
by a much greater number of bacteria in the soils inoculated 
with the treated soil. This result, however, was not obtained ; 
on the contrary, the counts made at thirty and forty-five days 
after inoculation showed no practical difference between the 
numbers of bacteria in the two soils, as is shown in Table XXXVI I. 



TABLE XXXVII 

The number of bacteria developing in sterilized soils inoculated with normal and 

with toluened soils 





NUMBER OF BACTBBIA PER GRAM 


POT 


35 days 


45 days 




Normal 


Toluened 


Normal 


Toluened 


1 

2 


127,000,000 
208,000,000 


112,000,000 
148,000,000 


126,000,000 
110,000,000 


102,000,000 
104,000,000 


Average 


190,000,000 


130,000,000 


118,000,000 


103,000,000 



176 



JAMES M, SHERMAN 



The effect of carbon bisulphide on the number of bacteria in sterilized 
soils reinoculated with normal soil and with protozoa-free soil 

Another experiment performed in order to detect the presence 
of the "harmful factor" was to inoculate soils sterilized by 
steam with normal soil and with the protozoa-free soil described 
in an earher part of this paper. These soils were allowed to 
stand three weeks and were then treated with 1 per cent of car- 
bon bisulphide. Bacterial counts were made before the soils 
were treated and then at fifteen and thirty days after treatment. 
According to the phagocytic theory, it would be expected that 
the number of bacteria in the soil inoculated with normal soil 
would subsequently be greatly increased while the soils inoculated 
with the protozoa-free soil should not be appreciably affected. 

As in the previous experiments, the results of this test give 
no indication that there exists in soil a biological factor which 
is harmful to the bacterial flora. It will be seen upon examina- 
tion of Table XXXVIII that the soils free of protozoa and those 
containing protozoa behaved in exactly the same way. 

TABLE XXXVIII 

Effect of CS2 upon sterilized soils inoculated with normal soil and with protozoa- 
free soil 





NUMBER OP BACTERIA IN MILLIONS PER GRAM 


POT 


Before treatment 


15 days 


30 days 




Without 
protozoa 


With 
protozoa 


Without 
protozoa 


With 
protozoa 


Without 
protozoa 


With 
protozoa 


1 

2 


178 
172 


120 
110 


228 
182 


140 
142 


166 
144 


109 
91 


Average 


175 


115 


205 


141 


155 


100 



The results of this test add further weight to the preceding 
experiments all of which point to the non-existence of a detri- 
mental biological factor in soil. The fact that volatile antiseptics 
have no appreciable effect in soils which have been steriHzed 
by steam and then reinoculated with normal soil would appear 
to indicate that the beneficial effects derived by the use of these 



STUDIES ON SOIL PROTOZOA 177 

substances are due to some action of the antispetics on the soil 
itself rather than to a simpUfication of its micro-organic popula- 
tion. 

VIII. RESUM^ 

Discussion 

The results of the foregoing experiments appear to establish 
quite definitely that protozoa in the soils which have been 
studied do not have a detrimental effect upon the bacterial 
flora. It is difl&cult to see how the action of an important phag- 
ocytic agent could have escaped detection by the methods 
employed unless the factor is unable to increase in soils which 
have been previously sterihzed with steam or partially sterilized 
with volatile antiseptics when again introduced into these 
soils with an inoculum of normal soil. This restricted power 
of growth would be very different from the properties of micro- 
organisms in general, either of animal or plant nature, and 
there is no evidence, as far as we are aware, that a group of 
organisms with such peculiar characteristics exists in the soil. 
As has been poiiited out, the soil protozoa, at least those types 
which appear in liquid cultures, grow better in soil which has 
been previously subjected to steam sterilization just as do the 
bacteria. Aside from the evidence that soil does not contain 
a biological factor which is inimical to the bacterial flora, the 
facts that volatile antiseptics do not exterminate the soil pro- 
tozoa, and that partial sterilization of soil under conditions 
unfavorable for the action of protozoa (e.g., at 37°C.) is followed 
by the characteristic rise in the number of bacteria, would ap- 
pear to cast serious doubt upon the theory of Russell and Hutch- 
inson as an explanation for the effect of volatile antiseptics upon 
the soil bacteria. 

Cunningham (1914) has recently pubhshed the results of 
some work which he thinks proves that protozoa act as a limit- 
ing factor upon the bacterial flora in soil. The fact that his 
data on this point are derived from only two experiments, one 
of which gave negative results, would preclude his conclusions 



178 JAMES M. SHERMAN 

from very serious consideration. A study of the methods he 
used indicates, however, that the difference found in the soils 
with and without protozoa might have been due to a difference 
ill the complexity of the two flora, as was the case in the experi- 
ments reported in Part IV (see Table XI) of this paper. In 
fact, his manner of attack was very similar; sterilized soils were 
employed as a substratum, and inoculations were then made 
into these soils of cultures containing protozoa and free of pro- 
tozoa. "One flask was inoculated with bacteria plus protozoa 
from a culture of protozoa from soil, the other received as nearly 
as possible an equal inoculation from the same culture of bac- 
teria alone." It is not clear from this statement how he obtained 
the bacterial culture free from protozoa, but it is very certain 
that a protozoa-free culture could not be obtained which would 
contain as complex a bacterial flora as did the original culture 
fi-om which it was derived. As was previously pointed out, a 
difference in the complexity of the bacterial flora in different 
soils may cause a great disparity in the counts obtained by the 
plate culture method. This fact was apparently not recognized 
by Cunningham as he concluded that "the reduction in bac- 
terial numbers in the soils inoculated with protozoa is very 
marked and lies well outside the limits of experimental error." 
A review of the data in Part IV of this paper will show, on the 
contrary, that his results may fall well within the limits of ex- 
perimental error. 

It is believed that the conclusions drawn from the work herein 
reported will hold in general for the cultivated soils i]i this 
country, but it is not desired to make too broad an application 
of them. Many of the "sick" soils which have been studied 
at the Rothamsted Experimental Station are very different from 
the ordinary American soil. Martin and Lewin (1914) describe 
a sick cucumber bed which was made up of one part of light 
pasture soil, one part of heavy pasture soil and two parts of 
horse manure, and had an optimum moisture content of 62 per 
cent. The assumption that the biological conditions in such 
a soil are the same as in the average soils of the United States 
(which contain about 2 per cent organic matter and the optimum 



STUDIES ON SOIL PROTOZOA 179 

moisture content of which is from 16 to 18 per cent) would be 
obviously unwarranted. That a difference in the micro-fauna 
does exist under various soil conditions is indicated by the fact 
that Martin and Lewin have found amoebae to be the predominat- 
ing types of protozoa in the soils they have studied, which are 
very rich in organic matter, while the results reported liere, 
as well as the data obtained by Cunningham on German soils, 
indicate that the flagellates occur in greater numbers than do 
the amoebae. It appears possible that in the rich soils and 
green-house beds, which have been studied extensively at the 
Rothamsted Station in connection with soil sickness, there 
might be a phagocytic agent which is not active in ordinary soils. 
This possibility, however, should not make us unmindful of 
the fact that no direct evidence has as yet been produced which 
indicates that such a factor exists in any cultivated soil. It 
should also be remembered that the beneficial effects of partial 
sterilization of soil — for the explanation of which the protozoan 
theory was advanced — have been observed in all localities in 
which the problem has been studied and in nearly all types of soil. 
The question of the activities of the protozoa which lead an 
active existence in soil is a problem upon which much work 
could profitably be done. The active protozoa which occur 
in soils in large numbers certainly have functions there, some 
of which in fact may be very important. It is not desired to 
give the impression that because the protozoa which have been 
studisd do not exert a limiting action on the bacteria in soil 
that it is thought that they do not ingest bacteria at all. Some 
in all probabihty do not, while others (e.g., Monas) it would 
appear undoubtedly do. Why active protozoa which feed upon 
bacteria should not cause a measurable decrease in the number 
of bacteria in soil is diflScult to explain. It would seem that the 
excretory products of the protozoa which feed upon the soil 
bacteria would increase the amount of available energy for the 
rest of the bacteria so that a condition of metabiosis would be 
established which might offset the antagonistic action of the 
protozoa. This hypothesis does not appear unreasonable when 
it is remembered that the chief limiting factor upon the bacteria 



180 JAMES M. SHERMAN 

in the soil is the food supply. In liquid cultures, on the other 
hand, the limiting factor is not the food supply but the accumu- 
lation of detrimental by-products; the number of bacteria soon 
reaches its maximum and then begins to decline gradually. 
It can readily be seen that if predatory protozoa are added to 
liquid cultures, in which the bacterial flora is in a comparatively 
inactive condition due to the presence of harmful by-products, 
a very striking reduction in bacterial numbers will be noted. 
Whatever the effect of protozoa on bacteria in solutions may be 
the results herein reported appear to indicate that imder ordi- 
nary conditions they are not able to Hmit the bacterial flora 
when acting in soil. 

Summary 

1. Determinations made by means of the dilution method 
indicate that the normal fertile soil has a protozoan content 
approximating 10,000 per gram. 

2. In the soils studied the flagellates were the predominating 
type of protozoa and not the ciliates nor amoebae. 

3. Colpoda cucullus apipears to be the most widely distributed 
cihate in soil and is occasionally found in numbers approxi- 
mating 1,000 per gram. 

4. Certain of the soil flagellates are active in soils of normal, 
and even subnormal, moisture contents. 

5. Tests made with the ciliates Colpoda cucullus, Balantio- 
phorus elongatus and Oxytricha sp. show that these organisms are 
not active under ordinary soil conditions. 

6. Colpoda cucullus is probably active whenever the moisture 
content is much above normal, but not under ordinary conditions 
of moisture. 

7. Active soil protozoa attain greater numbers when inoculated 
into previously sterilized soil than in normal soil. 

8. Sterile soils when inoculated with normal soil and with 
an artificial soil culture which is free of protozoa show a differ- 
ence in the total number of bacteria as determined by the plate 
culture method, due to a difference in the complexity of the two 
flora. 



STUDIES ON SOIL PROTOZOA 181 

9. A great difference may exist in the number of bacteria 
as determined by the plate culture method, due to a difference 
in the complexity of the flora, between soils which are free of 
protozoa. 

10. Experiments with soils containing protozoa and free of 
protozoa showed that the bacterial flora in the two soils behaved 
in exactly the same way when exposed to different conditions 
of temperature and moisture content. 

11. The data obtained indicate that soil does not contain 
a biological factor which is harmful to bacteria. 

12. Pure culture tests with the ciliates, Colpoda cucullus and 
Balantiophorus elongatus, showed that these organisms are very 
detrimental to bacteria in solutions. In soil, since the cihates 
are inactive, they are unable to affect the bacterial flora. 

13. Pure culture tests with four types of active soil flagellates 
showed that these organisms were not capable of limiting the 
number of bacteria when acting in soil. One of the cultures, 
however, had a very marked limiting action upon the bacteria 
when tested in soil extract. 

14. Treatment of soil with the ordinary amoimts of volatile 
antiseptics (1 to 2 per cent) does not appear to simplify the proto- 
zoan fauna. A complex mixture of cihates, flagellates and amoe- 
bae is to be found in cultures made from soils partially sterihzed 
with volatile antiseptics. 

15. As much as 10 per cent of carbon bisulphide and toluene 
when added to soil fails to extermmate the protozoa entirely. 

16. The active soil protozoa which are at first suppressed by 
treatment with volatile antiseptics soon begin to multiply so 
that they are again found in numbers equal to those of untreated 
soil within one month after treatment. 

17. The maximum number of bacteria in partially sterilized 
soil is not found while the protozoa are suppressed but after 
they have again returned to their normal level. It appears 
that the development of these two classes of micro-organisms 
subsequent to treatment with volatile antiseptics runs parallel. 

18. The reinoculation of partially sterilized soils with 1 per 
cent of normal soil fails to decrease the number of bacteria. 



182 JAMES M. SHERMAN 

19. The treatment of soil with carbon bisulphide at 37°C. 
gives a very marked increase in the number of bacteria in the 
soils treated. 

20. Sterilized soils which are reinoculated with normal soil 
and with partially sterilized soil show no essential difference 
in the numbers of bacteria which develop. 

21. Wlien volatile antiseptics are applied to sterilized soils 
reinoculated with and without protozoa no difference is to be 
noted between the behavior of the bacteria in the different soils. 

22. No evidence has been obtained which indicates that the 
beneficial effect of partial sterilization is due to the elimination 
of a biological factor which is harmful to the bacteria. 

Acknowledgment is due Professors E. G. Hastings, A. S. 
Pearse and E. B. Fred of the University of Wisconsin, from 
whom criticisms and suggestions have been obtained during the 
progress of this work. 

LITERATURE CITED 

BuiJERiNCK, M. W. 1896 Kulturversuche mit Amoeben auf festem Substrate. 
Centbl. Bakt. (etc.), Abt. 1, 19, 257-267. 

1897 Amoebenkultur auf festen Substraten. Centbl. Bakt. (etc.), 
Abt. Z, 21, 101-102. 

1901 Ueber oligonitrophile Mikroben. Centbl. Bakt. (etc.), Abt. 2, 
7, 561-582. 

Berliner, E. 1909 Flagellaten-Studien. Arch, fiir Protistenkunde, 15, 297- 
325. 

BoLLEY, H. L. 1910 Conservation of the purity of soils in cereal cropping. 
Science n. s., 32, 529-541. 

1913 The complexity of the micro-organic population of the soil. Sci- 
ence n. s., 38, 48-50. 

1913 Cereal cropping: sanitation, a new basis for crop rotation, 
manuring, tillage, and seed selection. Science n. s., 38, 249-259. 

BoTTOMLEY, W. B. 1911 Some effects of bacteriotoxins on soil organisms. 
Report British Assoc, for the Advancement of Science, 1911, 608. 

BuDDiN, W. 1914 Partial sterilization of soil by volatile and non-volatile anti- 
septics. Jour. Agr. Sci., 6, pt. 4, 417-451. 

Calkins, G. N. 1901 The protozoa. New York: MacMillan and Company. 

Cameron, F. 1910 Introduction to the study of the soil solution. Jour. Phys. 
Chem., 14, 393-451. 

Cauda, A. and Sangioro, G. 1914 Untersuchungen iiber die Mikrofauna der 
Boden aus Reisgegenden. Centbl. Bakt. (etc.), Abt. 2, 42, 393-398. 



STUDIES ON SOIL PROTOZOA 183 

Celli, a. 1896 Die Kultur der Amoeben auf festem Sunstrate. Centbl. Bakt. 

(etc.), Abt. 1, 19, 536-538. 
Celli, A., and Fiocca. 1894 Beitrage zur Amoebenforschung. Centbl. Bakt. 

(etc.), Abt. 1, 16, 329-339. 
Cunningham, A. 1914 Studies on soil protozoa II. Centbl. Bakt. (etc.), Abt. 

2, 4^, 8-27. 
Cunningham, A. and Lohnis, F. 1914 Studies on soil protozoa I. Centbl. 

Bakt. (etc.), Abt. 2, 39, 596-610. 
Egorov, M. a. 1908 The effect of carbon bisulphide on soils and plants. 

Zhur. Opuitn. Agron. 9, 34-95. Abs. Expt. Sta. Rec, W, 518. 
Emmerich, R., Leiningen, W. and Loew, O. 1912 tlber Bodensauberung. 

Centbl. Bakt. (etc.), Abt. 2, 31, 466-477. 
France, R. H. 1911 Studien fiber edaphische Organismen. Centbl. Bakt. 

(etc.), Abt. 2, 32, 1-7. 
Frank, B. 1888 Ueber den Einfluss welchen das Sterilisiren des Erdbodens 

auf die Pflanzen-Entwickelung ausiibt. Ber. d. Deutsch. Bot. Ges. 

(Generalversammlungs Heft) 6, 87-98. 
Fred, E. B. 1911 tjber die Beschleunigung der Lebenstatigkeit hoherer und 

niederer Pflanzen durch kleine Giftmengen. Centbl. Bakt. (etc.), 

Abt. 2, 31, 185-245. 

1915 Relation of carbon bisulphide to soil organisms and plant growth. 

Report of Director, 1914, Wis. Agr. Expt. Sta., Bui. 250, 18-19. 
Frosch, p. 1909 Beitrag zur Biologic saprophytischer Amoeben. Zeitschr. f. 

Krebsforschung. 8, 183. Abs. Centbl. Bakt. (etc.), Abt. 1, 45, 347-349. 
Gainey, p. L. 1912 The effect of toluol and CS2 upon the microflora and fauna 

of the soil. Ann. Rpt. Mo. Bot. Garden, 1912, 147-169. 
GooDEY, T. 1911 A contribution to our knowledge of the protozoa of the soil. 

Proc. Roy. Soc. (London), Ser. B, 84, 165-180. 

1914 A preliminary communication of three new proteomyxan rhizo- 

pods from soil. Arch. Protistenk., 35, 80-102. 
Grieg-Smith, R. 1911 The bacteriotoxins and the agricere of soils. Centbl. 

Bakt. (etc.), Abt. 2, 30, 154-156. 

1912 The inactivity of the soil protozoa. Proc. Linn. Soc. N. S. 
Wales, 1912, 655-672. 
HiLTNER, L. 1907 Uber neuere Ergebnisse und Probleme auf dem Gebiete der 

landwirtschaftlichen Bakteriologie. Jahuesber. d. Vereinig. f. Angew. 

Bot., 5, 200-222. 
HiLTNER, L. AND Stormer, K. 1903 Studien iiber die Bakterienflora des Acker- 

bodens, mit besonderer Berucksicktigung ihres Verhaltens nach einer 

Behandlung mit Schwefelkohlenstoff und nach Brache. Arb. K, 

Gesundheitsamt. Biol. Abt., 3, 445-528. 
HuNTEMtJLLER, O. 1905 Vcrnichtung von Bakterien im Wasser durch Proto- 

zoen. Arch, fi'ir Hygiene, 54, 89-100. 
Hutchinson, H. B. 1913 The partial sterilization of soil by means of caustic 

lime. Jour. Agr. Sci. 5, 320-330. 
Hutchinson, H. B. and MacLennan, K. 1914 The relative effect of lime as 

oxide and carbonate on certain soils. Jour. Agr. Sci. 6, 302-322. 



184 JAMES M. SHERMAN 

Killer, J. 1913 Die Zahlung der Protozoen im Boden. Centbl. Bakt. (etc.), 

Abt. 2, 37, 521-524. 
Koch, A. 1899 Ueber die Ursachen der Rebenmudigkeit mit besonderer Be- 

riichsichtigung der Schwefelkohlenstoffbehandlung. Arbeiten der 

Deutschen Landw. Gesellschaft, Jfi, 7-44. 

1911 tlber die Wirkung von Ather und Schwefelkohlenstoff auf hohere 

und niedere Pflanzen. Centbl. Bakt. (etc.), Abt. 2, SI, 175-185. 
KoRSHUN, S. W. 1907 Zur Frage der Verbreitung des Abdominal typhus durch 

Trinkwasser. Arch, filr Hygiene, 61, 336-347. 
Kruger, W. and Schneidewind, W. 1899 Ursache and Bedeutung der Sal- 

peterzersetzung im Boden. Landw. Jahr., ^8, 217-252. 
LiPMAN, J. G., Blair, A. W., Owen, I. L. and McLean, H. C. 1910 Experi- 
ments relating to the possible influence of protozoa on ammonification 

in the soil. N. J. Agr. Expt. Sta. Bui. 248. 
Lodge, C. A. and Smith, R. G. 1912 Influence of soil decoctions from sterilized 

and unsterilized soils upon bacterial growth. 24th Ann. Rpt. Mass. 

Agr. Expt. Sta., 126-134. 
Loew, O. 1911 Are protozoa concerned in soil sickness? Ann. Rpt. Porto 

Rico Agr. Expt. Sta., 1910, 15-17. 

1913 Protozoan life in soils of Porto Rico. Ann. Rpt. Porto Rico 

Agr. Expt. Sta., 1912, 13-14. 
Lyon, T. L. and Bizzell, J. A. 1910 Effect of steam sterilization on the water 

soluble matter in soils. Cornell Agr. Expt. Sta. Bui. 275. 
Martin, C. H. 1912 A note on the protozoa from sick soils, with some account 

of the life-cycle of a flagellate monad. Proc. Roy. Soc. (London), 

Ser. B, 85, 393-400. 

1913 The presence of protozoa in soils. Nature, 91, HI. 
Martin, C. H. and Lewin, K. R. 1914 Some notes on soil protozoa. Phil. 

Trans. Roy. Soc. London, Ser. B, 205, 77-94. 
MiNCHiN, E. A. 1912 An introduction to the study of the protozoa. London: 

Edward Arnold. 
Moore, G. T. 1912 Micro-organisms of the soil. Science n. s., S6, 609-616. 
MtJLLER, P. E. 1887 Studien iiber die naturlichen Humusformen. Berlin: 

Julius Springer. 
NXgler, K. 1909 Entwicklungsgeschichtliche Studien fiber Amoben. Arch. 

fur Protistenk., 15, 1-52. 
NoBBE, F. AND Richter, L. 1904 tjber die Behandlung des Bodens mit Ather, 

Schwefelkohlenstoff, Chloroform, Benzol und Wasserstoffsuperoxid 

und deren Wirkung auf das Wachstum der Pflanzen. Landw. Ver- 

suchs-stat. 60, 433-448. 
Peck, S. S. 1910 Some bio-chemical investigations of Hawaiian soils. Expt. 

Sta. of the Hawaiian Sugar Planter's Assoc. Bui. 34. 
Pfepper, T. and Franke, E. 1896 Beitrag zur Frage der Verwertung elemen- 

taren Stickstoff durch den Senf. Landw. Versuchs-stat., li.6, 117-151. 
Pickering, S. U. 1910 Studies of the changes occurring in heated soils. Jour. 

Agr. Sci., S, 258-276. 

1910 Plant growth in heated soils. Jour. Agr. Sci., 5, 277-284. 



STUDIES ON SOIL PROTOZOA 185 

Rahn, O. 1913 Methode zur Schatzung der Anzahl von Protozoen im Boden. 

Centbl. Bakt. (etc.), Abt. 2, S6, 419-421. 
RussKLL, E. J. AND GoLDiNG, J. 1912 Investigations on "sickness" in soil. 

Jour. Agr. Sci., 5, 21-47. 
Russell, E. J. and Hutchinson, H. B. 1909 The effect of partial sterilization 

of soil on the production of plant food. Jour. Agr. Sci. 3, 111-144. 

1913 The effect of partial sterilization of soil on the production of 

plant food. Jour. Agr. Sci., 5, 152-221. 
Russell, E. J. and Petherbridge, F. R. 1912 Investigations on "sickness" 

in soil. Jour. Agr. Sci., 5, 86-111. 
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U. S. Dept. of Agr., Bur. of Soils, Bui. 40. 

1907 Certain organic constituents of the soil in relation to soil fer- 
tility. U. S. Dept. of Agr., Bur. of Soils, Bui. 47. 
Sherman, J. M. 1914 The number and growth of protozoa in soil. Centbl. 

Bakt. (etc.), Abt. 2, 41, 625-630. 
Stone, G. E. 1912 The present status of soil sterilization. 24th Ann. Rpt. 

Mass. Agr. Expt. Sta., 121-125. 
StOrmer, K. 1907 tlber die Wirkung des Schwefelkohlenstoffs und ahnlicher 

Stoffe auf den Boden. Jahresber. d. Vereinig. f. Angew. Bot., 5, 113- 

131. 
TsujiTANi, J. 1908 tJber die Reinkultur der Amoeben. Centbl. Bakt. (etc.), 

Abt. 1, 24, 666-670. 
Whitney, M. and Cameron, F. 1904 Investigations in soil fertility. U. S. 

Dept. of Agr., Bur. of Soils, Bui. 23. 
Wilson, G. W. 1914 Studies of plant growth in heated soil. Biochem. Bui., S, 

202-209. 
Wolff, M. 1909 Der Einfluss der Bewasserung auf die Fauna der Ackerkrume 

mit besonderer Berucksichtigung der Bodenprotozoen. Mitt. Kaiser 

Wilhelm Instit. fiir Landw. (Bromberg), 382-401. 

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320. 
Woodruff, L. L. 1908 Effects of alcohol on the life cycle of infusoria. Biol. 

Bui., 15, 8^104. 



ARE SPORE-FORMING BACTERIA OF ANY SIGNIFI- 
CANCE IN SOIL UNDER NORMAL CONDITIONS?^ 

H. JOEL CONN 

Agricultural Experiment Station, Geneva, New York 

Among the best known of the soil microorganisms are the 
spore-forming bacteria. They have been described as soil- 
bacteria ever since the first bacteriological investigations of 
soil were made; and a more thorough taxonomic study has been 
made of them than of any other bacteria except those which 
have sanitary significance. It is seldom, however, that they 
comprise more than 10 per cent of the total flora of soil. Hiltner 
and Stormer (1903)2 recognizee^ three groups of colonies upon 
gelatin plates made from soil: hquefiers, non-Hquefiers and 
Streptothrix forms. The Hquefiers averaged about 5 per cent 
of the total flora. The ordinary spore-forming bacteria in 
soil are all rapid liquefiers and must have been included in this 
5 per cent mentioned by Hiltner and Stormer. Similar results 
have been obtained by various other investigators. 

The spore-forming bacteria, B. mycoides, B. cereus, and B. 
megatherium, are practically always present in soil aid have 
always been considered characteristic and important soil or- 
ganisms. These bacteria develop on gelatin or agar plates 
much more rapidly than those which comprise the othei 90 to 
95 per cent of the soil flora, and form large, striking cdonies. 
They are among the largest of all bacteria and have an unusually 
interesting morphology, so it is not surprising that they have 
been studied most extensively of all the soil organisms groying 

' Presented at Seventeenth Annual Meeting of the Society of American l3ac- 
teriologists, Urbana, Illinois, December 29, 1915. 

- Hiltner, L. and Stormer, K. Studien fiber die Bakterienflora des Acler- 
bodens, mit besonderer Beriiclsichtigung ihres Verhaltens nach einer Beha\d- 
lung mit Schwefelkohlenstoff und nach Brache. Kaiserliches Gesundheitsai^t, 
Biol. Abt. Land- u. Forstw. 3; ?. 445-545. 1903. 

187 • 



188 H. JOEL CONN 

on ordinary media, in spite of the fact that they are not very 
abundant in soil. In nitrogenous culture media these bacteria 
grow rapidly and cause a vigorous amonification. For this 
reason they have been assumed to be the important ammonifiers 
of the soil. 

This assumption was accepted as reasonable when I began 
to study the bacteria of soil. The first suspicion to the contrary 
came when it was noticed that the numbers of these spore- 
formers in the soil remained almost constant under all conditions, 
while the other bacteria varied in number according to the mois- 
ture content, aeration of the soil, or other conditions. The most 
natural explanation for this seemed to be that these bacteria 
lived over unfavorable conditions in the form of spores. It was 
soon realized, however, that this argument could not be carried 
to its logical conclusion without assuming that spore-formers 
were normally present in soil only as spores ; in which case natur- 
ally their nximbers would not vary. 

A series cf tests to investigate this matter has been made at 
the New York Experiment Station during the past year. The 
method used depended upon the fact that spores can resist 
higher temperatures than the vegetative forms. To determine 
the number of spores and vegetative rods present in any soil, 
one lot of diluted soil-infusion was plated in the ordinary manner, 
while a parallel lot of the diluted infusion was heated before 
plating ;or fifteen or twenty minutes at 75 to 85°. Then the 
colonies of the three spore-bearers, B. nycoides, B. cereus and 
B. megitherium, appearing on each set of plates, were counted. 
The colonies that developed from the heated infusion were as- 
sumed to arise from spores only; while in the case of the un- 
heatel infusion colonies might arise from vegetative rods as well. 

T^e culture medium used in these tests was gelatin.^ On 
this medium each of the three organisms investigated produced 
a farly characteristic colony, so that it was ordinarily possible 
to distinguish them with little difficulty from non-spore-formers 
on the plates made from unheated infusion. Plates were in- 

* Twelve per cent of Gold Label gelatine dissolved in tap-water and clarified 
wth white of egg. 



SPORE-FORMING BACTERIA 189 

cubated at 18°C. for seven days. This length of incubation 
was necessary in order to allow the late colonies (particularly 
of B. megatherium) to appear. The chief disadvantage of such 
a long incubation was that B. mycoides and B. cereus often had 
time to liquefy the plate completely unless high dilutions were 
used. Dilutions of 1-20,000 and sometimes even 1-100,000 
or 1-200,000 were necessary in order to avoid this trouble. 
At such dilutions the numbers of colonies of the spore-forming 
bacteria were so few that a long series of plates had to be made 
in order to obtain a reliable count; and even then no signifi- 
cance could be attached to variations in the count unless they 
were quite large. 

In the first of these tests a temperature of 85° was used; but 
later it was learned that at temperatures only about 10° higher 
than this large numbers of the spores were killed and it was 
suspected that even 85° might destroy some of them. For this 
reason 80° was used instead for a while, and in the last tests 75° 
was used. To test the efficiency of this last temperature the 
bacteria developing on the plates after heating the infusion 
were studied, and it was found that nothing but spore-bearing 
bacteria had survived (leaving out of account an occasional 
colony of some non-spore-forming type that might easily be 
due to air contamination). 

The greatest source of error in this method, which could not 
well be avoided, is the possibility that the bacteria investigated 
may occur in soil in clumps or chains instead of as isolated indi- 
viduals. There is some evidence that clumps of bacterial spores 
can be broken up by the action of heat, which would tend to 
increase the count in the heated infusion provided clumps do 
occur in the soil. No increase of any appreciable amount has 
ever been observed, however; and indeed, so far as microscopical 
examinations of soil have been made, no evidence has been ob- 
tained of chains or clumps of organisms of this type. For this 
reason this possibility of error did not seem great enough to 
invalidate the conclusions. 

A series of twenty-six tests was made. The results are given 
in the following table. The most striking fact to be observed 



190 



H. JOEL CONN 





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192 H. JOEL CONN 

at first glance is the regularity of the numbers of these organ- 
isms in the unheated infusion. The highest count is 1,500,000 
and the lowest 400,000. Compared with bacterial counts in 
general, these show remarkable regularity, especially when it 
is considered that the soils varied from poor sand to richly man- 
ured loam and that the counts were made on plates of such 
high dilution that comparatively few colonies were obtained on 
each plate. The counts obtained from the heated infusion 
are not quite as regular; but if the first eight tests are excluded — 
in which the use of 85° may have killed a few spores — there is 
scarcely any more variation than in the case of the unheated 
infusion. 

Because of this regularity in the counts it is possible to ob- 
tain general averages that can be fairly compared with each 
other. The average count from the unheated infusion is 
788,000, from the heated infusion 712,000. This slight differ- 
ence indicates that there are very few if any of the organisms 
present in soil in a form that can be killed by the temperatures 
used. Studying the figures more closely it will be noticed that 
the greatest differences between the two counts occurred in 
the first eight tests, in which 85° was used. The average count 
in these first eight tests, unheated, was 670,000, while the average 
count, heated, was 445,000. In the last eighteen tests, how- 
ever, both counts averaged nearly the name, 844,000 and 833, 
000, respectively. 

A more careful analysis of the data yields similar results. 
The last column of the table shows the difference between the 
two counts with a plus sign before it if the count obtained from 
the unheated infusion was the higher, with a minus sign if that 
from the heated infusion was the higher. It will be seen 
that there are eighteen cases in which the plus sign is used, 
and in these cases the greatest difference was 530,000, or if the 
tests are excluded in which 85° was used, it is 400,000. On 
the other hand in the eight tests in which a greater count 
was obtained from the heated infusion there is one difference as 
large as 400,000. The average difference between the two 
counts is 76,000 in favor of the unheated infusion; while if the 



ino 

SPORE-FOKMING BACTERIA ^"^ 



first eight tests are excluded it is only 5,300, an almost negUgible 

"^Tmight be concluded from these data that some vegetative 
forms do exist in normal soil and for this reaon a higher count 
was obtained eighteen times from the -"Seated mtusion; wh^e 
in the other eight cases a higher count was "btmned from the 
heated infusion because clumps were broken up by the heat. 
ItTs improbable, however, that these two factors sl-^W orf " 
narily so nearly neutraUze each other; nor is it possible, if tins 
explanation is used, to account for the greater average difference 
rthrfirst eight tests than in those in which lower tempera^^ 
were used. It seems more reasonable to explam most of the 
Ifferences in either direction as lying withm the experiment^ 
erroi^a perfectly plausible assumption m view of the high 
dilutions used— or if this is not enough to explam all the cases 
fn wMchX Ugher count was obtained from the unheated in- 
fusfon to assume that an occasional less resistant spore wa^ 
S'by the temperatures employed. The evidence all seems 
to ndicate that the three organisms investigated do not occu 
in soil under normal conditions as active vegetative forms but 
a" spores It is true that there are other spore-forn^ng bacter a 
fn Toil besides these three types, in regard to which definite 
data could not be obtained because their eol-- "^f ^-^f 
teristic enough to be recognized with certainty, but none ot 
them ar as constantly present as the three types studied, and 
what evidence is at hand suggests that the same facts are true 
to regard to them as in regard to B. mycotdes, B. cereus and 

^■rsTandft: reason, however, that these bacteria, so univer 
sallv present in soil, must grow and multiply under some natural 
Tonditions It is known that they ordinarily thnve m «ie pre. 
ence of organic matter; so it seemed not improbabeha^thy 
would multiply if manure were added to soil. A single experi 
menthrbeen undertaken to test out this point, but with negative 
Results In a pot of soil, mixed with a heavy application of 
rel horse manure, kept under observation for two months, 
hi was at first a' very great increase in the number of non- 



194 H. JOEL CONN 

Spore-bearers, but no appreciable multiplication of spore-formers ; 
nor was there any decrease large enough to be detected in the 
number of actual spores. Meanwhile the odor of the soil was 
enough to show that ammonification was vigorous. It is per- 
fectly possible that a repetition of this test might yield different 
results; but evidently this experiment did not furnish the right 
conditions for the growth of the spore-forming bacteria. Also 
it is plain that ammonification can take place without them. 

These results leave our knowledge as to the significance of 
spore-forming soil bacteria in a rather unsettled state. It has 
been quite generally taken for granted in the past that they 
are active in soil and of great importance. Perhaps their strik- 
ing appearance in plate culture has led to the assumption that 
they could grow equally vigorously in soil. Yet they com- 
prise but a small part of the soil flora, and even at that they do 
not seem to be present in vegetative form under normal con- 
ditions. Spores are generally regarded as inert. 

Never the less these spore-forming bacteria of the soil do not 
decrease in numbers, and spores cannot live forever. Their 
occurrence in soil cannot be due to accidental contamination, 
or their numbers would not be so constant. If it is true, as 
these results indicate, that they are of practically no importance 
under normal field conditions, it becomes a matter of much 
interest to learn under what conditions they can become active 
and multiply. 

SUMMARY 

1. The number of spore-forming bacteria in soil is relatively 
constant and is about the same in all the soils studied. Three 
of the spore-forming bacteria aways present in soil — B. rnycoides, 
B. cereus, and B. megatherium — were selected for the purpose 
of comparison, because their colonies on gelatin plates are quite 
readily distinguishable. The total number of these three or- 
ganisms, as determined by means of gelatin plates, proved to 
be between 400,000 and 1,500,000 per gram in the soils studied. 
They always comprised less than 10 per cent and usually less 
than 5 per cent of all the colonies developing on gelatin. 



SPORE-FORMING BACTERIA 195 

2. When soil-infusion was heated before plating at a tempera- 
ture (75-85°C.) high enough to kill the vegetative forms of 
bacteria, nearly if not quite as many colonies of these spore- 
forming bacteria developed as when it was plated unheated. 
In about one-third of the cases, indeed, their numbers were 
actually shghtly higher on the plates made after heating; al- 
though all such differences undoubtedly lay within the Umits of 
the experimental error. This suggests that these bacteria occur 
in normal soil as spores rather than in a vegetative state. 

3. No increase in the total number of these organisms nor 
decrease in the number of their spores could be detected in a 
pot of soil to which fresh manure had been added, 

4. These results throw considerable doubt on the common 
assumption that these organisms are important ammonifiers 
in the soil. They raise the question as to what possible soil 
conditions favor their growth and multiphcation. 



A POSSIBLE FUNCTION OF ACTINOMYCETES 

IN SOID 

H. JOEL CONN 
Agricultural Experiment Station, Geneva, New York 

It is not generally agreed whether Actinomycetes are to be 
classed with bacteria or with molds. They are thought to be- 
long with the Hyphomycetes by some mycologists; but those 
that occur in the soil have generally been considered in con- 
nection with the bacterial flora rather than with the soil fungi. 
The reason why they have been studied by soil bacteriologists 
may be partly because Actinomycetes can be handled by much 
the same methods as the lower bacteria; and partly because both 
of these groups seem to be much more numerous than molds 
proper in normal soil. 

The abundance of Actinomycetes in soil has been recognized 
for some time. In 1903 Hiltner and Stormer (1903) showed 
that of the colonies developing on gelatin plates from normal 
soil, 5 per cent were ordinarily liquefiers, 70 per cent non- 
liquefiers, and 20 per cent Streptothrix forms (a name often, al- 
though incorrectly, applied to this group). Probably every- 
one who has plated soil in gelatin, provided he has incubated 
his plates long enough for the slow-growing organisms to ap- 
pear, will recognize these figures as typical of ordmary soil. 

Perhaps the most interesting recent work on soil Actinomycetes 
is that of Krainsky (1914). It contains a valuable classification 
of these organisms and shows that the reason why few species 
have been distinctly recognized in the past is because the Actino- 
mycetes require special media in order to bring out their specific 
characteristics. His further contention, however, that these 
special media are necessary in ord6r to show the abundance of 

1 Presented at Seventeenth Annual Meeting of the Society of American Bac- 
teriologists, Urbana, Illinois, December 29, 1915. 

197 



198 H. JOEL CONN 

Actinomycetes in soil is not correct. With his special media 
he claims to have found as many as 20,000 per gram of soil; 
but he overlooks the fact that Hiltner and Stormer (1903) found 
as many as 2.5 millions per gram. Moreover, in the work that 
forms the basis of the present paper, 2 or 3 milHon per gram has 
proved to be a very common figure; while on certain occasions 
the number has reached 12 to 14 milUons. Occasionally over 
half the colonies developing on gelatin have been Actinomycetes 
— this in spite of the fact that Krainsky claims their growth 
to be suppressed by ordinary media. 

The great abundance of Actinomycetes in soil has led to many 
speculations as to their significance. It has often been stated 
that they are active agents in the decomposition of organic 
matter; but their part in this process has not been definitely 
studied. Beijerinck (1900) showed that one type was often 
present in the corky layer of various roots. He called this type 
Streptothrix chromogena after Gasperini (1894) (who, however, 
had called it Actinomyces chromogenus) . This type is one of 
the most numerous in soil; yet in the fight of recent work it 
must be regarded as a group rather than a species. To this group 
belongs the causal organism of potato scab. Lutman and Cun- 
ningham (1914), indeed, have recently attempted to show that 
the cause of this disease must be renamed Actinomyces chromo- 
genus because it agrees in every particular with Gasperini's 
description of that organism. This is plainly impossible; for 
Krainsky (1914) has shown that at least four separate species 
agree with the descriptions that have been given to A. chromo- 
genus. 

This fact brought out by Krainsky is very evident to anyone 
who uses his methods for studying the group. In fact it has 
proved possible, by the use of other special media^ besides those 
described by him, to recognize many more types than those 
listed in his article. Work is now in progress along this fine. 

- The medium which has given the best results of any yet investigated con- 
tains: 1000 cc. water, 15 g. agar, 10 g. glycerin, 1 g. sodium asparaginate, 1 g. 
glucose, 1.5 g. NH4H2PO4, 0.2 g., MgSOi, 0.1 g. CaCU, 0.1 g. KCl, trace FeCU, 
Further media are now being tested out that may prove even more satisfactory. 



FUNCTION OF ACTINOMYCETES IN SOIL 199 

This complexity in the group and the confusion in nomen- 
clature, however, must not hide the fact that an Actinomyces 
causes potato scab, nor that Beijerinck, approaching the sub- 
ject from an entirely different angle, has shown them to be 
associated with the roots of other plants. It is also to be re- 
membered that Actinomycetes are thought to be concerned in 
the decomposition of organic matter. Some recent observa- 
tions at the New York Experiment Station bear on this point. 

In the course of a qualitative study of the bacteria in certain 
New York State soils, it was early recognized that there was a 
great similarity between different soils in the relative numbers 
of Actinomycetes and lower bacteria present, provided the soils 
were in the same state of cultivation. Later it also became 
evident that the Actinomycetes were practically always present 
in greater abundance in old sod soil than in soil recently cul- 
tivated. This difference is shown in Table I, in which the num- 
bers of Actinomycetes found in 20 samples of various sod soils 
are compared with the numbers occurring in an equal number 
of samples of cultivated soil. Although it is possible to pick 
out numerous cases in which the number occurring in some 
one of the cultivated samples is greater than in some of the 
sod samples, nevertheless the average number in sod soil is 
twice that in the cultivated soil. The table also shows that 
the Actinomycetes averaged 39.4 per cent of the total flora of 
sod soil, but only 21.3 per cent of the flora of cultivated soil. 
There is only one instance (October 22, 1913) in which the 
percentage of these organisms in sod soil is as low as their aver- 
age percentage in cultivated soil, and only one (January 4, 1911) 
in which their percentage in cultivated soil is as high as their 
average percentage in sod soil. 

These figures furnish a strong indication that Actinomycetes 
are more numerous in sod than in cultivated soil; but even be- 
fore all the data given in Table I were collected the importance 
of making a more satisfactory comparison was realized. To 
do this, a study was made of a considerable variety of soil types,' 

^ The soil nomenclature of the Bureau of Soils has been used in this work. 
The soils mentioned are described in the Soil Surveys of Ontario and Tomp- 
kins Counties, New York, published by this Bureau. 



200 



H. JOEL CONN 








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FUNCTION OF ACTINOMYCETES IN SOIL 201 

and in order to avoid as many as possible of the other factors 
that might be involved in a comparison of miscellaneous soils 
two samples were always collected on the same date, from spots 
in the same soil not more than a few yards apart, one in old sod, 
the other in a cultivated field. In this series of tests thirty- 
eight pairs of samples were taken. Also a second shorter series 
of tests was made to compare the Actinomyces flora of three 
neighboring spots in a single soil type (Dunkirk silty clay loam), 
one spot in fallow soil, one in old sod and the third in a field 
which had been lq grass for two or three years only. 

All of the counts in these tests were made by means of gelatin 
plates, because in the earlier work gelatin had been found the 
best of the various media used for distinguishing Actinomyces 
colonies from those of the lower bacteria. The gelatin used 
sometimes contained soil-extract and sometimes tap-water 
alone.^ Plates were always incubated for seven days at 18°C. 
before counting. 

The results of the first series of tests are given in Table II. 
It will be seen that the average number of Actinomycetes in sod 
soil is nearly twice as high as the average number in cultivated 
soil and that they averaged 37.5 per cent of the total flora in 
sod soil but only 20.5 per cent of the flora of cultivated soil. 
These general averages are much like those given in Table I, 
but they tell only a part of the story, as it is possible for individual 
exceptions to obscure the differences in the average. In order 
to show the differences more plainly, the individual ratios were 
determined and averaged. In the sixth column of Table II 
is given the ratio of the actual number of Actinomycetes in the 
sod soil to the number in the corresponding samples of cultivated 
soil; in the last column of the table is given the ratio of the per- 
centage of Actinomycetes in sod soil to the percentage in the 
corresponding samples of cultivated soil. A study of these 
ratios brings out some information not shown by the general 
averages. 

* For the composition of these media see : Conn, H. J. Culture Media for 
Use in the Plate Method of Counting Soil Bacteria. N. Y. Agric. Exper. ta. 
Tech. Bui. 38, 1914. 



202 



H. JOEL CONN 



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FUNCTION OF ACTINOMYCETES IN SOIL 



203 



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204 H. JOEL CONN 

In making a comparison of these ratios it seemed reasonable 
to assume that those falHng between the hmits of 1.2: 1 and 
1:1.2 were so near imity as to indicate no real difference in 
numbers between the sod and cultivated samples. In the 
sixth column, giving the ratios of the actual numbers per gram, 
sixteen cases are hsted that fell within these limits. There 
were only two cases (May 19, 1914 and the third on November 
4, 1915) when the numbers in cultivated soil were enough greater 
than in sod to give a ratio outside these limits; and of these the 
greatest ratio was only 1: 1.8. There were nineteen cases, 
however, in which the numbers in sod were sufficiently greater 
than in cultivated soil to give a ratio exceding 1.2: 1; and of 
them the maximum ratio was 6.4: 1. The average ratio of 
all thirty-eight cases was 2.15: 1, which is larger than the ratio 
between the general averages of columns three and four. The 
figures which show what percentage of the total flora consisted 
of Actinomycetes are somewhat more striking. The average 
ratio, it is true, (as shown in the last column of the table) was 
2.1: 1 or practically the same as the ratio between the actual 
numbers per gram; but there were only four cases that fell 
between the hmits 1.2: 1 and 1: 1.2 and only two (May 21, 1914 
and the third on November 4, 1915) when the numbers in cul- 
tivated soil were enough greater than in sod to give a ratio out- 
side these limits. These two cases both showed a ratio of 1 : 1.3 
which is hardly to be compared with the maximum ratio, 7.2: 1, 
in favor of sod soil. 

The conclusion to be drawn from this comparison is that the 
few exceptional cases in which there were more Actinomycetes 
in the cultivated soil a,re completely overbalanced by the numer- 
ous cases in which there were more in the sod soil. In some of 
the border-line cases, moreover, the number of lower bacteria 
was greater in the cultivated soil than in the corresponding 
sod sample, with the result that the percentage of Actinomycetes 
was sometimes greater in the sod sample even though the actual 
number was the same in both samples. 

The last four cases in the table are of special interest because 
they were analyses of the same samples collected on November 



FUNCTION OF ACTINOMYCETES IN SOIL 



205 



4, 1915, made after keeping the samples in the laboratory twelve 
Weeks. On the date of collection the ratio obtained in the case 
of one pair of samples was in favor of the cultivated soil, while 
in two of the others case it was nearly unity. At the time of 
the later analysis the ratios in these three cases were still all 
near unity, although none of them were actually in favor of 
cultivated soil. 

The results of the other series of tests, comparing three neigh- 
boring spots in a single soil type, are given in Table III. The 

TABLE III 
Number of Aclinomycetes in three neighboring spots of a single soil type. A com- 
parison of old sod, neio sod, and cultivated soil. Numbers determined by means 
of gelatin plates 



DATE 


ACTUAL NUMBER PER GRAM 


PER CENT OP TOTAL 
FLORA 




Old sod 


New sod 


Cultivated 


Old 

sod 


New 
sod 


Culti- 
vated 


May 29, 1914... 
September 1, 1914... 
September 5,1914... 
September 10, 1914... 
October 23, 1914... 
September 16, 1915... 


8,000,000 
12,000,000 

8,500,000 

8,500,000 

12,000,000 


5,000,000 
7,500,000 
7,800,000 
6,600,000 


*4,000,000 
*2,400,000 
3,000,000 
2,500,000 
3,000,000 
2,500,000 


40.0 
34.4 

47.2 
38.6 
48.5 


20.8 
21.7 
25.2 
23.0 


*ig.o 

*14.G 
15.2 
13.8 
10.0 
12.5 


Average 


9,800,000 


6,600,000 


2,900,000 


41.7 


23.6 


14 1 







* The first two samples of cultivated soil were taken from a different spot 
from the rest, although similar in kind of soil and in state of cultivation. 

numbers obtained in this test were so constant that the few 
analyses mean as much as longer series of irregular results. 
The average number of Aclinomycetes in the old sod was 9,800,000 
per gram, in the new sod 6,600,000 and in the cultivated soil, 
2,900,000; or in percentages, they averaged 41.7, 23.6 and 
14.1 per cent, respectively, of the total flora in these three spots. 
The lowest count (of Actinomycetes) in old sod was higher than 
the highest in new sod, and the lowest in new sod higher than 
the highest in cultivated soil. These figures indicate that the 
number of Actinomycetes in sod soil increases as the age of the 
sod grows greater. 



206 H. JOEL CONN 

The interpretation of the figures hinges upon the question 
whether these organisms should be regarded as filametous fungi 
producing spores or as unicellular bacteria occurring in filaments. 
On ordinary culture media they exist as branched filaments 
that break up under certain conditions into short rods or coccus- 
like bodies, known as conidia because of their similarity to the 
conidia of molds in method of formation. When such cultures 
are plated, each colony ordinarily comes from one conidium or 
group of conidia. If they grow similarly in the soil and if the 
conidia are actually spores, an increase in the number of colonies 
on the plates may indicate merely an increase in spore-produc- 
tion. A few observations are at hand, however, to indicate 
that Actinomycetes occur in the soil not as filaments but as 
chains of short rods or cocci closely resembling ordinary bacteria. 
If this is the normal mode of growth in the soil and if these 
bodies are individuals instead of spores, an increase in the num- 
ber of colonies on the plates may be regarded as more nearly 
representing a true increase in the number of the organisms in 
the soil. 

Making the assumption that the latter condition actually 
exists in the soil, which seems justified so far as the facts are 
known, there are two explanations of the higher numbers ob- 
served in sod soil that seem sufficiently probable to be considered. 
One is that sod soil becomes more compact in time than culti- 
vated soil and that poor aeration favors the Actinomycetes in 
some way, in spite of the fact that they ordinarily seem to like 
a good supply of oxygen. This explanation does not well fit 
the facts, however; for it has been found that sod soil, dug up 
and well aerated and then kept in a pile for three months, may 
still retain its high Actinomyces content. The other explanation 
which has been considered is that the Actinomycetes are active in 
the decomposition of grass roots or perhaps of plant roots in 
general. In view of the past observations as to the association 
between Actinomycetes and plant roots, this explanation seems 
worth bearing in mind. Experiments are now being carried 
on which are designed to show whether or not this is the true 
function of Actinomycetes in soil. 



FUNCTION OF ACTINOMYCETES IN SOIL 207 

SUMMARY 

1. In general more colonies of Actinomycetes develop on plates 
made from sod soil than on those from cultivated soil. The 
average ratio between their numbers in neighboring sod and 
cultivated spots in the same soil type is sHghtly over 2:1. The 
maximum ratio is about 6:1. 

2. Actinomycetes average about 38 per cent of the total flora 
of sod soil, as determined by means of gelatin plates, but only 
about 20 per cent of the total flora of cultivated soil. 

3. In a study of three neighboring spots in a single soil type 
it has been found that Actinomyces colonies not only appear in 
greater numbers from sod than from cultivated soil, but also 
in greater numbers from old sod than from sod only two or three 
years old. 

4. This relation has been found to hold with very few exceptions 
In the isolated cases where more Actinomyces colonies have 
developed from a sample of cultivated soil than from the corre- 
sponding sample of sod soil, the ratio has never been greater 
than 1.8: 1. 

5. Although the reason for this difference in numbers has not 
been learned, a probable explanation seems to be that Actinomy- 
cetes are active in the decomposition of grass roots. 

BIBLIOGRAPHY 

Beijerinck, M. W. 1900. Uebei* Chinonbildung durch Streptothrix chromo- 
gena und Lebensweise dieses Mikroben. Centbl. f. Bakt. Abt. 2, 6, 
2-12. 

Gasperini, G. 1894. Versuche iiber das Genus "Actinomyces." Paper pre- 
sented at the Eleventh International Medical Congress at Rome. 
Abstract, Centbl. f. Bakt. Abt. 1, 15, 684. 

HiLTNER, L. AND Stormer, K. 1903. Studicn fiber die Bakterienflora des Ac- 
kerbodens, mit besonderer Beriicksichtigung ihres Verhaltens nach 
einer Behandlung mit Schwefelkohlenstoff und nach Brache. Kaiser- 
liches Gesundheitsamt, Biol. Abt. Land- u. Forstw. 3, 445-545. 

Krainsky, a. 1914. Die Aktinomyceten und ihre Bedeutung in der Natur. 
Centbl. f. Bakt., Abt. 2, 4I, 649-688. 

LuTMAN, B. F. AND CUNNINGHAM, G. C. 1914. Potato Scab. Vermont Agric. 
Exper. Sta., Bui. 184. 



PRACTICAL OBSERVATIONS ON THE TITRATION 
AND ADJUSTMENT OF CULTURE MEDIA 

BERTHA VAN HOUTEN ANTHONY and CLARENCE V. EKROTH 

Bureau of Laboratories, Department of Health, City of New York 

Any one who studies the methods given in the various text- 
books for the titration and adjustment of culture media, must 
be struck by the lack of uniformity of opinion. Not only is the 
beginner in media preparation bewildered but even the more 
experienced worker may be led into error. The difficulty arises 
from the fact that the complex nature of the materials dealt 
with is by no means fully understood, even in the case of the 
most fundamental culture media. In addition to this the 
changes that occur under even slightly different conditions and 
treatment are most confusing. As a result each laboratory 
is compelled to adopt the methods best adapted to its work and 
requirements, and each laboratory makes changes m these 
methods as need arises. 

The many requests constantly made for information regard- 
ing the methods employed in our laboratories for the titration 
and adjustment of both general and certain special culture media 
seem to indicate the need for a detailed account of such pro- 

cedures. 

In this paper we have tried to incorporate the practical in- 
formation gained after a number of years of experience. In 
addition, we have described experiments carried on with a 
view of clearing up, in a systematic manner, certam pomts upon 
which Httle, if any, information is available. 

The Standard Method' of titrating media for water and milk 
analyses was devised in an attempt to secure uniform prepara- 
tions of media at all times so that comparable results might 
be obtained. 

1 Committee on Standard Methods, 1905, 1913). 

209 



210 BERTHA VAN H. ANTHONY AND C. V. EKROTH 

The directions are as follows: 

Phenolphthalein shall be the standard indicator in obtaining reac- 
tion of all media. Tumeric paper possesses similar properties and its 
use advised where phenolphthalein is not available. Titrations and 
adjustments of reactions shall be made as follows: 

Put 5 cc. of media to be titrated in 45 cc. of distilled water. Boil 
briskly one minute. Add 1 cc. phenolphthalein solution (5 grams of 
commercial salt in one liter of 50 per cent alcohol.) Titrate while 
hot (preferably while boiling) with ^ caustic soda. A faint but dis- 
tinct pink marks the true end point. This distinct pink color may be 
described as a combination of 25 per cent of red (wave length approxi- 
mately 658) with 75 per cent of white as shown by the disks of the color 
top, described under Records of Tints and Shades of Color, p. 10. 
(The Standard^ color disks used in teaching optics may be used for 
this purpose.) 

In practice, titration is continued until the pink color of alkaline 
phenolphthalein matches that of the fused disks. All reactions shall 
be expressed with reference to the phenolphthalein neutral point and 
be expressed in percentages of normal acid or alkaline solutions re- 
quired to neutralize them. 

One of the objects of this paper is to consider whether the 
desired results are actually obtained by the Standard method 
or by modifications of this method. 

In our laboratory one modification, that is, titration of broth 
at room temperature (about 20°C.) and of agar at a temperature 
of about 30°C. has given good results for a number of years past. 

These and other experiences have led us to investigate further 
the following subjects: 

a. The effect of prolonged heating on meat infusions and 
beef extract solutions as shown by the titration curves of both 
adjusted and unadjusted portions. The results of boiling 
samples of media in the casserole for titration. 

b. The adjustment of broth and agar, including the remelt- 
ing of solid media. 

* A small sized top and disks costing only a few cents may be obtained from 
Milton Bradley Educational Company, Springfield, Mass. 



TITRATION AND ADJUSTMENT OF CULTURE MEDIA 211 

c. The reaction of peptone solutions and the effects upon 
them of prolonged heating. 

d. The question of indicators with consideration of the 
significance and sensitiveness of their end points indifferent 
media — also the method of choosing the one most suitable 
with reference to the hydrogen electrode as a standard. 

MEAT INFUSIONS 

It is a well known fact that each time a medium is heated to 
the boiling point, or above it, the reaction changes and be- 
comes more and more acid, depending on the length of time 
and the degree of heating. On this subject Eyre (1915) says: 

Meat extract [meat infusion] is acid in reaction owing to presence 
of acid phosphates of potassium and sodium; weak acids of the gly- 
colic series and organic compoimds in which an acid character pre- 
dominates. 

Owing to the nature of the substances from which it derives its 
reaction, the total acidity of meat extracts [infusion] can only be esti- 
mated accurately when the solution is at the boiling point. Prolonged 
boiling [as in media preparation] causes it to undergo hydrolytic changes 
which increase the acidity. 

He states further that meat extract [infusion] becomes stable 
in reaction after being heated at the boiling point for forty-five 
minutes so that no additional increase of acidity occurs on 
further heating. 

To procure more definite data as to the effect of heat on the 
acidity of media the following work was carried out: 

Preparation of the meat infusions. Chopped lean veal was 
soaked over night in tap^ water in the proportion of one pound 
of meat to one liter of water. It was then heated at 45° to 55° 
C. for one hour. At this point it was brought to a boil. Then 

3 Weekly analyses of the Croton water supply shows it to contain a negligible 
amount of mineral matter (only about 40 parts per million, expressed as total 
hardness.) Where the water supply is at all "hard," it is advisable to employ 
distilled water exclusively. 



212 BERTHA VAN H. ANTHONY AND C. V. EKKOTU 

the material (meat and watery infusion) was divided into three 
lots: 

B* 30 was kept at the boiling point 30 minutes. 
B 60 was kept at the boiling point 60 minutes. 
B 120 was kept at the boiling point 120 minutes. 
(Volumes being made up by addition of tap water.) 

After being boiled, each lot was strained through cheese- 
cloth, then filtered through paper (S. & S. "Falten" filter) and 
cotton (first moistened with cold water to hold back the fatty 
substances.) 

Each lot was titrated^ and then divided again into two parts 
and one series (B 30, B 60, B 120) was run in the autoclave at 
least six successive times, without the addition of soda. 

The second series (B 30 C, B 60 C, B 120 C) was corrected 
with normal sodium hydroxid to 1 per cent acidity ( + 1) and 
then run in the autoclave with the other set and under the 
same conditions. 

In all the tests made the autoclaving was done at a pressure 
between 15 and 17 pounds as indicated by a Bristol recording 
pressure gauge. The heating was carried on up to a total of 
eight hours and titrations performed at one-half hour intervals 
for the first four half -hours; then at one hour, two hour and 
three hour intervals. 

After each autoclaving the six samples were titrated and the 
corrected series (B 30 C, B 60 C, etc.) was adjusted again when 
necessary to plus one (+ 1). 

Method of titration. Freshly boiled and cooled distilled water 
was used for all titrations. A 5 cc. sample of meat infusion was 
drawn off by means of a 5 cc. pipette and added to 45 cc. of dis- 
tilled water in a casserole to which 1 cc. of a 1 per cent^ solution 

* Preliminary titrations on samples before boiling had been labelled "A." 
As these had no significance they are omitted in this article. 

'See: "Method of Titration." 

" These are also variations from the standard method of using 0.5 per cent 
solution of phenolphthalein and twentieth normal sodium hydroxid solution. 
They are, however, in accordance with the methods in use for years in this labora- 
tory and to preserve uniformity they were adhered to. 



TITRATION AND ADJUSTMENT OF CULTURE MEDIA 



213 



of phenolphthalein had been added. While stirring the mix- 
ture, deci-normaP soda solution from a burette was run in with- 
out any heating whatever. The end point taken was the first 
delicate pink tinge, observable throughout, which did not dis- 
appear after stirring the solution — and should not disappear for 
at least one minute. The figures were then recorded. The cas- 
serole with the mixture was then set over the flame, brought 
to a boil and boiled one minute by the watch. 





'B30 




B 60 




B 120 




B 30C 




B 60C 




B 120 C 


Meat infusions 




First lot 




of veal 


B, 60 




Bi 120 



Outline of Experiments 

= preliminary boiling of 30 min. 
= preliminary' boiling of 60 min. 
= preliminary boiling of 120 min. 



preliminary boiling of 30 min. 
preliminary boiling of 60 min. 
preliminary boiling of 120 min. 



preliminary boiling of 60 min. 
preliminary boiling of 120 min. 



Bi 60 C 
Bi 120 C 



preliminary boiling of 60 min. 
preliminary boiling of 120 min. 





B, 30 




B2 60 




B2 120 


eat infusions 




Second lot < 




of veal 


B2 30 C 




B2 60 C 




Bi 120 c 



preliminary boiling of 30 min. 
preliminary boiling of 60 min. 
preliminary boiling of 120 min. 



preliminary boiling of 30 min. 
preliminary boiling of 60 min. 
preliminary boiling of 120 min. 



Corrected t o 
plus one ac- 
cording to 
room tempera- 
ture titration 
after each au- 
toclaving. 



Cor rected to 
plus one ac- 
cording to boil- 
i n g tempera- 
ture titration 
after the first 
autoclaving. 
No further ad- 
justments 
were made be- 
cause of error 
in adding too 
much soda. 



Corrected to 
plus one ac- 
cording to 
boiling tem- 
perature titra- 
tion after each 
autoclaving. 



214 BERTHA VAN H. ANTHONY AND C. V. EKROTH 

Since the boiling had caused the faint pink color to disappear, 
the hot mixture was then promptly titrated again, the same 
end point being approximated^ as closely as possible, and the 
figures recorded. The room temperature figure plus that ob- 
tained after boihng one minute gave a total which represents 
the boiling titration figure as recorded in the charts. 

In the first lot of veal, (series B 30, B 60, B 120), the correc- 
tions were made to plus one at the room temperature figures. 
A second lot of veal infusions (B2 30, B2 60, B2 120) were pre- 
pared in the same manner as above. The corrections in this 
lot were made to plus one at the boihng figures. 

DESCRIPTION OF CHARTS 

In charts 1 and 2 are shown the uncorrected portions of meat 
infusions titrated after successive heatings in the autoclave 
and plotted according to both the room temperature and the 
boihng temperature figures. It will be seen that in each lot 
of meat the room temperature titrations fall into one group and 
the boiling titrations into another; also that the boiling figures 
are the higher. 

Chart 3 is a sample^ chart showing not only the uncorrected 
portion of B 120 as given in Chart 1, but also the corrected por- 
tion, B 120 C. This portion was corrected to plus one (+1) 
according to the room temperature titration figures and read- 
justed to plus one after each autoclaving, as shown by the a 
line. The a line shows the figures of this same material when 
boiled one minute in the casserole and titrated hot. This line 
is hypothetical and shows only the amount of soda that would 
have been needed had the boiling figures been used for adjust- 
ment to plus one. 

The Q line shows the total amount of acidity produced in 
the corrected portion even after the addition of soda, accord- 
ing to the room temperature figures. 

^ The difficulty of catching the first color change of phenolphthalein in hot 
solutions will be discussed under "Indicators." 

* This chart is typical also of the B 30 and B 60 sets. 



TITRATION AND ADJUSTMENT OF CULTUKE MEDIA 



215 



Key to Curue Notation. 



Letter 



Line: 



Deinotes 



= corrected at room temperature 



"(boiling fJKures) 



'W. 



" boiling temperature 



:b: 



"(room temp, figures) 



= xinoorrected. (room temperature 



boiling temperature) 



Total acidity, corrected at room temp. 



-w 



" boiling temp 



CHART I 



Si 

















J 
















^ ' 














































_,'' 


































> 












^ 














^ 


Y 




^^\/^ 






y 


i- — 


Biao-r' 




. ''^ 


'^^ 












,; 


^^ 





























^y^ 




. 




U-v 


- 


-BSOT* 


-^-^-^ 






^■^^^ 


r' 


/ 
/ 


" ^ 


" 






-^ss^ 






















— ^ y 






^j 








r' 


/ 




^____^ 


■ — ' 
























' ^ 


k*' 


^ 




























^ , 


— ^ 
















^'' 







































CHART 2 
























/■ 


n 
























SiJToV- 


f' .y 






-/ 


X 
/^^ 




r'' 








^ 


;^ 


^' 








^ 




^ 
















^ 



































rime. IN HOUKS 



216 



BERTHA VAN H. ANTHONY AND C. V. EKROTH 



In Chart 4^ is represented Bg 120 (second lot of veal) just as 
B 120 is shown in Chart 3 except that here the corrected por- 
tion (Bz 120 C)30 was adjusted to plus one ( + 1) according to 
the boiling titration figures. The ^ line shows the figures ob- 
tained each time at room temperature before the sample was 
boiled one minute in the casserole to give the boiling titration 



CHART 3. 



CHART A 



5 

it- 

















J 
















• 






























































/ 
















/ 














, 
















































/ 




J/ 










I 


'' 




y^ 






/ 


' 






y 








/ 








y 






, 


( 






/ 






^r^" 


«*" 




Siao^ 


^ 


/ 




,/ 


•' 


/ 


r^ 








.-^' 






/ 








^ 


^ 






/ 








,/ 












,-- -^ 


6;*5*^* 






> 


} 1 


jVi 


A 








4 




// 


/ 




y 

y 


/ 




'^'^ 


^r 


1 Ai / 


/'- 




--^ 


/^^ 
























TIME tH HOURS 



figures. The room temperature curve is inserted here for com- 
parison. 

The Q' fine shows the total acidity produced in the portion 
corrected according to the boiling titration figures. 

In chart 5 all the total acidity lines of the various corrected 
portions are compared. As with the uncorrected portions as 



» This chart is typical also of the B2 30 and B26O sets. 



TITRATION AND ADJUSTMENT OF CULTURE MEDIA 



217 



noted in charts 1 and 2, the total acidity Unes fall into two groups 
according to the method of titration. 

If chart 5, showing the total rises in acidity of the series B30 C, 
B 60 C, B 120 C and the series Ba 30 C, B2 60 C, B2 120 C, were 
applied successively to the corresponding curves of the uncor- 
rected series in charts 1 and 2, so that the point of origin in each 
set were the same, it would be seen that in every instance the 



7 HART ,5 



/, 



■7^' 



/ 



./ 



y 



^ 



■^ 



r56W 



# 



■r/ 






*^ 



"'«> 



-^c* 

i^ 



560C» 






'/.f 



■^ ;- 






H=^* 



total rise of acidity of the corrected curve equals or exceeds that 
of the uncorrected portion. 

From this it is plainly evident that, in spite of successive 
corrections of acidity with normal sodium hydroxid, hydrolysis 
not only continues on the application of heat but there is pro- 
duced in meat infusion media approximately as much acidity 
as would be developed were no correction made. 

Chart 6 shows the actual acidity of the different corrected 
portions after the successive adjustments and periods of heat- 
ing. As can be seen, those portions adjusted according to the 




«0 


1 — 


^^™ 






^^^^^^^p 




1 


^^^^^^ 



r 




VD0ZI9 
r>D099 
y>D0S8 



3 / 

218 



TITRATION AND ADJUSTMENT OF CULTURE MEDIA 



219 



room temperature titration are in general nearer to the de- 
sired reaction of plus one (+1), especially in the first two half- 
hours — the periods of time of greatest practical interest. 

In chart 7 are shown not only the uncorrected portions of 
Bi 60 (y and 7') and Bi 120 (7 and 7') (duphcate material from 



CHART 7. 



!:"«■ 



? ^- 

k ' 

Q 


















/ 
















/ 














/ 
















/ 














1 


/ 


Z' 








,.-- 


^-i^^^ 


r 


/■■■ 


y 


— •' 


> 


' 


</ 


' > 


r 








// 






y 








^ 


---rf 




/ 










> 




b;60"^ 












'%. 




5't-»TH..r 

•food*. 






^' 


^^-' 


*.***' 

^^-> 


^ \ 




' 


lU- 












^-^ / 




ifl 




r' . 


'^^'■' 


.^' 





first lot of veal), but also the efi'ect on the corrected portions of 
the addition of too much normal sodium hydroxid. By mis- 
take after the first half hour in the autoclave and the subse- 
quent titration, there was added to Bi 60 C /3' approximately 
five times the amount of normal sodium hydroxid necessary 
to correct it to plus one, and to Bi 120 C jS' also about five times 



220 



BERTHA VAN H. ANTHONY AND C. V. EKROTH 



the right amount. These amounts were in accordance with 
the boiling titration figures in both cases. The quantities needed 
for correction were, in round numbers, twice as much for the 
latter as for the former. The relation of the quantities of alkali 
added may, therefore, be expressed by the numerical relation 
of ten to five. The portions of meat infusion (without further 
additions of soda) were then run as usual in the autoclave with 
the uncorrected portions and titrated at the same intervals. 

Curiosity led us to continue these tests rather than discard 
them. In consequence, an interesting fact was brought out. 
To our surprise both over-corrected portions recovered the 
acidity of plus one (and more) but at different intervals of time. 
Bi 60 C/3' gained plus one at the end of the fifth hour while 
Bi 120 /3' reached plus one at about the seventh hour. 

In all the tests the curves show a distinct and steady rise in 
acidity. This rise is due to hydrolysis caused by heat and in- 
creases continually as more heat^" is applied. Further, it is 
plainly evident that a preliminary heating to the boiling point 
for at least forty-five minutes as advocated by Eyre (1915) 
does not produce a stable reaction uninfluenced by further heating. 



TABLE I 



MEAT INFUSION 


REACTION BEFORE 
AUTOCLAViNQ 


AFTER 8 HOURS IN 
AUTOCLAVE 


AFTER 14 HOURS IN 
AUTOCLAVE 




R.T.' 


B.T.t 


R.T. 


B.T. 


R.T. 


B.T. 


BjSO 


1.7 

1.9 
2.1 


2.7 
2.9 
3.1 


4.9 

4.6 
4.3 


6.5 
6.3 
6.5 


7.4 

7.3 
6.9 


10.5 


B26O 


10.4 


B2I2O 


9.3 







*R. T. = room temperature titration. 
*B. T. = boiling temperature titration. 

In the above tests we have gone outside the limits of inter- 
est from the practical standpoint. We were led to this, however, 
in an attempt to locate the point of complete hydrolysis or maxi- 
mum acidity of meat infusions. This goal was not reached, 

10 Three of the uncorrected portions, B2 30, B2 60, B2 120 were run an additional 
six hours, making fourteen hours all told in the autoclave at 15 pounds pressure. 
The results are shown in last colimins of Table I. 



TITRATION AND ADJUSTMENT OP CULTURE MEDIA 221 

as stated above, even after fourteen hours autoclaving. We 
are continuing this work. 

In the usual preparation of media from meat the total amount 
of heating in the autoclave varies from one half hour, at about 
15 pounds pressure, for sterilization of ordinary broth, to two 
and one-half hours in the preparation and sterilization of agar. 

It developed in the tests made on the corrected meat infusions 
that the change in acidity in the first half hour in the auto- 
clave (the usual time for the steriHzation of finished media) 
varied from nothing to 0.3 per cent at the room temperature 
figures, while in boiling temperature figures the change in acidity 
was 0.3 to 0.4 per cent (see chart 6). 

The nature of the acid products which are formed as a result 
of the hydrolytic decomposition on boiling with sodium hydroxid 
may be different from those produced by hydrolysis alone when 
boiling unadjusted media. Therefore, while the reaction may 
be adjusted in each to the same point of acidity, the behavior 
of the media toward the various organisms may not be the same. 

Similar tests to those above were made on Liebig's beef ex- 
tract dissolved in tap water, filtered and titrated in a similar 
fashion. These tssts showed no change in the reaction of the 
uncorrected series even after eight hours heating. Although 
a beef extract solution is undoubtedly quite stable," as com- 
pared with meat infusions, a sample corrected^^ ^o neutral, after 
two hours in the autoclave, rose 0.2 per cent in acidity. This 
was corrected to neutral once more and did not change again 
although heated four hours longer. 

While the above change is almost negligible, the addition of 
peptone to this as to other media, raises the change in acidity 
further. This must be taken into consideration in the prepara- 
tion of media with beef extract when delicate end points are 
desired, 

" This stability is due probably to very prolonged heating in the preparation 
of the beef extract itself. 

^2 Correction based on room temperature titration. 



222 BERTHA VAN H. ANTHONY AND C. V. EKROTH 

BROTH '2 

The tests on the meat infusions were carried out before any 
peptone or salt had been added. In the preparation of broth 
the choice of titration methods must of course be governed by 
the manner of preparing the meat juice in the preHminary steps. 
The relative merits of pressing out the meat juice before or 
after heating the soaked meat must be determined by experiment 
in the kinds of work for which the media are destined. 

For those workers whose needs and experience lead them to 
express the meat juice in the cold state and then dissolve the 
peptone and salt with very little preliminary heating, the use 
of the boiling titration for correction is essential, in order to 
approximate the future conditions due to further heating and 
sterilization after the reaction of the batch of medium has been set. 

In this laboratory the best toxin production has seemed to be 
obtained when the meat juice is pressed out after heating large 
amounts (20 liters) for one hour at 45° to 55°C. and then boil- 
ing up strongly until the meat coagulates. The meat infusion^^ 
is then strained through cheese-cloth. After the requisite 
amount of peptone and salt have been dissolved by further heat- 
ing up to the boiling point and the mixture boiled one half hour, 
the reaction is set according to a room temperature titration. 

If the specimen is boiled'^ in the casserole before titration, 
it no longer represents the lot of broth in the kettle but has 
risen somewhat in acidity. Consequently, if the large lot be 
adjusted according to the boiling titration a false correction 
is made. To be sure, after adding the normal soda solution, 

^' Broth = meat infusion plus peptone and, usually, salt. 

'^ This method proves useful in other lines of work for at this point the in- 
fusion can be filtered, sterilized and stored. It is ready for further use on the 
addition of any suitable peptone and may be set at any desired reaction; or it 
is ready as a basis for making agar. 

1^ At this point may be mentioned the length of time recommended for boil- 
ing by different authors. Heinemann (1911) heats "to boiling." Jordan (1914) 
MacNeal (1914) and the Standard Method (1913) boil one minute. Park and 
Williams (1914) boil two minutes, Abbott (1915), Abel (1914), Hiss and Zinsser 
(1914), Mallory and Wright (1915), Swithinbank and Newman (1903) boil for 
three minutes. 



TITRATION AND ADJUSTMENT OF CULTUEE MEDIA 223 

any further boiling of the large lot, together with the final sterili- 
zation raises the acidity but not to just the desired point as 
shown in the tests on meat infusions (chart 6). For example, 
the final reaction of + 1.2 in the case of diphtheria toxin broth, 
is found to be uniformly obtained by titrating at room temper- 
ature and setting the reaction to + 1. This allows 0.2 rise due 
to heating if the broth is to be sterihzed at 15 pounds pressure 
(121. 6°C.) for one half hour. If the sterilization is to be carried 
on at only 5 pounds pressure (108.8°C.) for one hour on three 
successive days, or in the Arnold sterilizer, streaming steam 
(100°C.), for the same length of time, the reaction is set at + 1.1 
as the more moderate heating raises the broth only about 0.1 
per cent in acidity, making the finished product + 1.2 in reac- 
tion. The use of this method for the diphtheria and tetanus 
toxin broths for a number of years has shown fully its value and 
it is the method still employed in our laboratory. 

On the other hand Eyre (1915) states that the correct esti- 
mation of acidity present can be made only by titration at the 
boiling point. Judged from our results as shown in chart 4 
by the /3' and <S curves, this statement is erroneous. The ^' 
curve shows the reactions and successive adjustments to plus 
one (+1) based upon the boiling temperature figures. The re- 
actions at room temperature of this material are shown by the 
/3 curve. This latter is far below the /3' curve (from 0.5 to 1 
per cent), and gives the actual reaction of the medium at a tem- 
perature nearer that of the incubator (37.5°C.)^^ 

To make this point clear, let us assume for example that a 
medium is to be adjusted to a definite acidity of + 1, accord- 
ing to the boiling titration, as stated in the Standard Method. 
The real reaction at which the bacteria will then be grown in 
the incubator is not that indicated by the boiUng titration 
figure but a reaction which is lower in acidity to an extent of 
about one per cent — in other words, almost neutral. 

On the other hand in chart 3 (a curve), there is shown a simi- 
lar meat infusion, adjusted to plus (+ 1) at room temperature 

i""' It is only after four hours' autoclaving that the room temperature reaction 
of plus one (+1) is reached. 



224 BERTHA VAN H. ANTHONY AND C. V. EKROTH 

titration. The boiling titration figures are plotted as curve 
a. The room temperature titration curve (a) comes so near 
the desired reaction of plus one ( + 1) that even a mere glance 
will suffice to convince one that the room temperature titration 
approaches more closely the one per cent line, which is the acid- 
ity we set out to secure. 

This was not surprising to us as our practical experience for 
several years past had indicated such a condition. The modifica- 
tion, (page 212), devised at that time and now further sub- 
stantiated by these experiments, has proved to be so very useful 
in its results that it is employed in these laboratories for nearly 
all the routine preparation of some fifty different kinds of media, 
aggregating over 8000 liters per year. 

STERILE SODA 

In order to avoid the complications of further hydrolysis 
and precipitation after the addition of soda to a medium which 
must later be sterilized, it has been suggested that sterihza- 
tion be done first and that sterile soda be carefully added after- 
ward according to the titration of samples withdrawn under 
sterile conditions. This has been practised by some workers, 
apparently with success. So far, in our laboratory, it has 
shown no advantages in the production of diphtheria toxin 
broth. Further work in this line is contemplated. 

AGAR 

When the need arose of supplying large amounts of neutral 
veal agar for the growing of the gonococcus, streptococcus and 
other organisms in bulk for antigens, difficulty was expericxiced. 
To grow these organisms in large lots with unfailing success 
is not always easy. Our trouble seemed due chiefly to the 
reaction of the medium. Finally the modified titration method 
was adopted for agar also. 

Since agar solidifies at a little below 40°C. the room temperature 
titration was not suitable. At first any temperature between 



TITRATION AND ADJUSTMENT OF CULTURE MEDIA 225 

40° and 50°C. was used. This was reduced later to about 30°C.^' 
Five cubic centimeters of the hot agar are added by means of 
a pipet to 45 cc. of distilled water — temperature about 30°C. 
(verified with a thermometer) . One cubic centimeter of phenol- 
phthalein (1 per cent solution in 50 per cent alcohol) is added 
and the titration performed at once. 

It is now our custom to titrate each batch of neutral veal 
agar at least twice during its preparation making the necessary- 
adjustments of reaction. Here, as with the broth, allowance 
must be made for further heating in the autoclave. Experience 
shows that agar made from meat infusion rises in acidity usually 
about 0.3 to 0.4 per cent, at 15 pounds pressure during one and 
one-half to two hours. ^^ Therefore, 3 to 4 cc. of normal soda 
should be added per liter in excess of the amount required to 
secure the phenolphthalein neutral point at the time of the 
first titration. The second titration is made just after filtra- 
tion, before tubing and sterilizing. If the amount of soda needed 
does not exceed 0.2 per cent, little if any precipitate occurs 
on heating further. If more than the above amount is needed 
in adjusting the reaction, the medium should be heated in the 
Arnold for half an hour and the precipitate filtered out, before 
tubing and sterilizing. 

In very careful work the medium is also titrated a third time 
as it comes from the autoclave. For the last test a tube or small 
bottle of neutralized glassware should be used in order that the 
reaction of the agar may be unaffected by its container. This 
sample is tested before it hardens — as remelting would raise its 
acidity further. On the addition of phenolphthalein, it should 
show a very dehcate shade of pink if it is "neutral." 

REMELTING OF SOLID MEDIA 

An important factor to be considered in the adjustment of 
media is the remelting of sohd media for the addition of sterile 
substances such as blood, serum, etc., or for the purpose of 

1^ This slight difference of temperature had no noticeable effect on the results 
of the titration. 

18 This time is necessary for melting (and clearing with egg) of large batches 
of agar (5 to 12 liters). 



226 



BERTHA VAN H. ANTHONY AND C. V. EKKOTH 



immediate use in plating. If, for example, the whole is to be 
neutral to phenolphthalein when entirely finished an over neu- 
tralization is necessary to allow for the acid changes during the 
re-heating, as in making Bordet Gengou medium. 

Since, in spite of the addition of soda for the correction of a 
medium further hydrolysis occurs when heat is applied, especi- 
ally in the autoclave, it is impossible to know the exact reaction 
a medium will have when sterilization is complete or when the 
medium is re-melted. In practical work, however, it has been 
found that an over neutralization of 0.1 to 0.3 per cent has given 
good results when the titration is performed at 30°C. 

The re-sterilization of media without suitable correction to allow 
for the effects of heating is to be avoided if a very definite end 
point is desired. 

PEPTONES 

The present necessity of finding substitutes for Witte's pep- 
tone, so long the standard in bacteriological work, has led us 
to test the reaction of various peptones on the market. A 1 
per cent solution of each in distilled water was boiled one minute 
and then filtered through cotton and filter paper. When cool, 
each was titrated at room temperature and then again after 
the same sample had been boiled one minute, that is, the same 
procedure was followed as in titrating the meat infusions (page 
214). 

The following table shows the reactions of eight peptones, 
including Witte's. 

TABLE II 

Reaction of peptones {titrated with phenolphthalein) 



Armour 

Atkinson 

Difco 

Eimer & Amend. 
Fairchild culture 

Leitz 

Squibb 

Witte 



ROOM TEMPER- 
ATURE PIQURE 
(AT 20 °C.) 



+ 0.6 
+ 0.4 

+0.6 
+ 1.0 
+0.7 
+0.4 
+0.3 
+0.3 



RISE AFTER 
BEING BO LED 

ONE MINUTE 
IN CASSEROLE 



+0.4 
+0.2 
+0.2 
+0.4 
+0.4 
+0.3 
+0.1 
+0.1 



BOTIINQ 

TEMPERATUKB 

FiaURB 



+ 1.0 
+0.6 
+0.8 
+ 1.4 
+ 1.1 
+0.7 
+0.4 
+0.4 



TITRATION AND ADJUSTMENT OF CULTURE MEDIA 227 

The peptone solutions were then divided into two sets (as 
with the meat infusions), one corrected and the other uncor- 
rected. These were given successive treatments in the auto- 
clave and titrated at the same intervals. As with the meat 
infusions there was a steady increase in acidity though not in so 
marked a degree. Three uncorrected portions of peptone solu- 
tions (Fairchild's, Eimer and Amend, Squibbs) were also run 
an additional six hours, making fourteen hours in all in the 
autoclave. As with the meat infusions the hmit of hydrolysis 
was not reached. 

In the corrected portions the total amount of acidity, developed 
by heating after successive additions of normal soda, again 
paralleled closely the rise in acidity of the corresponding un- 
corrected portions. 

From the above it is apparent that the introduction of a 
peptone into a medium will affect the reaction to some extent. 

INDICATORS 

The shade of phenolphthalein suitable for a correct end point 
varies greatly in the opinion of different authors. 

Miur and Ritchie (1913) give "the first trace of pink." 

Hiss and Zinsser (1914) — "faint but clear and distinct pink." 

Stitt (1913) — "a delicate pink (hot titration) a purplish violet 

color (cold titration)." 

Jordan (1914) — "Faint but distinct pink color." 

Park and Williams (1914) — "Faint, bub distinct pink which remains 

on re-heating." 

Heinemann (1911) — "Faint but decided and stable pink." 

Abel (1912)— "Brilliant red." (Translation.) 

Abbott (1915)— "Pink color." 

MacNeal (1914) — "Faint but distinct and permanent pink." 

Swithinbank and Newman (1903)— "Clear bright pink color." 

Mallory and Wright (1915) — "Bright pink color" not "the pinkish 

darkening of the fluid which preceeds it." 

Eyre (1915) uses a "pinkish tinge" or "a faint rose-pink which 

cooled to 30° or 20°C., becomes more distinct and decidedly deeper 

and brighter" resembling a "deep magenta color." 



228 BERTHA VAN H. ANTHONY AND C. V. EKROTH 

These shades given differ more or less from the Standard 
Method (1913). 

That no two people seem to titrate to exactly the same shade 
has often been shown in our laboratory when a different worker 
in the media room has attempted to set the final reaction of 
some special medium. If the method of titrating is to be at 
all accurate, it is necessary to assume a shade of pink for phenol- 
phthalein. This necessity is brought out by such great dis- 
crepancies between different workers titrating the same sub- 
stance as are given by Clark (1915).*^ As a help, a practical 
color scale may be of aid in determining the most suitable shade 
for a certain kind of work and approximating it as closely as 
possible. This should eUminate the personal factor to some 
extent. 

As stated before, in our opinion, the correct shade for a deli- 
cate end point in pale broth or other solutions with little color 
is the first most delicate pink tinge observable throughout, 
remaining at least one minute. 

With us, when titrating agar, a mixture of 5/20 red, 3/20 
orange and 12/20 white on the color-top (see page 210) has 
proved to be a desirable shade for the first and second titrations 
of neutral agar;^" while the third titration, when the medium 
comes out of the autoclave, should give (on the addition of 
phenolphthalein) a shade consisting of 3/20 red, 3/20 orange 
and 14/20 white on the color-top. These shades differ from 
the ones given above both in the Standard Method of titrating 
at boiling point (page 210) and our own definition (page 213) 
but they have yielded very good results. However, it may be 
as difficult to decide on an end point by means of a color-top 
or scale as to imagine an end point from the descriptions of the 
various writers. 

LITMUS 

Since any medium with meat infusion as a basis and peptone 
added is a most complex mixture, no one indicator shows all 

1' (16) page 117. Such relatively great discrepancies are surprising especially 
among the chemists. 

" The deeper color of agar as compared with the usual coloi of broth makes 
necessary the use of some orange in this scale. 



TITRATION AND ADJUSTMENT OF CULTURE MEDIA 229 

of its varying acid constituents. In the last 10 or 15 years 
phenolphthalein has been largely employed, yet it cannot be 
relied upon in every instance. 
Park and Wilhams-^ state: 

Different indicators differ not only in delicacy but in the substances 
to which they react. A medium alkaline to litmus is acid to phenol- 
phthalein showing that there are present substances possessing a char- 
acter which litmus does not detect, weak organic acids and organic 
compounds, theoretically amphoteric but in which an acid character 
predominates. 

Thus a liter of bouillon becomes, on the addition of 1 per cent of 
peptone, more alkaline to litmus but decidedly more acid to phenol- 
phthalein; 1000 cc. of water with 1 per cent peptone is acid to phenol- 
phthalein to such an extent that 3.5 cc. of deci-normal NaOH is re- 
quired to neutralize it. To litmus it is alkaline and requires 3.4 cc. 
of deci-normal HCl. Two per cent peptone doubles the difference. 
The same figures hold approximately true for peptone broth. 

Eyre (1915) states that although meat infusion is always 
acid to phenolphthalein it may react neutral or even alkaline 
to litmus; again, if rendered exactly neutral to litmus, it still 
reacts acid to phenolphthalein; that this is due to the facts: 

(1) Litmus is insensitive to weak organic acids whose presence is 
readily indicated by phenolphth ilein. 

(2) Dibasic sodium phosphate which is formed during process of 
neutralization is a salt which reacts alkaline to litmus but neutral to 
phenolphthalein . 

On the other hand, MacNeal (1914) considers litmus the more 
useful: 

The neutral point indicated by litmus is very nearly the actual 
point in respect to acidity and alkalinity, and this point is not appre- 
ciably displaced in either direction by the addition of a neutral mixture 
of a feebly dissociated acid and its salts to the solution. The end re- 
action indicated by phenolphthalein when it turns pink is actually a 
point at which there is a slight excess of alkali. This is so nearly the 
neutral point in inorganic solutions, when electrolytic dissociation 

21 Third and fourth editions— 1908 and 1910. 



230 BERTHA VAN H. ANTHONY AND C, V. EKROTH 

is marked, that the error is not appreciable. In solutions of organic 
substances, especially when considerable amounts of feebly dissociated 
substances such as are contained in peptone or gelatin, are present, 
this error becomes very appreciable. The discrepancy between the 
end point for litmus and for phenolphthalein will vary for different 
lots of media. 

Naturally those media which contain litmus as an indicator 
to show acid production by the growth of bacteria, must be 
alkaline to litmus yet not too alkaline or the indicator is ren- 
dered useless. The testing of such media by the use of litmus 
paper is an unsatisfactory and crude method useful for only 
the roughest work. The use of a litmus solution (Merck's 
purified in 5 per cent aqueous solution) is far more satisfactory. 

"Neutral to litmus" is "so and so" acid to phenolphthalein, 
the figure given varying with the writer. Muir and Ritchie 
place it at + 2.5, Stitt at + 1.5 boiling titration and about 
+ 0.7 with the cold titration. Abbott gives + 2.5, Abel + 1.5 
to 2.5 and Heinemann + 2, all depending on the shade of pink 
considered by the worker as suitable and the length of time 
the sample is boiled in the casserole. 

In the modified method used in our laboratory, the figure is 
as low as + 0.6 or + 0.7 with media prepared with 1 per cent 
peptone. (This figure rises to + 1 when the boiling titration 
is used.) A 1 per cent peptone (Witte) solution in water is 
about + 0.2 to + 0.3 with phenolphthalein at room tempera- 
ture and about + 0.4 at boiling figure. 

It has been shown by Hildebrand (1913) and others (Clark, 
1915; Bovie, 1915) that an indicator does not indicate the point 
of actual neutrality but merely a definite degree of hydrogen 
ion concentration. Where these two points coincide, i.e., where 
the hydrogen ion concentration at which the indicator changes 
color, is within the zone of absolute neutrality for a particular 
mixture of substances, this change of color in an indicator will, 
of course, be of significance. It would therefore seem desirable 
to select a specific indicator for each class of media. This 
could be accomplished only by determining the effect of added 



TITRATION AND ADJUSTMENT OF CULTURE MEDIA 231 

alkali upon the electrical conductivity of the particular medium. 
Such measurement should be made by means of the hydrogen 
electrode. The measurements of potential are usually expressed 
in terms of hydrogen ion concentration, that is, acidity. 

For an apparatus as described by Bovie (1915) the details 
of manipulation are as follows: A mixture of the medium with 
water in the same proportions as used for ordinary titrations 
is placed in a beaker kept at 30°C., and the precaution of ex- 
cluding CO2 observed. The indicator is added and the stand- 
ard electrodes are immersed. Successive portions of deci- 
normal sodium hydroxid solution are then added. After each 
addition of the alkali the potential of the mixture is observed 
and recorded. 

The point at which the indicator gives its first change of color 
is marked. The additions of alkali should be continued and 
the potentials further noted until the curve changes its shape — 
that is, from concave to convex or vice versa. The actual 
point at which this change takes place is known as the point 
of inflection and is a true neutral point. The nearness of 
the indicator's point of change to this point of inflection de- 
termines its suitability for this particular class of media. The 
indicator in which the change comes nearest to this point should 
be selected for practical use. Work in choosing such indicators 
according to this method is planned. 

When titrations are performed under ordinary circumstances 
at the boiling point, it is noticeable that making a decision as 
to the correct end point to phenolphthalein is much more diffi- 
cult than when the temperature of 20° to 30° C. is used. This 
is due to the changes of ionization in the mixture caused by this 
considerable change in temperature. 

Besides this, the constant presence of colloidal substances" 
in peptones, 23 phosphates and sugars in all media gives rise to 

22 Hildebrand (1913), Clark (1915) and others have shown that colloidal sub- 
stances affect the sharpness of indicator end points. 

" In the titration of the peptone solutions it was very difficult to get a sharp 
end point even at 20°C. The indicator (phenolphthalein) seemed to "flare" 
suddenly from the colorless state to a deeper pink than the one showing a really 
delicate end point. 



232 BERTHA VAN H. ANTHONY AND C. V. EKROTH 

a further depression of ionization, especially when these sub- 
stances are decomposed by boihng. 

The meagre results from the few investigations conducted 
in this field lead us to suspect that the presence of sugars in 
media may have a considerable influence on the effective acidity, 
that is, the dissociated acid principles (Hildebrand 1913). Work 
in these lines is to be continued. 



SUMMARY 

Marked and continued hydrolysis, resulting in the formation 
of acid principles, occurred on successive heatings of meat in- 
fusions in the autoclave. The state of complete hydrolysis, 
i.e., the point at which no further acidity is produced, was not 
reached with these meat infusions (which had been subjected 
previously to boiling over the open flame for one to two hours) 
even after prolonged autoclaving at fifteen pounds pressure 
for eight^^ hours. 

Hydrolysis did not occur in solutions of Liebig's beef ex- 
tract subjected to similar heatings. In the portion which had 
been adjusted to the neutral point, however, shght hydrolysis 
did take place. 

Those portions of meat infusion in which the natural acidity 
had been adjusted with normal sodium hydroxid to plus one 
(phenolphthalein) showed that hydrolysis occurred on applica- 
tion of heat and continued to do so after successive adjustments 
and autoclavings. There was produced in these portions as 
much acidity, approximately, as was developed on heating the 
corresponding uncorrected portions. 

The further production of acidity after the addition of sodium 
hydroxid is due to the hydrolytic effect of heat in the presence 
of water, upon these portions of the medium unbound by alkali. 

The fact that hydrolysis is promoted by heat makes inaccurate 
the estimation of acid ions in a batch of medium when there is 

** With three of the meat infusions the time was extended from eight to four- 
teen hours. 



TITRATION AND ADJUSTMENT OF CULTURE MEDIA 233 

taken as an index a sample^^ titrated at the boiling point with 
phenolphthalein as the indicator. Boiling has also a marked 
effect on the ionic concentration in media mixtures, hence the 
boiling titration called for in the Standard Method and followed 
more or less closely by many workers and recommended in the 
various text books, is subject to greater error than titrations 
performed at a temperature of 20° to 30° C. 

In the adjustment of culture media not only is the desired 
end point more closely approximated by titration at 20° to 
30° C. (see chart 6), but the misleading information of the boil- 
ing titration is avoided (see chart 4). Although according to 
Eyre (see page 211) certain acids are detected only at the boil- 
ing point, the actual reaction of media at the temperatures at 
which they are used is the object of vital importance. The 
conditions prevailing at these temperatures (incubator, 37.5°C., 
for some organisms and room temperature fui- others) arp morft 
closely indicated by titrations conducted at room temperature. 

As shown in chart 4 the actual reaction of a medium 
titrated by the boiling method is really from 0.5 to 1 per 
cent lower than is indicated by the boiling titration figures. 

The tests made on the reaction towards phenolphthalein of 
the various peptones on the market showed them to differ greatly 
in acidity. The effect on peptones of prolonged heating in 
tests similar to those on the meat infusions showed that hydroly- 
sis occurred on the application of heat. The development of 
acidity took place after successive adjustments of reaction with 
sodium hydroxid, as with the meat infusions and the total 
amount of hydrolysis approximated closely that of the uncor- 
rected portions. Here, too, prolonged heating of fourteen hours 
in the autoclave did not give complete hydrolysis. 

In the usual titration methods, no one indicator gives all the 

'6 That is, if the titration is performed after a preliminary boiling of the whole 
batch of meat infusion. When a titration is made on meat juice pressed out in 
the cold and containing added peptone dissolved at a low temperature, the boil- 
ing of the sample in the casserole is necessary to approximate future conditions 
after the boiling of the whole batch. 



234 BERTHA VAN H. ANTHONY AND C. V. EKROTH 

evidence desired in every case, e.g., the differences between 
litmus and phenolphthalein. 

The necessary dependence on the change in color of an indi- 
cator to show the reaction of a solution, especially in the case 
of culture media, gives rise to error. This is due partly to the 
depressing effect of colloids, phosphates and some sugars which 
affect the sensitiveness of indicators; and also to variations of 
judgment with different workers as to the correct shade of 
color for an end point. Where it is desirable to avoid these 
influences titrations may be made by measurement of the elec- 
trical potential. 

REFERENCES 

Abel, Rudolph. 1912. Laboratory Handbook of Bacteriology, 2d ed. (trans- 
lated by M. H. Gordon), p. 20. 

Abbott, A. C. 1915. Principles of Bacteriology, 9th ed., p. 109. 

BovTE. W T 1015. A Uiiecl, reading potentiometer. Jour, of Med. Research, 
33, 295. 

Clark, W. M. 1915. Reaction of bacteriologic culture media. Jour, of Infect. 
Dis., 17, 109. 

Committee on Standard Methods of Water Analysis. 1905. A. P. H. A. 
Jour. Inf. Dis., Suppl. No. 1, 106. 

Committee on Standard Methods of Water Analysis. 1913. A. P. H. A., 
p. 126. 

Eyre. 1915. Elements of Bacteriological Technique, 2d ed., p. 149. 

Heinemann, p. G. 1911. Laboratory Guide in Bacteriology, 2d ed., p. 19. 

Hildebrand, J. H. 1913. Some applications of the hydrogen electrode in analy- 
sis, research and teaching. Jour. Am. Chem. Soc, 35, 847. 

Hiss and Zinsser. 1914. Text-book of Bacteriology, 2d ed., p. 117. 

Jordan, Edwin O. 1914. Text-book of General Bacteriology, 4th ed., p. 29. 

MacNeal, Ward J. 1914. Pathogenic Microorganisms, p. 85. 

Mallory and Wright. 1915. Pathological Technique, 6th ed., p. 83. 

MuiR AND Ritchie.' 1913. Manual of Bacteriology, 4th ed., p. 33. 

Park and Williams. 1914. Pathogenic Bacteria and Protozoa. 5th ed., p. 88. 

Stitt. 1913. Practical Bacteriology, 3d, ed., p. 18. 

Swithinbank and Newman. 1903. Bacteriology of Milk, 1st ed., p. 32. 



A NEW SPECIES OF ALCOHOL FORMING BACTERIUM 
ISOLATED FROM THE INTERIOR OF STALKS OF 
SUGAR CANE INFESTED WITH THE CANE-BORER 
DIATRAEA SACCHARALIS 

WM. L. OWEN 
Louisiana Sugar Experiment Station, Audubon Park, New Orleans, La. 

Among the injuries sustained by sugar cane from various in- 
sects, that resulting from infestation with the sugar cane moth 
borer, Diatraea saccharalis, is generally regarded in Louisiana 
as of greatest importance. This pest is very widely dis- 
tributed throughout the sugar cane growing countries of the 
world. Holloway (1912) who has made a very extended study 
of the occurrence of this insect in Louisiana, reported an aver- 
age infestation of the crop of 1911 to be about 38 per cent. This 
infestation varied in degree in different sections, ranging from 
0- to 78 per cent. The nature of the injury wrought upon 
sugar cane by this parasite is manifold. Barber (1911) attributed 
a greatly impaired germinating power of the cane to the injury 
of its eyes from the burrows made by the borer. Borer infesCed 
cane is also stunted in growth, and is rendered less able to with- 
stand high winds, while its value is further impaired by the 
secondary infection of its interior by various fungi. 

Of these various injuries caused by borer infestations, the 
last named is perhaps the most serious. Barber (1911) found 
the decrease in the purity of such cane to amount to 5.6 per cent. 
On the basis of recoverable sucrose per acre of cane, the above 
deterioration would amount to over 1000 pounds of sucrose 
for every 25 tons of cane. 

Van Dine (1912) in investigating the borer injury to sugar 
cane in Porto Rico, reports a decrease of 5.8 in the purity of 
the juice, resulting from infestation with this parasite. 

235 



236 



WM. L. OWEN 



Among the secondary injuries to borer infested cane induced 
by fungi, those resulting from the infection with the Red Rot 
disease, is of first importance in this State. Of this injury, as well 
as of the general nature of the disease and the fungus, Edgerton 
(1911) has made a very extended study. That this fungus 
plays a most important part in the deterioration of the juice 
of canes infested with the borer, may be judged from the fol- 
lowing analyses in Table III of the pubHcationof the above author. 



CONDITION OF CANE 



Sound 

Borer cane 

Borer cane with red rot 



SUCROSE 


GLUCOSE 


per cent 


■per cent 


10.50 


2.30 


11.40 


1.90 


7.80 


3.03 



As the greatest infestation of cane with the red rot disease 
occurs as a sequel to borer attacks the economic importance of 
the control of this insect is obviously very great. 

It would appear that the burrows made by the cane borer 
might offer suitable surroundings for the development of many 
species of bacteria which would inevitably find their way into 
the interior of the cane stalks. With a view of determining the 
part played by bacteria in causing a deterioration of the juice 
of borer infested cane, the writp.r began a prpliminary investi- 
gation of this subject in the fall of 1914. At that time a large 
number of borer infested canes were examined, and an attempt 
made to isolate the species of bacteria occurring therein. The 
canes were brought to the laboratory, cut into short sections, 
and the portion of the stalk surrounding the borer wounds, 
washed off in a 1 :1000 bichloride of mercury solution. A sterile 
platinum loop was then inserted as far into the interior of the 
v/ound as possible, and the injured tissue transferred to tubes 
of sterile culture media. As it happened the media employed 
for this purpose contained 10 per cent of sucrose. After a short 
incubation period, an examination of these tubes showed that 
practically every one of them was undergoing a vigorous fer- 
mentation. The presence of yeast cells in the tubes was quite 



A NEW SPECIES OF ALCOHOL FORMING BACTERIA 237 

naturally expected, but a microscopical examination showed 
that bacteria rather than yeasts were predominant. Transfers 
to sterile plates, and the isolation of the predominating bacterial 
species, resulted in obtaining a culture, which showed a marked 
abihty to ferment sucrose solutions. The copious amount of 
gas given off, which was tested for CO2 with positive results, 
and the odor of alcohol, suggested the capacity of the species 
to induce an alcoholic fermentation. When transferred to 
sterile glycerine bouillon, the species also induced a vigorous 
fermentation, indicating its possible relationship or identity 
with other species of bacteria described in the literature. Al- 
though several species of alcohol forming bacteria have been 
isolated, this property may be regarded as rare. Among the 
most prominent of these species the following may be mentioned. 
B. A'temnws was isolated from a cold hay infusion by Fitz (18£0) 
and later more closely studied, and named by Buchner. This 
species forms ethyl alcohol from glycerine. Frankland and Fox 
(1889) isolated from the solid excreta of sheep an alcohol form- 
ing species of bacteria to which they gave the name B. ethaceiicus. 
This species forms ethyl alcohol and acetic acid from glycerme. 
According to the work of Friedlander (1911) B. pneumoniae has 
the power of forming ethyl alcohol and acetic acid in nutrient 
solutions containing sucrose. Kruis and Rayman (1895) isolated 
from sour yeast a lactic acid bacterium that forms ethyl alcohol 
as a by-product. 

Other species of alcohol forming bacteria are Duclaux's (1895) 
Amylobader ethylicus isolated from garden soil, B. butylicus 
isolated by Fitz (1884) and two species isolated from malt 
decoctions by Henneberg (1909). There seems to be much in 
common between the characteristics of these species and our 
Louisiana organism, yet there are sufficient differences clearly 
to differentiate the latter from the former. 

The characteristics of the sugar cane bacterium are as follows: 

Morphological. Short thick rods with rounded ends, the 

individual cells averaging 2.8 m in length and 1.0 m m breadth. 

The rods occur chiefly in pairs, are frequently single, never in 

chains. The cells stain readily by aqueous and alcoholic solu- 



238 



WM. L. OWEN 



tions of aniline dyes, and are Gram positive. The rods are 
non-motile, non-flagellated, and do not form endospores. 

Physiological characteristics. This species does not liquefy 
gelatine at all. Milk is rendered slightly acid, and gas is devel- 
oped after an incubation of 24 hours at 35°C. The consistency 
of the milk is unchanged in three days. Nitrates are not re- 
duced. A fairly good growth occurred in a Novy jar from 
which all of the air was exhausted by means of a vacuum pump, 
and with the bottom covered with pyrogallic acid solution. The 
species is therefore a facultative anaerobe. 

The following sugars are fermented by this species: 





SUC- 
ROSE 


GLU- 
COSE 


lEVU- 
LOSE 


MAN- 
NITE 


LAC- 
TOSE 


GALAC- 
TOSE 


RAFFI- 
N08B 


MALT- 
OSE 


GI YC- 
ERIN 


Gas 

Growth in closed 
arm 


+ 
+ 


+ 
+ 


+ 
+ 


+ 
+ 


+ 
+ 


+ 
+ 


+ 
+ 


+ 
+ 


+ 
+ 



Cultural characteristics. On plain agar the colonies are small, 
rounded, but slightly raised, with entire edges. The surface 
is smooth and moist, with an amorphous interior structure. 
On glycerin agar the colonies are round greyish white, with a 
more glistening surface, but otherwise similar to the colonies 
on the plain agar. The sub-surface colonies are surrounded 
by gas bubbles resulting from the fermentation of the glycerin. 

On agar streaks the growth is exceedingly rapid. Inoculated 
tubes show a marked growth along the line of the needle after 
six hours' incubation at 35°C. 

On potato the organism forms a dirty white echinulate growth, 
slightly raised, with a glistening lustre. The growth is of a 
butyrous consistency. In bouillon the growth is very rapid, 
and the solution quickly becomes cloudy throughout. No film 
is produced. In bouillon containing 2 per cent of sucrose a 
vigorous fermentation follows inoculation with this species. 
The medium is rendered acid, and the acidity on the third day 
gives an acidifying coefficient of 3.8. 

In its morphological and physiological characteristics the 
species in question strikingly resembles other species previously 



A NEW SPECIES OF ALCOHOL FORMING BACTERIA 



239 



isolated. In the table given below and in that on page 240 will 
be found the most prominent distinguishing features. 





SUGAR CANE 

ORGANISM 

B. SACCHABALIS 


BAC. PITZIANUS 


BAC. 


BAC. ETHACETO 
SUCCINIC US 


HENNBBERG BACILLI 


CHARACTERS 


(fitz) 


(prankland) 


1 


2 


Size I 


2.8 m long 
1.0 m broad 


Very large; 
s i m i 1 ar 
to B. 
subtilis 


1.5-5.1 M L 
0.8-I.Om B 


1.7-1.5M L 
0.5-I.Om B 




Small short 
rods 


Spore for- 
mation — 


- 


+ 


— 


— 


— 




Flagella 


— 


+ 
(Probable) 


+ 

(Motile) 


+ 
(Probable) 




(Motile) 
(+) 



From the foregoing table of characteristics of the various 
species of alcohol forming bacteria, and from that on page 240, 
it will be noted that the species isolated from sugar cane, differs 
from the others in the following essential points. 



FROM 
BAC. FITZIANUS 



In smaller size 
and absence of 
spores 



FROM 
BAC. ETHACETICU8 



Not liquefying 
gelatine ; ab 
sence of mo 
tility 



FROM 

BAC. ETHACETO 

8UCCINICU8 



Growth on agar 
and gelatine; 
absence of 
motility 



FROM HENNBBBRO BACILLI 



Non - liquefac 
tion of gela 
tine; absence 
of motility 



Absence of mo- 
tility; strong 
fermentation 
of sucrose, 
glucose, and 
levulose 



The observed differences in the characteristics of this species, 
seem sufficient to constitute it as a new species. Owing to its 
prevalence in borer infested sugar cane, the name Bacillus sac- 
charalis seems appropriate. , , -u- 

In order to determine the amount of alcohol formed by this 
species in the fermentation of glycerin, a solution was pre- 
pared according to the formula of Frankland and Fox (1889), 

which is as follows: ^^^^ 

60 

Glycerm 2 

Peptone • 2q 

Calcium carbonate (precipitated) 



240 



WM. L. OWEN 



O 



H " 

a 


a 


1 1 






1 

O 




1 


- 


1 1 




1 




1 


o 

►J p 


Yellow. No liquefac- 
tion. Colonies thin 
spreading 


1 


13 

o 

o3 
^ to 


CO 

O 
c3 

03 

2 ° 
.2 .2 


03 > 
t< o 

to >> 

§^ 
tH -^ 
o ^ 
bC o 

> bD 


1 


0) 

P 
u 

CO «! 

tj « 

n 


Thin veil-like growth 
almost invisible 

Small white dots. Me- 
dium liquefied 






a c 

o3 O 

a, (u 
.2" 

~ tr 


Dirty white shining 
groAvth covering en- 
tire surface 


O 

13 

"jo 
s -"^ 

<D a 
p,o 

«^ ;^ 


01 

D 
Z 

■<! 

D 

J 

5 

-<! 


? ^ >=! 

p P o 

P3 


1 


1 


1 


1 


1 


o 
n 


Round white raised, 

glistening 
Small round white; no 

liquefaction 


White glistening 
growth. Confined to 
needle track 


Non-characteristic. 
Surface growth 
white. Line at punc- 
ture echinulate* 


a 

o 

^ a 
S.2 

-^ 2 

to V 

o ;^ 

si 


'5 

a 
.22 1; 

to ^ 

§^ 

bO ^ 
> 


Cloudy white sediment 
deposited on bottom 
of tube 


H 
H 

K 

a 
o 




° ^ ^ 

bC <u 
m '^ m O 

.2 c .2 '-^ 
o ^ o "o 

O O 


<» 
I-) 

CO 

<3 


a 

00 

u 

bO 


CO 

.s 
o 


O 

■+J 
o3 

+i 
O 

Oh 


a 
'B 

o 



A NEW SPECIES OF ALCOHOL FORMING BACTERIA 241 

Dissolved in 2000 cc. of the following salt solution: 



dis'OTjVed in 
5000 cc. 

WATER 



grarns 

Potassium phosphate 5 

Magnesium sulphate 1.0 

Calcium chloric! 0.5 

The solution was divided into one liter portions sterilized 
by the intermittent method, and inoculated with a pure culture 
of Bacillus saccharalis. After an incubation period of two weeks, 
an alcohol determination was made. The liquor was evaporated 
down to about a third of the original volume, until the distil- 
late gave only a faint reaction with iodoform. After repeated 
distillations, the specific gravity of the 50 cc. portion was found 
to be 0.99744 which corresponds to 1.707 per cent of alcohol 
by volume. By dehydrating a small portion with fused car- 
bonate of potash a solution was obtained which distilled at 79° 
to 80°C. showing it to be ethyl alcohol. 

The residue was tested for acids and acetic acid was found 
to be present, using the ethyl acetate test, A mannite solu- 
tion made up according to the same formula as the glycerin 
solution, except that 3 per cent of mannite was substituted 
for an equal weight of glycerine, was next tried. It yielded 
50 cc. of a distillate with a specific gravity of 0.99836 correspond- 
ing to an alcohol per cent of 0.55 by volume. The presence of 
acetic acid was also detected in the residue. 

Thinking that the low yields of alcohol in the two cases was 
due to the small quantity of assimilable nitrogen in the solution, 
3 per cent of glycerin was added to plain bouillon, and the 
flask sterilized and inoculated as before. In this case the solu- 
tion yielded 50 cc. of distillate of a specific gravity of 0.9864, 
which corresponds to an alcohol per cent of 4.895, which was 
much higher than in the previous experiment. The higher 
yield in the latter case indicated that there was a lack of nitro- 
gen in the solution previously used. Frankland and Fox in 
their experiment with B. ethaceticus obtained a yield of 11.41 
grams of alcohol from 60 grams of glycerin. In the experiments 
of the above investigators it was found that B. ethaceticus formed 



242 



WM. L. OWEN 



1.63 parts of alcohol to one part of acetic acid, from mannite, 
while from glycerin the ratio of alcohol and acid was 2.11 to 1. 
Although B. saccharalis also forms alcohol and acetic acid from 
mannite, the ratio in which these products are formed was not 
determined in our experiments. 

ACTION ON SUGAR CANE JUICE 

B. saccharalis grows vigorously in cane juice, and apparently 
induces a strong fermentation of its sugars. In order to deter- 
mine its effect upon the composition of this substance, sterilized 
cane juice was inoculated and the following results were ob- 
tained. To one flask 1 per cent CaCoa was added, in order to 
neutrahze acids formed during fermentation. 



I 

II 

III 
IV 



TREATMENT 



Inoculated + 1 per 
cent CaCOs 

Control + 1 per cent 
CaCo3 

Inoculated 

Control 



TOTAL 
SOLIDS 


INVERT 
SUGAR 


SUCROSE 


ACIDITY 


AI.CO- 
IIOL 
PER 
CENT 
VOL- 
UME 


Single 
polari- 
zation 


Clerget 






per ceni 


per cent 






7.75 


0.73 


4.4 


4.86 


1.2 


0.53 


12.73 


3.7 


5.5 


6.91 


1.2 




8.75 


1.3 


4.2 


5.92 


2.8 


0.79 


12.43 


4.27 


5.7 


7.36 


1.8 





56 

43 
47 
45 



It will be noted from the above table that while some of the 
sucrose of the juice is inverted by the organism, a larger quanity 
of reducing sugars is destroyed. This results in an apparent 
increase in the purity of the inoculated flasks over the controls. 

The question of what role this species plays in growing cane, 
and what effect its presence exercises on the composition of 
the juice of such cane, led to some inoculation experiments 
being conducted in 1914 and 1915 in the fields of the Sugar Experi- 
ment Station. In the first series of experiments the cane was 
inoculated by means of a small cork borer, and a pipette. The 
inoculations were made in the following manner. Holes were 
made in the cane with the cork borer, and 5 cc. of a water sus- 



A NEW SPECIES OF ALCOHOL FORMING BACTERIA 243 

pension of a 24 hour-agar streak of the organism, was then 
introduced. The controls were treated in a similar manner, 
except that 5 cc. of sterile water was used instead of a culture. 
The holes in the cane were then sealed with grafting wax. All 
of the canes sleeted for the experiment were first examined for 
borer infestion, and only the borer free canes were used. About 
twenty inoculations were made in the first experiment, the 
varieties D. 74 and D. 95 being selected for the purpose. The 
inoculations were made on the 12th of October, and the canes 
were analyzed about the first of December, allowing nearly 
two months for the bacteria to develop. When the canes 
were analyzed, they were split through lengthwise, and transfers 
made with a sterile platinum loop, from the inoculation wounds 
to sterile glycerine bouillon. In the majority of cases the B. 
saccharalis was recovered from the inoculated portion of the cane, 
showing that it had remained in a living condition within the 
cane. The analyses of the canes were so variable, that it was 
decided to repeat the experiment the following year, using a 
slightly different method of inoculation. In September of the 
following year two rows of D. 74 cane were inoculated. Instead 
of a water suspension of the organism, a three days'old culture 
grown on sterile mashed potato was used as the inoculating ma- 
terial, and a blackleg vaccine injector was employed for the 
inoculations. The analysis of the cane was made in November, 
thus allowing an incubation period of two months for the organi- 
ism to carry on its activities within the cane. The results of 
the analyses again showed that there was no marked deteriora- 
tion of the juice of the inoculated cane. The juice of the in- 
oculated cane, it is true, showed an average purity of 64.3 as 
against 67.4 for the control, but there were as many cases where 
the purity of the inoculated cane was higher than its control, 
as where it was lower. In this experiment, just as in the pre- 
vious one, the organism was recovered from the inoculated 
portion of the cane, showing that it had remained in a living 
condition during the entire period. It is possible that the 
variations in the composition of the juice from different canes, 
even though they may be of the same size and in the same 



244 WM. L. OWEN 

stool, may have accounted for the negative results of the inocula- 
tion experiments. It is likely, however, that B. saccharalis does 
not induce any marked deterioration of the juice of growing 
cane, and indeed the apparently negative results which indicated 
a higher purity in the inoculated canes is well within the range 
of possible results from the action of the species. We have 
seen in the experiment on the action of this species upon cane 
juice how an increase of the purity of the juice may result from 
the fermentation of the invert sugar. It seems very probable 
that a similar result might follow from the presence of the species 
in growing cane. The occurrence of B. saccharalis in borer in- 
fested cane, and its survival in the interior of cane artificially 
inoculated with it, suggests a certain ability on its part to pro- 
tect itself against the defensive properties of the plant. Sugar 
cane, hke all other plants, possesses protective enzymes which 
tend to prevent the invasion of its tissues with organisms and 
their development therein, once they succeed in gaining an 
entrance. Browne (1906) reports a distinctly germicidal property 
of freshly extracted cane juice. He says: 

The darkening of vegetable tissues on the exposure to the air has 
been explained by Bertrand, to be due to the action of an oxidizing 
enzjTne upon various tannin bodies, all more or less related to the 
polyphenols, and the query naturally arises does cane juice itself 
exercise any germicidal properties in connection with the natural 
phenomenon of darkening. The conclusion which we have reached 
in investigating this point is that cane juice does acquire for a time 
such germicidal characteristics. Counting the bacteria in the expressed 
juice of the cane at regular periods usually shows for several hours a 
uniform decrease in numbers; with juice from sterilized canes on the 
other hand, the bacterial content increases from the very start. 

Again the author referred to states, that 

The Hving plant therefore does appear to protect itself against the 
invasion of microscopic parasites by forming toxic products. 

The relation between the germicidal power of cane juice and 
the enzymes it contains, is suggested in the following obser- 
vation by Browne, viz., 



A NEW SPECIES OF ALCOHOL FORMING BACTERIA 245 

The test for oxydase and catalase in cane juice becomes very feeble 
after ten or twelve hours, and with the disappearance of enzymic 
power, the number of bacteria begins to undergo a sudden increase. 
But it is more especially within the body of the cane itself that this 
germicidal action is most evident, and this we might expect not only 
from the colloidal and adherent character of the enzymes which ren- 
ders them resistant to expression, but from the facts of localization, etc. 

From this we must conclude that B. saccharalis possesses cer- 
tain defensive properties which enable it to develop in spite of 
exposure to the germicidal action of the enzymes within the 
interior of the sugar cane. The prevalence of this interesting 
species in the interior of borer infested cane, and its predomi- 
nance therein, further emphasizes the ability of B. saccharalis to 
overcome the defensive properties of the plant. 



246 WM. L. OWEN 

REFERENCES 

Barber, T. C. 1911. Damage to sugar cane in Louisiana by the sugar cane 

borer. Bureau of Entomology Circular No. 139. 
Browne, C. A. 1906. Fermentation of sugar cane products. Journal of 

American Chemical Society, 28, No. 4. 
Dtjclaxtx, E. 1895. Sur la nutrition intercellulaire. Ann. Inst. Pasteur 9. 
Edgerton, C. W. 1911. La. Exp. Station Bulletin No. 133. 
FiTz. 1880. Uber Schizomyceten Garungen. Ber. d. Deutsch. Chem. Gesellsch. 

9, 17. 
Frankland, p. F. and Fox, J. J. 1889. On a pure fermentation of mannite 

and glycerine (B. ethaceticus). Proc. of the Royal Society, London, 

Friedlander. 1911. Jorgensen. Microorganisms and fermentation, 134. 
Henneberg, W. 1909. Garungsbakteriologische Praktikum, 594. 
HoLLOWAY. T. E. 1912. Field observations on sugar cane insects in the United 

States Bureau of Entomology Circular No. 171. 
Kruis, K. AND Ratman, B. 1895. Chemische Biologische Studien, 11 Bulletin 

internal Acad. Science de I'Emp. Frangois Joseph. Prague. 
Van Dine, D. L. 1912. Damage to sugar cane juice by the moth stalk borer. 

Experiment Station of the Sugar Producers Association of Porto Rico. 

Circular No. 1. 




Fig. 2. A twenty-four hour growth of B. saccharalis on plain agar. 




Fig. 1. A photomicrograph from a twenty-four hour agar growth of B. 
saccharalis. 




Fig. 3. A twenty-four hour growth of B. saccharalis on glycerin agar. 

247 



ABSTRACTS OF AMERICAN BACTERIOLOGICAL 
LITERATURE 

ANIMAL PATHOLOGY 

The Maintenance of Virulence of Bacillus abortivus equinus. E. S. 

Good and W. V. Smith. Jour. Med. Res,, 1916, 33, 493-498. 

The authors present a note on the abihty of the above bacillus to re- 
tain its virulence when kept under artificial cultivation over a long 
period. Inoculation of 1 cc. of a mixture of eight strains, represent- 
ing only one-fifth of an agar slant, produced typical abortion in a mare 
protected with 200 cc. of hyperimmune serum. The strains used in 
this experiment had been isolated for periods varying from three to 
five years. — H. W. L. 

Studies to Diagnose a Fatal Disease of Cattle in the Mountainous Regions 

of California. K. F. Meyer. Jour. Am. Vet. Med. Assoc, 1916, 

48, 552-560. 

Discussion of the subject is divided into symptomatology, anatomical 
findings, bacteriological examinations and epidemiology. 

Pieces of organs forwarded to the laboratory were subjected to mi- 
croscopic examination without revealing bipolar organisms that could 
be regarded as Bacterium bovisepticum nor did ordinary cultm-e methods 
give satisfactory results. Of about twenty-five rabbits inoculated with 
emulsions of liver infarcts, lymph nodes and spleen material, two died 
from a typical bipolar infection. The cultures isolated from these rab- 
bits gave all the cultural identity reactions recognized as typical for 
Bacterium bovisepticum. The pathogenicity tests were characteristic 
except that large doses were necessary to produce fatal results. A 
three weeks old calf succumbed 22 hours after the intravenous injec- 
tion of 3 cc. of a 20 hour old broth culture. Both strains were identi- 
cal and serologically protected against each other and against strains 
of Bacterium bovisepticum from various sources in the United States. 

Inoculation of guinea pigs with liver and infarct material caused 
death from infections with an undetermined anaerobe. Bacillus coli 
and diplococci. The anaerobe was not pathogenic to calves. Most of 
the mice inoculated with similar material remained alive or succumbed 
to the same anaerobe as did the guinea pigs. 

The writer does not feel that the evidence thus far collected is suffi- 
cient to make a conclusive diagnosis of hemorrhagic septicemia but 
as a working hypothesis has assumed that the disease in all proba- 
bility is hemorrhagic septicemia. The difficulties experienced brought 
forward again the fact that the bacteriological diagnosis of hemor- 
rhagic septicemia is not as easy a procedure as is generally considered. — 
A. R. W. 

249 



250 ABSTRACTS 

Vaccination Experiments Against Ayithrax. A. Eichhorn. Jour. Am. 

Vet. M3d. Assoc, 1916, 1^8, 669-686. 

The writer reviews the development of measures for protecting ani- 
mals from anthrax by such means as Pasteur's vaccination, spore vac- 
cines, and injection of a serum as prepared by Sobernheim. The 
latter showed that the injection of an immune animal with increasing 
amounts of virulent virus would produce a serum possessing great pro- 
tective value against anthrax. The author draws the following 
conclusions from his work with spore vaccines and serum: 

1. Horses are suitable for the production of highly potent anthrax 
serum. Serum of such horses should protect large animals in 10 cc. 
doses. 

2. The use of the serum treatment alone is indicated in cases where 
the infection has already occurred in a herd. Since the serum confers 
only a passive immunity it is advisable to revaccinate the herd in 
from three to five weeks by the simultaneous method. 

3. The serum possesses great curative value. Depending on the 
severity of the infection, the curative dose is from 30 to 100 cc; the 
injection may be repeated if necessary. 

4. For the simultaneous treatment, a spore vaccine carefully stand- 
ardized, is preferable to the ordinary Pasteur vaccine. 

5. Spore vaccine should be employed in preference to the Pasteur 
vaccines for immunization with vaccine alone. The possibility of more 
accurate dosing of the spore vaccine and the better keeping qualities 
of the same, give this product a decided advantage over the other. 

6. Experiments with concentrated serum and dry spore vaccine are 
very promising. This method would greatly simplify the vaccination 
process and also insure the product against subsequent contamination 
and deterioration. — A. R. W. 

BACTERIOLOGY OF WATER AND SEWAGE 

The Fundamental Principles of the Activated Sludge Process of Sewage 
Treatment. T. Chalklet Haltox. Indiana San. and W. S, Assn., 
1916, Eng. Contrg. 45, 235-236. 
The activated sludge process depends on the presence of biological 

life in the sludge under aerobic conditions. — L. P. 

Sanitary Features of Los Angeles Aqueduct. B. A. Heinley. Mun. J., 

1916, 40, 35-37. 

The water is brought 233 miles from Owens River to Los Angeles. 
The density of the population in the Watershed is 1.4 per square mile. 
In addition to the time in the aqueduct, reservoirs increase the normal 
storage period of 65 days under present conditions to 468 days. B. 
coli noted were traced to ducks. The mineral content ranged from 
15 to 22 grains per gallon. Algae growths occasionally cause odors 
and taste, despite covered reservoirs on distribution system. — L. P. 



ABSTRACTS 251 

The Activated Sludge Process of Sewage Treatment. G. J. Fowler. 

Can. Eng. 1916, 80, 227-228. 

The author sketches the historical development of the process, and 
dwells on the "M7" process of adding bacterial cultures in the pres- 
ence of iron in solution. Activated Sludge has 3 general effects (1) a 
clotting or clarifying action (2) a rapid oxidation of carbon and (3) 
nitrification. Much research is still required. — L. P. 

Hartford (Conn.) Waterworks Notes. C. M. Saville. Report Bd. 

Water Commrs., 1915, Mun. J., 1916, 40, 333-334. 

On account of the proximity of highways to reservoirs the water is 
sterilized before delivery. The raw water shows bacterial counts as 
high as 39,000 per cubic centimeter with B. coli found from 9 to 23 
times in 10 cc. or less, every month. Treatment with 0.95 part per 
million available CI has removed B. coli and 99.8 per cent of total bac- 
teria. With CaOCl2, 1 part per million available CI was used or 25 
pounds of bleach per milhon gallons. Liquid CI used, 0.65 p.p.m. or 
5.4 pounds per million gallons. — L. P. 

Vitality of the Cholera Vibrio in the Water of New York Bay. A.J. 

Gelarie. Medical Record, 1916, 89, 236. 

The question whether the cholera vibrio dies or survives in native bay 
water is of importance in view of the fact that the waters about the 
Quarantine Station in New York may at any time be open to infection. 
Accordingly, a series of experiments was carried out to determine the 
viability of the cholera vibrio in native bay water. 

Preliminary work demonstrated that the subjection of cholera vibrios 
to the osmotic pressure of bay water had no apparent effect. 

Other organisms present in bay water were found to have an in- 
hibitory influence upon the growth of the cholera vibrios. Vibrios not 
previously enriched with peptone were eliminated after 48 hours, those 
receiving preliminary enrichment after 7 to 47 days, the period vary- 
ing according to the strain of cholera employed, the character of the 
water, and the quantity of bacteria added. Cholera vibrios added to 
sterihzed bay water were found alive in some cases at the end of 285 
days. 

The demonstration of live vibrios in native bay water after a period 
of 7 to 47 days proves conclusively that every precaution should be 
taken to prevent pollution of bay waters. — M. W. C. 

IMMUNOLOGY 

Tuberculin Therapy. Henry L. Shively. New York Med. Jour., 

1916, 103, 51. 

General discussion of tuberculin therapy with report of three cases. — 
M. W. C. 



252 ABSTRACTS 

Experimental Study of the Effect of Emetinized Blood on the Typhoid 
Bacillus. Marcus Beekman. Medical Record, 1916, 89, 284. 
The subcutaneous administration of emetine hydrochloride in 0.5 
grain doses every six hours does not impart to the blood any bacteri- 
cidal properties for the typhoid bacillus. — M. W. C. 

Newer Practical Points in the Treatment of Typhoid Fever. Beverley 

Robinson. Medical Record, 1916, 89, 311. 

In a discussion of the newer methods of treatment of typhoid fever, 
the advantages of vaccine treatment are considered as still questionable. 
— M. W. C. 

Note on a Skin Reaction in Pneumonia. Richard Weil. Jour. Exp. 

Med., 1915, S3, 10-14. 

The intradermic injection of pneumococcus autolysate in patients suf- 
fering from pneumonia produced such variations in the skin reactions 
that the author concludes that from a diagnostic standpoint, at least, 
the test has no significance. — B. W. 

The Preparation aiid Preservation of Complement. Loyd Thompson. 

Jour. A. M. A., 1916, 66, 652. 

Fresh guinea pig complement is diluted 1: 1 with an 8.1 per cent 
sodium chlorid solution. It is sealed in small tubes, 2 cc. to the tube. 
Before use 8 cc. of water is added to a tube, giving an isotonic 1 : 10 
dilution of guinea pig serum. Complement held under these condi- 
tions is active for two weeks or longer. — G. H. S.- 

Frontal and Maxillary Sinusitis and Sequelae. Due to Staphylococcus 
pyogenes albus. Ralph Opdyke. Medical Record, 1916, 89, 18. 
An account of a case in which a prolonged series of severe and ob- 
stinate involvements was found to be due solely to Staphylococcus 
alhus. An autogenous vaccine, prepared and administered immediately 
after the beginning of the disease, was used without beneficial re- 
sults.— M. W. C. 

Recent Developments in the Treatment of Leprosy. Victor G. Heiser. 

New York Med. Jour., 1916, 103, 289; 

In reviewing the methods which have been used in the treatment 
of leprosy, the author states that vaccine treatment has apparently 
caused improvement in some cases, but in his own experience it has 
proved unreliable. In his opinion, the most satisfactory treatment con- 
sists of the subcutaneous administration of a mixture of chalumoogra 
oil, camphorated oil, and resorcin. The use of this mixture has caused 
cures in some cases, marked improvement in many. Examination of 
material taken from cured cases did not reveal the presence of leprosy 
baciUi.— M. W. C. 



ABSTRACTS 253 

Vaccine Therapy. G. A. Ehret. Medical Record, 1916, 89, 328. 

Bacterins were used in a variety of infections — colon cystitis, bron- 
chopneumonia, lobar pneumonia, chronic gonorrheal cystitis and pros- 
tatitis, gonorrheal arthritis, chronic articular rheumatism, neuritis, 
bronchial asthma, and otitis media — with successful results in every 
instance except one case of bronchial asthma. In the majority of 
cases, stock vaccines were used. The number of administrations and 
duration of treatment varied with the character of the case. — M. W. C. 

Immunological Studies in Pneumonia. Richard Weil and John C. 

ToRREY. Jour. Exp. Med., 1916, 23, 1-10. 

The authors injected guinea pigs subcutaneously with 4 cc. of the 
inactivated serum of pneumonia patients and from two to six days later 
tested the animals for hypersensitiveness by applying a pneumo- 
coccus autolysate to the excised uterus according to the method of Dale. 
The tests were controlled with serum from normal individuals or from 
patients suffering from diseases other than pneumonia. Of twenty 
cases of pneumococcus infection only two failed to produce sensitiza- 
tion, while in none of the control cases was a positive reaction obtained. 
The sensitizing antibody is present in the blood early in the disease 
and is found rarely after crisis. From the results it would appear that 
while the reaction is specific for the genus it cannot be used for group 
differentiation. — B. W. 

Progress in the Treatment of Skin Diseases. G. M. MacKee. New 

York Med. Jour., 1916, 103, 441-444. 

An interesting summary is given of the progress made during the last 
few years in the treatment of skin diseases. 

Most important advances have been made in work upon the etiology 
of many of the dermatoses, particularly eczema. 

The results reported with vaccine treatment are not uniform. Vac- 
cine therapy has met with success in ringworm of the scalp, and in 
some cases,' though not generally, in acne vulgaris. Bazin's disease, 
known as erythema induratum, and lupus vulgaris have been aided by 
tuberculin therapy. 

TubercuUn is of no service, however, in the tubercuUdes nor in lupus 
erythematosus. — M. W. C. 

The Treatment of Typhoid Fever with Bacterins. Edward Waitz- 

FELDER. New York Med. Jour., 1916, 103, 407. 

Of sixteen cases of typhoid fever fourteen were treated with bac- 
terins while two were used as controls and treated symptomatically. 
The bacterins used were prepared by the New York City board, of 
health and were given intramuscularly in doses varying from 66 to 100 
millions. It was found that the larger doses were the more effective. 

The treatment was successful in that in the bacterin treated cases 
thfixe was less fever, cardiac weakness, delirium, and exhaustion than 



254 ABSTRACTS 

in the control cases. The period of convalescence as well as the period 
of acute illness was shortened, and in no cases were there any untoward 
results.— M. W. C. 

Treatment of Rheumatic Fever. Beverley Robinson. Medical 

Record, 1916, 89, 11. 

In discussing methods of treatment of rheumatic fever, the author 
mentions the use of vaccines and serums. Serums have proved to be 
without success and the advantages of vaccine treatment are still ques- 
tionable. Whenever tried, vaccines should be used with great caution. 
Polyvalent vaccines should not be administered, as there is too great 
a risk of overburdening the system with non-specific antibodies. A 
further difficulty in the way of vaccine treatment is the fact that in the 
acute stage of the disease, the only time when bacteria can be isolated 
from the joints, vaccines do the least amount of good and their use is 
accompanied by greater local and general reactions than at a later 
period.— M. W. C. 

Antiblastic Immunity. A. R. Dochez and 0. T. Avery. Jour. Exp. 

Med., 1916, 23, 61-68. 

Ehrlich's side-chain theory, comprehensive as it is, fails to account 
for certain phenomena observed in immunological studies. Its author 
postulated a "third factor" to cover this decrepancy. Dochez and 
Avery now find that antipneumococcus serum possesses the power 
not only of inhibiting for a certain period the multiplication of pneu- 
mococci but also of inhibiting in varying degree their proteolytic and 
glycolytic functions. This power is present to a limited extent in the 
sera of certain normal animals, and, inhuman serum during the course of 
lobar pneumonia it appears or increases markedly at the critical period 
of the disease. The hypothesis that this retardation of bacterial growth 
is dependent upon the inhibition of metabolic function due to the pres- 
ence of anti-enzymotic substances in antipneumococcus serum offers a 
possible explanation of the so-called "third factor" as well as a promis- 
ing suggestion for further investigation. — B. W. 

The Complement Fixation Reactions of the Bordet-Gengou Bacillus. M. 

P. Olmstead and O. R. Povitzky. Jour. Med. Res., 1916, 33, 379- 

392. 

Testing fourteen typical and four atypical strains of Bacillus per- 
tussis, and nine strains of strictly hemoglobinophilic bacilli, by means 
of complement fixation, the authors report further confirmatory evi- 
dence of the individuality of B. pertussis, particularly their ability to 
differentiate between it and Bacillus influenzae. No differences in 
ability to bind complement were observed among twelve typical per- 
tussis strains. Some cross reaction, although weak, was observed in 
two atypical strains and two strains of hemoglobinophilic bacilli. 

The work was done with immune sera produced by the inoculation 
of rabbits with live cultures of the various organisms. The original 



ABSTRACTS 255 

Wassermann technic reduced to one-tenth volume was used. The an- 
tigen which was found to give the best results was prepared as fol- 
lows: A forty-eight hour growth on Bordet-Gengou medium was taken 
up in neutral distilled water and shaken for three to four hours in an 
electric shaker, the resultmg emulsion allowed to stand at 56°C. over 
night, filtered through a Berkefeld, and the supernatant fluid used 
after being rendered isotonic with 9 per cent salt salution. — H. W. L. 

Pollen Extracts and Vaccines in Hay Fever. Solomon Strouse and 

Ira Frank. Journ. A. M A., 1916, 66, 712-715. 

That pollen is the etiologic agent in hay fever cannot be questioned, 
but that it is the only factor is not certain. 

It is possible that hay fever is a pollenosis associated with bacterial 
subinfection. It may be that the inhalation of pollen in susceptible 
individuals irritates the nasal mucosa rendering it more liable to bacteiial 
infection and that this infection in turn favors the absorption of more 
pollen. . 

Cultures from the nose yielded in most mstances pure cultures ot 
StapJujlococcus alhus, although the pneumococcus and Micrococcus ca- 
tarrhalis were occasionally found. Autogenous bacterial vaccines were 
prepared from the organisms isolated. Thirteen patients were treated 
with the bacterial vaccines; of these, 64 per cent showed signs of 
improvement. 

A series of patients treated prophylactically with pollen ejctract alone 
showed decided improvement. The administration of vaccines follow- 
ing a previous pollen treatment resulted in seasonal cures. G. H. S. 

Equilibrium in the Combination and the Dissociation of Precipitates. 

Richard Weil. Proc. N. Y. Pathol. Soc, 1915, 15, 132-134. 

If a serum or other similar antigen be mixed with its specific precipi- 
tating anti-serum, the resulting precipitate never exhausts completely 
either of these two factors. Furthermore the serum of immunized ani- 
mals sometimes contains both precipitin and precipitinogen. When a 
chemically pure antigen, namely crystallized egg albumen, is mixed 
with its specific anti-serum, a precipitate forms. The supernatant 
liquid can always be shown to contain one of the two factors, either egg 
albumen or antibody, but never both at the same time. Therefore it 
is concluded that under proper experimental conditions the precipita- 
tion reaction goes on to complete exhaustion of one factor and that equi- 
librium in the sense of mass action, does not exist. The results of earlier 
observations are therefore explained by the presence of a multiplicity 
of antigens and antibodies, as was first suggested by Von Dungern. 
Furthermore the presence of a third colloid, such as rabbit serum, does 
not interfere with the completeness of the reaction. 

In the subsequent discussion Dr. Weil stated that by heating a pre- 
cipitin to 72° it is possible to deprive it completely of its precipitating 
property while the sensitizing value is retained almost unimpaired.— 
W. J. M. 



256 ABSTRACTS 

LABORATORY TECHNIQUE 

An Electrical Furnace for Sterilizing Inoculating Loops. H. J. Corper. 

Journ. A. M. A., 1916, 66, 187. 

The author describes the construction of an electrical furnace for 
sterilizing platimun loops. — G. H. S. 

Two Laboratory Suggestio7is. Geo. B. Lake. Medical Record, 1916, 

89, 422-423. 

An eye shade for microscopical work is recommended. 

By the addition of a small quantity of acid or alkali, tone may be 
restored to Wright's stain, which has deteriorated with age. — M. W. C. 

A Method of Obtaining Suspensions of Living Cells from the Fixed 
■^Tissues, and for the Plating Out of Individual Cells. Peyton Rous 
and F. S. Jones. Proc. Soc. Biol, and Med., 1916, IS, 73. 
Bits of tissue are cultivated in plasma and the growing cultures 

flooded with trypsin dissolved in Locke's solution. The fibrin network 

is dissolved and the spherical living cells released. These are washed 

and plated anew. — W. J. M. 

A Simple Method for Blood Cultures. Paul G. Weston. Jour. A. M. 

A., 1916, 66, 507. 

An ordinary vaccine ampule is half filled with culture medium. The 
neck is drawn to a capillary tube. A vacuum is obtained in the ampule 
and the capillary tube is sealed. A rubber tube, with needle for in- 
sertion into the vein, is placed over the capillary tube. The appara- 
tus is then sterilized. 

After puncture of the vein the capillary tube is broken. After the 
collection of blood no sealing is necessary as a firm clot plugs the 
needle. — G. H. S. 

A Stain for Tubercle Bacilli. Emanuel Klein. New York Med. 

Jour., 1916, 103, 217. 

The author suggests as a substitute for the usual carbol-fuchsin, acid 
alcohol, methylene blue stain for tubercle bacilli, the following: 

(1) 3 per cent alcoholic solution of crystal violet. 

(2) 1 per cent aqueous solution of ammonium carbonate. 

(3) 10 per cent solution of nitric acid (C. P.). 

(4) 95 per cent alcohol. 

(5) Saturated alcoholic solution of Bismarck brown of which enough 
is added to water to make a tincture of iodine color. 

(1) and (2) are mixed in proportion 1:3. This is placed upon smear, 
which has been fixed in the usual manner, and allowed to steam and cool 
three successive times. Excess stain is poured off, slide washed in tap 
water. (3) and (4) are added alternately with rinsing after each, until 
specimen is perfectly colorless. Without washing, (5) is added for three 
minutes. Slide is dried and examined. The chief advantage of this 
stain is the contrast obtained with tubercle bacilli stained violet upon a 
light brown background. — M. W. C. 



ABSTRACTS 257 

MEDICAL BACTERIOLOGY 

Present Views in Respect of Modes and Periods of Infection in Tubercu- 
losis. Mazyck p. Ravenel. Jour. A. M. A., 1916, 66, 613. 
A general review of the literature on the subject. — G. H. S. 

Influenza. A. H. Doty. Medical Record, 1916, 89, 455-456. 

A general discussion of influenza with special emphasis upon means 
of prevention. M. W. C. 

Chronic Tonsillitis. Louis Fischer. New York Med. Jour., 1916, 

103, 147. 

Bacteriological examinations of the throats of cases of chronic tonsil- 
litis have shown the presence of Staphylococcus aureus, and an occa- 
sional streptococcus, never the Klebs-Loffler bacillus. — M. W. C. 

Peritonitis Following Acute Ovaritis of Anginal Origin. Russell M. 

Wilder. Jour. A. M. A., 1916, 66, 659. 

In the authors opinion many cases of so-called primary peritonitis 
result from infection of the throat passing to the ovaries and finally 
causing peritonitis. In the author's case diplococci and streptococci 
were found. — G. H. S. 

Two Unusual Strains of Diphtheroid Bacilli. Ralph R. Mellon. 

Medical Record, 1916, 89, 240. 

A preliminary note briefly describing the cultural and biological char- 
acteristics of two strains of diphtheroid bacilli, both of which are patho- 
genic for animals. One of the strains is of especial interest culturally 
because of a most marked pleomorphism. — M. W. C. 

Common Affections of the Eye. S. D. Risley. New York Med. Jour., 

1916, 103, 145. 

Bacteriological examinations of the discharges from a large number 
of cases of ophthalmia neonatorum indicate that the disease is not al- 
ways due to the gonococcus, but frequently to a variety of other micro- 
organisms. Gonococcus is present in from 50 to 65 per cent of the 
cases.— M. W. C. 

The Control of Diphtheria Epidemics. W. D. Stovall. Jour. A. M. 

A., 1916, 66, 804-806. 

The author reports an epidemic of diphtheria in which the Schick 
test was employed. The use of the skin reaction and throat swabs 
together with prophylactic administration of antitoxin where indi- 
cated presents a most satisfactory method of combating epidemics of 
diphtheria. — G. H. S. 

Removal of Tonsils and Adenoids in Diphtheria Carriers. S. A. Fried- 
berg. Jour. A. M. A., 1916, 66, 810. 
Report of 6 cases of diphtheria carriers m which the condition could 

not be remedied by the local application of kaolin. 



258 ABSTRACTS 

Removal of the tonsils and adenoid tissue resulted in the prompt 
disappearance of the organisms upon culture. — G. H. S. 

A Study of the Etiology of Chronic Nephritis. P. K. Brown and W. T. 

Cummins. Journ. A. M. A., 1916, 66, 793-797. 

From the study of a large number of cases of nephritis the authors 
conclude that venereal and other serious infections, chiefly strepto- 
coccus and pneumococcus, have a very definite bearing on the occurrence 
of advanced kidney disease. — G. H. S. 

Experiynental Syphilis in the Rabbit Produced by the Brain Substance of 
the Living Paretic. Udo J. Wile. Jour. Exp. Med., 1916, S3, 
199-202. 

Brain tissue from living paretics easily produces experimental syphilis 
in rabbits and the spirochaetes contained in this living tissue consti- 
tute a virulent strain with a shorter period of incubation for the rabbit 
than exists with other strains. — B. W. 

The Incidence of Syphilis Among Juvenile Delinquents. Thomas H. 

Harris. Journ. A. M. A., 1916, 66, 102. 

Wassermann tests performed on the sera of 365 juvenile delinquents 
taken without selection, gave positive results in about one-fifth of the 
cases. The author regards much of the infection as acquired rather 
than congenital. The relation of syphilitic infection to mental status 
is discussed. — G. H. S. 

Cultural Experinfients with the Spirochaeta pallida Derived from the 
Paretic Brain. Udo J. Wile and Paul Henry De Kjriuf. Jour. 
A. M. A., 1916, 66, 646. 

Rabbit inoculation with paretic brain has yielded pure cultures of 
pallida. The strains may be cultivated in artificial media. The or- 
ganisms from brain tissue are morphologically identical with spiro- 
chaetes derived from cutaneous syphilids, although the growth of the 
latter is much more luxuriant. — G. H. S. 

Diphtheria Carriers. J. C. Geiger Frank L. Kelly, and Violet M. 

Bathgate. Jour. A. M. A., 191b, 66, 645. 

Nose and throat cultures were taken from all contacts in six inves- 
tigations. Nose cultures gave 42.2 in the percentage average of posi- 
tives, throat cultures 7.9. Of all positive cultures 72 per cent were 
nose and 28 per cent throat. 

The Schick test applied in one investigation proved of value in dis- 
tinguishing between contacts and carriers. — G. H. S. 

Rdle of the Lymphatics in Ascending Renal Infection. Daniel N. 

Eisendrath and Jacob V. Kahn. Jour. A. M. A., 1916, 66, 561. 

In a series of experiments on dogs and rabbits the authors have 
demonstrated that infection of the bladder with the Bacillus coli, 



ABSTRACTS 



259 



Staphylococcus aureus and Proteus vulgaris may result in infection of the 
kidney with these organisms. 

Infection travels by way of the lymphatics in the wall of the ureter 
and not along the mucous membrane. — G. H. S. 

The Etiology of the Current Epidemic of Respiratory Infections in Chi- 
cago. George Mathers. Jour. A. M. A., 1916, 66, 30. 
Cultures obtained from the sputum, nasal discharge and the pharyn- 
geal mucosa of twenty-four cases of respiratory infection revealed the 
fact that in seventeen instances the predominating organism was a 
hemolytic streptococcus, culturally resembling the Streptococcus pyo- 
genes type. Pigment-producing streptococci and pneumococci were 
uniformly found. B. influenzae and M. catarrhalis were not present in 
any of the cultures. — G. H. S, 

The Cause of Rat-Bite Fever. Kenzo Futaki, Etsuma Takaki, Tenji 
Takiguchi, and Shimpachi Osumi. Jour. Exp. Med., 1916, 2S, 
249-250. 

A preliminary note in which is announced the finding of a spirochaete 
in the skin and in a l}anph gland of patients suffering with rat-l^ite 
fever. The skin tissue and blood drawn from a patient when injected 
into monkeys, guinea pigs and white rats produced infection and the 
disease could be transmitted from these to other animals. Further 
details are promised. — B. W. 

The Etiology of Rat-Bite Fever. Francis G. Blake. Jour. Exp. Med., 

1916, 23, 39-60. 

A case of rat-bite fever coming under the author's observation, ter- 
minating fatally and coming to autopsy, afforded an excellent oppor- 
tunity for studying the etiology of this disease. An organism, which 
the author identifies as the Streptothrix muris-ratti, was demonstrated 
in a mitral vegetation and isolated in pure culture from the blood. 
The patient's serum contained strong agglutinins for the Streptothrix.— 
B. W. 

Influenza and Grippe in Infants and Children. Carl G. Leo-Wolf. 

Medical Record, 1916, 89, 226.' 

A discussion of influenza and grippe in children. All phases of the 
subject, such as history, etiology, pathology, treatment, etc., are 
treated in detail. 

Emphasis is placed upon the fact that the two diseases are mani- 
festly distinct entities. Both are due to bacterial infection, influenza 
being caused by the cocco-bacillus of Pfeiffer, grippe by one or more 
of a number of bacteria-pneumococcus, Micrococcus catarrhalis, bacillus 
of Friedlander, streptococci, and bacteria living as saprophytic para- 
sites in the mouth. 1 1, i_ 1 -J 

In both influenza and grippe particular stress should be laid upon 

prophylaxis. — M. W. C. 



260 ABSTRACTS 

Rocky Mountain Spotted Fever. Henry C. Michie, Jr. and Houston 

H. Parsons. Medical Record, 1916, 89, 266. 

A comprehensive investigation of Rocky Mountain spotted fever, 
which includes a resume of all work done upon the disease as well as an 
account of the results of an extensive research on the fever as it occurs 
in the Bitter Root Valley, Montana. The report gives in detail the 
history, geographical distribution, etiology, symptoms, pathology, diag- 
nosis, treatment, prophylaxis, prognosis, and epidemiology of the 
disease.— M. W. C. 

Remarks on B. Welchii in the Stools of Pellagrins. W. H. Holmes. 

Arch. Int. Med., 1916, 17, 453-458. 

In a study of the stools of pellagrins an abnormally large number of 
organisms of the B. Welchii group have been found. These organisms 
are able to produce diarrhea in the presence of a high carbohydrate 
diet, which can be cured by the substitution of a protein diet. Since 
he believes that pellagra is caused by a high carbohydrate diet, the 
writer suggests a further investigation of the r61e of B. Welchii in this 
disease. — G. H. R. 

The Treatment of Infections of Accessory Sinus. A. M. MacWhinnie. 

New York Med. Journ., 1916, 103, 213. 

Theoretically, the treatment of ethmoiditis should consist in the ad- 
ministration of an autogeneous vaccine made from all types of bacteria 
isolated. In most cases these vary in number from three to five. The 
use of such a vaccine has met with marvelous results in a few cases, 
but the average of successful treatments is 30 per cent. 

The author recommends a system of cleansing to be used in con- 
junction with the application of his suction piunp. — M. W. C. 

Salvarsan in Primary Syphilis. Alexander A. Uhle and Wm. H. 

Mackinney. New York Med. Jour., 1916, 103, 6. 

Treatment of primary syphilis with salvarsan is most successful in 
cases in which a diagnosis is established sufficiently early to allow 
prompt administration of the drug. The earliest positive diagnosis of 
syphilis can be made by examination by dark field illumination of the 
expressed serum of a suspected sore. A Wassermann reaction is not 
positive until the seventh to fourteenth day after the appearance of the 
chancre. The Wassermann reaction is of value in the diagnosis of 
syphilis, as a positive reaction means the onset of systemic syphilis, 
while a negative reaction, for practical purposes, means a local infection 
only.— M. W. C. 

The Prompt Cure of Gonorrhea. George A. Wyeth. New York Med. 

Jour., 1916, 103, 244. 

Treatment with a 0.25 to 0.5 per cent solution of protargol, if begTm 
within twenty-four hours after the appearance of a purulent discharge, 



ABSTRACTS 261 

has resulted in a cure within five to seven days in 60 per cent of the 
author's cases of gonorrhea. 

New, well developed cases, where no evidence of phagocytosis is 
shown, are more stubborn in yielding to treatment than cases where 
the gonococci are mostly intracellular. In the latter group of cases, 
the use of vaccines is indicated. — M. W. C. 

A Study of the Bacteriology of Chronic Prostatitis and Spermatocystitis. 

Harry B. Culver. Jour. A. M. A., 1916, 66, 553. 

Review of literature. Detailed account of technic employed by the 
author. 

34 cases examined organisms were recovered from 70 per cent. 
Twelve different organisms were isolated — Staphylococcus alhus, Strep- 
tococcus hemohjticus, Gonococcus, diphtheroid bacillus. Micrococcus tetra- 
genus and M.catarrhalis, Bacillus profews, a colon-like bacillus, anaerobic 
staphylococus and streptoccoccus and unidentified gram-negative cocci 
and diplococci. 

Skin tests, agglutination and opsonic determinations showed that in 
66 per cent of the cases tested the organisms isolated appeared to be 
specific for the infected individual. Vaccine treatment was apparently 
beneficial. — G. H. S. 

Gallbladder Diseases. C. H. Mayo. New York Med. Jour., 1^16, 103, 

433-436. 

Diseases of the gallbladder are of infectious origin. Cultures made 
from the tissues of actively diseased gallbladders, and inoculated in- 
travenously into experimental animals caused disease of the gall- 
bladder, even to occasional stone formation, in 61 per cent of 41 ani- 
mals. (Rosenow.) - . 

Stones removed from the gallbladder may retain living bacteria for 
years. The stone is the result of the infection, not the cause of the 

The mode of infection is not yet known. Several theories have been 
advanced, the most probably being that of Rosenow— that the tissues 
of the gallbladder are open to infection through the vascular system. 
Typhoid bacteremia is frequently the etiological factor and in this case 
the attack is undoubtedly through the vascular system. 

Several methods of treatment are described.— M. W. C. 

The Bacteriology of the Recent Grip Epidemic. Charles Halpin 

Nammack. Medical Record, 1916, 89, 369. 

Cultures made from 50 cases, which had been clinically diagnosed 
as grip, revealed the following findings: 

Influenza-like bacilli in 19 cases, in 6 cases alone and m 13 asso- 
ciated with other microorganisms. 

Pneumococcus F ^^ ^^^^^ 

Hemolytic streptococcus P ^ ^^^^^ 

Friedlander's bacillus ^'^ ^ ^^^^^ 

Staphylococcus P f ^^^^^ 

Micrococcus catarrhalis "^ ^ ^^^^ 



262 ABSTRACTS 

These organisms were isolated by means of sputum and nasal 
cultures. 

Two cases are reported in which after recovery there were isolated 
pure cultures of capsulated pneumococci in one and Bacillus influenzae 
in the other. Such findings emphasize the necessity of taking pre- 
cautions against infection from persons who are undoubtedly carriers 
of organisms that may cause grip. — M. W. C. 

So-Called Grippe. J. B. Rucker, Jr. New York Med. Jour., 1916, 

103, 294. 

Bacteriological examinations of 20 cases of so-called grippe presented 
findings as follows: 8 containing pneumococcus, typical at least in 
morphology; 6 containing atypical pneumococcus or Streptococcus niu- 
cosus; 20 containing small gram positive biscuit-shaped diplococci; 2 
containing the bacillus of Pfeiffer, in smear only. 

Of all organisms isolated, pathogenicity for animals was established 
only with the small gram positive diplococci. These organisms caused 
death of mice in 2 cases, and typical grippe-like symptoms in a rabbit 
in 1 case. Results of examination of these 20 cases suggest that the 
etiological factor in the recent epidemic of so-called grippe is the small 
gram positive diplococcus isolated from all cases. — M. W. C. 

Routine Wassermann Examinations of Four Thousand Hospital Pa- 
tients. I. C. Walker and D. A. Haller. Jour. A. M. A., 1916, 
66, 488. 

Routine examinations of 4000 hospitals admissions were made. The 
prevalence of unsuspected syphilis and the frequency of positive reac- 
tions in various diseases were studied. 

The reaction was positive in 600 cases. Of the 600 positive patients 
48 were in the very early stages of syphilis, 306 were in a later stage, 
and 120 were in cases having involvement of the central nervous sys- 
tem. 13 cases were congenital. 

There were 54 positive cases with aortic disease, 10 with epilepsy, 
10 with disease of the liver, 10 with disease of the kidney, 9 with pneu- 
monia, 7 with diabetes, and 13 distributed among miscellaneous diseases. 
The authors conclude that syphilis is more prevalent than is ordi- 
narily supposed and that infectious diseases, such as typhoid fever, 
pneumonia, tuberculosis and scarlet fever, do not cause false positive 
reactions. — G. H. S. 

The Treatment of Gastric Ulcer. A. F. R. Andresen. Medical Record, 

1916, 89, 4.57-459. 

A rational, specific therapy of gastric ulcer should be based upon the 
fact that this pathological conditions is due to an infective process, the 
etiological agent of which is Streptococcus viridans. 

Treatment in cases of simple ulcer should consist first of removal of 
the cause of infection, second of rest of the infected part, and third of 
efforts to overcome the infection and to repair the injured tissues. 



ABSTRACTS 263 

Under the third heading, the use of autogenous vaccines is of chief 
importance. Such vaccines have proved of great assistance in clearing 
up foci of infection, as well as in exerting a beneficient action on ulcer 
symptoms. Vaccines were used by the author in 38 cases with con- 
stantly successful results. — M. W. C. 

The Treatment of Diphtheria Carriers with Iodized Phenol. W. O. Ott 

and K. A. Roy. Jour. A. M. A., 1916, 66, 800-802. 

The treatment of 17 diphtheria carriers by the use of iodized phenol 
is reported. 

The solution (60 per cent phenol, 20 per cent iodine crystals and 20 
per cent glycerin) was swabbed over the tonsils, uvula and posterior 
wall of the phar^Tix in pharyngeal cases and over the entire anterior 
part of the nasal cavity in nasal cases. Applications were made every 
48 hours until negative cultures were obtained. 

No bad results were noted from the use of the preparation al- 
though the application is painful for half a minute or less until the 
anesthetic action of the phenol takes effect. 

Negatives cultures were obtained in 35 per cent of the cases after one 
application; in 29 per cent after the second; in 12 per cent after the 
third; in 6 per cent after the fifth and in 12 per cent after the sixth. 
One case required 9 applications. 

Fifteen cases were followed from one to three weeks after leaving the 
hospital and yielded negative cultures. — G. H. S. 

The Extent and Significance of Gonorrhea in a Reformatory for Women. 

Elizabeth A. Sullivan and Edith R. Spaulding. Journ. A. M. 

A., 1916, 66, 95. 

An exhaustive study of 500 women delinquents with respect to the 
prevalence of gonorrheal infection; the nature, duration and extent of 
the infection, together with its susceptibility to treatment; the effect 
of the infection in producing other pathologic conditions; its relation to 
birthrate; and general considerations of an economic and sociologic 
nature. 

Among 522 cases examined, 395 or 75.7 per cent were found to be 
gonorrheic. The average duration of infection was 4 years, 5 months, 
the case of longest duration being 26 years. In 82.7 per cent of the 
cases there had been no cessation of clinical symptoms since the initial 
infections. With respect to treatment during the course of the infec- 
tion, the clinical history of 378 cases showed that but 1 per cent had 
received adequate medical treatment. 

A comparison of the birthrate among gonorrheic and non-gonorrheic 
women showed that the average number of children among the former 
was 1.1, among the latter, 2.8. 

The amount of treatment required to eradicate the clinical symptoms 
depends on the duration of the disease previous to treatment. After 
disease has lasted from 4 to 6 months without treatment, 10 to 12 
months' treatment is usually necessary. — G. H. S. 



264 ABSTRACTS 

The Epidemiology of Tuberculosis. F. C. Smith. Journ, A. M. A., 

1916, 66, 77. 

A general discussion of the subject. 

The climate and topography of the country cannot of themselves 
constitute immune zones. Such areas are simply uninfected territory. 

Infection of a majority of all persons occurs before the age of 12 
years. Such factors as street dust, flies, water and fomites are prob- 
ably of less moment in causing infection then direct contact. The 
lymph glands as avenues of infection are significant. 

The importance of infection with the bovine type is indicated by 
the fact that 8 per cent of deaths from tuberculosis are due to this 
agent. 

While it is granted that certain occupations predispose to tubercu- 
losis and that age, social condition, economic state, and race may be 
potent factors, It is most certain that physical exhaustion, whatever 
may be its cause, entails the failure of some of the natural defenses, 
and latent infection becomes active. 

Gross infection should be avoided; a diagnosis should be made at 
the earliest possible time, but in the eradication of tuberculosis the 
greatest problem is the economic one. — G. H. S. 

Tuberculosis. Hermann M. Biggs. New York Med. Jour., 1916, 

103, 168. 

In reviewing the progress made in the treatment and control of tu- 
berculosis during the past 20 years, the author states that while much 
has been done toward eradicating the disease, researches upon tubercu- 
losis have not added anything essentially new to the knowledge of the 
subject. A clearer definition has been given to certain phases of the 
disease, such as the establishment of the facts that bovine infections 
play practically no part in the production of pulmonary tuberculosis, 
but do cause 30-35 per cent of the tuberculosis of lymph nodes of chil- 
dren under five years; that pulmonary tuberculosis is practically al- 
ways the result of the direct transmission of tubercle bacilli from the 
sick to the well; and that the disease is definitely preventable. 

Neither a specific treatment nor an eft'ective method of producing 
insusceptibility for tuberculosis has been discovered. Tuberculins and 
various forms of modified vaccines are receiving less recognition than 
formerly. The evidence of the wide dissemination of tuberculous in- 
fections in early life renders the use of tuberculin of little value as a 
diagnostic agent. 

With the use of the X-ray, some progress has been made in diagnosis 
and with the aid of an earlier diagnosis a larger per cent of recoveries is 
probable. 

As a constructive program for further eradication of tuberculosis, 
emphasis should be laid upon extensive improvements in preventive 
measures. Among these are disinfection of tuberculous material, 
caution in disseminating the disease, increased facilities for bacterio- 
logical diagnosis, adequate provision for institutional care of tuber- 



ABSTRACTS 



265 



culous cases, the extension of nursing service of the type now done by the 
Visiting public health nurse, insistence upon pasteurization of milk 
supplies, and vigorous prosecution of the educational compaign. — M. 
W. C. 

The Period of Life at which Infection from Tuberculosis Occurs most 

Frequently. S. Adolphus Knopf. Medical Record, 1916, 89, 47. 

A study of several still unsolved problems of tuberculosis brings 
forth the following facts, based upon the opinions and statistical evi- 
dences of a large number of authorities upon tuberculosis and children's 
diseases. 

Tuberculous diseases in childhood, compared with tuberculous in- 
fection, is relatively rare (36 per cent). Tuberculous infection in in- 
fants and young children is exceedingly frequent and the majority of 
cases in the adult can be traced to a childhood infection. Such an in- 
fection is most apt to become active about the fifteenth year ;_ if not 
then, between 18 and 30. A tuberculous infection contracted in later 
life usually occurs between the ages of 20 and 35. It is probable that 
prenatal infection is more frequent than has been generally believed. 
The frequency of infection increases with the age of the child and is 
affected by environment. 

Lungs and l>Tnph nodules are the organs most frequently mvolved 
in children; secondly, bones; thirdly, intestines; and fourthly, meninges. 

The most common sources of infection are contact with tuberculous 
individuals and infected food; especially milk from tuberculous cows. 

The most successful means of combating tuberculosis is to dimmish 
the source of infection in childhood. In order to do this, there must 
be a radical change in our present regulations in regard to the disease 
and a much more extensive provision for the care of the infected. 
Particular attention should be given to preventive measures, especially 
the establishment of an extensive educational system and the im- 
provement of social conditions. These changes can be best accom- 
plished by a Federal Commission on Tuberculosis.— M. W. C. 

The Epidemic of Typhus Exanthematicus in the Balkans and in the 
Camps of Europe. Bert. W. Caldwell. Jour. A. M. A., 1916, 66, 

A general discussion of the epidemic, its causes and ext.ent; and the 
means employed in its control. .. i j u 

One person out of every five of the population was attacked by 
typhus, the fatal cases numbering 135,000. The hospital mortality 
rantred from 19 to 65 per cent. Conditions (cold weather and con- 
gestion of population) peculiarly favorable to the distribution of the 

disease obtained. , , , i ui + • 

The body louse is a certain, and the head louse a probable, agent m 
its transmission. Evidence of any other mode of transmission is 
entirely lacking. With proper hygienic precautions non-immunes are 
practically safe from mfection. The incubation period of the disease 



266 ABSTRACTS 

is about fourteen days. Eruption follows the onset closely and reaches 
its maximum intensity on the fifth day. It is during this five-day 
period that there is greatest danger of infection. The disease seems 
to be a general septicemia, the on.y discovery relative to its pathology 
being the recovery from the spleen of an organism resembling the 
Plotz organism. 

In the eradication of the epidemic the American Red Cross Sanitary 
Commission employed such measures as fumigation of all hospitals, bar- 
racks, schools and other foci of infection, bathing patients and steriliz- 
ing their clothing, maintaining quarantine of patients, and the institu- 
tion of measures of general sanitation. 

The treatment of typhus fever is unsatisfactory and is supportive 
and symtomatic in character. The serum prepared by Nicolle, or the 
vaccine prepared from the Plotz organisms tends to abort the disease 
and apparently has therapeutic value. The prophylactic value of the 
Plotz vaccine is problematic. — G. H. S. 

PROTOZOA AND OTHER ANIMAL PARASITES 

Trichinosis. Arthur R. Elliott. Jour. A. M. A., 1916, 66, 504. 

A case of trichinosis is reported from which actively motile trichina 
larvae were foimd in the spinal fluid. — G. H. S. 

Filaria Sanguinis Hominis. Codis Phipps. Journ. A. M. A., 1916, 

^^,266 

The author reports a case of infection with Filaria sanguinis hominis 
{Filaria nocturua, Filaria bancrofii) in which a cure was effected by the 
administration of salvarsan. — G. H. S. 

Dermatitis Herpetiformis. M. F. Engman and Robert Davis. Jour. 

A. M. A., 1916, 66, 492. 

It is probable that the endameba is an etiologic factor in a certain 
percentage of cases of dermatitis herpetiformis. 

In such cases the administration of emetin hydrochloride has proved 
of value.— G. H. S. 

Trichiniasis. Michael G. Wohl. Medical Record, 1916, 89, 98. 

A general review of the disease with the report of one case. 

In discussing methods of treatment, the author states that the admin- 
istration of vaccines prepared from trichinae derived from infected hogs 
would be a logical step, as specific antibodies have been demonstrated 
in the blood of patients suffering from trichiniasis. — M. W. C. 

Thionin as a Diagnostic Stain for Pyorrhea Alveolaris. Martin Dupray. 

Jour. A. M. A., 1916, 66, 507. 

An excellent diagnostic stain for endamebae may be prepared as 
follows : 

Thionin 0.5 gm. 

Distilled water 100.0 cc. 

Phenol crystals 2.0 gm. 



ABSTRACTS 267 

The smear is air-dried, fixed in flame, stained for a few seconds while 
warm, washed and dried. 

The cytoplasm of the endamabae stains a light purplish violet, the 
nuclei a deeper reddish violet. Ingested blood corpuscles are nearly- 
black. Pus cells are a light blue. Bacteria are well stained, the fusi- 
form bacilli and spirilla being especially plain. 

The stain deteriorates in three to four months. — G. H. S. 



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JOURNAL OF BACTERIOLOGY 

OFFICIAL ORGAN OF THE SOCIETY OF AMERICAN BACTERIOLOGISTS 



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CONTENTS 

Frontispiece Picture of Professor T. J. Burrill. 

In Memoriam. Thomas J. Burrill. Erwin F. Smith 269 

Resolutions adopted at the Urbana Meeting of the Society of American Bac- 
teriologists in regard to the work of Professor Burrill 271 

W. W. Ford: Studies on Aerobic Spore-bearing Non-pathogenic Bacteria, 
Part I. Introduction. J. B. Lawrence and W. W. Ford: Spore-bearing 
Bacteria in Milk 273 

Robert S. Breed and W. W. Dotterrer: The Number of Colonies Allow- 
able on Satisfactory Agar Plates 321 

Harriet Leslie Wilcox: A Modification of the Hygienic Laboratory 
Method for the Production of Tetanus Toxin 333 

Horry M. Jones: A Method of Anaerobic Plating Permitting Observation 
of Growth 339 

Ivan C. Hall: Testicular Infusion Agar. A Sterilizable Culture Medium 
for the Gonococcus 343 

Gary N. Calkins: Book Review. Der Erreger der Maul-und Klauen- 

seuche, by Heinrich Stauffacher 353 

Abstracts of American Bacteriological Literature: 

Animal Pathology 357 

Bacteriology of Air and Dust 360 

Bacteriology of Foods 361 

Bacteriology of Soils 361 

Bacteriology of the Mouth 362 

Bacteriology of Water and Sewage 363 

Classification of Bacteria 364 

Disinfection 364 

Immunology * 364 

Industrial Bacteriology 372 

Laboratory Technique 373 

Medical Bacteriology 376 

Paleontology 384 

Plant Pathology 384 

Number one of volume one of the Journal of Bacteriology, dated January, 
appeared April 22; number two, dated March, appeared May 17. 

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IN MEMORIAM THOMAS J. BURRILL 

ERWIN F. SMITH 

In the recent death (April 14) of Prof. Thomas J. Burrill of 
the University of Illinois, there passed away, at a ripe old age 
but still in possession of all his faculties, a lovable man of un- 
common personality, and one who contributed materially during 
his earher years to the advancement of American science. In 
America we have a peculiar way of treating all those who have 
demonstrated the possession of research ability of a high order, 
which may be designated as a method of extinction by promo- 
tion. As soon as a man becomes conspicuous through his re- 
searches, boards of control find other things for him to do, more 
in keeping with their ideas of efficiency and eternal fitness, and 
he ceases to contribute further, except perhaps very indirectly, 
to the advancement of science. Professor Burrill was no ex- 
ception to this rule. He never lost his interest in science and 
having a high order of mind he was peculiarly fitted to be a 
productive research worker, but from middle life on it was his 
misfortune, recognized by him as such, but borne with cheerful- 
ness, to have his time absorbed by administrative duties con- 
nected with his university, of which he was at one time the 
acting head. His actual contributions, however, were amply 
sufficient to perpetuate his memory. 

In addition to his mycological studies, which he pursued with 
great eagerness and with good results ("Fungi of Illinois' ') he 
studied the bacteria at a time (1870-1882) when literature was 
scanty, methods were crude, and microscopes were not what they 
are today. Into this field of darkness, or at best of dim half- 
lights and perplexed gropings, which he has described to me in 
memorable words, Burrill projected his keen intellect and brought 
forth the beginnings of a whole new science, i.e., he discovered 
and demonstrated in ''pear blight" the first bacterial disease 

269 



270 ERWIN F. SMITH 

of plants. To Burrill and America belongs this honor, whatever 
other honor belongs elsewhere! Just as Pasteur's contribu- 
tion to science is more vital than Koch's, because it was earlier 
and was pioneer work, so Burrill's discovery was more difficult 
to make and hence more worthy of praise, than anything that 
has come after. Anyone of ordinary capacity can follow a 
blazed trail, but only a great man can hew a path through the 
unbroken wilderness to be a highway for all men who come after ! 

Burrill did not publish on pear blight fully, in the modern 
sense of that word, for he was a pioneer, but in studying the 
freshly diseased tissues (and he had the wisdom to select just 
those) he saw clearly in many sections that fungi were not there 
and that swarms of bacteria (called by him Micrococcus amylovorus) 
were always present and were therefore probably the cause of 
this mysterious disease. Acting on this assumption he took 
masses of these bacteria which his microscope had shown to 
be free from fungi (with a multitude of whose forms he was al- 
ready very familiar) and with them by inoculation reproduced 
the pear disease, not once but many times. Others, elsewhere, 
in these same early days made similar announcements, but 
were less fortunate or less painstaking, since no one in later 
days has been able to confirm their findings, whereas Burrill's 
discoveries have been confirmed a hundred times, and relate 
to one of our most serious orchard diseases, known for a hun- 
dred years, and for the control of which the nation and the or- 
chard states are still spending much time and money. 

Professor Burrill was born at Pittsfield, Mass., April 25, 
1839. He was educated at the Illinois State Normal School, 
and was always a teacher, and a good one. He held honorarj^ 
degrees from the University of Chicago (Ph.D., 1881) and The 
Northwestern University (LL.D.,1893), and was a member of 
various scientific societies. I remember seeing him first at 
meetings of the American Association for the Advancement of 
Science, of which he was long a member, and this year president 
of Section G (Botany). He had also been president of the 
American Microscopical Society, 1885-86, and was president 
of the Society of American Bacteriologists at the time of his death. 



IN MEMORIAM THOMAS J. BURRILL 271 

Professor Burrill was very companionable and very helpful 
to his students. He was also much respected by his colleagues 
the country over. At one of the last scientific gatherings he 
attended (the twenty-fifth anniversary of the Missouri Botanic 
Garden held at St. Louis in 1914), when his name was incidentally 
mentioned by one of the speakers there was a round of applause 
from the crowded room. I did not see him after this time 
but he was then (at 75) very well preserved and intellectually 
keen. 

The ancient Greeks had a proverb "Let not a man boast that 
he has had a happy life until the day of his death." Professor 
Burrill would not have boasted of anything since he was quiet 
and unassuming rather than loud and aggressive, but it may be 
said for him, that he represented the best type of scientific mind 
and now that he has gone we may say as we close the ranks 
and turn away: Happy was this man because he lived unob- 
trusively, serenely and usefully, and because he died full of years 
and of honor, loved by all his intimates, and respected by all who 
knew him. 



RESOLUTIONS ADOPTED AT THE URBANA MEETING OF THE SOCIETY 
OF AMERICAN BACTERIOLOGISTS IN REGARD TO THE WORK OF 
PROFESSOR BURRILL. 

Whereas, It is rarely possible for a scientist to make a discovery 
of such fundamental importance that it serves to develop an entirely 
new branch of science, and 

Whereas, One of our hosts at this time, Dr. T. J. Burrill, made 
such a discovery when he worked out the cause of pear blight and 
thus founded the science of bacterial plant pathology, 

Therefore, he it resolved, that the Society of American Bacteriolo- 
gists regards it as a peculiar privilege to congratulate him for his pioneer 
and epoch-making work, and expresses its appreciation of his vigorous 
enthusiastic and inspiring address of welcome. 

Be it further resolved, That a copy of these resolutions be engrossed^ 
signed by the officers of the Society, and presented to Dr. Burrill. 



STUDIES ON AEROBIC SPORE-BEARING NON-PATHO- 
GENICi BACTERIA 

Part I 

From the Laboratory of Hygiene and Bacteriology, Johns Hopkins University 

INTRODUCTION 

BY W. W. FORD 

One of the most important problems of modern hygiene is libkarv 
the identification and classification of the bacteria in our environ- new yOk.x 
ment. Microorganisms of various kinds exist everywhere in fiv"f *.s\r. * • 
nature and influence profoundly all sorts of substances which o av, . 
affect man's physical condition. This is true of food-stuffs in 
general and especially true of milk which is markedly altered in 
its chemical composition by the bacteria which multiply in it. 
The microorganisms in our environment are of various sorts, 
pigmented bacteria, spore-bearing bacteria, yeasts, moulds, 
etc. Some of these forms are identified without great difficulty 
but our knowledge of the spore-bearing bacteria is still in a 
state of chaos. The reason for this lack of knowledge is not far 
to seek. The science of bacteriology developed primarily among 
physicians whose interest naturally lay in the disease-producing 
properties of the various parasites which infect man and the 
animals. Non-pathogenic bacteria were of importance chiefly 
as laboratory contaminations to be avoided. With the de- 
velopment of industrial bacteriology those species were again 
most carefully studied which seemed to serve some distinct 
purpose in nature, as for example, the nitrifying bacteria of the 

' The term "non-pathogenic" is used here in the sense of "lacking in disease- 
producing properties." Many spore-bearing bacteria are at times pathogenic 
to small animals and instances are reported in which they may produce inflam- 
matory reactions when vegetating on mucous surfaces. The organisms here 
described are however in no instances capable of producing definite diseases 

273 



274 J. S. LAWRENCE AND W. W. FORD 

soil and the lactic acid bacteria in milk. In consequence the 
bacteria found in nature which seem to be lacking in any 
definite function have been largely neglected. At various times 
many species of spore-bearing organisms have been described 
and recorded in the literature and in many instances these 
cultures have been kept alive in laboratories both in Europe 
and in America. It would seem an easy task therefore to collect 
the spore-bearing bacteria from different institutions, make a 
careful study of their properties and arrive at some conclusion 
as to their identity and classification, just as is done with the 
pathogenic species. This method of solving the difficulty is 
open to serious objections, however, and has not thus far proved 
of great value. In the first place the descriptions originally 
given of many of these species are meager and the original 
cultures have not been saved. In consequence the literature 
of bacteriology is thickly strewn with names of spore-bearing 
organisms which have absolutely no meaning. The term 
Bacillus suhtilis for instance is applied to almost any large 
microorganism which forms spores readily and grows abundantly 
on artificial media, and cultures identified as Bacillus subtilis 
by different bacteriologists are often found to have little or 
nothing in common. Again the cultures which have been kept 
alive have in many instances so changed in character as no longer 
to give the reactions orginally described. Thus Migula (1897) 
found that of some six hundred cultures obtained by him from 
the laboratories in Germany only a small number had the 
characteristics first ascribed to them. Finally, pure strains of 
spore-bearing bacteria are more difficult to keep in direct descent 
in the laboratory than are other species. When cultures become 
contaminated it frequently happens that the contaminating 
species is picked up from the plates made to purify the strain 
and carried on as the original. This has happened a number of 
times in our own laboratory during the past few years and in 
consequence we have become very sceptical of the value of any 
conclusions based upon a comparison of existing stock cultures. 
A number of years ago an attempt was made, in the laboratory 
of Dr. Adami in Montreal (Ford, 1903) to separate and classify 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 275 

the sporulating organisms by the use of carbohydrates. The 
result of this work was not entirely satisfactory because of the 
difficulty of estabUshing the fundamental species from which to 
build up our system of classification. During the past few years, 
however, a number of very valuable papers on spore-bearing 
bacteria have appeared in the literature and have cleared up 
some of the most difficult points. Of especial importance is the 
work of Meyer (1903) and his collaborators, Gottheil (1901) 
and Neide (1904) in Germany, and the work of Chester (1903) 
in this country. As a result of the efforts of these authors we 
now have accurate descriptions and definite means of identi- 
fication of a small number of our most common spore-bearing 
species. 

Some four years ago a large number of spore-bearing bacteria 
was obtained from raw milk and from milk heated to various 
temperatures from 60° to 100° and so much difficulty was en- 
countered in their identification that it seemed as if the time was 
ripe for a more extensive investigation of the subject, based 
upon the work above referred to. The problem was first under- 
taken by Mr. Lawrence and myself with the organisms from 
milk. After a working basis had been obtained for the classi- 
fication of these species a study of the spore-bearing bacteria of 
water was undertaken by Dr. Laubach, and of the soil by Dr. 
Laubach and Mr. Rice with the object of testing the classifi- 
cation already adopted for milk bacteria and of adding to it such 
species as had not previously been encountered in our work. 
Finally stock cultures of well-known species were obtained from 
the Krai collection in Vienna, the Winslow collection in the 
American Museum in New York, from the laboratories of 
hygiene of the University of Pennsylvania, and of the University 
of Chicago, and the bacteriological laboratory of the Sheffield 
Scientific School, and our cultures were compared with them. 
From the start of the work however, our object has been to 
establish clearly the different types of spore-bearing organisms 
in our own laboratory and then to link these types up with types 
already established by other observers. Altogether over 1700 
cultures have been studied from various sources, milk, soil, dust, 



276 J. S. LAWRENCE AND W. W. FORD 

water, intestinal contents and contaminated plates. From this 
number we have obtained 28 distinct types of which 22 are 
clearly to be identified as well known species, 2 are distinct 
varieties of old types and 4 are evidently new species. In general 
our aim has been to clarify our knowledge in regard to old species 
and not to establish new types except when our isolations showed 
certain characteristics not already referred to in the literature 
and of distinguishing importance. 

The media employed in this work were the standard media 
of the laboratory. A great deal of emphasis was laid upon the 
reactions with gelatin, with litmus milk, with glucose, saccharose, 
and lactose broth, with glucose htmus agar and with Loeffler's 
blood serum. The morphology was studied from smears made 
from plain and glucose agar cultures 6 to 8 hours old and 22 to 
24 hours old, and from cultures 1 to 2 weeks old, the organisms 
being always stained with Gentian violet. The same preparations 
were used later for measurements and for illustrations. The 
method of sporulation and the size, shape and position of the 
spore were observed with great care. A study of the spore wall, 
and its differentiation into the exine and intine of Gottheil and 
Chester, while interesting and important, proved of little help 
in classification. The method of spore-germination was like- 
wise found relatively valueless. Nearly every type of spore- 
germination could eventually be found with most species and 
our observations were so inconstant as not to furnish any basis 
for classification. Micro-chemical reactions, while undoubtedly 
of great value, could not be worked out with any degree of 
thoroughness and were eventually discarded. Careful obser- 
vations were made upon the thermal death points which were 
established with broth cultures subjected to various degrees of 
temperature in the Arnold sterilizer and in the autoclave. In 
general our classification may be said to rest upon morphological 
and tinctorial properties, spore-formation and cultural reactions. 
How valuable our results are can only be determined by the 
extent to which other workers may be able to utilize this classi- 
fication in subsequent investigations. 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 277 

SPORE-BEARING BACTERIA IN MILK 
BY J. S. LAWRENCE AND W. W. FORD 

It has been pointed out by a number of observers (Fliigge, 
1904; Ford and Pry or, 1904) that milk heated to any tempera- 
ture above 60°C, if kept in a warm place, shows an excessive 
development of spore-bearing bacteria which are ordinarily 
inhibited by the lactic acid bacteria universally present. Hueppe 
(1884) was possibly the first to call attention to the presence of 
aerobic spore-bearing forms in milk but it is impossible to say 
now what his Bacillus butyricus (an aerobe) really was. Sub- 
sequently Loeffler (1887) described an organism from boiled 
milk which had been allowed to clot, under the name Bacillus 
lactis albus, now known as Bacillus albolactus Migula. Con- 
siderably later Flugge (1894) took up the question at some length 
and described eleven different species found in boiled milk and 
to them he ascribed an etiological role in the summer diarrhoea 
of infants. Several of the organisms described by Flugge are 
now considered identical with such common saprophytes as 
Bacillus viigatus and Bacillus mesentericus while others can be 
identified with diflSculty or not at all, as their originals have been 
lost. During the past three years we have worked out the 
morphological and cultural reactions of 250 spore-bearing 
bacteria obtained from raw milk and from milk subjected to 
various temperatures. The two most common species proved 
to be Bacillus cereus of Frankland and Bacillus subtilis of Cohn. 
In this differentiation we follow Chester who has given us a 
definite and clear conception of Cohn's species and has taken up 
at length the somewhat involved discussion concerning the two 
organisms. As a result of his work Chester decided that the 
real Bacillus subtilis of Cohn is one of the smallest of the spore- 
bearing species, forms central or slightly excentric spores which 
have a characteristic appearance and gives definite cultural 
reactions. The reactions as outlined by Chester we are able to 
confirm in the main but we disagree absolutely from him in his 
contention that this species is identical with Bacillus vulgatus, 
{B. mesentericus vulgatus) the old fashioned "potato bacillus." 



278 J. S. LAWRENCE AND W. W. FORD 

The cultures identified by us as Bacillus subtilis corresponded 
in all particulars to a culture sent us several years ago by Chester 
and kept in the laboratory since then. The particular points 
by which Bacillus subtilis may be differentiated from Bacillus 
vulgatus are the development on glucose litmus agar where it 
forms a dry hard warty growth made up of dense masses of 
material clinging firmly to the medium, in which may be ob- 
served numerous blebs or blisters containing milky fluid, and 
on Loeffler's blood serum where a similar growth appears, often 
however with a distinct red color. On both glucose litmus 
agar and blood serum Bacillus vulgatus develops as a soft 
wrinkled friable mass easily broken and lifted from its sub- 
stratum. On potato the subtilis differs from the vulgatus. 
The former produces at first a rather dense whitish or greyish 
mass often showing blebs similar to those on agar and blood 
serum, a distinct red line appearing in the potato a little dis- 
tance from the growth, from which characteristic the name 
Bacillus subtilis-ruber is frequently employed. After 48 to 72 
hours a wrinkling appears, the growth later becoming moist and 
homogeneous. B. vulgatus produces a wrinkled growth from the 
start, this becoming extremely abundant in 3 to 4 days and 
frequently assuming a decided pink color. The differences 
between the two species are somewhat difficult to describe but 
when potato cultures of the organisms are placed side by side 
the points of differentiation become clear and definite. In 
general the subtilis cultures are dry and hard on solid media and 
produce firm tenacious scums on fluids, while the vulgatus cul- 
tures are soft and mealy and their scums friable and easily broken. 
On a morphological basis it is extremely puzzling to attempt the 
differentiation of the two types. In general the rods of Bacillus 
vulgatus are longer and thinner than those of Bacillus subtilis 
while the spores are flatter and bulge the organism only a little 
if at all. 

This view of Bacillus subtilis of Cohn and the interpretation 
put on Cohn's work by Chester is not entirely accepted 
by bacteriologists but we feel convinced of its correctness 
except in regard to the differentiation from Bacillus vulgatus 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 279 

already mentioned. The organisms frequently regarded as 
Bacillus subiilis which are characterized by their greater size, 
their soft mealy growths on hard media, and their thick friable 
scums we agree with Chester in referring to the "cereus" group 
the principal type of which, Bacillus cereus, was first described 
by the Franklands. There are two strains of Bacillus cereus 
differentiated by their action on saccharose but it does not 
aeem wise at the present time to divide the species. Our identi- 
fication of Bacillus cereus rests upon Chester's work and upon 
cultures sent us by him several years ago. In addition a num- 
ber of strains of Bacillus cereus have been received from American 
laboratories and from the Krai collection in Vienna, all of them 
agreeing with Chester's in their reactions and thus furnishing 
us a distinct type by means of which our own strains were identi- 
fied. Bacillus cereus is the most widely distributed aerobic 
spore-bearing organism in nature in Baltimore and vicinity, 
as it seems to be in other localities, and possibly has more syno- 
nyms than any other species. With these types of Bacillus 
suhtilis and Bacillus cereus clearly outlined the task of identify- 
ing the other spore-bearing organisms became somewhat simpler. 
Bacillus vulgatus was soon found so frequently as to enable us 
to recognize it without difficulty. One strain of this organism 
was obtained from the Winslow collection in New York. When 
freshly isolated the vulgatus is very characteristic and differs 
entirely from other species. The strains isolated in Baltimore 
were identical with the organisms found in Montreal several 
years ago and regarded there as Bacillus vulgatus and give 
reactions ascribed to the widely distributed ''potato bacillus." 
Bacillus mesentericus {B. mesentericus fuscus) was recognized by its 
morphology and its cultural reactions. In this species we follow 
Chester. In one instance we obtained a stock culture of Bacillus 
mesentericus which gave the correct reactions as outlined by 
Chester, this culture coming from the laboratory of hygiene of 
the University of Pennsylvania. Bacillus pumilu^ of Gottheil 
we do not regard as a distinct species. Bacillus aterrimus (B. mes- 
entericus niger) was identified by its production of a black or 
grey-black pigment, its cultural reactions resembling those of 



280 J. S. LAWRENCE AND W. W. FORD 

B. vulgatus. Another organism producing a black pigment and 
evidently belonging to the mesentericus group was sent us by 
Winslow as Bacillus lactis-niger. It corresponds culturally to 
Bacillus mesentericus. It was not encountered in our work but 
is included here for the sake of completeness. The same holds 
true of the organism described originally as Bacillus mesenteri- 
cus-ruber (properly B. globigii) a culture of which was obtained 
from the Krai collection in Vienna. Evidently this is an extreme- 
ly rare organism in this country as it was never obtained 
in Baltimore either from milk or from any other source. 

One of the most difficult organisms to identify was a species 
frequently isolated in Baltimore from milk which after boiling 
clots and peptonizes. In morphology and in its chief cultural 
reactions it corresponds closely to Bacillus cereus but is differ- 
entiated from this species by its acid fermentation of lactose 
and its coagulation of milk. This organism was evidently 
first described by Loeffler in 1887 as Bacillus lactis albus {Bacillus 
albolactus Migula). We have been unable to obtain a culture 
of Loeffler's organism but in his original description Loeffler 
differentiates this species clearly from several other organisms 
found in milk particularly the ones now known as Bacillus 
vulgatus of Fliigge, Bacillus liodermos of Fliigge, and Bacillus 
butyricus of Hueppe. Since Loeffler was the first to call attention 
to the presence of an organism in boiled milk which acidifies 
and clots it and which he differentiated from other spore-bearing 
bacteria, we feel that similar organisms from boiled milk which 
correspond to Loeffler's description should be regarded as identical 
with his species. We therefore propose to utilize the name 
Bacillus albolactus Migula, (synonym Bacillus lactis albus Loeffler) 
for the organisms isolated from the source studied by Loeffler. 
This organism is undoubtedly isolated from time to time by 
other bacteriologists and must exist in a number of laboratories. 
It was apparently described recently by Neide (1904) in Meyer's 
laboratory as Bacillus teres. Bacillus albolactus is, we believe, 
the common cause of the clotting and peptonization occasionally 
seen with boiled milk. It is undoubtedly also a contributing 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 281 

factor to the changes seen in milk pasteurized at lower tempera- 
tures, 60° to 65°C., which subsequently develops a bitter taste. 

Bacillus mycoides was identified without difficulty by the 
classical descriptions and by the work of Chester whose con- 
clusions were based upon a culture which was sent him from our 
laboratory several years ago. The felted growths in the depths 
of agar are very characteristic and are given by but one other 
species, Bacillus ramosus-liquefaciens oi Prausnitz. 

For a long time we were in doubt as to the identification of the 
very large microorganisms which are placed in a heterogeneous 
group and sometimes called Bacillus megatherium, sometimes 
Bacillus petasites, and sometimes Bacillus tumescens. The 
first member of this group was described by De Bary (1884, 
1887) whose illustrations are very characteristic. Many of the 
cultures identified and sent to us as Bacillus megatherium differed 
radically from De Bary's description, and Chester's conclusions 
in regard to the ill-defined character of the group seemed to be 
entirely justified. These large organisms were very abundant 
however and soon resolved themselves into two distinct types. 
One type agreed with De Bary's original description in aU 
essential particulars and this type agreed also with an isolation 
of Bacillus megatherium by Kellermann sent us from the Winslow 
collection. Two cultures of Bacillus tumescens of Zopf agreed 
closely with this Bacillus megatherium and there seems to be no 
reason to regard it as a distinct species. The other type has 
almost the same morphology and the same cultural reactions as 
Bacillus megatherium but produces an intense yellow pigment. 
This type corresponds to the organism recently described as 
Bacillus petasites by Gottheil. All the organisms thus far en- 
countered with the morphology referred to can thus easily be 
divided into these two main forms. The Bacillus graveolens of 
Gottheil, not the Bacillus graveolens of Bordoni-Uffreduzzi, 
seems to be merely a strain of Bacillus megatherium in which the 
bacilli have a pecuHar property of growing in short spirals. It 
has not been encountered in our work. 

The Simplex-cohaerens group of Chester proved possibly the 



282 J. S. LAWRENCE AND W. W. FORD 

most difficult of all to clarify. Two organisms were originally 
described by Gottheil as distinct species, regarded by Chester 
however as practically identical. Strains of Bacillus simplex 
and Bacillus cohaerens received by us from Krai were quite differ- 
ent morphologically and while it is evident that we lack 
many of those pronounced cultural and morphological re- 
actions which render species and groups easy to recognize yet 
we must not therefore place organisms together which are clearly 
different. On one occasion we found in milk an organism evi- 
dently identical with the strain of Bacillus cohaerens received 
from Krai and corresponding to Gottheil's original description. 
Subsequently this species was found five times by Dr. Laubach 
in soil. These organisms gave us a fairly clear idea of the species 
and its differentiation from Bacillus simplex whose description 
we also give here. This latter description while made from a 
strain isolated by Gottheil, applies also to a species subsequently 
found in dust by Dr. Laubach. On two occasions we isolated 
from milk the species described as Bacillus fusiformis by Gottheil. 
Our isolations were identical with Gottheil's in every partic- 
ular. Finally on one occasion we obtained a strain with prop- 
erties practically the same as those of the species described by 
Fliigge as No. XII and now known as Bacillus terminalis Migula. 

The 250 cultures studied were from 68 samples of milk, 12 
of raw milk, 12 of milk pasteurized at 60°C., 32 of milk heated 
to 85°C., and 12 of boiled milk. These cultures may thus be 
held to represent so many various conditions in the development 
of the bacteria of milk as to give an accurate idea of the spore- 
bearing organisms of milk in Baltimore and they probably rep- 
resent conditions met with elsewhere. By their combined 
development in heated milk they give rise to the putrid decom- 
position so frequently observed. As can be seen from their 
cultural reactions these organisms are in the majority of instances 
energetic protein-splitters and in practically every case rapidly 
dissolve the casein in milk either before or after a preliminary 
coagulation. 

After the various types of spore-bearing organisms were es- 
tablished by the study of 250 cultures from the 68 samples 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 283 

mentioned above another series of milks, also subjected to vari- 
ous treatments, was investigated with the object of testing the 
preliminary classification adopted. In this second portion of 
our work the types previously established were abundantly 
confirmed but no new organisms were isolated. We believe 
therefore that the organisms first worked out represent the spore- 
bearing organisms usually present in Baltimore milk. Our 
original 250 cultures show the various species in the following 
proportions. 

Baltimore Milk 

Bacillus cereus Frankland 124 

Bacillus subtilis (Ehrenberg) Cohn 79 

Bacillus alholactus Migula 25 

Bacillus vulgatus (Fliigge) Trevisan 15 

{Bacillus mesentericus vulgatus Fliigge.) 

Bacillus mesentericus (Fliigge) Migula 2 

(Bacillus mesentericus fuscus Fliigge.) 

Bacillus fusiformis Gottheil 2 

Bacillus petasites Gottheil 1 

Bacillus cohaerens Gottheil 1 

Bacillus terminalis Migula 1 

In addition to these the following species were isolated from 
other sources during the work on milk and made the basis for 
comparison. We have reason to believe that they may occur 
in milk, partly from the work of others and partly because they 
are not infrequent in milk products. We introduce them here 
for completeness. 

Bacillus mycoides Fliigge. 

Bacillus megatherium De Bary. 

Bacillus simplex Gottheil. 

Bacillus aterrimus Lehmann & Neumann (Bacillus mesentericus 
niger Lunt). 

Bacillus niger Migula (Bacillus lactis niger Gorini) . 

Bacillus globigii Migula. (Bacillus mesentericus ruber Globig). 

Finally a brief note is added in regard to certain other cul- 



284 J. S. LAWRENCE AND W. W. FORD 

tures sent us which we do not regard as entitled to specific 
rank, namely 

BaciVus pumilus Gottheil. 

Bacillus graveolens Gottheil. 

Bacillus tumescens Zopf. 



\/ 



Bacillus cereus Frankland 1887 



This organism was first described by the Franklands in 1887 
(Franklands, 1887). It has since been described under a host of 
names and it is impossible to say how many different species 
are identical with it. It fs the most widely distributed organism 
of this group in Baltimore, being found abundantly in milk, 
soil, dust, water, and in the intestinal contents. It is partic- 
ularly common as a laboratory contamination. The present 
description applies to cultures received from the Krai collection, 
from the American Museum, and from a number of American 
laboratories and to over a hundred of our own isolations. 

Morphology. Regular bacilli with homogeneous protoplasm 
and rounded ends, in young cultures measuring about 0.75 by 
2.25 to 4 microns. Many of the organisms show peculiar re- 
fractile bodies of various sizes as the cultures get older, presenting 
a characteristic appearance. The nature of these bodies is 
not clear as they do not give reactions for starch or volutin. 
They can usually be differentiated from the beginning spores. 
On glucose agar the bacilli are thicker and longer measuring 
0.75 to 1 by 3 to 6 microns. Here the entire protoplasm of the 
organism is converted into the bodies mentioned above. They 
are globular, highly refractile, and are often as thick as the 
organism. (Figures 25, 26 and 27.) 

Motility. Actively motile in young cultures. 

Staining properties. Gram-positive. 

Spore-formation. Spores are formed early on both plain and 
glucose agar, often appearing within 24 hours or even in less 
time. They may be central in position, excentric or even sub- 
terminal but the latter location of the spore is rare. The spores 
are usually wider than the organisms from which they spring 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 285 

and thus bulge the rods slightly. The free spores retain their 
protoplasm at the ends for some time, usually in equal amounts. 
Often, however, the protoplasm is greater at one end than at the 
other and the spore then has a characteristic appearance like 
an enlarged mesentericus spore. The free spores are cylindrical, 
soon shed their protoplasm and measure 0.5 to 0.75 by 1.125 to 
1.5 microns. 

Agar slant. Abundant, thick, white mealy growth along the 
line of inoculation sometimes with arborescent edges. In older 
cultures the growth is much thicker, yellowish white and may 
show pellucid areas surrounded by more highly refractive patches. 

Agar stab. Little growth along line of inoculation but luxuri- 
ant surface growth spreading over entire surface of agar and 
extending to the walls of the tube. 

Agar colonies. Round, raised, dense, highly refractive sur- 
face colonies. If slight amount of water of condensation be 
present the colonies may be amoeboid. Under low power the 
colonies consist of dense central nuclei with spreading peripheries 
made up of numerous curling and parallel chains. The colonies 
are soft and easily hf ted from the agar. Deep colonies punctiform, 
stellate or rhizoid. Under the low power they are fuzzy, irregu- 
lar and may resemble a chestnut burr. 

Litmus glucose agar slant. Thick, yellowish-white growth 
along the line of inoculation and spreading out over entire surface. 
The medium is acidified and the growth is sometimes distinctly 
yellow. Typical cultures rapidly decolorize the litmus and then 
become alkaline and the agar turns deep blue. Occasionally 
the cultures are less active alkali-producers and the medium 
remains permanently acid. Such cultures, however, can usually 
be stimulated to alkali production by plating and they then give 
characteristic growths. 

Glucose agar colonies. Surface colonies round or bizarre, 
heaped up, with irregular margins, smaller than plain agar 
colonies. Under low power granular, with dense central nuclei 
and irregular margins, showing fine parallel strands. Deep colo- 
nies small irregular or round. Under low power they consist of 
dense central nuclei with fine, irregular or parallel strands in the 
periphery. 



286 J. S. LAWRENCE AND W. W. FORD 

Gelatin stah. Uniform growth along entire line of inoculation 
with a liquefaction also along entire line. The liquefaction be- 
comes cup-shaped or sacculated with a surface scum. It is rapid 
and frequently in two days the entire gelatin tube is liquefied. 

Gelatin colonies. Loosely filamentous colonies with dense, 
central nuclei and spreading irregular margins, often very thin, 
edges entire. Gelatin liquefied rapidly. 

Broth. Very turbid in 24 hours with no scum except occasion- 
ally a slight ring growth. In two days a heavy friable scum is 
produced which is entirely precipitated within a short time. 
The medium gradually clears while a heavy flocculent precipi- 
tate is deposited. 

Peptone. Very turbid in 24 hours. Scum appears usually 
on the second day and is soon precipitated. It is like that pro- 
duced in broth but is more friable. 

Potato. Thick, white, mealy growth in a few days becoming 
yellowish or brown with a discoloration of the potato. This 
brownish growth may become very moist and sHmy and is 
occasionally measley but never vermiform. It never assumes an 
appearance similar to that seen with cultures of Bacillus subtilis 
or Bacillus vulgatus. 

Litmus milk. With the majority of cultures peptonization 
begins immediately and progresses rapidly in three zones. Sur- 
face zone is amber, middle zone violet, the lowest zone blue. 
Peptonization continues until the entire milk tube is converted 
into an amber fluid with a slight sediment. Milk does not coag- 
ulate. With some cultures the three-zone appearance does not 
show but the milk is gradually changed to a muddy gray-colored 
fluid. Eventually, however, the same clear amber-colored fluid 
is produced. 

Blood serum. Thick, white, dry, smooth growth. No lique- 
faction. 

Fermentation tubes. Glucose. Abundant growth in bowl and 
arm. Friable scum forms which is soon precipitated. Turbidity 
gradually disappears and a flocculent precipitate is deposited. 
Reaction highly acid. 

Saccharose. Abundant growth in bowl and arm. Filmy and 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 287 

friable scum forms which is soon precipitated. In many cul- 
tures the reaction is acid. Reaction alkaline in the majority of 
cultures. 

Lactose. Turbidity in bowl with scum formation. Arm 
clear. Reaction alkaline. 

Thermal death point. The spores survive steaming one hour 
in the Arnold sterihzer and autoclaving at 19 pounds pressure. 
Killed by 20 pounds pressure. 

1/ 

Bacillus alholactus Migula 1900 

This organism was apparently first obtained in pure culture 
in 1887 by Loeffler who found it in boiled milk which had soured 
and clotted and who named it Bacillus lactis-alhus. It is possibly 
identical with Bacillus corrugatus Migula (1900) (Bacillus No. 
II Fliigge), with Bacillus bernensis Lehmann and Neumann, 
(1901) and with the organism described recently by Neide as 
Bacillus teres which was also obtained from boiled milk which 
had subsequently soured. It is common in boiled milk in 
Baltimore and produces the souring, clotting, and subsequent 
peptonization seen so frequently in this material. 

Morphology. These organisms are identical morphologically 
with Bacillus cereus. In young cultures 6 to 24 hours old, on 
plain agar, they have round ends and measure 0.5 to 0.75 by 
2.25 to 4 microns. The protoplasm may be homogeneous or 
may show globular bodies of various dimensions. On glucose 
agar the globular bodies are much more abundant and give the 
organism a characteristic appearance. Here the rods measure 
0.75 to 1 by 2.5 to 4 microns. (Figs. 28, 29, and 30.) 

Motility. Actively motile in young cultures. 

Staining properties. Gram-positive. 

Spore formation. Spores are formed readily on plain and on 
glucose agar. They are abundant in 24 to 48 hours and have 
the same appearance as the spores of Bacillus cereus. They are 
usually central or sUghtly excentric and a trifle wider than the 
organisms from which they spring thus bulging the rods some- 
what. The free spores may retain equal or unequal bits of 



288 J. S. LAWRENCE AND W. W. FORD 

protoplasm at the ends and thus have a characteristic appearance. 
They are oval to cylindrical and measure 0.5 to 0.75 by 1.5 to 
2.125 microns. The spores are frequently seen in pairs attached 
by their protoplasmic remnants, and also sometimes in chains. 

Agar slant. Luxuriant, thick, white growth with a smooth 
and glistening surface, spreading over the entire surface of the 
agar. Some cultures show a delicate transverse wrinkling. 

Agar stab. Fine, slightly arborescent growth along line of 
inoculation. Thick, white, wrinkled surface growth. 

Agar colonies. Surface colonies thick, raised, round or bizarre, 
frequently show'ng dense, central nuclei. Under low power 
granular with dense, central nuclei and spreading peripheries 
made up of curved parallel strands. Deep colonies small, round 
or irregular. Under low power irregular, mossy, with irregular 
fuzzy margins. 

Litmus glucose agar slant. Thick, yellowish-white, moist 
growth, spreading over the entire agar and wrinkling slightly 
at the base when the culture is very active. Reaction in medium 
acid in first few days but gradually alkali is produced and the 
agar turns dark blue. 

Litmus glucose agar colonies. Surface colonies thin, translu- 
cent, somewhat smaller than plain agar colonies. Under low 
power granular with thin peripheries made up of curling parallel 
strands. Deep colonies round or irregular. Under low power 
irregular, mossy with irregular, fuzzy margins. Medium first 
acidified and then made alkaline. 

Gelatin colonies. Surface colonies round, spreading concen- 
trically and composed of a central loose mass of filaments denser 
than the surrounding zone. Deep colonies are composed of 
spherical masses of loose filaments with irregular, mossy or 
bristling margins. Rapid iquefaction. 

Gelatin stab. Growth along hne of puncture with a rapid cup- 
shaped liquefaction and scum production. 

Broth. Turbidity with ring growth in 24 hours and scum 
formation in 2 to 3 days. Scum quickly precipitated. 

Peptone. Turbidity with scum formation on the second day. 
Scum usually persists. 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 289 

Potato. Thick, white, moist growth later becoming yellowish 
brown. Never wrinkled or vermiform, rarely measley. Med- 
ium discolored. 

Litmus milk. Acid production and coagulation, usually within 
24 hours. The coagulum is at first firm but gradually undergoes 
peptonization, and is usually completely dissolved at the end of 
three weeks. Odor distinctly faecal and very disagreeable, with 
a suggestion of indol. 

Blood serum. Thick, white growth. No hquefaction. 

Fermentation tubes. Glucose. Abundant growth in bowl ex- 
tending up into closed arm which becomes turbid. Flocculent 
precipitate forms but usually no scum. Reaction highly acid. 

Saccharose. Turbidity in bulb extending up into the closed 
arm. Flocculent precipitate. No scum. Reaction highly acid. 

Lactose. Turbidity in bowl extending up into the closed arm. 
No precipitate but usually a thick scum is formed. Reaction 
highly acid. 

Thermal death point. Organisms have survived 1 hour in the 
Arnold sterilizer and autoclaving to 15 pounds pressure. Killed 
by 16 pounds pressure. 

Bacillus suhtilis (Ehrenberg) Cohn 

Synonyms. Vibrio subtilis Ehrenberg 1838; Bacillus suhtilis 
Cohn 1872; Bacillus suhtilis (Ehrenberg) Cohn, Migula 1900. 

Considerable difference of opinion exists as to the correct in- 
terpretation of the somewhat puzzhng literature concerning 
this organism. In this paper we have followed the views of 
Chester who has identified a number of organisms isolated in this 
country as the real Bacillus suhtilis of Cohn, and who sent one of 
his isolations to our laboratory several years ago. It is one of the 
commonest organisms in milk, soil, dust and water. In mor- 
phology it is one of the smallest of the aerobic spore-bearing 
bacteria and is thus easily distinguished from Bacillus cereus 
with w^hich it is most often confused. 

Morphology. Small, thin, homogeneous bacilli measuring 
0.375 by l.o to 2.5 microns in 24 hour agar cultures. Some- 



290 J. S. LAWRENCE AND W. W. FORD 

what thicker and longer on glucose agar measuring 0.5 by 1.5 
to 4 microns. Does not usually form threads on this medium. 
(Figures 4 and 5.) 

Motility. Sluggishly motile in young cultures. 

Staining properties. Gram-positive. 

Spore formation. Spores are formed early appearing within 
24 hours on plain and glucose agar. They arise in the center or 
towards one end of the rods and are slightly greater in diameter 
than the rods, thus causing a distinct bulging. The free spores 
may retain bits of protoplasm at each end, often unequal in 
amount, giving the spore a characteristic appearance. Such 
spores measure about 0.5 by 0.875 microns. The spores rapidly 
lose their protoplasm, become more oval and measure about 0.5 
by 0.75 microns. 

Agar slant. Weakly refractive, glassy, membranous growth 
along line of inoculation, later spreading out over entire surface 
of agar. The surface is usually dry and hard, but in old cul- 
tures it becomes soft and smeary, but is always firmly attached 
to the agar from which it cannot be scraped off. 

Agar stab. Little growth along the line of inoculation but a 
spreading, dry, membranous growth on the surface of the agar, 
extending to the wall of the tube. 

Agar colonies. Surface colonies weakly refractive, spreading 
concentrically or in amoeboid fashion from small dense nuclei. 
Under the low power edges may be complete or finely crenate. 
If water of condensation be present one or two colonies frequently 
overgrow the entire plate. Under the low power the colonies 
are homogeneous and granular or irregular and gyrose. The 
deep colonies are punctiform and under the lower power lichen- 
like with irregular margins myceleoid in character. The colonies 
are usually membranous dry, hard, and glassy, and can be sepa- 
rated from the agar only with great difficulty. 

Glucose litmus agar slant. Highly refractive growth verrucose 
or vesicular, with milky liquid in vesicles, not spreading. Parts 
of growth show distinct red pigment. Acid is produced in 24 
hours, but is replaced by alkali in about ten days, medium turn- 
ing deep blue. 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 291 

Litmus glucose agar colonies. Irregular, spreading, bizarre 
surface colonies, usually more luxuriant than plain agar colonies. 
Under low power, irregular with entire edges or fuzzy, with 
myceleoid outgrowths from dense central nuclei. Deep colonies 
slightly irregular or punctiform. Under low power irregular 
myceleoid with filamentous edges. Medium first acidified then 
made alkaline. 

Gelatin stab. Slow growth along line of inoculation and rather 
slow cup-shaped, surface liquefaction with scum production. 

Gelatin colonies. Surface colonies round, homogeneous, spread- 
ing, thin and granular. Deep colonies yellowish brown, highly 
refractive. Under low power granular. Colonies may also 
show dense central nuclei and thin myceleoid filamentous growth 
extending in every direction through the medium. Gelatin 
liquefied. 

Broth. Single isolated pelHcles appear on the surface in 24 
hours. In 48 hours these unite to form a thin branching scum, 
which gradually becomes more dense and tough. Medium 
grows turbid in first 24 hours, but later clears. Scum is pre- 
cipitated as a whole in about ten days. This manner of scum 
formation is characteristic of Bacillus subtilis. 

Peptone. Turbidity in the first 24 hours and gradual clearing 
with a flocculent precipitate. Scum on the surface formed in 
the same manner as on broth, but not so dense or tough. The 
pellicles often show chains and branching figures. Frequently 
the scum has a delicate pink color after about five days' growth. 

Potato. Growth on potato characteristic. It is luxuriant 
and warty, having the appearance of many large and small dew 
drops scattered along the fine of inoculation. In 48 hours a 
pink pigment collects on top of this growth and persists. In 
older cultures a decided rose-red fine in the substance of the 
potato marks the limit of the growth. In ten days the vesicles 
dry down and only a reddish-brown dry growth remains on the 
discolored medium. Later the growth is moist and sticky. 

Litmus milk. No change in 24 hours and sometimes none in 
48 hours except that the milk becomes more alkahne. In three 
days the medium begins to clear from the surface, the deeper 



292 J. S. LAWRENCE AND W. W. FORD 

parts remaining unchanged. Clearing progresses slowly, the 
supernatant fluid persisting as a grayish, pinkish or yellowish 
muddy medium. After a month at room temperature the 
medium may becorne very alkahne and turn deep blue-purple. 
Milk never coagulates. 

Blood serum. Vesicular, dew-drop growth with pink color 
often very marked, in 24 hours. Vesicles dry down eventually 
leaving a hard wrinkled growth. Medium is not liquefied. 

Fermentation tubes. Glucose. Turbidity in bowl and arm. 
Scum formation like that seen in broth. Highly acid. 

Saccharose. Turbidity in bowl and arm with a fragile scum 
forming from pelhcles in about two days. Acid production but 
not so marked as in glucose. 

Lactose. Turbidity in bowl and extending up in the arm to the 
level of the medium in the bowl. Rest of the arm clear. Dense 
tough scum. Reaction alkahne. 

Thermal death point. Spores survive steaming l\ hours in 
the Arnold sterilizer. Survive autoclaving up to and including 
19 pounds pressure but usually destroyed by 20 pounds pressure. 



/ 



Bacillus vulgatus (Flligge) Trevisan. 



Synonomy. Bacillus mesentericus vugatus F'iigge 188G; Bacil- 
lus vulgatus Trevisan 1889; Bacillus vulgatus Eisenberg 1891; 
Bacillus vulgatus (Flugge) Migula 1900. 

This organism was first described by Flugge in 1886 (Flugge, 
1886) and is commonly known as the "potato bacillus." Ac- 
cording to Chester it is identical with Bacillus suhtilis. By 
the use of glucose agar and blood serum and by the careful 
observation of the cultural reactions, particularly in broth and 
on potato the species is easily separated from this organism. It 
is fairly common in Baltimore but by no means as frequent an 
isolation as are many of the other spore-bearers. 

Morphology. Small homogeneous organisms usually distinctly 
larger than Bacillus suhtilis, measuring 0.5 by 2 to 3 microns. 
Occasionally short forms 1.125 and long forms measuring 4 
microns are seen on plain agar. On glucose agar the organisms 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 293 

are thicker and much longer measuring often nearly 0.75 microns 
in thickness and 5 microns in length. (Figures 6 and 7.) 

Motility. Active progressive and rotatory motiUty in young 
cultures. 

Staining properties. Gram-positive. 

Spore formation. Spores are formed early appearing in 24 
hours on plain and glucose agar. They arise in the center or 
towards one end of the rods but do not ordinarily bulge the rods 
appreciably. When free they are elongated and flattened and 
retain tags of protoplasm at each end. At times the protoplasm 
at one end is greater in amount then at the other. Such spores 
measure about 0.5 by 1.125 microns. As they lose their protoplasm 
they become cylindrical measuring about 0.5 by 1 micron. In 
general the spores are about the same width a.s the vegetative 
rods or only very shghtly wider. 

Agar slant. Moist profuse thick growth on agar, easily 
hfted or brushed from the surface of the medium with the plati- 
num wire. Growth is usually white or cream white, spreads 
but little from the hne of inoculation and is whitest at the edge 
where it is heaped up. When water of condensation washes 
over the agar many small, round colonies develop apart from 
the main growth. In some strains the agar growth is dry and 
fine wrinkles develop but the growth can always be lifted from 
the agar w'th a platinum loop. 

Agar stab. Little growth along line of inoculation but rather 
dry wi'inkled rooty growth spreads over the surface of the agar. 

Agar colonies. Surface colonies round, waxy, highly refrac- 
tive or spreading and amoeboid with greatest refraction at the 
edge of the advancing growth, where colonies are thickest. 
Under low power of the microscope edges entire. Deep colonies 
punctiform, round or elliptical. Under low power they are 
irregular, brown, slightly granular with entire or fuzzy edges. 

Litmus glucose agar slant. Characteristic appearance. Luxu- 
riant diy hTown and abundantly wrinkled growth develops 
within 24 to 48 hours. The medium is acidified. After a few 
days the growth usually becomes moist and the wrinkles are 
obUterated while the medium becomes alkahne and turns deep 
blue. 



294 J. S. LAWRENCE AND W. W. FORD 

Litmus glucose agar colonies. Superficial colonies are thick, 
highly refractive, waxy, with entire edges or spreading with 
irregular edges. They soon become dry and wrinkled in the 
center. Under low power opaque with entire edges. Deep 
colonies are punctiform, round, oval or irregular with crenated 
margins. Under low power opaque with irregular margins. 
Medium acidified at first then turned alkaline. 

Gelatin. Stab gives cup-shaped and surface liquefaction with 
heavy scum production. 

Gelatin colonies. Colonies round with highly refractive cen- 
ters occasionally showing beautiful concentric rings. Under 
the low power the colonies have a granular appearance. Medium 
liquefied. 

Broth. Tiu-bidity within 24 hours and scum formation usually 
on second day. Medium gradually clears. 

Peptone. Turbidity within 24 hours. Thin fragile scum 
after the lapse of several days. Medium gradually clears. 

Potato. Characteristic appearance. Thick, white, gray or 
pink folds or wrinkles are formed within 24 to 48 hours often 
covering entire cut surface of potato. Later these folds dry 
down to a brown, reticulate mass. Potato usually discolored. 

Litmus milk. Slight clearing of the milk just beneath the 
cream layer usually appears in 24 hours. This reaction rapidly 
intensifies with the production of a clear fluid colored* deep 
Chinese-blue or purple. No acid production or coagulation. 
As the milk gets older complete peptonization occurs with the 
formation of a clear amber-colored fluid. 

Blood serum. Thin dry abundant growth usually smooth, 
but sometimes wrinkled and pink. Growth later becomes 
moist and gives a suggestion of liquefaction. 

Fermentation tubes. Glucose. Luxuriant growth in bulb gradu- 
ally extending into the closed arm. An abundant scum is 
formed which may be quite wrinkled. Reaction acid. 

Saccharose. Growth luxuriant in bowl but scanty in arm. 
Very thin scum may be formed after several days, but this may 
be lacking. Reaction varies from slight to marked acidity. 

Lactose. Abundant growth in bowl with late scum produc- 
tion. No growth in closed arm. Reaction alkaline 



AEEOBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 295 

Thermal death point. Organisms survived heating in broth 
in the Arnold steriHzer for one hour. Survived autoclaving 
up to and including 19 pounds pressure, but were destroyed 
by 20 pounds pressure. 

Bacillus mesentericus (Fliigge) Migula 1900 

This organism was first described by Fliigge in 1886 (Flugge, 
1886) as a species distinct from Bacillus mesentericus-vulgatus 
and named Bacillus mesentericus-fuscus. We have isolated a 
number of organisms which correspond to the description given 
by Flugge and also by Chester. It is one of the less common 
of the aerobic spore-bearing bacteria but occurs in milk, soil, 
dust and water. 

Morphology. Organisms about the same in morphology as 
Bacillus mesentericus-vulgatus. On agar cultures in 24 hours 
they are homogeneous rods measuring 0.5 by 1.5 to 3 microns. 
Sometimes shorter forms predominate in the cultures, a little 
over a micron in length. On glucose agar they are thicker 
and longer measuring 0.75 by 2 to 5 microns, with many long 
forms measuring 6 to 8 microns in length. (Figures 8 and 9.) 

Motility. Active motility, progressive and rotatory, in young 
cultures. 

Staining properties. Gram-positive. 

Spore formation. Spores begin to form in 24 hours on plain 
and on glucose agar. By the end of 48 hours they are very 
abundant. They appear in the center or towards one end of 
the rods and do not bulge the organism appreciably. The free 
spores are cylindrical and may retain equal bits of protoplasm 
at each end or this protoplasm may be unequal in amount 
giving a characteristic appearance to the spore. They measure 
about 0.5 by 1.125 microns. They rapidly lose their protoplasm 
and become slightly more oval, measuring 0.5 by 0.75 microns. 

Agar slant. Soft white or cream-white growth somewhat 
translucent when old, spreading but little from ohe line of inocula- 
tion except in the presence of water of condensation. Easily 
Ufted from the agar. Edges of growth irregular or serrate. 
Growth does not become dry or wrinkled. 



296 J. S. LAWRENCE AND W. W. FORD 

Agar stab. Little growth along line of puncture, luxuriant 
growth on surface. 

Agar Colonies. Superficial colonies round highly refractive 
with entire edges, or spreading and amoeboid. Under low power 
opaque with crenated edges. Deep colonies round and regular. 
Under low power slightly granular with crenated margins. 

Litmus glucose agar slant. Tliick, abundant, white or cream 
white to yellow growth spreading along the line of inoculation. 
Medium first turns acid but as growth becomes older it again 
becomes deep blue. 

Litmus glucose agar colonies. Superficial colonies round, 
highly refractive, with entire edges or spreading and amoeboid 
with densest part of the growth along the advancing edge. 
Under low power of the microscope edges crenated. Deep 
colonies round or oval and under low power slightly granular 
with crenated margins. Medium first acidified and then made 
alkaline. 

Gelatin stab. Cup-shaped or surface liquefaction and scum 
production. 

Gelatin colonies. Colonies dense with liquefaction centers 
and granular ring at the edges of a cup-shaped liquefaction. 

Broth. Turbidity and a rather fragile scum appears late. 
Medium then clears. 

Peptone. Turbidity with small patches of surface growth. 
Medium soon clears. 

Potato. Growth abundant, moist, brown with finely wrinkled 
or lichen-like appearance in the majority of instances. At times 
the fine wrinkling is lacking and only a thick, moist, brown, 
mealy growth is produced. 

Litmus milk. Slow peptonization with the production of a 
lilac color turning to amber. In a few weeks digestion is com- 
plete and only a white sediment is left behind. No acidification 
No coagulation. 

Blood serum. Thin, white, dry, at times finely wrinkled growth 
which later becomes yellowish and moist. Suggestion of lique- 
faction, but this is never complete. 

Fermentation tubes. Glucose. Turbidity and scum in bulb 
and turbidity in closed arm. Reaction acid. 



AEKOBIC SPOKE-BEAKING NON-PATHOGENIC BACTERIA 297 

Saccharose. Turbidity in open bulb and usually no scum. 
Turbidity in closed arm. Reaction acid. 

Lactose. Turbidity in open bulb. No scum. Arm clear. 
Reaction alkaline. 

Thermal death point. Spores survive one hour's heating in 
Arnold sterilizer and autoclaving at 19 pounds pressure. Killed 
by 20 pounds pressure. 

Bacillus globigii Migula. 

This organism was originally described by Globig (1888) 
as Bacillus mesentericus-ruber. A culture was obtained from 
Krai's Laboratory in Vienna which has the same cultural re- 
actions as those given by Globig. 

Morphology. Homogeneous bacilU measiuing 0.5 by 2 to 3 
microns in 24 hours agar cultures. On glucose agar the organisms 
are longer and slightly thicker often growing out into long 
chains but short forms are also frequently seen (Figure 14). 

Spore formation. Spores are formed very sparsely and at 
a late period in the present culture. They are usually seen only 
in 16 to 18 days giowth and are then characteristic mesentericus 
spores. 

Motility. Actively motile in 24 hour cultures. 

Staining properties. Gram-negative. 

Agar slant. Thin, spreading, glassy, soft, yellowish-white 
growth along line of inoculation. 

Agar stab. Slight uniform growth along Hue of puUcture with 
spreading amoeboid surface growth. 

Agar colonies. Dense, soft, white amoeboid colonies similar 
to those of Bacillus mesentericus. 

Litmus glucose agar slant. Thick, narrow, white growth 
along line of inoculation. The medium shows an acid reaction. 

Litmus glucose agar colonies. Thick, round, raised, soft 
colonies later turning yellowish and rarely pinkish. Medium 
acidified. 

Gelatin stab. Growth along hne of inoculation and shght 
surface growth with liquefaction of the gelatin. 



298 J. S. LAWRENCE AND W. W. FORD 

Gelatin colonies. Surface colonies round, granular, punctiform 
with slow liquefaction. Some of the larger colonies are spreading 
and have a glassy surface. Deep colonies punctiform, spherical 
with dense centers. 

Broth. Slight turbidity with no surface growth but a floccu- 
lent precipitate. Medium is eventually turned dark yellow. 

Peptone. Slight turbidity with no surface growth and no 
precipitate. 

Potato. Yellow, moist growth becoming a reddish brown. 
Medium is discolored. 

Milk. No change in 48 hours. In twenty days the medium 
shows an acid reaction with the precipitation of a white sediment. 

Blood serum. Thick, transparent spreading growth with 
irregular edges. 

Fermentation tubes. Glucose. Turbidity in open bulb. No 
scum. No growth in closed arm. Reaction acid. 

Saccharose. Turbidity. No scum. No growth in closed 
arm. Reaction alkaline. 

Lactose. Turbidity. No scum. No growth in closed arm. 
Reaction alkaUne. 

Thermal death point. Spores withstood one hour's sterihzing 
in the Arnold sterilizer, survived autoclaving at 15 pounds 
pressure but were killed by 16 pounds pressure. 

Bacillus aterrimus Lehmann & Neumann. 

This organism was originally described by Biel (1896) and 
named by Lunt (1896) Bacillus mesentericus-niger. It is not 
uncommon in milk, soil, and the intestinal contents of man. 

Morphology. Bacilh similar to Bacillus vulgatus in morphol- 
ogy. On plain agar they are homogeneous with blunt ends and 
measure about 0.5 by 2 to 3 microns in dimensions. On glucose 
agar they are thicker and longer measuring 0.75 by 2 to 4 microns 
but at the same time shorter forms are frequent measuring 0.75 
by 1.5 microns. (Figures 10 and 11.) 

Spore formation. Spores are formed earljf appearing in 24 
hours on plain agar. They form in the center or towards one 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 299 

end of the rods which do not swell appreciably. When free 
they may retain spurs of protoplasm at each end unequal in 
quantity and measure about 0.5 by 1.5 microns. The spores 
rapidly lose their rims of protoplasm and are then oval to cylin- 
drical measm'ing 0.5 by 0.75 microns. 

Motility. Actively motile in 24 hour cultures. 

Staining properties. Gram-positive. 

Cultural reactions. This organism is identical with Bacillus 
vulgatus in all its cultural reactions except that it imparts a dis- 
tinct color to the various media. This color varies from a steel 
grey to a brown or black and is best seen on solid media. It is 
very pronounced on potato where the characteristic folds of the 
"vulgatus" are converted to thick black wrinkhng bands. 

Thermal death point. The spores resist an hour's steaming in 
the Arnold sterihzer and 15 pounds pressure in the autoclave. 
They are destroyed by 16 pounds pressure. 

Bacillus niger Migula 1900 

This organism was first described by Gorini (1894) in 1894 as 
Bacillus lactis-niger and is closely related to the preceding 
organism. A culture obtained from Krai's Laboratory shows 
the following reactions. 

Morphology. Bacilli with homogeneous protoplasm and blunt 
or rounded ends measuring 0.375 to 0.75 by 1.5 to 3 microns in 
24 hour agar cultures. No change in morphology on glucose agar. 
(Figures 12 and 13.) 

Spore formation. Spores are formed in 24 hours on plain agar 
and in 48 hours on glucose agar. They appear in the center or 
towards one end of the rods and are oval or cylindrical in shape. 
The free spores may retain protoplasm at both ends and are 
typical of the ^'mesentericus" group. They measure 0.75 to 1 
by 1.125 to 1.25 microns in dimensions. 

Motility. Active motihty in young cultures. 

Staining properties. Gram-positive. 

Cultural reactions. This species has the general cultural 
reactions of Baciltus mesentericus. It grows on agar as a rather 



300 J. S. LAWRENCE AND W. W. FORD 

thick moist mass with a silvery sheen which shows black areas 
at the edges and in old cultures imparts a black tone to the agarr. 
It liquefies gelatin rapidly, produces a faint acidity in milk 
which it first coagulates and then slowly digests. On glucose 
agar it tends to wrinkle sHghtly. It produces a faint acid in 
glucose, saccharose and lactose fermentation tubes. On potato 
it grows as a raised brown mass and it also produces a 
brownish growth on blood serum. 

Thermal death point. The spores withstand boiling one hour 
in the Arnold sterihzer and a pressure of 20 pounds in the auto- 
clave. They are destroyed by a pressure of 22 pounds. 

Bacillus pumilus Gottheil 1901 

An organism described by Gottheil (1901) in 1901 as Bacillus 
pumilus is regarded by Chester as identical with Bacillus mesen- 
tericus. A culture of Bacillus pumilus received from Krai's 
collection in Vienna has all the morphological, tinctorial, de- 
velopmental and cultural reactions of this species. 

Bacillus mycoides Fliigge 1886 

This organism was first described by Fliigge (1886) in 1886 
and has since then been given other names by various authors. 
It is not the same as Bacillus ramosus-liquefaciens of Prausnitz 
which is a distinct species. Bacillus mycoides is quite common 
in Baltimore and is present in milk, water, soil, and dust. 

Morphology. In young cultures 6 to 8 hours old on plain agar 
the organisms are homogeneous with square ends and measure 
usually a little more than 0.5 micron in width by 3 to 6 microns in 
length. They are distinctly thinner and longer than Bacillus 
cereus. As the organisms mature the protoplasm appears more 
granular and a characteristic arrangement in short and long 
chains is seen. They then resemble the anthrax bacillus. On 
glucose agar the bacilH are thicker, 0.75 to 1 micron, and usually 
about the same length. On this medium the protoplasm is 
converted into globular bodies which do not take the stain and 
which are similar to those seen in Bacillus cereus. In certain 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 301 

instances the organisms seem to be made up of a network of 
fine strands in which the globular bodies hang suspended. Often 
the chains are curled or curved upon themselves. Old cultures 
show an abundance of swollen involution forms, which seem to 
have a skein-like arrangement. (Figures 22, 23 and 24.) 

Motility. Active motility in young cultures. 

Staining properties. Gram-positive. 

Spore formation. Spores begin to form early, appearing first 
as small retractile bodies in the centers or towards one end of the 
organisms usually at the end of 24 to 48 hours. Gradually the 
organisms swell and the spores at the same time increase in size 
and at this stage a long chain of organisms each containing a 
spore may often be seen. The protoplasm soon disintegrates 
leaving a rim about the spore which is round or oval or slightly 
rectangular. Such spores measure 0.75 to 1 by 1.125 microns. 
Other spores are more definitely elongated and may measure 
0.75 to 1 by 1 to 2 microns. The spores often remain attached 
to each other in short or long chains. The spores vary more 
in size than do others of this group and may show small forms 
0.375 to 0.5 by 0.5 to 1 and large forms measuring 1.125 by 2 
lying side by side. 

Slant agar. Filamentous rhizoid growth spreading from the 
line of inoculation and extending into the agar. This growth is 
at first glassy and glistening, but later grows dull and soft. Ap- 
pearance on agar characteristic. 

Agar stab. Faint arborescent growth along line of inoculation 
with a surface development in concentric zones. "^ 

Agar colonies. Surface colonies spread from dark dense 
nuclei and show dense, rhizoid peripheries extending into the 
agar on all sides. Under low power the periphery of the colony 
is found to be composed of parallel strands of growth. Deep 
colonies have almost the same appearance and always exhibit 
the spreading peripheral myceleoid outgrowths. 

Litmus glucose agar. Thin membranous myceleoid growth 
later becoming branched and reticulate. Growth at first 
moist and white, later becoming pale yellow. Medium first 
acidified and then turned deep blue. 



302 J. S. LAWRENCE AND W. W. FORD 

Litmus glucose agar colonies. Surface colonies thin, round or 
irregular. Under low power found to consist of masses of 
matted filaments with usually dense central nuclei, from which 
single or parallel strands extend into the agar in every direction 
for long distances. Deep colonies exhibit the same small, 
punctiform and matted myceleoid growth, under lower power. 
Medium first acidified and then made alkaline. 

Gelatin stab. Filamentous growth along line of inoculation 
with surface liquefaction. 

Gelatin colonies. Colonies consist of dense central nuclei 
with matted edges from which long strands emerge. The 
colonies present a peculiar appearance like a chestnut burr. 

Broth. No turbidity but a firm scum forms which is soon 
precipitated. 

Peptone. No turbidity, but a flocculent suspension and a 
firm scum which is soon precipitated. 

Potato. Mealy white, later becoming brownish. 

Litmus milk. Slow peptonization to an amber-colored fluid. 
No acidification. No coagulation. 

Blood serum. Dry, myceleoid interlacing luxuriant growth. 
No liquefaction. 

Fermentation tubes. Glucose. Flocculent growth in bowl and 
arm. Scum forms and is soon precipitated. Reaction acid. 

Saccharose. Flocculent in bowl and in arm. Scum is formed 
and precipitated. Some cultures produce moderate acidity. 
Others produce no acid. 

Lactose. Growth in open bulb with a slight extension into 
arm. Scum formed and soon precipitated. Reaction alkaline. 

Thermal death point. Spores survived one hour in the Arnold 
sterihzer and 15 pounds pressure in the autoclave. Destroyed 
by 16 pounds pressure. 

'^Bacillus megatherium De Bary 

This organism was originally found and named by De Bary 
(1884, 1887) and has since beeen described under a variety of 
names by a number of authors. It is one of the most conmaon of 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 303 

the spore-bearing bacteria and has been found in dust, soil, 
milk, water, and as a laboratory contamination. The present 
description applies to cultures obtained from the Krai collection, 
and from the American Museum, and to over a hundred of our 
own isolations. 

Morphology. These are the largest of the spore-bearing 
organisms. On plain agar in young cultures, from 8 to 24 hours 
old, they are long and thick with homogeneous or shghtly granu- 
lar protoplasm, measuring 0.75 to 1.25 by 3 to 9 microns. On 
glucose agar they are even thicker measuring 1.25 to 1.5 in 
width. On both media long forms occur but especially on glu- 
cose agar. These may measure 30 to 45 microns in length and 
may show homogeneous protoplasm without evident segmen- 
tation. The protoplasm of the organism is at first homogeneous, 
but by the end of 24 to 48 hours it is converted into a mass of 
globular bodies resistant to the stains. These globular bodies 
are clear, highly refractile, bulge the organism somewhat, and are 
quite numerous six to eight appearing in each rod. They thus 
give the organism a peculiar and characteristic appearance. 
They show most markedly on glucose agar but are also present 
on plain agar where they can best be demonstrated by decoloriz- 
ing an over-stained preparation. Their nature is not clear as 
they do not take any special bacterial stains. Shadow or 
transparent forms appear in Bacillus megatherium early, both 
on plain and glucose agar. These measure 1.125 to 1.5 by 4 to 
10 microns, take the stain very faintly and show peculiar bodies 
of agglomerated protoplasm at the sides or sometimes at the 
ends. These transparent forms are often thicker and longer 
and may even measure 2 by 40 to 45 microns. Occasionally 
they are distinctly oval with rounded ends measuring about 
1.5 by 4 microns and show a small bunch of cytoplasm at the 
side. When these forms are in chains they are exactly like the 
original pictures of De Bary. (Figures 31, 32, 33, 34, 35, 36, 37). 

Motility. Active progressive and rotatory motility in young 
cultures. 

Stainifig properties. Gram-positive. 

Spore formation. Spores are formed abundantly on plain 



304 J. S. LAWRENCE AND W. W. FORD 

agar in 24 hours and on glucose agar in 48 hours. They appear 
in the center or shghtly towards one end of the rods and are 
usually of the same diameter but may be slightly thicker. Some- 
times two spores seem to arise in one rod but these may possibly 
be in a rod just prior to division. In general each rod has a 
single spore. The spores occasionally lie obliquely in the rods. 
Frequently two spores are at opposite ends of rods lying in 
juxtaposition and these may remain attached in chains and 
present a characteristic appearance. The free spores retain 
protoplasm at the ends for some time. When this is unequal 
in amount the spore has somewhat the shape of a tennis racket 
and handle. The free spores are oval to cylindrical and measure 
0.75 to 1.125 by 1.5 to 2 microns. They are often flattened on 
one side having an appearance described as kidney shaped or 
reniform. The spores show great variations in size more so 
than do those of the other members of this group. 

Slant agar. Thick, raised, soft, white or cream-colored growth 
which shows a pink tinge by reflected light, with many small, 
minute pellucid areas. As the cultures get older the growth 
becomes pale yellow. 

Agar stab. Shght growth along line of inoculation, heaped 
up and spreading slightly on surface. Later surface growth 
becomes slightly pinkish. 

Agar colonies. Surface colonies round, thick, white or cream- 
colored, highly refractive, turning pale yellow or yellowish- 
brown in old cultures. Under low power slightly granular, 
brownish yellow, with entire margins. Deep colonies puncti- 
form. Under low power round or irregular with entire edges, 
brown and granular. 

Litmus glucose agar slant. Thick, luxuriant growth along 
line of inoculation, at first white and then pale yellow or cream- 
colored. Medium is first acidified but later becomes alkaline 
and changes from a dark blue to a smoky brown while the 
growth becomes a dark gray or smoky brown. 

Litmus glucose agar colonies. Large, round, raised surface 
colonies, cream colored to pale yellow, with heaped up central 
nuclei. Under low power dark, slightly granular with entire 



AEROBIC SPOEE-BEARING NON-PATHOGENIC BACTERIA 305 

edges. Deep colonies punctiform. Under low power dark, 
irregular, bizarre, with entire edges. Medium is acidified and 
then made alkaline. 

Gelatin stab. Funnel-shaped hquefaction. No scum. 

Gelatin colonies. Round colonies with concentric zones of 
growth. Under low power cloudy central nuclei with filamen- 
tous peripheries. 

Broth. Turbidity but no scum. 

Peptone. Turbidity but no scum. 

Potato. Thick, white, mealy growth later becoming pale or 
cream yellow. 

Litmus milk. No change within 24 hours then a gradual 
peptonization with the production of a port-wine colored fluid. 
No acidification. No coagulation. 

Blood serum. Thick, white, moist, heavy growth cream white 
to yellow in color. No liquefaction. 

Fermentation tubes. Glucose. Turbidity in open bulb. No 
scum. No growth in closed arm. Acid production feeble. 

Saccharose. Turbidity in open bulb. No scum. No growth 
in closed arm. Acid production feeble. 

Lactose. Shght turbidity in open bulb. No scum. No 
growth in closed arm. Reaction alkaline. 

Thermal death point. Spores withstood 1 hour steaming in the 
Arnold sterilizer and 18 pounds pressure in the autoclave. 
Killed by 19 pounds pressure. 

V 

Bacillus petasites Gottheil 1901 

This organism was described originally by Gottheil (1901) in 
1901. Its chief point of differentiation from Bacillus megatherium 
is that it produces a distinct yellow pigment on artificial media, 
particularly on plain agar and on potato. It is extremely com- 
mon, having been found in dust, soil, water, milk, and various 
milk products. The present description applies to a culture 
from the Krai collection and to over a hundred of our own 
isolations. 

Morphology. The organisms do not differ appreciably in 
morphology from Bacillus megatherium. They are homogeneous 



306 J. S. LAWRENCE AND W. W. FORD 

or slightly granular rods measuring 0.75 to 1.5 by 3 to 6 microns 
on plain agar in young cultures (8 to 24 hours), and 1 to 1.75 by 
3 to 6 on glucose agar. Long forms measuring 12 to 25 microns 
are seen on plain and on glucose agar. Shadow forms with 
faintly staining protoplasm, like those seen in Bacillus megatherium 
are common as well as the peculiar refractile globular bodies. 
(Figures 38, 39, 40, 41 and 42.) 

Motility. Active progressive and rotatory motility in young 
cultures. 

Staining reactions. Gram-positive. 

Spore formation. Spores are formed abundantly in 24 hours 
on plain and on glucose agar. The spores are oval to rectangular, 
of about the same width as the rods from which they spring 
and frequently form long chains. The free spores may retain 
tags of protoplasm but soon lose them and then show great vari- 
ations in size and shape. They may be nearly round, oval, 
rectangular and reniform and measure 0.75 to 1 by 1.5 to 2 microns. 

Agar slant. Thick, moist, abundant mealy growth at first 
slightly pinkish by reflected hght, then becoming bright lemon 
yeKow. Agar slightly discolored. 

Agar stab. Slight growth along line of inoculation with heaped 
up yellowish growth on surface. 

Agar colonies. Surface colonies thick, white or yellow, highly 
refractive. Under low power dark, granular with entire or 
myceleoid edges. Deep colonies punctiform. Under low power 
irregular, with irregular edges showing myceleoid, rooty or fuzzy 
edges. 

Litmus glucose agar. Luxuriant, thick, heaped-up growth at 
first yellow then assuming an orange and then a dark-brown color. 
Reaction of medium first acid then alkaline. It eventually 
becomes smoky-brown. 

Litmus glucose agar colonies. Surface colonies round, regular 
and thick or thin and spreading. Under low power granular 
with entire edges. Deep colonies punctiform. Under low 
power granular, irregular, with fuzzy edges. Reaction of medium 
acid at first then alkaline. 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 307 

Gelatin stab. Growth along line of inoculation with funnel- 
shaped surface hquefaction. No scum formation. 

Broth. Turbidity. No scum. Medium eventually becomes 
yellow. 

Peptone. Turbidity. No scum. 

Potato. Thick, mealy, bright yellow growth gradually be- 
coming dark yellow. 

Litmus milk. No change in 24 hours then a gradual pep- 
tonization with the production of a port-wine-colored fluid. 

Blood serum. Thick, dry, yellowish, moist growth becoming 
pale to bright yellow. No liquefaction. 

Fermentation tubes. Glucose. SHght turbidity in bulb. No 
scum. No growth in closed arm. Feeble acid production. 

Saccharose. Slight turbidity in bulb. No scum. No growth 
in closed arm. Faint acid production. 

Lactose. Shght turbidity in bulb. No scum. No growth in 
closed arm. Reaction alkaline. 

Therynal death point. Spores survived steaming in Arnold 
sterihzer 30 minutes, but were killed by 1 hour exposure. With- 
stood 19 pounds pressure in autoclave but were killed by 20 
pounds pressure. 

Bacillus tumescens Zopf 1885 

This organism was described by Zopf in 1885 (Zopf, 1885). 
A culture received from the Krai collection and another received 
from the American Museum agree in their morphological, 
developmental, tinctorial and cultural features all of which are 
identical with those of Bacillus megatherium. (Figures 45, 46 
and 47.) 

^Bacillus graveolens Gottheil 1901 

This organism was described in 1901 by Gottheil (1901) as a 
new species. A culture from the Krai collection in Vienna has 
all the cultural reactions of Bacillus megatherium. Morphologi- 
cally it is about the same size, forms spores in the same way, is 
Gram-positive, produces globular bodies on plain and glucose 
agar and undergoes involution with the formation of shadow or 



308 J. S. LAWRENCE AND W. W. FORD 

washed-out forms. It shows however a distinct tendency to 
produce curved or spiral forms. On the basis of this one char- 
acteristic it is hardly justifiable to make it a distinct species. 
It should be noted that this use of the term "graveolens" is 
probably incorrect since a Bacterium graveolens was described 
by Bordoni Uffreduzzi (1886) in 1886. This was a small non- 
sporulating bacillus producing a green pigment. (Figures 43 and 
44.) 

v Bacillus cohaerens Gottheil 1901. 

This organism was described by Gottheil (1901) in 1901 but 
according to Chester it is identical with Bacillus simplex. The 
culture of Bacillus cohaerens received from the Krai collection 
is distinct from Bacillus simplex and is represented by four 
organisms isolated in Baltimore, one from milk and three from 
soil. The present description applies to all five strains. 

Morphology. Small, rather uniform homogeneous organisms 
with rounded ends, measuring 0.375 to 0.5625 by 0.75 to 2.25 mi- 
crons in 24 hour cultures on plain agar. On glucose agar the bacilli 
are thicker and longer measuring 0.5625 to 0.75 by 2 to 5 microns. 
On both media shadow forms appear early often in 24 hours. 
These are made up of faintly-staining protoplasm with deeply- 
staining particles in various positions, at the ends, towards the 
center, or at the periphery. (Figures 15, 16, and 17.) 

Motility. Actively motile in 24 hour cultures. 

Staining properties. Gram-positive. 

Spore formation. Spores were formed slowly and sparsely 
in the Krai culture and in one of ours. They appeared in about 
10 days, were oval or elliptical, arose in the centers of the rods 
which were slightly bulged on sporulation. The free spores 
were very delicate and stained with difficulty. They measured 
about 0.5625 by 0.75 microns. In a more recent isolation of 
our own the spores appeared in 48 hours, were central or excen- 
tric, bulged the rods and later retained distinct rims of protoplasm, 
measuring 0.75 by 1.5 to 1.5 microns. Later the spores lost 
their protoplasm, became more oval and measured 0.5 to 0.5625 
by 0.9375 to 1.25 microns. Rarely the spores retained unequal 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 309 

bits of protoplasm at the ends and then they resembled the 
mesentericus spores slightly. 

Agar slant. Thin, soft spreading, whitish growth later be- 
coming yellow. Easily scraped off the agar. 

Agar stab. Faint growth along line of inoculation and spread- 
ing on the surface, thick and whitish in old cultures. 

Agar colonies. Surface colonies round or bizarre, thick, 
white. Under low power granular with dense central nuclei. 
Edges entire. Deep colonies punctiform. Under low power 
irrregular, with entire edges. 

Litmus glucose agar. Thick, soft, whitish growth along line of 
inoculation becoming yellowish and irregularly heaped up. 
Medium quite markedly acidified. 

Litmus glucose agar colonies. Surface colonies round or ir- 
regular, thick, whitish. Under low power granular and fre- 
quently show dense central nuclei with thin peripheries showing 
regular edges. Deep colonies punctiform. Under low power 
irregular with irregular edges. Reaction of medium acid. 

Gelatin stab. Faint growth along line of inoculation with 
surface hquefaction and scum production. 

Gelatin colonies. Thin, circular colonies, under low power 
granular. 

Broth. Turbidity at first, then the medium clears and a dense 
surface growth appears which shows many clear, globular masses 
like globules of fat floating on the surface. 

Peptone. Turbidity with a faint fragile jseum. 

Potato. Thin, spreading, moist, yellow growth. 

Litmus milk. Slow decolorization of the Utmus with peptoni- 
zation and the production of an amber-colored fluid. 

Blood serum. Thin, whitish growth. No Hquefaction. May 
appear finely wrinkled. 

Fermentation tubes. Glucose. Turbidity in bowl with surface 
growth and flocculent precipitate. Arm clear. Reaction acid 
at the end of 2 to 3 days. 

Saccharose. Turbidity in bowl with sHght surface growth. 
Arm clear. Acidity at the end of 2 to 3 days. 

Lactose. Turbidity in bowl with very shght surface growth. 
Arm clear. Reaction alkahne. 



310 J. S. LAWRENCE AND W. W. FORD 

Thermal death point. In one isolation the spores survived 
one hour steaming in the Arnold sterilizer; and withstood 18 
pounds pressure in the autoclave but were killed by 19 pounds 
pressure. In another isolation from soil the spores survived 
14 pounds pressure in the autoclave but were killed by 16 pounds. 
They survived one hour steaming in the Arnold. 

^Bacillus simplex Gottheil 1901 

This organism was described by Gottheil (1901) in 1901 as a 
distinct species. According to Chester it is the same as Bacillus 
cohaerens of Gottheil. Cultures of both organisms have been 
received from Krai's Laboratory in Vienna and can easily be 
differentiated. The present description applies to the Krai 
culture and to an organism obtained from soil by Dr. Laubach. 
The species is evidently one of the rare spore-bearing organisms. 

Morphology. In the Krai culture the organisms are large 
homogeneous rods with rounded ends, measuring usually 0.5625 
to 0.75 by 3 to 4.5 microns. At times much thicker forms are 
seen approximating 1.125 micron in thickness while longer forms 
6 microns in length are not uncommon. The organisms often 
grow out into long threads or filaments 10 to 12 microns in length, 
especially on glucose agar. Even in young cultures the homogen- 
eous rods lose their protoplasm and are converted into peculiar 
shadow forms. These are made up of a very faintly staining 
protoplasm in which denser aggregations of cytoplasm appear. 
Such forms measure 1. '125 to 1.25 by 12 to 15 microns in dimensions. 
On glucose agar the organisms have the same morphology but 
may show an abundance of shadow forms. Involution and 
shadow forms are very abundant in old cultures. In our own 
isolation the organisms, while somewhat smaller, did not differ 
appreciably in morphology, measuring 0.5 to 0.5625 by 1.5 to 
2.5 microns but also showing both the thicker and longer forms 
seen in the Krai culture and the characteristic shadow and in- 
volution forms. Long forms were also very common on glucose 
agar. (Figures 18, 19, 20, and 21.) 

Motility. Actively motile in young cultures. 

Staining properties. Gram-positive. 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 311 

Spore formation. In the Krai culture the spores were at first 
formed very slowly appearing only after the lapse of 15 to 16 
days. Subsequently after repeated transfers, spore formation 
became more active and spores were often formed in 24 hours. 
They appeared in the centers or towards one end of the rods, 
were no thicker than the rods from which they sprung, and were 
cylindrical or almost rectangular in shape. They retained rather 
thick walls of protoplasm for some time and measured 0.5625 by 
1.125 to 1.25 microns. In our own isolation the spores were 
formed in 48 to 72 hours in the same way as in the Krai culture but 
were a trifle smaller measuring 1.375 to 0.5 by 0.75 to 1 micron. 

Agar slant. Thin, translucent, sHghtly yellowish gelatinous 
growth, gradually becoming denser and developing occasionally 
a dry slightly wrinkled surface. Single accessory colonies not 
uncommon at the edges of the main growth. 

Agar stab. Slight uniform growth along line of puncture with 
a thick circular surface growth. 

Agar colonies. Surface colonies thin, translucent, amoeboid 
developing from pin-point centers. Under low power granular. 
Deep colonies round or oval, regular, granular, with clean or 
rarely irregularly fuzzy edges. 

Litmus glucose agar. Thick, abundant yellowish-white, heaped 
up growth with serrated margins. Medium faintly acidified 
in old cultures. 

Litinus glucose agar colonies. Superficial colonies thin, smooth, 
white and soft. Under low power granular, edges irregular 
but entire. Deep colonies punctiform. Under low power 
irregular with irregular rarely fuzzy margins. A trace of acid 
usually produced. 

Gelatin stab. Faint growth along fine of inoculation with 
eup-shaped surface liquefaction. 

Gelatin colonies. Round, thick, whitish colonies with con- 
centric rings and sharply defined edges. Medium liquefied. 

Broth Faint turbidity, shght sediment, no scum but rarely a 
faint ring growth along side of tube. 

Litmus milk. Gradual clearing with production of straw- 
colored fluid in the Krai culture. In our own isolation a gradual 



312 J. S. LAWRENCE AND W. W. FORD 

clearing to a port-wine fluid. No coagulation. Later straw- 
colored. 

Peptone. Faint turbidity and sediment with rarely a slight 
ring growth. 

Potato. Thick, moist, abundant, gelatinous, yellowish-brown 
growth. 

Blood serum. Thin, spreading, whitish growth. No lique- 
faction. 

Fermentation tubes. Glucose. Turbidity in open bulb. No 
scum, arm clear. Reaction neutral or slightly acid. 

Saccharose. Faint turbidity in bulb. No scum. Arm clear. 
Reaction alkaline. 

Lactose. Faint turbidity in bowl. No scum. No growth 
in closed arm. Reaction alkaline. 

Thermal death point. In the Krai culture the spore survived 
steaming in the Arnold steriUzer for 15 minutes. They with- 
stood a pressure of 15 pounds in the autoclave but were destroyed 
by 16 pounds pressure. In our own isolation the spores sur- 
vived 10 pounds in the autoclave but were killed by 12 pounds 
pressure. They survived 15 minutes steaming in the Arnold 
sterihzer but were killed by 30 minutes steaming. 

Bacillus fusiformis^ Gottheil 1901 

This organism was first described by Gottheil (1901) in 1901. 
A transfer from Gottheil's original was obtained from Krai's 
Laboratory in Vienna. Fourteen organisms corresponding closely 
to Gottheil's isolation were obtained in Baltimore, two from 
milk, four from dust, two from water, five from soil and one 
from contaminated hirudin. The present description apphes 
to all of them. 

* Bacillus fusiformis has practically the same morphology and the same cul- 
tural reactions as the organism described in 1909 by Jordan and Harris as the 
cause of milksickness and named by them Bacillus lactimorhi (Journal of In- 
fectious Diseases, Vol. 6, No. 4, September 20, 1909, p. 401). A culture of Bacillus 
lactimorhi received from the Winslow collection in New York does not differ 
appreciably in its reactions from the strains of Bacillus fusiformis in our labora- 
tory. Without a thorough study of pathogenicity, however, it is impossible to 
state whether the organisms found by us are identical with Bacillus lactimorhi 
or not. 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 313 

Morphology. Thick stubbed homogeneous organisms with 
round or pointed ends usually appearing as single cells or in twos. 
No chain formation. On 24 hour plain agar cultures they 
measure 0.5 to 0.75 by 1.5 to 2 microns. Organisms not in- 
creased in size on glucose agar and protoplasm remains homo- 
geneous. Sometimes long forms 6 to 8 microns appear in old 
cultures. (Figures 48, 49, 50 and 51.) 

Motility. Active progressive and rotatory motihty in 24 
agar cultures. 

Staining properties. Gram-negative. 

Spore formation. Spores form early appearing in 24 hours on 
both plain and glucose agar. They are round, greater in 
diameter than the organisms from which they spring, and are 
usually located at the ends of the rods in a terminal or sub- 
terminal position. They thus give a clavate or club-shaped 
appearance to the rods which resemble somewhat the tetanus 
bacillus. The spores may also be central and the rods thus be- 
come fusiform in shape. The free spores may retain spurs of 
protoplasm assuming a peculiar diamond shape or may appear 
naked. They vary in diameter from 0.5 to 1 micron and are 
occasionally swoHen equaling 1.5 microns in thickness. 

Agar slant. Thick white rather dry growth in 24 hours, 
becoming distinctly yellow or cream-colored in old cultures. 
Easily scraped from medium. 

Agar stab. Faint line growth and non-spreading surface 
growth. 

Agar colonies. Superficial colonies may be round, regular 
thick and opaque, or thin and spreading. Under low power 
they show dark central nuclei and thinner margins with clean- 
cut edges. Older cultures thick and heaped up. Deep colonies 
small and fine, under low power dark, opaque, round or irregular. 

Glucose agar. Thick dry growth with heaped-up edges be- 
coming thicker and granular in old cultures. Reaction alkaline. 

Glucose agar colonies. Superficial colonies thick, irregular 
spreading and heaped up. Under low power granular with 
irregular fuzzy margins. Deep colonies opaque under low power 
showing irregular fuzzy edges. Older colonies thicker and more 
bizarre-shaped. Reaction alkaline. 



314 J. S. LAWRENCE AND W. W. FORD 

Gelatin stab. Growth along line of inoculation with cup-shaped 
or funnel-shaped liquefaction. Dense turbidity in the liquefied 
gelatin with a thick scum. Gelatin may be faint pink in color. 

Gelatin colonies. Small fine colonies round and regular or 
irregular and spreading. Under low power they show fine 
hairy outgrowths. Gelatin slowly liquefied. 

Broth. Turbidity and fine sediment. No scum. 

Peptone. Turbidity and fine sediment. No scum. 

Potato. Faint yellow growth becoming yellowish brown in old 
cultures. 

Litmus milk. Gradual reduction of the litmus and slow but 
complete digestion of the proteins. No coagulation. 

Blood serum. Non-spreading cream yellow growth becoming 
yellowish brown in old cultures. No liquefaction. 

Fermentation tubes. Glucose. Turbidity in bowl. Arm clear. 
No scum. Reaction alkaline. 

Saccharose. Reactions the same. 

Lactose. Reactions the same. 

Thermal death point. Spores destroyed by steaming 15 min- 
utes in the Arnold sterilizer. They survive 7| pounds in the 
autoclave but are destroyed by 10 pounds pressure. 

^Bacillus terminalis Migula 1900 

This organism was first obtained by Fliigge (1894) in 1894 
and called by him, No. XII. It was subsequently correctly 
named Bacillus terminalis by Migula and still later named 
Bacillus lacteus by Chester (1901). On two occasions we have 
isolated organisms which have the same morphology and method 
of spore-formation as Bacillus terminalis but differ slightly in 
cultural reactions. It does not seem wise to make a new species 
since our strains may represent merely attenuated varieties of 
Fltigge's organism. The following description is taken from 
our own isolations and the points of differentiation between them 
and Fltigge's original isolation are indicated. 

Morphology. Long thin bacilli with slightly granular pro- 
toplasm measuring 0.375 by 2.25 to 4 microns in 24 hour agar cul- 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 315 

tures. On glucose agar the organisms retain the same diam- 
eter but grow out into long chains which often assume spiral 
arrangements. (Figure 52.) 

Spore formation. Spores are formed slowly seldom appearing 
before 48 hours. They are cyhndrical, thicker than the rods 
from which they spring, terminal or sub-terminal, giving the 
organisms a clavate or club-shaped appearance. Free spores are 
0.75 by 1.5 microns in dimensions. 

Motility. Active motiUty in 24 hour cultures. 

Staining properties. Gram-negative. 

Agar slant. Thin spreading smooth glistening growth with 
gradual darkening of the agar. 

Agar stab. Faint growth along line of puncture and on the 
surface at the point of inoculation. 

Agar colonies. Colonies grow slowly appearing only after 
3 to 4 days. They are round, regular, under low power showing 
central nuclei with thin spreading peripheries. Deep colonies 
apt to be irregular under low power, showing clean-cut or entire 
edges. 

Glucose agar. Faint white filmy growth with an alkahne re- 
action. 

Gliicose agar colonies. Thin slow-growing spreading surface 
colonies, under low power showing dense central nuclei and thin 
margins. Deep colonies punctiform, under low power shghtly 
granular with irregular margins. Reaction alkahne. 

Gelatin stab. Growth along Une of inoculation and slow cup- 
shaped liquefaction. 

Gelatin colonies. Colonies on the surface show dense central 
nuclei and concentric spreading peripheral margins. Deep 
colonies punctiform and tend to show same arrangement. Under 
low power edges entire. 

Broth. SHght turbidity. No scum. No sediment. Fragile 
scum described by Fliigge. 

Peptone. Slight turbidity. No scum. No sediment. 

Potato. No visible growth in our isolations. Faint moist 
growth gradually becoming thicker and yellowish, noted by 
Fliigge. 



316 J. S. LAWRENCE AND W. W. FORD 

Milk. No change produced by our strains. Slow pepton- 
zation described by Fliigge. 

Blood serum. Thin transparent spreading growth, pale yellow 
to yellowish-brown. No Uquefaction. Slight sinking-in of the 
growth mentioned by Fliigge. 

Fermentation tubes. Glucose. Faint turbidity in bowl. No 
scum. No growth in closed arm. Reaction alkaline. 

Saccharose. Appearance the same. Reaction alkaline. 

Lactose. Appearance the same. Reaction alkaUne. 

Thermal death point. Spores survived 10 pounds in autoclave 
but were killed by 15 pounds pressure. 

BIBLIOGRAPHY 

BiEL. (1896) Centralbl. f. Bakt., 2 Abt., 2, 137. 
Bordoni-Uffreduzzi. (1886) Fortschr. der Med., 157. 

Chester. (1903) Fifteenth Annual Report of the Delaware College Agricul- 
tural Experiment Station. 

(1901) Manual of Determinative Bacteriology, 291. 
DeBary. (1884) Vergleichende Morphologic und Biologie der Pilze, Myceto- 

zoen und Bakterien, 500. 

(1887) Vorlesungen fiber Bakterien, 2 Aufl., 13. 
FLtJGGE. (1886). Die Mikroorganismen, 2 Aufl., 400, 403. Watson Cheyne 

Translation. 

(1894) Zeitschr. f. Hyg., 17, 272. 
Ford and Pryor. (1904) Johns Hop. Hosp. Bull., 25, 270. 
Ford. (1903) Studies from the Royal Victoria Hospital, 1, no. 5. 
Frankland, Grace and Percy. (1887) Phil. Trans. Roy. Soc. London, 87, 297. 
Globig. (1888) Zeitschr. f. Hyg., 3, 323. 

GoHiNi. (1894) Giornale della Reale Societa Italiana d'Igiene, 16, no. 1. 
Gottheil. (1901) Centralbl. f. Bakt., 2 Abt., 7, 430. 
HuEPPE. (1884) Mittheil. aus. dem. Kais. Gesundheitsamte, 2, 309. 
Lehmann and Neumann. (1901) Atlas and Principles of Bacteriology, 2d 

edition. Translation by G. H. Weaver. Part II, 320. (W. B. 

Saunders.) 
LoEFFLER. (1887) Berl. Klin. Wchnschr., 24, 607. 
Lunt. (1896) Centralbl. f. Bakt., 2 Abt., 2, 572. 
Meyer. (1903) Practicum der botanischen Bakterienkunde. Jena. 
MiGULA. (1897) System der Bakterien, 1, 252. 

(1900) System der Bakterien, 2, 577. 
Neide. (1904) Centralbl. f. Bakt. 2 Abt. 12, 1. 
ZoPF. (1885) Spaltpilze, 3 Aufl., 82. 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 317 

ILLUSTRATIONS 

The illustrations were drawn by Mrs. W. W. Ford and Mr. W. P. Didusch 
from preparations illustrating the different phases in the morphology of the 
various species. The preparations were always stained by gentian violet and 
drawn from a Zeiss microscope with a 1/12 oil immersion lens and a compensating 
ocular No. 8, giving a magnification of 1080 diameters. The attempt was made 
to show the morphology of the vegetative cells which comes out best in certain 
species at 6 to 7 hours and in others at 22 to 24 hours, the method of spore-for- 
mation which varies in the time in which it appears, and the changes which occur 
in the organism when grown on carbohydrate media. 

EXPLANATION OF PLATES 

Plate 1 

Fig. 1. Bacillus coli. Plain agar, 24 hours 

Fig. 2. Bacterium anthracis. Plain agar, 24 hours 

Plate 2 

Fig. 3. Bacterium anthracis. Plain agar, 4 days 

Fig. 4. Bacillus subtilis from milk. Plain agar, 20 hours 

Plate 3 

Fig. 5. Bacillus subtilis from milk. Glucose agar, 24 hours 
Fig. 6. Bacillus vulgatus from milk (Bacillus mesentericus vulgatus). Plain 
agar, 20 hours 

Plate 4 

Fig. 7. Bacillus vulgatus from milk (Bacillus mesentericus vulgatus). Glu- 
cose agar, 24 hours 

Fig. 8. Bacillus mesentericus from soil (Bacillus mesentericus fuscus). 
Plain agar, 72 hours 

Plate 5 

Fig. 9. Bacillus mesentericus from soil (Bacillus mesentericus fuscus). Glu- 
cose agar, 48 hours 

Fig. 10. Bacillus aterrimus from human intestinal contents (Bacillus 
mesentericus niger). Plain agar, 20 hours 



S 



Plate 6 

Fig. 11. Bacillus aterrimus from human intestinal contents (Bacillus 
mesentericus niger). Glucose agar, 48 hours 

Fig. 12. Bacillus niger from Krai (Bacillus lactis niger). Plain agar, 48 
hours 



318 I J. S. LAWRENCE AND W. W. FORD 

Plate 7 

Fig. 13. Bacillus ntger from Krai {Bacillus lactis niger). Glucose agar, 
48 hours 

Fig. 14. Bacillus globigii from Krai {Bacillus mesentericus ruber). Plain agar, 
20 hours 

Plate S 

Fig. 15. Bacillus cohaerens from milk. Plain agar, 7 hours 
Fig. 16. Bacillus cohaerens from soil. Plain agar, 6 hours 

Plate 9 

Fig. 17. Bacillus cohaerens from soil. Plain agar, 24 hours 
Fig. 18. Bacillus simplex from Krai. Plain agar, 5 hours 

Plate 10 

Fig. 19. Bacillus simplex from Krai. Plain agar, 20 hours 
Fig. 20. Bacillus simplex from soil. Plain agar, 24 hours 

Plate 11 

Fig. 21. Bacillus simplex from soil. Plain agar, 3 days 

Fig. 22. Bacillus mycoides from cow dung. Plain agar, 5 hours 

Plate 12 

Fig. 23. Bacillus mycoides from cow dung. Plain agar, 24 hours 
Fig. 24. Bacillus mycoides from cow dung. Plain agar, 5 days 

Plate 13 

Fig. 25. Bacillus cereus from milk. Plain agar, 7 hours 
Fig. 26. Bacillus cereus from milk. Plain agar, 24 hours 

Plate 14 

Fig. 27. Bacillus cereus from milk. Glucose agar, 24 hours 
Fig. 28. Bacillus albolactus from milk {Bacillus lactis albus). Plain agar, 7 
hours 

Plate 15 

Fig. 29. Bacillus albolactus from milk {Bacillus lactis albus). Plain agar 
plate, 24 hours 

Fig. 30. Bacillus albolactus from milk {Bacillus lactis albus). Glucose 
agar, 24 hours 

Plate 16 

Fig. 31. Bacillus megatherium from American Museum. Plain agar, 7 hours 
Fig. 32. Bacillus megatherium- horn American Museum. Plain agar (plate), 
24 hours 



AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 319 

Plate 17 

Fig. 33. Bacillus megatherium from American Museum. Glucose agar, 24 
hours 

Fig. 34. Bacillus megatherium from American Museum. Glucose agar, 48 
hours 

Plate IS 

Fig. 35. Bacillus megatherium from Krai. Plain agar, 7 hours 
Fig. 36. Bacillus megatherium from Krai. Plain agar, 24 hours 

Plate 19 

Fig. 37. Bacillus megatherium from Krai. Glucose agar, 20 hours 
Fig. 38. Bacillus petasites from milk. Plain agar, 7 hours 

Plate 20 

Fig. 39. Bacillus petasites from milk. Plain agar, 20 hours 
Fig. 40. Bacillus petasites from milk. Plain agar, 48 hours 

Plate 21 

Fig. 41. Bacillus petasites from Krai. Plain agar, 7 hours 
Fig. 42. Bacillus petasites from Krai. Plain agar, 20 hours 

Plate 22 

Fig. 43. Bacillus graveolens from Krai. Plain agar, 7 hours 
Fig. 44. Bacillus graveolens from Krai. Plain agar, 20 hours 

Plate 23 

Fig. 45. Bacillus tumescens from Krai. Plain agar, 7 hours 
Fig. 46. Bacillus tumescens from Krai. Plain agar 20 hours 

Plate 24 

Fig. 47. Bacillus tumescens from Krai. Glucose agar, 48 hours 
Fig. 48. Bacillus fusiformis from dust. Plain agar, 24 hours 

Plate 25 

Fig. 49. Bacillus Jusiformis from dust. Plain agar, 48 hours, showing long 
threads 

Fig. 50. Bacillus fusiformis from contaminated hirudin. Plain agar, 24 hours 

Plate 26 

Fig. 51. Bacillus fusiformis from contaminated hirudin. Plain agar, 48 hours 
Fig. 52. Bacillus terminalis from milk. Plain agar, 17 days 



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(Lawrence and Ford: Aerobic Spore-bearing Non-pathogenic Bacteria) 



JOURNAL OF BACTERIOLOGY VOL. I 



PLATE 21 




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JOURNAL OF BACTERIOLOGY VOL. 1 



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JOURNAL OF BACTERIOLOGY VOL. I 



PLATE 23 









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JOURNAL OF BACTERIOLOGY VOL. I 



PLATE 24 



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JOURNAL OF BACTERIOLOGY VOL. I PLATE 25 



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JOURNAL OF BACTERIOLOGY VOL. I 



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THE NUMBER OF COLONIES ALLOWABLE ON 
SATISFACTORY AGAR PLATES 

ROBERT S. BREED and W. D. DOTTERRER' 

New York Agricultural Experiment Station, Geneva, New York 

INTRODUCTION 

A point which is of much importance in making bacteriological 
counts is the hmit in the number of colonies that may be allowed 
to grow on a plate without introducing serious errors. Probably 
every bacteriological worker has this point in mind in making 
counts and has his own opinion based on experience; but there 
are few pubhshed data on the subject. The matter has been 
specially under discussion in connection with the proposed re- 
vision of standard methods of milk analysis. This investiga- 
tion was made in order to increase the amount of information 
available for the use of the Committees who have undertaken 
the work of revision. 

HISTORICAL 

It is interesting to note the published opinions of different 
workers on this point. In 1895 Neisser (1895) pubhshed an 
article in which he reached the conclusion that plates should 
be so made that they will have about 10,000 colonies per plate, 
which numbers should then be estimated by means of the low 
power lenses of a compound microscope. He undoubtedly 
believed that each bacterial cell put into an agar plate would 
produce a colony regardless of overcrowding. Three years 
later Hesse and Niedner, (1898) reahzing, to some extent at 

' The senior author of this paper is responsible for the original suggestion of 
this problem, for direction in carrying it out, and aid in preparing the esults 
for publication. The junior author has carried out the laboratory work and has 
helped in preparing the results for publication. 

321 



322 ROBERT S. BREED AND W. D. DOTTERRER 

least, the true state of affairs published an article in which 
they claim that plates having more than 100 colonies should be 
disregarded and that under these conditions the microscope 
should not be used for counting. In 1897, Hill (Hill and Ellms, 
1897) contended that overcrowded plates would not give re- 
liable results in water analysis. In 1899, Jordan and Irons 
(1899) independently urged the same thing. Again Hill (1908) 
called attention to the point in a paper read before the labora- 
tory section of the American Public Health Association in 1907, 
in which he pointed out that wide discrepancies in counts might 
be caused by different methods of computation and concluded 
that only those plates having numbers of colonies falling be- 
tween 40 and 200 per plate should be considered in reporting 
results. These figures were adopted in the report presented 
by the Committee on Standard Methods for the Bacterial 
Examination of Milk at the Richmond meeting of the Ameri- 
can Public Health Association (1910). In the Report pre- 
sented at the Rochester meeting in September 1915 (Comm. 
Stand. Meth. 1915) the lower limit in the number of colonies 
allowable on agar plates was changed from 40 to 30, and the 
limits of 30 and 200 were also accepted by the Committee on 
Standard Methods of Bacterial Water Analysis in their Report 
presented at the same meeting. 

STATEMENT OF PROBLEM 

It is generally recognized that the kind of bacteria present 
in the material under examination will have an influence on 
the size of the colonies, and, consequently, on the number that 
can develop on a plate. Some of the commonest and most 
important bacteria in milk do not produce colonies larger than 
pin points on ordinary agars even when only a few are present. 
Other colonies grow large and in the case of spreaders may cover 
the entire plate. 

Just what prevents the development of all the bacteria into 
colonies on crowded plates is not thoroughly understood. In 
some cases it may be because the food material is all used up; 



COLONIES ALLOWABLE ON AGAR PLATES 323 

in others it is clearly due to the fact that by-products of bacterial 
growth inhibit the growth of other colonies; and occasionally 
colonies fuse or overgrow each other and so reduce the count. 
On the other hand colonies growing side by side sometimes 
stimulate each other, a phenomenon which has been noted 
in this work on plates containing large numbers of B. bulgaricus 
with an occasional mold or bacterial colony of a different type. 
The molds and many bacteria so stimulate the B. bulgaricus 
that these organisms form visible colonies in the region of the 
larger colonies, faiUng to develop in all other parts of the plate. 
The same condition has been noted in plating material con- 
taining large numbers of long chained streptococci. This 
phenomenon naturally produces marked irregularities in count 
when it occurs. 

Because of these and other difficulties certain plates in any 
series made from a given sample are more satisfactory for use 
in computing a total count than are others. The matter of select- 
ing plates to be used in computing a count becomes therefore a 
matter requiring considerable judgment. 

EXPERIMENTAL DATA 

a. Analyses made in the Station Laboratory 

The object of this study has been to determine the hmits in 
the number of colonies on plates which are satisfactory for 
making bacterial counts. The data used have been obtained 
by plating market milk samples on standard agar in triplicate 
and in three different dilutions, incubating for five days at 21°C., 
following with an incubation for two days at 37°C. The plates 
were counted at the end of five days and again after the two 
days incubation at 37°C. The five day and seven day counts 
are tabulated separately and show the conditions for each period 
of incubation. 

In deciding which plate counts to select as probably nearest 
correct it became necessary to discard all of the counts on a 
few samples where no satisfactory average could be made be- 
cause of spreaders or because the milk contained more bacteria 



324 EGBERT S. BREED AND W. D. DOTTERRER 

than was anticipated and the dilutions were not carried far 
enough to give assurance that the count was not affected by 
overcrowding. In selecting individual plate counts which were 
to be tabulated as satisfactory, those counts were chosen which 
could be used in making an average without any individual 
figure varying more than 20 per cent from the average. All 
others were listed as discrepancies. For example, one sample 
gave the following counts per plate, 1: 100 dilution 1944, 1472 
and 1928 colonies; 1: 1000 dilution 484, 515 and 610 colonies; 
1: 10000 dilution 43, 45, and 46 colonies. The counts of 
484 and 515 from the 1: 1000 dilution were averaged with the 
1: 10000 counts of 43, 45 and 46; and this average was taken 
as the final count on the sample. The counts made on the 
1: 100 plates were all listed as discrepancies because they are 
more than 20 per cent lower than the average, and the count 
of 610 from one of the 1: 1000 plates was also listed as a dis- 
crepancy because it was more than 20 per cent higher than the 
average. Occasionally all of the nine plates made from a sam- 
ple could be included in the final average. 

Table I gives the number of plate counts made after five days 
of incubation at 21°C., arranged in groups according to the 
number of colonies which appeared on the plates. Four hun- 
dred and thirty-nine of the 1435 plates had less than 10 colonies 
per plate. Only 22.3 per cent of these checked within the 20 
per cent limit. One hundred and eighty plates fell in the group 
having more than 10 and less than 20 colonies per plate. Of these 
53.9 per cent checked within the 20 per cent limit. Percent- 
ages calculated for the groups of plates having 20 to 30, 30 to 
50, 50 to 100, 100 to 200 and 200 to 400 colonies per plate were 
more or less variable, showing that from 66.3 per cent to 93.2 
per cent of the total number of plates agreed within the 20 per 
cent Hmit. The best percentage of agreement is shown by the 
group having more than 100 and less than 200 colonies per plate, 
and the next highest by the group having between 50 and 100 
colonies per plate. There were decidedly fewer plates giving 
satisfactory results among those which had more than 400 colo- 
nies per plate, the percentage of plates which checked within 
20 per cent being 44.4. 



COLONIES ALLOWABLE ON AGAR PLATES 



325 



The results given in the lower part of table 1 were calculated 
from the same counts, the groups of plates having been arranged 
differently. From this part of the table it will be seen that 
the percentage of discrepant plates is practically the same 
for the groups of plates having 20 to 400, 30 to 400, 20 to 200, 
30 to 200, or 40 to 200 colonies per plate, the best showing being 
made by the group of plates having more than 40 and less than 

TABLE 1 

Plate counts after incubation at 21 ° C. arranged to show the number and percent- 
age of counts in groups according to (he number of colonies per plate 





CHECKED WITHIN 20 PER 


DISCREPANT PLATES, DID NOT CHECK WITHIN 




GROUP 


CENT OP AVERAGE 




20 PER CENT 


OF AVERAGE 


NUMBER 




















Number 


Per cent 


Too low 


Too high 


Total 
number 


Per cent 


IN GROUP 


to 10 


98 


22.3 


172 


169 


341 


77.7 


439 


10 to 20 


97 


53.9 


29 


54 


83 


46.1 


180 


20 to 30 


54 


72.9 


6 


14 


20 


27.1 


74 


30 to 50 


67 


66.3 


11 


23 


34 


33.7 


101 


50 to 100 


162 


84.8 


17 


12 


29 


15.2 


191 


100 to 200 


179 


93.2 


8 


5 


13 


6.S 


192 


200 to 400 


105 


78.9 


25 


3 


28 


21.1 


133 


Over 400 


100 


44.4 


114 


11 


125 


55.6 


225 


to 30 


249 


35.9 


207 


237 


444 


64.1 


693 


20 to 400 


567 


82.0 


67 


57 


124 


18.0 


691 


30 to 400 


513 


83.1 


61 


4S 


104 


16.9 


617 


20 to 200 


470 


82.9 


43 


54 


97 


17.1 


567 


30 to 200 


416 


84.3 


37 


40 


77 


15.7 


493 


40 to 200 


376 


86.0 


23 


28 


61 


14.0 


437 


Over 400 


100 


44.4 


114 


11 


125 


55.6 


225 



Total number of counts summarized in this table 1435. 



200 colonies per plate. Plates having less than 30 colonies or 
more than 400 colonies show very large percentages of dis- 
crepancies. 

Table 2 gives the results obtained by counting 1056 of the 
same plates as those whose counts are summarized in table 1 
after two days of additional incubation at 37°C. In general 
the results obtained from these counts are similar to those given 
in table 1. However the best showings are made in this case 
by groups of plates having more than 200 and less than 400 colo- 



326 



ROBERT S. BREED AND W. D. DOTTERRER 



nies per plate (87 per cent of satisfactory plates), the group 
of plates having 100 to 200 colonies (82.4 per cent) and the 
group having 30 to 400 colonies per plate (81.4 per cent). As 
in table 1 there is a marked increase in the number of discrepant 
counts from plates having less than 30 or more than 400 colonies 
per plate While the results in table 1 favor the 40 to 200 
group rather than the 30 to 400 group by 2.9 per cent., the same 

TABLE 2 

Plate counts after two additional days of incubation at 37° C. arranged to show the 
number and percentage of counts in groups according lo the number of colonies 
per plate 





CHECKED WITHIN 20 PER 


DISCREPANT PLATES, DID NOT CHECK WITHIN 




GROUP 


CENT OF AVERAGE 


20 PER CENT OF AVERAGE 


NUMBER 




















Number 


Per cent 


Too low 


Too high 


Total 
number 


Per cent 


IN GROUP 


to 10 


60 


28.4 


60 


91 


151 


71.6 


211 


10 to 20 


76 


60.0 


23 


28 


51 


40.0 


127 


20 to 30 


46 


63.0 


8 


19 


27 


37.0 


73 


30 to 50 


55 


72.3 


5 


16 


21 


27.7 


76 


50 to 100 


117 


81.0 


14 


12 


26 


19.0 


143 


100 to 200 


127 


82.4 


16 


11 


27 


17.6 


154 


200 to 400 


101 


87 


14 


1 


15 


13 


116 


Over 400 


78 


50 


71 


4 


78 


50 


156 


to 30 


182 


44.2 


91 


138 


229 


55.8 


411 


20 to 400 


445 


79.2 


57 


61 


117 


20.8 


562 


30 to 400 


399 


81.4 


49 


42 


91 


18.6 


490 


20 to 200 


353 


77 


45 


60 


105 


23 


458 


30 to 200 


307 


79.7 


37 


41 


78 


20.3 


385 


40 to 200 


277 


79.8 


36 


34 


70 


20.2 


347 


Over 400 


78 


50 


74 


4 


78 


50 


156 



Total number of counts summarized in this table 1056. 

comparison in table 2 shows an advantage of 1.6 per cent for 
the 30 to 400 group This indicates that there is little advantage 
in selecting one group of plates in preference to the other. 

In the fourth and fifth columns of these two tables, the num- 
ber of cases is shown in which the discrepancy was caused by 
having too few or too many colonies on the plate Arranging 
the plates in the groups to 10, 10 to 20, 20 to 30, 30 to 50, 50 
to 100, 100 to 200, 200 to 400 and more than 400 colonies per 



COLONIES ALLOWABLE ON AGAR PLATES 327 

plate, it is seen that there is a tendency for discrepancies caused 
by having too many colonies on a plate to occur in all groups 
having less than 50 colonies per plate (one exception to this 
statement is seen in the group to 10 in table 1). In all cases 
where more than 50 colonies occurred on the plates, the greater 
number of discrepancies was caused by having too few colonies 
on the plates. The tendency toward discrepancies caused by 
having too few colonies on the plates becomes very marked 
as soon as the limit of 200 colonies per plate is passed. 

These findings indicate that while the greater proportion of 
the discrepancies on plates having less than 50 colonies per plate 
are caused by the operations of the laws of choice and chance, 
yet there is some factor present which tends to cause more 
colonies to develop than should do so. In all probabihty this 
factor is chance contamination from the air which occurs during 
planting. As is well known, it is common for supposedly sterile 
check plates to develop one, two or more colonies on prolonged 
incubation. The presence of these colonies on inoculated plates 
having fewer than 50 colonies per plate causes a relatively large 
error in the counts which in some cases would cause the individual 
plate count to exceed the 20 per cent limit specified here as neces- 
sary before the plates were classed as satisfactory. 

The tendency for irregularities, due to having too few colo- 
nies on plates, to occur in counts having 50 or more colonies 
per plate is too well known to all bacteriologists to require ex- 
tended discussion. These are undoubtedly caused by the 
effect of overcrowding. The fact that not all of the discrepancies 
on plates having more than 400 colonies per plate were of this 
sort is more significant, for it shows that not all of the discrep- 
ancies on plates having numerous colonies are due to over- 
crowding. Irregularities in the number of bacteria used in 
inoculating or chance contaminations are two things which 
might produce plates having too many colonies even on crowded 
plates. 

When all of these things are taken into consideration, it 
becomes a difficult matter to decide upon the limits in number 
of colonies which should be allowed on plates. It is at once 



328 ROBERT S. BREED AND W. D. DOTTERRER 

clear that plates having less than 20 and more than 400 colonies 
are so apt to be widely discrepant that counts from plates of 
this sort should be disregarded. There are likewise clear indi- 
cations that plates having between 40 and 200 colonies per plate 
are as satisfactory as any that can be selected. However the 
results secured in this investigation do not indicate that serious 
errors would be introduced in routine work by extending these 
limits to 30 and 400, or even to 20 and 400, thereby lessening 
the amount of work necessary to secure acceptable counts. 

b. New York City analyses 

Another set of data which is more satisfactory in one way 
because of the fact that a very large number of plates were 
made from a single sample of milk but which is also less satis- 
factory in another way because of the fact that it is more limited 
in its application, has been secured from a set of analyses made 
on November 19, 1915, by five New York State laboratories, ^ 
under the supervision of Prof. H. W. Conn. In this series 20 
samples of the same milk were sent to each laboratory for analy- 
sis. Four laboratories made plate counts, one making them 
in duplicate, so that five sets of plate counts are available. These 
were made from two dilutions of 1 : 100 and 1 : 1000 each. Two 
plates were made for each dilution. Three laboratories made 
microscopic counts, one making them in duplicate so that four 
sets of these counts are available. 

The average of the accepted plate counts was 4250. The aver- 
age of the microscopic counts of clumps, or sources, was 5590. 
The close correspondence in results obtained by these two very 
different methods of counting makes it very probable that the 
total number of groups of bacteria in this milk was close to 5000 
per cubic centimeter. The 1: 100 dilution plates gave counts 
in which the average number of colonies on the two plates varied 
between 24 and 125. The 1 : 1000 plates gave counts in which 
the average number of colonies from the two plates varied be- 

2 Lederle Laboratories, North's Sanitary Laboratories, N. Y. City Board 
of Health Laboratory, Borden's Laboratory, N. Y. Agric. Exp. Sta. Laboratory. 



COLONIES ALLOWABLE ON AGAR PLATES 329 

tween 0.5 and 16.5 with a single case where the average of the 
two plates was 44. 

If we arbitrarily assume that plates giving a count more 
than 2500 above or below the average fail to check with the 
accepted count, we find that the averages of all but three of the 
100 pairs of 1: 100 plates check with the accepted count while 
there are 27 cases out of the 100 where the count from the 1 : 1000 
dilution fails to check within these limits. It is important to 
note also that 23 of these 27 cases are instances where the dis- 
crepancy was such as to give a higher count that the accepted 
count, indicating that chance contaminations were probably 
the chief cause of trouble. 

SUMMARY 

1. The work here reported includes a study of the counts 
made from 1435 agar plates inoculated from samples of market 
milk and incubated five days at 21°C.; and also a study of the 
counts made from 1056 of the same plates after two days addi- 
tional incubation at 37°C. The results obtained indicate that, 
for milk analyses, the counts made from plates having more 
than 30 and less than 400 colonies on the plates are very nearly 
as satisfactory as those obtained from plates having more than 
40 and less than 200 colonies, the latter being the limits in 
numbers originally recommended by the Committee on Standard 
Methods for the Bacterial Examination of Milk. 

2. Plates having less than 20 or more than 400 colonies on 
them are shown to be so frequently discrepant that counts obtained 
from them should never be trusted unless checked by compari- 
son with plates from different dilutions having more than 30 
or less than 400 colonies. The acceptance of counts from plates 
having 20 to 30 colonies per plate would not greatly increase 
the percentage of discrepancies. 

3. All groups of plates, regardless of the number of colonies 
showed a certain percentage of plates which gave counts which 
varied more than 20 per cent from the accepted count. The 
percentage of discrepant counts of this sort varied between 37 



330 ROBERT S. BREED AND W. D. DOTTERRER 

and 7 for all groups of plates having more than 20 and less 
than 400 colonies per plate, the worst showing being made by 
the plates having 20 to 30 colonies per plate and the best by 
the plates having 100 to 200 colonies per plate. 

4. The discrepancies which occurred in counts made from 
plates having less than 50 colonies per plate were more fre- 
quently caused by too many colonies on the plates than by too 
few colonies. This excess is undoubtedly due to the influence 
of chance air contaminations which took place during the plating. 
Where the plates have a small number of colonies on them a 
few extra colonies of this sort produce relatively wide discrep- 
ancies. 

5. The discrepancies in counts made from plates having more 
than 50 colonies per plate were more frequently caused by hav- 
ing too few rather than too many colonies on the plates. The 
frequency of this type of discrepancy became very marked 
where the number of colonies exceeded 200 per plate. The 
probable explanation of the excess of this type of irregularity 
is that of overcrowding. Since however there was always a 
certain percentage of discrepancies caused by having too many 
colonies on the plate even where there were more than 400 colo- 
nies per plate, it is evident that not all of the irregularities are 
caused in this way. 

6. Counts made from 20 dupUcate samples of the same milk 
in five series of analyses showed 27 out of a possible 100 wide 
discrepancies in the counts obtained from an average of two 
plates made from a 1: 1000 dilution. The number of colonies 
of these plates averaged more than 0.5 and less than 16.5 for 
the two plates, with one exception where the average was 44. 
Counts made from the 100 pairs of 1: 100 plates which had 
more than 24 and less than 125 colonies as the average of the 
two plates, showed only 3 out of a possible 100 wide discrepancies. 



COLONIES ALLOWABLE ON AGAR PLATES 331 

REFERENCES 

Hesse, W. und Niedner. (1898) Die Methodik der bakteriologischen Wasser- 

untersuchung. Zeitschr. f. Hyg. u. Infektionskrankh. 29: 454-462, 
Hill, H. W. and Ellms, J. W. (1897) Report on Brooklyn Water Supply. 

pp. 164-169. 
Hill, H. W. (1908) The Mathematics of the Bacteria' Count. Amer. Jour. 

Pub. Hyg. 18 (N. S. 4) : : 00-310. 
Jordan E. O. and Irons, E. E. (1899) Notes on Bacterial Water Analysis, 

Public Health Papers an 1 Reports Amer. Pub. Health Ass'n. 25: 

564-569. 
Neisser, Max. (1895) Die mikroskopisch- Plattenzahlung und ihre specielle 

Anwendung auf die Zahlung von Wasserplatten. Zeitschr. f. Hyg. 

u. Infektionskrankh. 20 119-146. 
Report of the Committee on Standard Methods of Bacterial Milk Analysis. 

1910. Amer. Jour. Pub. Hyg. 20 (N. S. 6) : 315-345. 
Report of the Committee on Standard Methods of Bacterial Examination 

of Milk. 1915. Amer. Jour. Pub. Health, 5: 1261-1262. 



A MODIFICATION OF THE HYGIENIC LABORA- 
TORY METHOD FOR THE PRODUCTION 
OF TETANUS TOXIN 

HARRIET LESLIE WILCOX 

Research Laboratories, New York City Department of Health 

In the November, 1915, issue of the Journal of Medical Re- 
search, ^ Anderson and Leake briefly describe the method used 
at the Hygienic Laboratory, Washington, D. C, for the produc- 
tion of a uniformly potent toxin. The method in use at the 
Research Laboratory is essentially that given by Anderson and 
Leake with a few shght variations. As there have been many 
inquiries as to how we obtain our potent toxin it was thought 
that the details of the exact procedure might be of sufficient 
value to pubhsh. 

STOCK CULTURES 

The stock cultures are grown on a semi-sohd medium made 
in the following way. 

Veal broth 1000 cc. 

Agar 5 grams 

Witte's Peptone 10 grams 

NaCl 5 grams 

Reaction Neutral to phenolphthalein 

About 8 CC. to 10 cc. of this medium are put into tubes which 
are autoclaved at 15 pounds pressure for one-half hour, the tubes 
being then ready for use. 

To transfer cultures, one of the semi-solid agar cultures is 
melted and 1 cc. is added to a freshly melted semi-solid agar tube, 
at least ten sub-cultures being thus made from one stock culture. 
After inoculation, the tubes are cooled, the plugs inmiersed in 

' Anderson and Leake, 1915. A Method of producing Tetanus Toxin, Jour- 
nal of Medical Research, 33, 239. 

333 



334 HARRIET LESLIE WILCOX 

paraffin and the tubes incubated at 37°C. After one week's 
incubation the cultures are stored in the ice chest, where they 
may be kept for six months without affecting their abihty to 
produce toxin. 

THE BROTH FOR TOXIN AND PRELIMINARY CULTURES 

The following broth is used both for the toxin and for the 
preliminary cultures for inoculating the toxin broth: 

To every pound of market veal, add 1000 cc. of water and place 
in ice chest over night. The next morning the infusion is placed 
over a free flame and raised to 45°C. and held at that temper- 
ature for one hour; it is then boiled briskly for one-half hour 
and the broth strained through cheesecloth. The amount of 
filtrate is measured and the following added: 

Witte's Peptone 1.0 per cent 

Glucose (anhydrous) 1.0 per cent 

NaCl 0.5 per cent 

When the above ingredients are added, the broth is boiled 
until the peptone, glucose, etc., are melted, and is then titrated 
with T^ NaOH. After the reaction has been corrected to +1 
to phenolphthalein, the flame is turned out. The broth is 
then filtered through two layers of absorbent cotton directly 
into 2 litre Erlenmeyer flasks, leaving only sufficient space in 
the flasks for the expansion of the broth during the sterilization 
in the Arnold. The flasks are sterilized for 1| hours on the 
first day and one hour on the second day. 

PRELIMINARY CULTIVATION 

Fill potato tubes with about 40 cc. of the glucose broth and 
sterilize for one and one-half hours on the first day and one 
hour on the second day. These tubes may be kept for two 
weeks when they will still give satisfactory growth. To make 
the first transfer for the preliminary cultivation, add 8 cc. of 
the melted semi-solid agar stock culture of the B tetani to two 
tubes of glucose broth from which the air has been previously 



* METHOD FOR PRODUCTION OF TETANUS TOXIN 335 

expelled by heating in the Arnold for fifteen to twenty minutes, 
and which have been cooled down to about 50°C. These tubes 
are incubated for 24 hours and the next day, two freshly heated 
tubes of glucose broth are inoculated with 5 cc. of the glucose 
broth cultures planted the previous day. On the third day, 
determine the number of flasks that are to be inoculated and 
inoculate as many freshly heated glucose broth tubes from the 
second glucose broth generation as there are flasks. Ander- 
son calls for at least six or seven generations in the glucose broth 
before the inoculation of the toxin broth, but at the Research 
Laboratory it has been found that three generations or rven 
two if need be, are sufficient for obtaining a toxicity of 1-25,000. 

INOCULATION OF TOXIN BROTH 

After the second sterilization in the Arnold, the flasks are 
ready for inoculation. The broth may be cooled down to 55°- 
60°C. by allowing the flasks to stand at room temperature, or 
in a more rapid way by placing the hot flasks in a large sink, 
to which cool, and then cold, water is added until the lower 
portions of the flasks are covered. When the bottoms of the 
flasks are cool to the hand, the portions above the water being 
still very hot, the inoculation may be made as follows: 

The plugs are carefully removed, the necks flamed and the 
plugs replaced. In a similar way, the mouths of the culture 
tubes are steriUzed and then, partly removing the plug of a 
flask, the contents of a potato tube is poured rapidly into a 
flask. If one prefers, the broth culture may be transferred 
by using a pipette, but the former method has been used here 
without subsequent contamination and found very satisfactory. 
After inoculation, the flasks are incubated for fifteen days at 
36°-37°C., care being taken to exclude all light from them. 

The flasks at the end of 24-48 hours show a diffuse cloudi- 
ness with the formation of gas bubbles on the surface of the 
broth. Toward the end of two weeks, the gas bubbles usually 
disappear, while the cloudiness persists and a light precipitate 
forms at the bottoms of the flasks. If it is not convenient 



336 HARRIET LESLIE WILCOX 

to filter on the fifteenth day, the cultures may be kept in the 
incubator until the twentieth day without a loss of toxicity 
but from the twentieth day to the twenty-fifth day, the toxin 
loses about 20 per cent in potency. 

FILTRATION 

All glassware, filters, etc., should be neutral to phenolphthalein 
and the greatest care should be taken to exclude light, either 
direct or indirect, by darkening the room and by covering the 
filtering apparatus with dark cloths, ordinary black cambric 
being used at the Research Laboratory. 

The broth cultures are first passed through Buchner filters 
about 8 inches in diameter, which have been packed with a 
layer of finely shredded paper pulp 0.25 inch in thickness. It 
is of importance that the pulp should be so well packed that the 
filtrate is absolutely clear, otherwise it will clog the Berkefeld 
filter. The first filtrate, about 200 cc, which passes through 
the Buchner is discarded, as it contains a considerable amount 
of water from the pulp, and then the filtering of the toxin may 
proceed. If, after passing 8 to 14 litres through the pulp, the 
filtrate begins to appear cloudy, the pulp must be discarded 
and the Buchner repacked. The clear filtrate is then passed 
through a sterile Berkefeld filter, and 10 per cent of a 5 per cent 
solution of carbolic acid solution is added to the toxin which is 
now placed in the ice-chest, ready for testing its potency. 

POTENCY TEST 

Two 350 gram guinea pigs are inoculated subcutaneously 
over the abdomen with 1 cc. of a dilution of 15;^ and 25:000 
of the toxin respectively. If the toxin has a potency of 25:000 
the pig receiving the rs^o dilution will die on the second to 
third day and the pig receiving the 257000 dilution should die 
on the fourth day. If both pigs die with symptoms of tetanus 
before the fourth day, the toxin is stronger than 25;^ and a 
higher dilution should be tested. 



METHOD FOR PRODUCTION OF TETANUS TOXIN 337 

Though no comparative tests have been made with toxin 
produced under the usual anaerobic conditions, the toxin pro- 
duced by the Hygienic Laboratory method showed a some- 
what unexpected stabihty. For example: 



Lot 25. 



Filtered 


Tested 


Toxicity 


7-24-14 


7-24-14 


1-25,000 




8-31-14 


1-25,000 




9-30-14 


1-20,000 




10- 4-14 


1-20,000 



The test was made on a small amount of toxin, about 30 cc, 
kept in an ordinary test tube in the ice-box and protected from 
the air only by a shallow covering of albolene. 

From the above, it will be seen that our method for growing 
the tetanus bacillus for toxin varies very slightly from that of 
the Hygienic Laboratory. Instead of beef, we use market 
veal which has given almost invariably a toxin of 25^00? even 
going as high as 40^100 occasionally. It is interesting to note 
that with the bob veal, that is, veal under the legal age limit for 
selhng, which has always given a more highly potent diphtheria 
toxin than the older veal, we have rarely obtained a tetanus 
toxin above 157000 whereas the market veal used as control has 
produced a toxin of 2X000 strength. ^ 

Anderson recommends that the preliminary cultures should 
be carried on from one to three weeks, by daily transfers, but 
with only three generations in glucose broth before inoculating 
the toxin flasks, we have obtained a toxicity of 1-40,000 show- 
ing that fewer generations may be sufficient. 

Here it might be well to state that we obtained different re- 
sults from those recorded by other writers in regard to the growth 
in the preliminary cultures. Anderson and Leake found that 
a good growth is obtained in the first generation in 48 hours, 
in 24 hours in the second and third transplants, and in 16 hours 

^ Furthermore, by inoculating the glucose broth directly with a melted semi- 
solid agar culture of B. tetani, we have secured a toxin of over - in strength, 

25,000 

the control test which had been inoculated from preliminary broth cultures 
giving the same degree of toxicity. Confirmatory tests are being made along 
this line. 



338 HARRIET LESLIE WILCOX 

in the fourth. At the Research Laboratory, we have found 
an abundant growth in 18 to 24 hours in the first generation 
in glucose broth from the semi-soHd; this profuse growth con- 
tinues in the subsequent cultures up to the third or fourth 
generations, when there is a diminution, until in the sixth or 
seventh, as frequently happened, no growth or very slight growth 
appears after 48 hours or more. That is, out of thirteen cultures 
transferred in the sixth generations, there may be only six or 
seven tubes which show signs of growth, even after several 
days incubation. 

At the Hygienic Laboratory, glucose stab cultures are used 
for growing the stock strains, but it is not stated whether these 
cultures are grown anaerobically or aerobically. The semi- 
solid agar that we are using for the stock cultures is especially 
satisfactory as no anaerobic conditions except such as the medium 
provides are necessary to produce a heavy growth of B. tetani 
with spore formation after a few days incubation. 

It is in the hope that other laboratory workers may find 
the above technique of practical aid in producing an uniformly 
potent toxin that these minute details have been given. 



A METHOD OF ANAEROBIC PLATING PER- 
MITTING OBSERVATION OF GROWTH^ 

HORRY M. JONES 
Research Laboratories of the Dairy Division, U. S. Department of Agriculture 

Because of the already numerous descriptions of methods 
for growing cultures anaerobically, one hesitates to add another 
method to the list without an apology. The method here 
described will however be found of advantage in the isolation 
of anaerobes from cheese, milk, soil or other material where 
it is desired not only to secure growth of the anaerobes, but also 
to obtain them in pure culture directly from the material under 
examination. The method is chiefly to be recommended 
because of the relatively small amount of inert gases necessary 
to replace the air in contact with the media — an advantage 
that will be appreciated in laboratories where generous supplies 
of these gases are not available and where the method of anaerobic 
plating is frequently employed on a small scale. Furthermore, 
the rate and character of growth of the colonies are easily ob- 
served from the beginning, so that it is not necessary to open 
the anaerobic chamber at any time until sub-cultures are to 
•be made. 

The apparatus consists of one half of a Petri dish sealed with 
paraffine, of relatively low melting point, on a square stone or 
metal base provided with an inlet for the inert gas and an out- 
let for the displaced air. The base is conveniently made as 
follows: Slabs of stone (such as Alberine stone, or soapstone) 
or of cast iron, of about 2 cm. thickness, are cut in squares 
about 1 cm. larger than the diameter of the Petri dish to be used. 
Cut in one face of these slabs, with a lathe, an annular groove 
3 mm. deep and 4 mm. wide, and of such a diameter that the 

^Published by permission of the Secretary of Agriculture. 

339 



340 HORRY M. JONES 

edges of the Petri dish will fit loosely into this groove. Within 
the circle described by this groove, about 2 cm. from an edge 
of the slab; i.e., about 5 mm. from the inner edge of the groove, 
drill a hole about 4 mm. in diameter and about half the thick- 
ness of the slab in depth. Drill a similar hole for the opposite 
side of the annular groove. Now drill horizontal holes from the 
corresponding surface edges of the slab to meet the vertical 
holes, and of such a diameter that small rubber stoppers may be 
used to stopper these holes securely. With this slab as a base 
for the Petri dish, the method of manipulation, provided it is 
desired to grow all of the colonies on the surface of the media, 
is somewhat as follows : Pour the agar or gelatin into the Petri 
dish as usual and allow to solidify. Then flow suitable water 
or broth dilutions of the material to be examined on the surface 



Petri Dish /Ocm. dia/vj. 






fuL ! '^ Tn — Jin 



Annular (Sroova 
3mm Deepx 



-4 1 I I 

J \ \ Air Oufht 



C/'OS'S ^Section i^/ew of stone base 
w/ttj petr/ djsh in po^i//on. 

of the medium. Tilt the dish to one side, and, with a sterile 
pipette, withdraw the excess of the diluting fluid. Invert the 
dish into its sterile cover and allow to drain. This draining 
will prevent contaminations on the stone base from spreading 
upward on the plate and so obviate the necessity of sterilizing 
the base. Now place the dish, edges down, into the annular 
groove of the slab, which has been previously warmed to a 
temperature sufficient to melt paraffine with a melting point 
of say 45 degrees Centigrade. The plate may be sealed to the 
stone by flowing melted paraffine into the groove either before 
or after the plate is put into position in the groove. The stone 
is then allowed to cool until the paraffine is thoroughly con- 
gealed. There are now, between the stone and the surface of 
the medium, only about 20 cc. of air to be replaced by the inert 



A METHOD OF ANAEROBIC PLATING 341 

gas, and by leading the gas from a suitable generator in through 
one of the rubber stoppers, (allowing the air to escape by way 
of the opposite loosely-stoppered opening) the oxygen pressure 
inside the inclosed space may be reduced to less than 1 mm. by 
the use of only about 200 cc. of the gas. The holes are then 
securely closed and the plates are ready for incubation. 

Oxygen- and C02-free air has been secured by the following 
method : Connect in series one or more of the sealed Petri dishes 
with two wash bottles containing a 5 per cent pyrogallic acid 
10 per cent caustic soda solution. Force air slowly through 
this train and allow the displaced air to escape byway of the op- 
posite outlet of the sealed Petri dish. 

When hydrogen is to be used for displacing the air, two wash 
bottles are required: One of AgNOa solution for traces of ASH3, 
and one of lead acetate solution for H2S. 

In general, satisfactory anaerobic conditions are obtained 
when the volume of H2 or N2 allowed to pass through the sealed 
plates amounts to ten times the volume of air inclosed by the 
plates and their connections. 



TESTICULAR INFUSION AGAR— A STERILIZABLE 
CULTURE MEDIUM FOR THE GONOCOCCUS 

IVAN C. HALL 

From the Hearst Laboratory of Pathology and Bacteriology, 
University of California^ 

INTRODUCTION 

The use of testicular infusion agar suggested by Hirschfelder 
(1914) aroused the hope that a medium for the cultivation 
of the gonococcus had been found which might be sterilized 
by steam, thus avoiding the addition of raw albumin (ascitic 
fluid or blood) to agar with its uncertain sterility and frequent 
failure to support growth even when sterile. Unfortunately 
I cannot agree that by the use of his formula all difficulties in 
cultivating the gonococcus (at least in pure culture) are removed, 
as he claims. I have however, determined some of the factors 
affecting successsful cultivation of gonococci in comparatively 
large quantities upon a sterilizable agar containing infusion of 
testicle. 

Vannod (1905) claimed that proper adjustment of the re- 
action with sodium carbonate facilitated cultivation of the 
gonococcus on so called ordinary media but the possible varia- 
tion from the optimum is so sHght that the method has not come 
into general use. One of Vannod's later contributions (1907) 
testifies to the general acceptance of the idea of the necessity 
of adding raw albumins. 

More recently some success in improving the media has been 
attained by Schwarz and McNeil (1912) in this country with so 
called ''salt free" veal agar, which is now generally used in the 
preparation of polyvalent antigens for the alexin fixation test. 

' The experimental work of this paper was carried out and its practical appli- 
cation made in The Cutter Laboratories, Berkeley, California, and it is pub- 
lished with the consent of the Director, Dr. H. E. Foster. 

343 



344 IVAN C. HALL 

Abroad, Lumiere and Chevrotier (1913) have advocated a mix- 
ture of beer wort and albumin sterilized in the autoclave, to 
which however, the addition of sterile horse serum is said to be 
advantageous, though not indispensable. Upon this medium 
gonococcus cultures were found to be viable at remarkably low 
temperatures (Lumiere and Chevrotier, 1914). 

Emile Weil and Noire (1913) have also suggested an agar 
containing whey, peptone, saccharose and urea. I have failed 
in several attempts to corroborate their claim that gonococci 
would grow upon this medium. 

I have not tried the cultivation of gonococci upon the egg broth 
of Besredka and Jupille (1913) nor according to the method of 
Ohlmacher (1915) upon Loeffler's blood serum but in several 
tests upon the starch agar of Vedder (1915) I have found it to 
be one of the most promising media. However, the growth, 
while possibly less long lived upon testicular infusion agar, 
is so much more abundant that the use of the latter is recommend- 
ed for the preparation of gonococcic vaccine. It should prove 
equally valuable for the preparation of antigens to be used in 
the alexin fixation test but this remains to be determined. 

CULTURES 

My strains of gonococci came originally from clinically typical 
cases of urethritis and epididymitis, having been isolated upon 
blood agar and cultivated in some cases as long as two years on 
ascitic agar. All were typical gram negative biscuit shaped 
diplococci, showing sparse growth upon rabbit blood or ascitic 
agar and failure of growth, at least in the second subculture, 
upon plain agar at 37°C. These have been our criteria and while 
we have had a realization quickened by the work of Broughton- 
Alcock (1914) that we might occasionally exclude true gonococci 
thereby we have not hesitated to insist that our media should 
be tested particularly on the less saprophytic strains. At the 
conclusion of each experiment therefore the purity of each 
culture was checked by gram stain and failure of growth upon 
plain agar at 37°C. 



TESTICULAR INFUSION AGAR 345 

Torrey's strains "C," "K," "L," ''N," ''0," and "S" kindly 
supplied by Dr. Charles Krumwiede, Jr., of the New York 
City Board of Health, conform to the above requirements and 
like our own were found to grow abundantly upon testicular 
infusion agar. 

The hope that opportunity might be found for making com- 
parative tests of this medium in the isolation of gonococci from 
lesions has already deferred pubUcation so long that it seems 
likely this will have to be left for some one more advantageously 
situated. Dr. H. E. Foster,^ has however succeeded sufficiently 
often in cultivating the gonococcus from cases of gonorrhea to 
warrant making such comparisons. 

EXPERIMENTAL WORK 

Successful cultures were first secured with a shghtly acid 
medium comprising aqueous infusion of beef testicle (500 grams 
per litre of distilled water), 2 per cent Witte's peptone, 2 per 
cent agar, 0.5 per cent glucose, and 0.3 per cent NaHa PO4, 
nearly neutrahzed with N/1 NaOH and sterilized by intermittent 
steaming in the Arnold steriUzer on three successive days for 
30 minutes at 100°C. 

This formula differs from Hirschfelder's particularly in its 
sugar content, the advisabiUty of which was shown by Elser 
and Huntoon (1909) and by Martin (1911). In each succeeding 
series of experiments a single factor was varied, the control 
consisting in a combination previously found successful; as 
the limits of variability of each factor involved were determined 
the preferable procedure was adopted for the following experi- 
ments. 

Thus it was quickly shown that steriHzation in the autoclave 
at 10 pounds pressure for 30 minutes is permissible. The sub- 
stitution of veal for testicle infusion was found to yield a less 
vigorous growth. The optimum amount of testicle was found 
to be 500 grams per liter although fair growth resulted when 
the proportion was as low as 125 grams per liter of water. The 

^ Personal communication. 



346 



IVAN C. HALL 



use of equal parts of veal extract seemed not to decrease the 
volume of growth but we have adhered to the use of testicle 
infusion alone. 



AGAR 



The amount of agar is important as shown in table 1. Media 
were made from the same testicular infusion in four lots with 
1, 2, 3 and 4 per cent agar. After sterilization the slanted tubes 
were left in the incubator at 37°C. for three days to dry out 



TABLE 1 
Optimum amount of agar 



AGAR % 


CULTURE 


16 HOURS 


40 hours' 


64 hours 


r 


G2 


None 


Poor 


Slight 


1 1 


G3 


None 


None 


None 




G5 


None 


None 


None 


' 


02 


None 


Fair 


Fair 


2 1 


G3 


None 


Fair 


Fair 




G5 


Slight 


Fair 


Good 




G2 


Slight 


Fair 


Good 


3 1 


G3 


Slight 


Fair 


Good 




G5 


Slight 


Fair 


Good 




G2 


Slight 


Fair 


Fair 


4 1 


G3 


Slight 


None 


Excellent 




G5 


None 


None 


Good 



* Patchy colonies respread on all tubes. 

Note. In this and other tables a "slight" growth approximates that of B. 
influenzae upon blood agar; a "fair" growth corresponds to that of B. typhi upon 
plain agar; "good" to that of B. coli; and "excellent" to that of Bact. pneumoneae. 



the surface of the slopes, a point shown to be necessary by ample 
experience. The slopes were inoculated from 48 hours ascitic 
agar cultures, incubated at 37°C. and observed daily. 

Soft testicular infusion agar has in our hands regularly yielded 
less satisfactory results than that which was more firm and 
less moist, a fact apparently at variance with the experience of 
McCann (1896) working with cyst fluid agar, and Van Saun 
(1913) with "salt free" veal agar. Firm testicular infusion 
agar is moreover not only favorable for growth but facihtates 



TESTICULAR INFUSION AGAR 



347 



the removal of the gonococci without the admixture of soHd 
particles of medium. 

Warden (1915) has recently pointed out that one of the fac- 
tors in the autolysis of gonococci is excessive moisture; it is 
suggested also that weak acids may inhibit autolytic disinte- 
gration. At any rate smears from testicular infusion agar 
cultures contain more whole cocci than those from ascitic agar, 
but whether the acid reaction due to fermentation of the glucose 
or the freedom of the media from excessive moisture, or both, 
may be held responsible in this case can not be stated with 
certainty. However, the factor of moisture in the media had 
to be reckoned with in all our experimental and practical work 
so that frequently where a clear cut result could not be obtained 
with fresh media there was httle difficulty after a few days drying. 

I have chosen 3 per cent agar as the most suitable for further 
use. 

GLUCOSE 

In the preparation of a portion of one lot of medium the usual 
glucose was omitted. The prepared slants were dried at 37°C. 
for 48 hours and afterwards at room temperature for four days. 
The media still appeared quite moist and the growths upon the 
controls where they appeared at all were patchy and unsatis- 
factory even on further incubation after respreading. Further 
drying at room temperature for ten days however led to a satis- 
factory result as shown in table 2, cultures being made from 24 
hour ascitic agar slants and incubated at 37°C. 





TABLE 2 
Omission of glucose 






MEDIA 


CULTURE 


16 HOURS 


40 HOURS 


60 HOURS 


With added glucose, 0.5 per 


cent \ 

[ 

. . . . -l 


Gl 
G2 
G3 
Gl 
G2 
G3 


Good 

Good 

Good 

None 

Fair 

Fair 


Excellent 

Excellent 

Excellent 

Slight 

Good 

Fair 


Excellent 

Excellent 

Excellent 

Slight 

Good 




Good 



348 IVAN C. HALL 

The usual control tests failed to show contamination in any 
tube so that the above result demonstrates that growth is possible 
without added glucose but is not so good as with it. 

That a modicum of carbohydrate is necessary is indicated 
by a lot of media made as usual with the exception of added 
glucose and from which only a portion^ of the tissue carbohydrates 
had been removed by the growth of B. communior, in which 
no growth of gonococci could be secured. But media which 
had been so fermented and then re-inforced by addition of 1 
to 2 per cent glucose yielded very excellent growth showing that 
inhibition in the fermented media could scarcely have been 
due to the accumulation of metabolic wastes from B. communior. 
Thus it was shown that for these strains the order of preference 
for added glucose content in media previously fermented by 
B. communior is 1 per cent, 2 per cent, and 3 per cent. Since, 
however, I have found no advantage in a prehminary fermenta- 
tion the addition of 0.5 per cent glucose to unfermented media 
has been retained. 

PHOSPHATES 

The use of unsaturated phosphates in culture media for bac- 
teria was recommended by Henderson and Webster (1907) 
for their stabiUzing effect upon the reaction, and a medium of 
this sort plus human serum was advocated by Martin (1911) 
for the cultivation of the gonococcus. 

My experiments upon the necessity of added phosphate have 
been inconclusive; at times excellent growth has been secured 
without its addition. In three separate double lots of testicular 
infusion agar made with and without the addition of inorganic 
phosphate the advantage has been in favor of that containing 
it. I have made no effort to determine the optimum amount 
or to attempt the cultivation of gonococcus in phosphate free 
media. 

' That the tissue sugar was not completely eliminated was proven by further 
gas production in deep tubes of the supposedly sugar free testicular infusion 
agar by B. communior. 



TESTICULAR INFUSION AGAR 



349 



REACTION 

The inclusion of 0.3 per cent NaH2P04 permits a considerable 
variation in the amount of sodium hydroxide added to reduce 
the titrable acidity. The results of a typical controlled experi- 
ment upon this point are shown in table 3. I might mention here 
having previously encountered some difficulty in the addition of 

TABLE 3 
Range of reaction of testicular infusion agar 



CUBIC CEN- 
TIMETERS 

XT /I "NT {~\13 


TITUE 


CULTURE 


16 HOURS 


40 HOURS 


64 HOURS 


N/1 Na OH 

ADDED 
IN 110 CC. 


Hypothetical 
end point* 


Actual 
end point 






( 


Gl 


None 


None 


None 





+ 6.0 


-6.0 


G3 


None 


None 


None 








G5 


None 


None 


None 






f 


Gl 


None 


None 


Slight 


2 


+ 4.0 


+4.2 \ 


G3 


Slight 


Fair 


Good 








G5 


Slight 


Fair 


Good 








Gl 


Fair 


Excellent 


Excellent 


4 


+2.0 


+3.2 


G3 


Fair 


Excellent 


Excellent 








G5 


Good 


Excellent 


Excellent 






f 


Gl 


Good 


Excellent 


Excellent 


6 





+ 1.7 


G3 


Fair 


Excellent 


Excellent 








G5 


Good 


Excellent 


Excellent 






c 


Gl 


Slight 


Good 


Excellent 


2 


-2.0 


?t 


G3 


Slight 


Good 


Excellent 








G5 


Fair 


Good 


Excellent 






( 


Gl 


None 


None 


None 


10 


-4.0 


Alkaline f { 


G3 


None 


None 


None 






^ 


Go 


None 


None 


None 



*Assuming no unsaturated compounds. 

t Media darkened by caramelization — end point uncertain. 



more than sufficient alkali to saturate the phosphate, the glucose 
being thereby caramelized on heating with resultant inhibition 
of growth of the gonococcus. 

A liter of medium was prepared with the usual testicular 
infusion, 2 per cent peptone, 3 per cent agar, 0.5 per cent glucose 
and 0.3 per cent Na H2PO4. Before neutrahzation 5 cc. titrated 
hot with phenolphthalein required 6 cc. N/20 NaOH to show 



350 IVAN C. HALL 

color. Six lots of 100 cc. each were separated and to each was 
added the amount of N/1 NaOH shown in table 3, and the total 
volume of each lot was then equalized at 110 cc. by addition 
of distilled water. The various media were tubed, sterilized 
in the autoclave at 10 pounds for 30 minutes, slanted, and dried 
at 37° C. for six days. Inoculation was made from 24 hour 
testicular infusion agar cultures; incubation was at 37°C. and the 
usual control tests confirmed the purity of the growth observed. 

It may be seen that "excellent" results may be expected be- 
tween the limits of actual titre to phenolphthalein from below 
+ 1.7 to +3.2 Normal acidity. There will be found little if 
any difficulty in the reaction when adjustment is made by addi- 
tion of N/1 NaOH as if the acidity were to be reduced to a titre 
of zero. It will still be found sufficiently acid, thanks to the 
phosphate, to obviate the difficulty of caramelization. 

PEPTONE 

The recent scarcity of Witte's peptone has necessitated experi- 
ments upon the substitution of an American product. These 
have shown "Difco" peptone of the American Digestive Fer- 
ments Co., Detroit, Michigan, to be equal to Witte's for this 
purpose. 

VIABILITY 

In contrast with the experience of Vedder (1915) with starch 
agar, prolonged viability cannot be claimed for cultures of gono- 
cocci upon testicular infusion agar. It was found that daily 
transfer of several strains for two weeks was eminently success- 
ful; planting every other day failed to keep some of the strains 
alive for more than four transfers and in all of these there was 
evidence of deterioration. 

Inoculating testicular infusion agar as well as ascitic agar, 
starch agar, ''salt free" veal agar or blood agar from another 
medium we have often found it necessary to coax the growth 
by repeated transfers and especially by respreading, before the 
maximum crop could be obtained. For this reason much em- 
phasis should be placed upon the importance of personal experi- 



TESTICULAR INFUSION AGAR 351 

ence in handling gonococcus cultures. I am pleased to thank 
Miss Vera Bennett who has prepared much of the culture media 
for me and Miss Lettie Watkins who has assisted in keeping 
the stock cultures alive and planting the experimental media. 

SUMMARY 

The formula now followed in preparing testicular infusion 
agar for the growth of the gonococcus in preparing suitable 
vaccines. 

1. Mix 500 grams ground beef testicle from which the tunica 
vaginalis has first been stripped, with 1000 cc. distilled water. 

2. Soak overnight at room temperature. 

3. Heat to 50°C. Keep warm for one hour by placing in the 
incubator at 37°C. 

4. Boil, strain, and restore to 1000 cc. with distilled water. 
If in excess do not reduce by boiling since overheating is in- 
jurious. 

5. Add 2 per cent peptone (Witte's or Difco), 3 per cent agar 
chopped fine, 0.5 per cent glucose, 0.3 per cent NaH2P04. 

6. Soak at least one hour to soften the agar. 

7. Melt in the autoclave at 10 pounds pressure for 30 minutes. 

8. Titrate with phenolphthalein and add N/1 NaOH sufficient 
to neutrahze if no unsaturated compounds were present. 

9. Check the titre by repetition. 5 cc. should require from 
1.0 to 2.0 cc. N/20 NaOH. to display color hot. 

10. Tube and sterilize in the autoclave at 10 pounds for 30 
minutes. 

11. Slant or pour into plates. 

This medium may be melted for plating, etc., but the less 
heating the better. Filtration for the purpose of removing the 
distinct turbidity of the medium also seems to be a disadvantage. 



352 IVAN C. HALL 

REFERENCES 

Besredka and .tupille. (1913) Le bouillon a I'oeuf. Ann. de I'lnst. Past., 
27, 1009. 

Broughton-Alcock. (1914) Studies of a strain of gonococcus on ordinary- 
media. Jour. Path, and Bact., 19, 214. 

Elser and Huntoon. (1909) Studies on the meningococcus. Jour, of Med. 
Research, 15, 377. 

Henderson and Webster. (1907) Preservation of neutrality in culture media 
with the aid of phosphates. Jour, of Med. Research, 16, 1. 

Hirschfelder. (1914) A new culture medium for the gonococcus. Jour. 
Am. Med. Assn. 62, 776. 

LuMiERE AND Chevrotier. (1913) Sur un nouveau milieu de culture eminem- 
ment propre au developpement du gonocoque. Compt. rend. Acad, 
d. sc, 157, 1097. 

(1914) Sur la resistance du gonocoque aux basses temperatures. 
Compt. rend. Acad. d. sc, 158, 139. 

McCann. (1896) Fluid in ovarian cysts as a medium for the cultivation of 
gonococcus and other microorganisms. Lancet, 1, 149. 

Martin. (1911) Isolation of the gonococcus. Jour, of Path, and Bact., 15, 76. 

Ohlmacher. (1915) A procedure minimizing the difficulties of transplanta- 
tions of a gonococcus culture. Jour. Am. Med. Assn. 64, 585. 

ScHWARZ and McNeil. 1912. Further experiences with the complement fixa- 
tion test in the diagnosis of gonococcus infections of the genito-urinary 
tract in males and females. Am. Jour. Med. Sci., 144, 815. 

Vannod. (1905) L'agar ordinaire, comme milieu culture du gonocoque. Cen- 
tralblatt f. Bakt. Abt. 1, Orig., 40, 162. 

(1907) Contributions k I'etude du gonocoque. Centralblatt f. Bakt. 
Abt. 1, Orig., 44, 10. 

Van Saun. (1913) Effects of variations in media on gonococci antigens. Col- 
lected studies. Dept. of Health, N. Y., 7, 101. 

Vedder. (1915) Starch agar — A new culture medium for the gonococcus. 
Jour. Inf. Dis., 16, 385. 

Warden. (1915) Studies on the gonococcus. Jour. Inf. Dis., 16, 426. 

Weil, Emile and Noire. (1913) Note sur un milieu de culture pour le gonoco- 
que. Compt. rend. Soc. de Biol., 74, 1321. 



BOOK REVIEW 

Der Erreger der Maul — und Klauenseuche. By Dr. Heinrich Stauf- 

FACHER. Wilhelm Engelmann, Leipzig, 1915. 57 pages, 29 text 

figures and 2 plates. M 2.80. 

The cause of hoof and mouth disease of cattle has been variously 
assigned, usually on a priori grounds, to bacteria, protozoa, and to 
ultra-microscopic organisms, but no one except the author of the present 
work has succeeded in satisfying the necessary postulates for the de- 
terinination of a disease-causing parasite. Dr. Stauffacher of Frauen- 
feld, Switzerland, has found definite bodies in the blood and diseased 
tissues of every animal with the disease examined ; he has grown these 
bodies in the condensation water of blood agar culture media and has 
observed many developmental stages; he has inoculated normal cattle 
with the organisms from the artificial cultures and produced the dis- 
ease in such previously unaffected animals, and he has recovered the 
organisms from the diseased tissues of the inoculated animal. 

Students of hoof and mouth disease have been bull-dozed by author- 
ity into the view that the cause must be ultra-microscopic, first, be- 
cause no trace of organisms can be found in diseased tissues treated 
and stained by the ordinary technical methods, and second, because 
the virus passes through the ordinary filters. When no less an authority 
than Loffler implies that it is a waste of time to look for the organisms 
of hoof and mouth disease with a microscope, it is to be expected that 
only those investigators who have authority-proof minds will under- 
take the task. Such men do not forget the history of Treponema 
pallidum. Nor is the fact that the virus of hoof and mouth disease 
will pass through a filter, necessary evidence of ultra-microscopic size; 
trypanosomes, to say nothing of spirochaetes, pass through ordinary 
bacteria filters. 

Stauffacher seems to have such an authority-proof mind; he argued 
that the organisms must be in the infected tissues, and that the fact 
of their not taking the ordinary stains is no guarantee that they may 
not stain with altered methods. So after vainly trying to stain sections 
with haematoxylin, fuchsin-methylen-blue, and other anilin combina- 
tions, finding no characteristic chromatin reaction in the infected cells, 
either in the nuclei or cytoplasm, he substituted acid fuchsin for the 
ordinary fuchsin and found, as Zschokke had found before, that in- 
fected tissues take up more acid fuchsin than do normal tissues. Even 
with this modification however, he was unable to get any trace of 
basichromatin material either in the nuclei or in the cell bodies. Finally, 
after "months of study of thousands of sections" he discovered the im- 
portant secret of treatment, after which the nuclei again took up the 

353 



354 BOOK REVIEW 

characteristic methylen-blue stain, while thousands of minute bodies 
in and around the cells, and not seen before, were revealed. The 
method used was very simple : the sections were first stained for from 
two to six hours in dilute aqueous acid fuchsin (0.2 per cent), rinsed 
in distilled water and then stained for from six to ten hours in Ehrlich's 
fuchsin-methylen-blue mixture, then rinsed thoroughly in distilled 
water after which they were left in absolute alcohol until no more 
color came out, and were cleared in xylol and mounted in balsam. 

The important part of Stauffacher's work centers in the cell inclusions 
and the similar bodies found in the lymph spaces and in the blood of 
infected animals. These are minute polymorphic structures with an 
average length of one micron (1 fj.), and either spherical, ellipsoidal, 
crescentic, chain-form, comma-form, or ring-form in shape. They 
were found in all of the infected animals (26) examined, and were never 
found in similar tissues of normal, healthy animals. The same bodies 
were found in the freshly-drawn blood, both free in the plasma and 
within the red blood corpuscles of infected animals, a fact which practi- 
cally excludes the possibility that they are products of nuclear and 
cellular degeneration brought about by the disease. 

Blood was drawn from the jugular vein of an infected animal under 
sterile conditions, and a few drops were added to sterile tubes of blood 
agar prepared according to Nicolle's formula for growing Leishmania. 
Vesicles on the tongues of infected cows were flooded with sterilized 
distilled water which was then withdrawn and placed in similar agar 
tubes, one cubic centimeter to each tube. On the fourth day after- 
wards, the tubes containing the vesicular lymph had a decidedly milky 
appearance. A drop of this, examined under the microscope, showed 
myriads of actively moving organisms. The tubes with the venous 
blood showed the same picture somewhat later. Two distinct types 
of organisms were observed; one, shorter and thicker, had the char- 
acteristic appearance of a flagellated protozoon, with a lancet-formed 
body which becomes sharply attenuated and drawn out into a long 
flagellum. The average length of these individuals was 45 n of which 
the body comprised from 20 to 25 ju with a diameter of about 3 m- The 
second type was much longer and more thread-like, v^ith maximum 
dimensions of 120 ju by 1 ju. Granules within the bodies of these two 
types were regarded as blepharoplast, nucleus and chromidia; the 
bodies themselves were not metabolic, nor was there any evidence of 
mouth or vacuoles. Reproduction by longitudinal division was common 
to all types. In addition to reproduction by division, another method 
analogous to spore-formation was described; in this the body becomes 
thickly strewn with chromidia, each of which becomes the minute 
nucleus of an excessively small spherical structure, similar to some of 
the intra-cellular stages, and which might well be able to pass a filter. 

Finally Stauffacher inoculated two normal cows with the uncon- 
taminated agar culture material. The first experiment was interrupted 
by the mobilization of the Swiss army in August, 1914, and we are not 
told what became of the animal. The second experiment was successful, 



BOOK REVIEW 355 

the animal developing symptoms of the disease on the fourth day after 
subcutaneous injection of culture material. From this animal the 
contents of a fresh vesicle was p aced in an agar tube, and in two days 
the fluid was swarming with the same kinds of organisms that had 
been introduced from the earher culture tube. 

From the standpoint of protozoology these results are entirely 
consistent with the established facts of the life histories of other cell- 
invading flagellated parasites. It is regrettable that the technique was 
such that no definite conclusions can be drawn in regard to the finer 
structures of the parasite. All of the tissues and smears were fixed 
in 70 per cent alcohol which is far from satisfactory for the interpretation 
of delicate structural details. Observations on the living organisms 
leave no doubt of the flagellum, but we do doubt a statement to the 
effect that this flagellum "is not a flagellum in the usual sense of the 
word, but rather a flagellum-hke appendage to the cell." A better 
fixation and more careful staining would probably reveal the nucleus 
and blepharoplast and the insertion of the flagellum base in or near the 
blepharoplast. The polymorphic structure of the organisms is not an 
uncommon feature of allied forms of protozoa; short and stumpy 
types mingled with long thread-like forms are characteristic of cul- 
tural stages of Crithidia, Leptomonas, Trypanosoma and Leishmania, 
and similar differences in form are met with in the normal hosts. 

The intra-cellular bodies with their varied forms, which, however, 
are reducible to one general type, are strikingly suggestive of Leish- 
mania donovani of kala azar. But here again, the finer structures 
must remain unknown until a better method of fixation is employed. 

On the whole, we are incUned to accept Stauffacher's interpretation 
of the organism which he names Aphthomonas infestans, as belonging 
to the group of simple flagellated protozoa (Monadida) and closely 
related to the genus Leishmania. If this work is confirmed on material 
fixed with better methods, we should naturally expect the next step 
to be the discovery of the transmitting agent in some form of tick or 
biting fly. 

Gary N. Calkins. 

Columbia University, 
March, 1916. 



ABSTRACTS OF AMERICAN BACTERIOLOGICAL 
LITERATURE 

ANIMAL PATHOLOGY 

The Bacillus enteritidis as the Cause of Infectious Diarrhea in Calves. 
K. F. Meyer, J. Traum, C. L. Roadhouse. (Jour. Am. Vet. Med. 
Assoc, 1916, 49, 17-35.) t. a • u i 

In the course of an experiment on feedmg calves at the Agricultural 
Experiment Station of the University of California, infectious diarrhea 
or scours occurred in severe form. The etiological agent was deter- 
mined to be of the paracolon type by all the identity reactions and sero- 
logical tests; and was regarded as identical with B. enteritidis (Gartner). 
Bacteriological findings were confirmed by feeding experiments with 
two calves, of which one succumbed and one recovered.— A. R. W. 

A Filterable Orgaiiism Isolated from the Tissues of Cholera Hogs. D. J. 
Healy and E. J. Gott. (Jour. Infect. Diseases, 1916, 18, 124-128, 
1 1 \ 

In the course of a previous attempt to isolate a filterable virus from 
the mesenteric glands of virus hogs, the technique involved the filtra- 
tion of the glands immediately after grinding. In the present investi- 
gation the gland tissue after grinding was suspended m 1 per cent glu- 
cose neutral beef broth at 4°C. for five days, then put through a tested 
Chamberland-Pasteur "F." The filtrate was divided between two 
flasks, one of which was placed in a Novy jar, the other sealed, and both 
incubated at 4°C. While the anaerobic preparation showed no growth 
after thirteen days, the other flask showed a distinct growth after four 
days. "This growth appeared as a fine sediment in the bottom of the 
flask. " Upon agitation it " ascended through the fluid in the shape of a 
small cloud. ..." This "filterable organism" grew best at 37 C. 
but also at 20°C. and at 4°C. Hanging drop preparations revealed 
clumps of a non-motile organism surrounded by a gelatinous material. 
Satisfactory stained preparations were obtained by the Giemsa method. 
In such preparations the organism appeared as a coccus or a small 
bacillus 0.2 to 0.3/z in diameter. Subcultures were not obtained. In 
tests with immune serum complement fixation was obtained with the 
culture fluid in which the organism had been grown.— P. B. H. 

A Report Upon an Outbreak of Fowl Typhoid. Walter J. Taylor. 

(Jour. Am. Vet. Med. Assoc, 1916, 49, 35-47.) 

The writer encountered in California an outbreak of the disease 
first described by Moore in 1895, as infectious leukemia, and by Daw- 

357 



358 ABSTRACTS 

son in 1898, and studied under the name, fowl typhoid, by Curtice in 
1902. 
His observations led Taylor to the following conclusions: 

1. Fowl typhoid is a specific disease of fowls caused by Bacterium 
sanguinarium occurring sporadically and causing heavy losses among 
affected flocks; which unless properly investigated may easily be mistaken 
for fowl cholera because of its high mortality. 

2. The specific morbid conditions consist of an enlarged liver con- 
taining necrotic areas, an enlarged spleen and a general anemic con- 
dition of the serous and mucous membranes together with a marked 
increase in leucocytes and a corresponding decrease of the red cell 
content of the blood. 

3. The increase in leucocytes seems to be confined to the poly- 
morphonuclear variety. 

4. Fat, well conditioned, adult fowls are more susceptible than young, 
nearly mature growing birds. 

5. Birds may contract the disease by the ingestion of pure cultures 
of Bacterium sanguinarium. 

6. Birds fed upon the offal of other birds dead of this disease show a 
mild non-fatal form of the disease tending to recovery. 

7. There is evidence that recovery from this mild form produces 
more or less of an immunity. Further investigation upon this point is 
needed. 

8. The power of some of the red corpuscles of the affected fowls to 
take the violet stain, when the blood is diluted in Toisson's fluid is 
especially noticeable in this disease. 

9. While the lesions produced in fowls which are infected with 
Bacterium sanguinarium resemble in many respects those produced by 
Bacterium pullorum, and although there is a still closer resemblance in 
the biological characters of the two organisms, there is enough difference 
to warrant the conclusion that they are distinctly different diseases. 

A. R. W. 

The Value of Virulent Salt Solution in the Production of Anti-Hog-Cholera 
Serum by the Intravenous Method. Robert Graham and L. R. 
HiMMELBERGER. (Jour. Infect. Diseases, 1916, 18, 118-123.) 
Craig and Robbins have each shown that salt solution rendered 
virulent by remaining for some time in the peritoneal cavity of virus 
pigs can be used advantageously by subcutaneous inoculation to pro- 
duce potent antisera in immune hogs. The present authors attempted 
to apply this method modified by the use of intravenous inocula- 
tion. A virulent salt solution was obtained by injecting into the intra- 
peritoneal cavity 25 cc. of a 0.9 per cent solution per pound weight of 
hog. The salt solution, recovered at time of killing five hours later, 
represented 40 to 70 per cent of the original volume. The volume of 
blood obtained at the same time was found to have increased on the 
average by 10 to 20 per cent. Salt virus and blood virus, mixed in the 
proportions of 1:1 or 3:1, and inoculated either subcutaneously or 



ABSTRACTS 359 

intravenously at the rate of 7 cc. per pound of body weight into immunes 
gave satisfactory results in the production of potent antisera when 
these sera were tested on susceptible shoats infected with 2 cc. of virus. 
The conclusions are reached that an immune serum may be produced 
and used more economically when mixed salt virus and blood virus 
are employed and injected by the intravenous method, since the total 
volume of available virus solution is raised by this procedure from 75- 
80 per cent.— P. B. H. 

Experiments to Determine the Relative Value of Trikresol and Carbolic 

Acid in the Preservation of Hog Cholera Serum. John Reichel. 

(Mulford's Vet. BuL, 1916, 7, 61-64.) 

Carbolic acid is generally used as the preservative of hog cholera 
serum, even though comparatively little is known of its value as a 
germicide in such a product as hog cholera serum in the form of de- 
fibrinated blood as originally prepared by Dorset, McBryde and Niles. 
That carbolic acid itself has little or no effect on the potency of the 
product is conclusively established. 

Whether trikresol is equally harmless and as effective as a preservative 
remains to be shown. 

Hog cholera serum as generally prepared is not sterile. The blood 
as drawn from the serum-producing animal is invariably contaminated 
and subsequent handling in defibrination allows for additional con- 
tamination, up to the time the preservative is added, varying in degree 
with the care exercised in its preparation. Even though it is possible 
to obtain sterile blood from a serum-producing hog, this can only be said 
of experimental trials and in producing hog cholera serum defibrinated 
blood, in a practical way, the question of sterility must be entirely 
sacrificed. 

Carbohc acid will not sterihze this contaminated product and the 
question naturally arises "does it hold the organisms in check whether 
present in large or small numbers?" 

The writer observes that: 

1. Carbolic acid must be used in less than 0.75 per cent to avoid 
changes in the physical appearance of hog cholera serum defibrinated 
blood. 

2. Trikresol must be used in less than 0.6 per cent for the same reason. 

3. Carbohc acid added in amounts up to 0.75 per cent to lightly or 
heavily contaminated defibrinated blood first caused a decrease in the 
number of bacteria followed by an increase exceeding the first bacterial 
count. 

4. Trikresol with a carbolic acid coefficient of 2.87 added in 
amounts up to 0.6 per cent was also followed by a decrease, then an 
increase practically equal to that which occurred in the carbolized 
samples. 

5. All of the controls, without any preservation, showed an increase 
in the bacterial count from the time the samples were first set aside 
along with those to which a preservative had been added. This 



360 ABSTRACTS 

increase was followed by a noticeable decrease, and the last count 
was on an average lower in the control samples than those treated. 
That putrefactive changes took place was appreciated by the odor and 
liquefied appearance of the product. 

6. No odor or putrefactive changes were observed in the carbolized 
and trikesoHzed samples. Both preservatives served well in this 
respect, but this alone must not be accepted as proof of the value of 
either preservative for defibrinated blood. 

7. From the limited number of examinations made in these experi- 
ments as to the types of bacteria which survive and then flourish in 
the carbolized and trikresolized samples, it can be said that the types 
were not limited to the spore-forming bacteria alone, as organisms of the 
colon type, staphylococci and streptococci were found as long as the 
samples were kept. 

8. Contaminated hog cholera serum defibrinated blood cannot be 
sterilized by the addition of carbolic acid and trikresol in practical 
amounts, and the numbers of bacteria are not kept in check by the 
preservative. 

9. No evidence is brought forth here to show that carbolic acid or 
trikresol would not serve well as preservatives when added to a sterile 
product. 

10. Hog cholera serum must be prepared in a sterile manner or 
sterilized by one means or another to enable carbolic acid or trikresol 
to serve as a satisfactory preservative. 

11. The physical nature of hog cholera serum defibrinated blood, 
probably has much to do with the limitations of carbolic acid and 
trikresol as preservatives, and it is highly probable that both would 
prove more effective if the insoluble, inert material, fibrin cellular debris, 
etc., were eliminated from hog cholera serum. These inert materials 
undoubtedly exert a large influence in the complications following the 
use of the product, and for this reason alone should not be allowed to 
remain in hog cholera serum on the market. — A. R. W. 

BACTERIOLOGY OF AIR AND DUST 

Recovery of Streptococcus viridans from New York Street Dust. W. C. 
Thro. (New York Med. Jour., 1916, 103, 444-445.) 
Cultures made from New York street dust, collected at the level 
of the second floor revealed the presence of Bacillus fluorescens once; 
a member of the colon group, probably paracolon, once; chromogenic 
Gram positive cocci several times; and Streptococcus viridans seven 
times. The strains of Streptococcus viridans were tested for their 
fermentative properties and their pathogenicity for mice and rats. 

M. W. C. 



ABSTRACTS 361 

BACTERIOLOGY OF FOODS 

Food Poisoning by the Bacillus Paratyphosus B. Harry S. Bern- 
stein and Ezra S. Fish. (Journ. A. M. A., 1916, 66, 167.) 
Report is made of an epidemic of food poisoning at Westerly, R. I. 

Sixty persons were made seriously ill, four of whom died. Symptoms 

of gastric disorder occurred within 4| to 19 hours after eating pie 

obtained from a local restaurant. 

In an analysis of the ingredients of the pies an organism was isolated 

possessing the morphologic, serologic and cultural characteristics of 

Bacillus paratyphosus B. — G. H. S. 

Indol in Cheese. V. E. Nelson. (J. Biol. Chem., 1916, 24, 533.) 
N. determined presence of putrefactive products, indol, skatol and 
phenol in different cheeses. Indol present in limburger and camem- 
bert, phenol only in former. Skatol absent in both. Cheddar, swiss 
and roquefort are free from these substances. Lactic and bulgarian 
bacilli and a liquefying coccus were grown in a medium containing 
tryptophan, 4 per cent lactose and salts. First two failed to pro- 
duce indol; last produced small amounts. N. believes that medium 
was probably unfavorable for growth of former. He apparently 
disregards the sparing effect of lactose. — I. J. K. 

BACTERIOLOGY OF SOILS 

Relation of Green Manures to the Failure of Certain Seedlings. E. B. 

Fred. (J. Agr, Res., 1916, 5, 1161-1176.) 

It has been observed that germination of seeds is poor on soil to 
which green manures have been recently added. The writer reports 
the result of an investigation to show whether this may not be due to 
micro-organisms (bacteria or fungi) that develop in large numbers in 
decomposing green manures. The indications point to fungi as the 
harmful agents. Some fungi have been isolated from decomposing 
green clover that are very destructive to seedlings. Oily seeds are 
easily damaged by the fungi, but starchy seeds are very resistant. 
Damage to seeds by green manures is generally confined to the first 
two weeks after their addition to the soil. Small applications of 
calcium carbonate seem to increase the injury. In all cases where 
the germination is slow, a high percentage of the seedlings prove to 
be diseased. — H. J. C. 

Relation of Carbon Bisulphid to Soil Organisms and Plant Growth. 

E. B. Fred. (J. Agr. Res., 1916, 6, 1-19.) 

It has been shown in the past that if soil is treated with carbon bi- 
sulphid there is an initial decrease in the number of micro-organisms 
which is followed by a large increase in their numbers and in the amount 
of nitrate and ammonia produced, as well as by increased plant growth. 
The writer confirms these conclusions. 



362 ABSTRACTS 

One theory commonly held to explain these results is that carbon 
bisulphid kills certain of the micro-organisms, thus allowing other 
kinds to increase to abnormal numbers and to supply the plants with 
an unusual amount of available nutrient matter. Another theory, 
held by A. Koch, is that the carbon bisulphid in the small quantities 
used is a direct stimulant to bacteria and to higher plants. 

The writer's data tend to support Koch's theory. Carbon bisul- 
phid does not act alike in all soils or toward all crops. There is 
an increased growth of plants in sand culture (pure silica sand with 
nutrient solution added) as well as in soil. Of all the crops investigated, 
mustard (which contains sulphur) receives the most striking benefit 
from the treatment (except in acid soils). Next to mustard comes 
rape, then red clover, then buckwheat, then oats, while corn is scarcely 
benefited if at all. — H. J. C. 

BACTERIOLOGY OF THE MOUTH 

Deep Seated Alveolar Infections. M. L. Rhein. (Surg., Gynecol., 

and Obstet., 1916, 22, 33-37.) 

One case of arthritis is mentioned. Streptococcus viridans was 
isolated from the pulp of an apparently sound tooth. Patient made 
good recovery. — C. P. B. 

The Dental Aspect of the Relation of Endamoeba to Pyorrhea Alveolaris. 

W. A. Price, D.D.S., M.S. (Surg., Gynecol., and Obstet., 1916, 

22, 37^3.) 

The author's subject was really Cinematographic Film studies 
showing the movements of mouth organisms, including endamoeba. 
This method should lend itself to studies of most bacterial forms. 

C. P. B. 

On the Cultivation of Entameha huccalis. Wm. B. Wherry and Wade 

W. Oliver. (Lancet Clinic, 1916, 115, 295.) 

Wherry and Oliver found that the Entameha huccalis grew best on 
"Martin's pleuritic" medium, made up with basic sodium phosphate, 
and pleuritic fluid in the proportion of about two of the fluid to three 
of the agar. The tubes were slanted and allowed to remain in the ice 
box, so that the water of syneresis could collect. The Entameha and 
bacteria from the margin of the tooth were introduced into the water 
of syneresis and incubated at 35° to 37°C. There was a profuse growth 
at the end of 48 hours; staining with Mallory's ferric chloride-hema- 
toxylin, showed characteristic nuclear structure. The authors hope 
to be able to throw some light on the life history of this organism and 
to make tests of its pathogenicity. — O. B. 

The Relation of Amoehiasis to Pyorrhea Alveolaris. A. H. Sanford, 
M.D. and Gordon B. New, M.D. (Surg., Gynecol, and Obstet., 
1916, 22, 27-33.) 
The authors studied 327 patients, dividing them into 5 groups. 

Material from pyorrheal pockets, also from sto Is was examined. Of 



ABSTRACTS 363 

73 patients with Entameba histolytica present in the stools only 31 
also had Entameba buccalis in the mouth while in 254 cases in which 
parasites were absent from the stools, 150 had Entameba buccalis present. 

Dogs were inoculated about the teeth with pus containing amoebae 
from pyorrheal pockets. In subsequent examination no signs of pyor- 
rhea were present and amoebae could not be found. 

Kittens given intracaecal inoculations of Amoeba histolytica developed 
typical dysentery while those inoculated with Amoeba buccalis showed 
no signs of dysentery. — C. P. B. 

The Dental Path: Its Importance as an Avenue to Infection. T. B. 

Hartzell, M. D. and A. T, Henrici, M.D. (Surg., Gynecol., and 

Obstet., 1916, 22, 18-27.) 

In a series of acute dental abscesses staphylococci were the active 
organisms, in 250 cases of chronic abscess Streptococcus viridans was 
the predominating organism. 

Heart lesions were produced in rabbits, injected with cultures of 
Streptococcus viridans and other streptococci obtained from pyorrheal 
pockets and saliva, while some strains seemed to have a predilection 
for the kidneys. 

In one case death was due to the fusiform bacillus, the organism 
being isolated from the blood. Another died of pneumonia, the pri- 
mary infection with the pneumococcus occurring in a bicuspid tooth. 

Comment. Many authors now classify cocci producing greenish 
color on blood agar, not pneumococci, as Streptococcus viridans. It 
would seem desirable that the authors should determine whether the 
organism is the classic Streptococcus viridans, that is a pin head grayish 
rough irregular colony, which gradually becomes brownish producing a 
greenish color, in the blood agar surrounding the colony and grows 
in very long chains in bouillon, the elements of which are diplococci; 
or simply belongs to the group which produces the green color when 
grown on blood agar. — C. P. B. 

BACTERIOLOGY OF WATER AND SEWAGE 

The Electrical Treatment of Water. T. A. Starkey, (American Medi- 
cine, 1916, 11, 181.) 

S. passed currents of electricity through water containing B. coli 
and B. prodigiosus. He found that the current as such had very little 
effect on the germs. He used both direct and alternating currents 
and varied the amperage from 0.1 to 2.0 and the voltage from 90 to 
40,000. 

He found that the gases produced by the electrolysis did have some 
germicidal power. In one case the count was reduced from 34,000 
to 2,000. In no case was the water made entirely sterile. S. does not 
state whether there was any salt in the water from which hypochlorite 
might have been made. 

In another series he tried to test the sterilizing value of any metallic 



364 ABSTRACTS 

salts that separated from the plates (electrodes) and obtained complete 
sterility in a few cases. He does not, however, state what material 
composed the plates or what salts passed out into the water. The 
report is so incomplete as to be of little value. — E. C. L. M. 

CLASSIFICATION OF BACTERIA 

Grouping of Meningococcus Strains by Means of Complement Fixation. 

Miriam P. Olmstead. (Proc. N. Y. Pathol. Soc, 1915, 15, 136- 

143.) 

Forty strains of meningococci were used but only twenty-nine were 
completely tested by complement fixation. Of these, fourteen fell 
into one group, eight into another, two cross-fixed with each other only, 
two failed to fix with any other strain and three acted irregularly. 
All strains could be clearly distinguished from the gonococcus. Cul- 
turally they were all alike. The para-meningococci are regarded as 
constituting a special strain among meningococci, not, however, a 
wholly homogeneous one. — W. J. M. 

DISINFECTION 

The Standardization of Disinfectants. J. T. Ainslie Walker. (New 

York Med. Jour., 1916, 103, 500-505.) 

A critical comparison of the Hygienic Laboratory and Rideal- Walker 
tests, leading to the conclusion that the Rideal-Walker test is superior 
to the Hygienic Laboratory method in every one of the points discussed. 

M. W. C. 

Soap. G. K. Dickinson. (Medical Record, 1916, 89, 556-558.) 
Work done up to the present time upon the antiseptic and bactericidal 
action of soap has not been sufficiently uniform in conditions or methods 
to draw from it hard and fast conclusions. 

In general, it may be said that all soaps possess some disinfectant 
power by virtue of the alkaline reaction alone. All bacteria exposed 
to soap solutions are not, however, killed in the same time, and a 
considerable interval is necessary before any practical disinfection can 
occur. Most so-called disinfectant soaps have no value beyond that 
of ordinary soaps. A combination of soap and biniodide of mercury 
is a useful disinfectant, but it does not produce complete sterility 

M. W. C. 

IMMUNOLOGY 

Therapy as Related to the Immunology of Tuberculosis. E. R. Baldwin. 

(New York Med. Jour., 1916, 103, 532-534.) 

A discussion of the relation of immunology to the methods of treat- 
ment now used in tuberculosis. — M. W. C. 



ABSTRACTS 365 

Vaccines of Favus and Ringworm. C. H. Lavinder. (Journ. A. M. A. 

1916, 66, 945-946.) 

A method is given for the preparation of vaccines from the fungi 
which cause the above infections. — G. H. S. 

Treatment of Cases of Epidemic Meningitis. J. B. Neal. (Journ. 

A. M. A., 1916, 66, 862-864.) 

The author places emphasis upon the value of continued injections 
of antimeningitis serum even though the patient shows improvement. 

Autogenous vaccines have been employed in cases which tended to 
become chronic. — G. H. S. 

The Effect of Moderately High Atmospheric Temperatures upon the 
Formation of Hemolysins. C.-E. A. Winslow, James Alexander 
Miller, and W. C. Noble. (Proc. Soc. Exp. Biol, and Med., 
1916, 13, 93-98.) 

Rabbits kept at a temperature of 29° to 32°C. were compared with 
control rabbits kept at 18° to 21 °C. by injecting them with washed 
sheep erythrocytes and subsequently testing the specific hemolytic 
activity of the serum. Hemolysin formation was relatively delayed 
in the animals kept at the higher temperature, but at the end of four 
weeks the titre was as high in these as in the controls. Considerable 
individual variation within the groups was observed. — W. J. M. 

An Allergic Skin Reaction to Diphtheria Bacilli. J. A. Kolmer. Proc. 

Soc. Exp. Biol, and Med., 1916, 13, 89-91. 

Diphtherin was prepared by suspending washed diphtheria bacilli 
in salt solution and sterilizing the suspension at 60°C. for an hour. 
Each cubic centimeter contained approximately two billion bacilli. 
An intracutaneous injection of 0.1 cc. of diphtherin was used for the 
test, and the usual Schick test was also made at the same time. The 
two tests agreed in 63 per cent of the patients tested. The diphtherin 
test is regarded as an index of bacteriolytic immunity whereas the 
Schick test is an index of antitoxic immunity. — W. J. M. 

The Influence of Typhoid Bacilli on the Antibodies of Normal and Immune 
Rabbits. C. G. Bull, Journ. Exp. Med., 1916, 23, 419^29. 
The subcutaneous, intraperitoneal, or intravenous inoculation of 
cultures of typhoid bacilli did not cause, as far as could be determined, 
a decrease in the antibody content of the blood serum of the rabbit. 
On the other hand, the intravenous inoculation of typhoid bacilH 
causes a rapid mobilization of antibodies, thus increasing their con- 
centration in the blood, to be followed somewhat later, by the pro- 
duction of so-called acquired antibodies. No such condition as the 
negative phase of Wright was discovered, although it was especially 
looked for in the experiments. — G. B. W. 



366 ABSTRACTS 

A Method for the Rapid Preparation of Anti-Meningitis Serum. H. L. 

Amoss and Martha Wollstein. (Jour. Exp. Med., 1916, 23, 

403^17.) 

The method described by the authors consists of three successive 
intravenous inoculations of many strains of living meningococci and 
parameningococci repeated at stated intervals. Anaphylactic dangers 
are obviated by preliminary desensitizing injections and the doses 
are adjusted according to the febrile reaction. The great advantage 
of the method is that a polyvalent serum of high titer can be produced 
in 8 to 12 weeks instead of in the 10 months required by the subcutane- 
ous method. This investigation offers a promising suggestion for the 
production of other immune sera. — G. B. W. 

Variations in the Strength of Positive Wassermann Reactions in Cases 

of Untreated Syphilis. D. A. Haller. (Journ. A. M. A., 1916, 

66, 882-884.) 

From an examination of over 6000 Wassermann reactions in which 
but one antigen was used it appears that amboceptor is the only con- 
stituent of the hemolytic system which is a constant. All other fac- 
tors may vary and will account for the difference in daily determinations 
of the fixing unit of a positive serum. 

The titer of sera from cases of untreated syphilis remains the same 
from day to day or from month to month. 

The administration of mercury may quickly change a positive to 
a negative reaction, and upon stopping the treatment the reaction va&y 
as quickly become positive again. — G. H. S. 

Continuous Transfusion; The Production of Immunity. An Experimen- 
tal Study. A. Kahn. (Medical Record, 1916, 89, 553-556.) 
Dogs were infected by opening the peritoneal cavity and inserting 
a small quantity of dust, gauze saturated with pus, or pure pus. After 
an interval, varying from 1 to 5 days, the donor and the infected dog 
were prepared for transfusion and a continuous flow of blood from one 
animal to the other was allowed to occur from ^ to 3 hours. 

In dogs that were not transfused following infection, death occurred 
in 24 to 48 hours; in transfused dogs death was deferred from 3 to 4 
days, or absolute recovery took place. 

Transfusion raises the vital resistance. Whether or not immunity 
is produced is not known. — M. W. C. 

Possible Reasons for Lack of Protection after Antitijphoid Vaccination. 
Henry J. Nichols. (The Military Surgeon, 1916, 38, 263-268.) 
Summaiy of article as given by author is: 

1. False failures in immunization may be due in some cases to the 
difficulties of exact clinical diagnosis. 

2. The uncertain duration of immunity following vaccination may 
account for some true failures. At present in the Army one revaccina- 



ABSTRACTS 



367 



tion of three doses is compulsory four years after the first course of 
three doses. 

3. The kind of vaccine used may account for some failures. 

a. Whole killed vaccine. 

(1.) The strain of bacillus used may not be suitable. 
(2.) The method of preparation may be faulty. 
(3.) The vaccine may be too old. 

b. Sensitized vaccine. 

(1.) Sensitization may diminish the immunizing properties of 
the vaccine. 

(2.) Discarding the supernatant fluid may lessen the immunizing 
power of the vaccine. 

4. The conditions of a soldier's life do not protect him from exposure 
to typhoid fever in his vicinity. 

5. The Army vaccine is probably superior to some of the vaccines 
available for the general population. 

The writer comes to the conclusion that soldiers are better protected 
than those who become infected after vaccination in civil life, and that 
this protection must be due either to better vaccination in point of 
numbers of doses and intervals of revaccination, or to a better vaccine. 
Of the two factors the vaccine is probably the most important. 

E. B. V. 

Methods of Using Diphtheria Toxin in the Schick Test and of Controlling 
the Reaction. Abraham Zingher. (American Journal of Dis- 
eases of Children, 1916, 4, 269-277.) 

The Schick reaction consists in the intracutaneous injection of one- 
fiftieth M.L.D. of well ripened diphtheria toxin and indicates the 
absence or presence of a protecting amount of antitoxin in the blood 
according to whether there is or is not produced a local inflammatory 
reaction. In some individuals there occurs a so-called pseudo-reaction 
which probably bears no relation to the free toxin but is the result of 
an anaphylactic reaction with the proteins of the diphtheria bacilli. 
The true and the false reactions can usually be distinguished by their 
appearance and time of occurrence but as a further control a super- 
heated (75°C.) toxin or one which has been over neutralized by the 
addition of two units of antitoxin to each L plus dose of toxin may be 
injected into the opposite arm. 

The author also emphasizes the importance of careful technique in 
giving the injection so that it is definitely intracutaneous. The value 
of the test now seems well established and especially prepared vial& 
containing undiluted toxin and with directions concerning its dilution 
are prepared by the New York City Board of Health and also by com- 
mercial laboratories. 

Of a thousand children admitted to the Willard Parker Hospital, 
who gave a negative Schick reaction and who were more or less exposed 
to diphtheria not a single one developed the disease. 

Tests on 2700 normal children in orphan asylums between the ages 



368 ABSTRACTS 

of 2 and 16 years show that from 17 to 32 per cent give a positive 
reaction and are therefore probably susceptible to the disease. 

R. M. T. 

Preliminary Notes on Skin Reactions Excited hy Various Bacterial 

Proteins in Certain Vasomotor Disturbances of the Upper Air Passages. 

J. L. GooDALE. (Boston Med. and Surg. Jour., 174, 223-226.) 

G. finds that many patients who suffer from perennial vasomotor 

disturbances of the nasal mucous membrane give a positive anaphylactic 

skin reaction to extracts of certain bacteria that are commonly found 

in vasomotor rhinitis. Among these are Staphylococcus albus, aureus 

and citreus, Micrococcus tetragenus and an unidentified bacillus somewhat 

like Friedlaender's bacillus. — E. C. L. M. 

Pollen Therapy in Pollinosis. S. Oppenheimer and M. J. Gottlieb. 

(Medical Record, 1916, 89, 505-508.) 

Hay fever, or pollinosis, is caused in persons having a predisposition 
to anaphylactic diseases, by irritation of any denuded surface of the 
body by proteins of pollen. One or more of a large variety of pollens 
may be responsible for an attack of pollinosis in a susceptible individual. 

The method most frequently employed for determining which pollen 
or pollens are operative in a given case is the skin scarification or cutane- 
ous method. Complement fixation tests may also be made both as 
an aid in diagnosis and as an indicator of immunity. 

Infections, caused by streptococci, pneumococci, etc., are often 
complicating factors in pollinosis. In such cases an autogenous vac- 
cine should be administered in conjunction with the specific pollen 
antigen. 

Treatment of pollinosis proper may consist either of active immuniza- 
tion with pollen extract or passive immunization with the blood serum 
of animals that have been actively immunized with pollen extract. 

The results obtained with treatment with pollen extract, in cases 
of spring pollinosis show 50 per cent of seasonal cures for 1913-1914, 
while of 32 cases treated in 1915 before the time of attack, only two 
had symptoms. 

Of 62 cases treated for fall pollinosis 52 began treatment early enough 
to acquire an active immunity before the usual time of attack. Of these, 
15 were free from symptoms, 2.5 were markedly improved, and 12 were 
in no way afi"ected by the treatment. Of the 10 cases that did not 
begin treatment until after the onset of the attack, 4 were favorably 
mfluenced by one or two injections. 

The authors caution against using "hay fever" vaccines which con- 
tain a mixture of a large number of pollen extracts, as patients should 
receive extracts of only those pollens which have been shown by diag- 
nostic means to be operative. Care should be taken in the dosage of 
pollen extracts, as in large doses they are extremely dangerous. 

M. W. C. 



ABSTRACTS 



369 



Complement Fixation in Intestinal Parasitism of Dogs. John A. Kol- 
MER, Mary E. Trist and George D. Heist. (Jour. Infect. Dis- 
eases, 1916, 18, 88-105.) 

The aim of the investigation was to ascertain by complement faxa- 
tion tests whether the absorption of foreign substances and conse- 
quent production of specific antibodies occurred in dogs infected with 
intestinal parasites. For antigens were used salt solution and alcoholic 
extracts of various species of Tenia, Dipylidium, Ascaris and Strongylus. 
The antisheep hemolytic system was employed. Each one of 172 
dog sera was tested with all of the antigens. Serum tests and feces 
examinations together were made in 110 cases. The results of feces 
examinations showed infections as follows: Ascaris (23 per cent), 
Ascaris and Trichocephalis (20 per cent). Tenia (6 per cent), Dipylidium 
(3.6 per cent), no infection (26 per cent). These results did not con- 
form with the serum examinations since (1) dogs showing the ova of 
certain parasites failed to react with the corresponding antigen, and 
(2) positive reactions were frequently obtained with antigens of types 
whose eggs were not found in the feces. The analysis of the data, 
however, leads the authors to conclude that the production of anti- 
bodies may occur after infestation with the common intestinal para- 
sites. Such antibodies were in special evidence in tapeworm infesta- 
tions, less so in round worm and only slightly in whip worm infestations. 
The reactions as a whole are stated to have suggested a biologic rela- 
tion between the tapeworms Tenia serrata and Dipylidium caninuyn, 
and between Ascaris canis and Strongylus gigas. The authors state 
that complement fixation tests may be of value in the diagnosis of 
intestinal parasitism of man. — P. B. H. 

Studies in Non-Specific Complement Fixation: I. Non-Specific Com- 
plement Fixation by Normal Rabbit Serum. John A. Kolmer and 
Mary E. Trist. (Jour. Infect. Diseases, 1916, 18, 20-26.) 
The authors direct attention to the fact that fresh active sera from 
normal rabbits, in doses of 0.1 cc. show non-specific fixation with 
lipoidal extracts in 5 to 15 per cent of sera tested. When the same 
sera were inactivated by heating fixation occurred in 38 to 49 per cent 
of sera. In the case of both active and inactivated sera the percentage 
of positive reactions increased in the following order when the sub- 
stances named were used as antigens: (1) alcoholic extract of heart 
muscle reinforced with cholesterin, (2) alcoholic extract of syphilitic 
liver, (3) extract of acetone insoluble lipoids. With bacterial anti- 
gens '(staphylococci, colon, typhoid) fixation occurred in some degree in 
31 to 42 per cent of cases, with active sera, and in 51 to 62 per cent when 
inactivated sera were used. The rabbits tested were conservative m 
their reactions, 80 per cent being persistently positive or persistently 
negative in successive examinations. The authors conclude by recom- 
mending that "when rabbits are to be employed for experimental studies 
with a view to using their sera for complement-fixation tests, their 
sera should be tested one or more times before inoculation preferably 



370 ABSTRACTS 

with the particular antigen to be used, and only those selected that 
react negatively." — P. B. H. 

Studies in Non-Specific Complement Fixation: II. Non-Specific Comple- 
ment Fixation by Normal Dog Serum. John A. Kolmer, Mary E. 
Trist and George D. Heist. (Jour. Infect. Diseases, 1916, 
18,27-31.) 

The study was undertaken to ascertain whether normal dog serum 
would fix or absorb complement with lipoidal and bacterial antigens 
as had been found to be the case with normal rabbit serum. The 
technique was that of the Wassermann reaction. It was found that 
the dog sera tested, whether active or inactivated, are capable of ab- 
sorbing complement in a large percentage of cases, the greater num- 
ber of positive reactions appearing in the case of bacterial antigens. 
When lipoidal antigens were used the order of positive reactions varied 
exactly as in the case of rabbit serum (vide supra). The best reactions 
with active dog serum were obtained when 0.05 cc. was used. Heat- 
ing the sera at 55°C. for 30 minutes greatly increased the power 
for fixation for both groups of antigens, while by heating at a higher 
temperature the power was lessened. The authors conclude that in 
complement fixation tests with dog serum, ''it would appear advisable 
to use the serum in a perfectly fresh and active condition in doses of 
0.01 to 0.2 cc, after heating the serum at 62°C. instead of 55°C. for 
half an hour, since this removes, or greatly diminishes the tendency 
toward non-specific fixation of the complement." — P. B. H, 

Studies in Non-Specific Complement Fixation: III. The Influence 
of Splenectomy and Anesthetics on the Non-Specific Complement 
Fixation Sometimes Shown by Normal Rabbit and Dog Sera. John 
A. Kolmer and Richard M. Pearce. (Jour. Infect. Diseases, 
1916, 18, 32^5.) 

The aim of the investigation was to gain some understanding of 
the part played by the spleen in hemolysis, and in the increased resist- 
ance of erythrocytes after splenectomy. Pre-operative and post- 
operative sera were tested in both active and inactivated condition 
in doses of 0.1 cc. against three lipoidal extracts (vide supra) and two 
bacterial antigens (Staphylococci and B. coli). Ether, chloroform and 
nitrous oxid were employed as anesthetics. The results of the experi- 
ment showed that anesthetics, as employed, weaken or remove tem- 
porarily the power of normal rabbit and dog sera of fixing or absorbing 
the complement with lipoidal and bacterial antigens in a non-specific 
manner. "This alteration usually is not apparent at once after the 
administration of the anesthetic but is found after one to three days; 
later the serum returns to its former power of causing this non-specific 
fixation." Ether-administration was not found to reverse the reaction 
of negatively-reacting sera. Nitrous oxid oxygen had no appreciable 
influence on the serum reactions of normal rabbits. "Splenectomy 
alone probably has no influence upon the property in normal rabbit and 



ABSTRACTS 371 

dog sera of fixing or absorbing complement with various non-specific 
lipoidal and bacterial antigens, the effect being in larger doses attribut- 
able to the anesthetic ; the changes observed in dogs following splenec- 
tomy under ether were somewhat more profound than those in rabbits." 

P. B. H. 

Studies in N on-Specific Complement Fixation: IV. The Relation of 
Serum Lipoids and Proteins to Non-Specific Complement Fixation 
with Normal Rabbit and Dog Sera. John A. Kolmer. (Jour. 
Infect. Diseases, 1916, 18, 46-63.) 

The aim of the present study was to determine the relation of serum 
lipoids to the process of non-specific complement fixation (1) by ex- 
tractions of serum with lipoid solvents (ether, chloroform, etc.), and 

(2) by feeding and immunization experiments with various lipoids. 
The method employed antilytic and Wassermann tests with rabbit 
and dog sera, both active and inactivated (56°C. for one-half hour), 
before and after extraction. It was found that both serum lipoids 
and proteins were concerned in the antilytic and non-specific comple- 
ment fixation; also, that extraction with ether or chloroform usually 
diminished the antilytic and complement-fixing powers of a serum, 
while enteral and parenteral administration of lipoids increased the anti- 
lytic and complement-fixing powers. Sera extracted with ether were 
rendered more antilytic, but heating an extracted serum reduced the 
antilytic titer compared with plain heated serum. It was further 
concluded "that both the globulin and albumin (filtrate) fractions 
of normal rabbit and dog sera possess thermostabile antilytic and 
complement-fixing properties .... The antilytic and com- 
plement-fixing substances of normal rabbit and dog serum are not 
dialyzable."— P. B. H. 

Studies in Non-Specific Complement Fixation: V. The Effect of Heat 
on Normal Rabbit and Dog Sera in Relation to Antilytic and Non- 
Specific Complement Fixatio7i Reactions. John A. Kolmer and Mary 
E. Trist. (Jour. Infect. Diseases, 1916, 18, 64-87.) 
The authors had already shown (1) the ability of normal rabbit and 
dog sera to yield non-specific complement fixation with various bac- 
terial and lipoidal antigens; (2) the influence of anesthetics upon this 
property and (3) the relation of serum lipoids to the process. The 
aim of the present investigation was to study the influence of cer- 
tam factors and methods for lessening its effects in complement fixa- 
tion tests. The tests were conducted with lipoidal extracts and with 
three bacterial antigens previously mentioned (vide supra), the doses 
being the same as used in Wassermann tests. Guinea pig comple- 
ment was used. The hemolysins were antisheep (rabbit), antihuman 
(rabbit) and antiox (rabbit). Tests for the antihemolytic properties 
of serum were performed by (1) incubating heated serum and com- 
plement for one hour; (2) adding the cells and two units of hemolysin; 

(3) re-incubating for one hour. The complement fixation tests were 



372 ABSTRACTS 

conducted by (1) incubating antigen, serum and complement for one 
hour; (2) adding cells and two units of hemolysin; (3) re-incubating 
for one hour. The authors conclude from their study as follows: 
"(1) Non-specific complement fixation by normal rabbit and dog sera 
is probably due primarily to thermolabil and thermostabil antilytic 
(anticomplementary) substances in the sera. (2) While fresh and 
active rabbit and dog sera may yield non-specific complement fixation 
the tendency is greatly increased as a result of heating the sera. At 
56°C. the changes may occur in 20 minutes or even less; at 62''C. for 
30 minutes the tendency for non-specific reaction is much decreased 
and is entirely removed by heating serum at 70°C. for 30 minutes. 
Changes may occur after exposure at 45°C. for 30 minutes, but the 
optimal temperature is between 55° and 60°C. (3) In complement 
fixation tests for specific antibodies with inactivated rabbit, dog and 
mule sera, it is advisable to heat the sera at 62'^C. for one-half hour 
and to use at least two units of complement or hemolysin and no more 
than one-quarter of the anticomplementary unit of antigen after it 
has been carefully titrated. (4) Complementoids and amboceptoids 
probably bear no relation to the process of non-specific complement 
fixation by rabbit and dog sera. (5) The blood corpuscles of various 
animals and various bacteria may absorb a portion of the antilytic 
substances from rabbit and dog sera, but they have much less influence 
on the complement fixation reactions. Digestion of fresh sera with 
corpuscles and bacteria not infrequently increases the anticomplemen- 
tary properties of the sera. (6) Bacteriolytic amboceptors are not 
responsible for non-specific complement fixation by normal rabbit 
and dog sera. (7) Parasitic infestations of rabbits and dogs bear no 
relation to the antilytic and complement fixing properties of the sera. 
(8) Single, large doses of salvarsan are without definite influence on 
the reactions with rabbit serum. (9) Quantitative factors in the 
hemolytic system and antigen are of considerable importance in rela- 
tion to these non-specific reactions. (10) If time permits, preliminary 
complement fixation tests should be performed with the sera of rabbits 
or dogs before immunization or inoculation is begun, and only those 
animals selected the sera of which react negatively with the antigen 
used."— P. B. H. 

INDUSTRIAL BACTERIOLOGY 

The Importance of Bacterium bulgaricus Groups in Ensilage. O. W. 
Hunter and L. D. Bushnell. (Science, 43, 318-320.) 
Various kinds of ensilage were examined at different stages of fer- 
mentation. On acidulated glucose agar only Bacterium bulgaricus 
and yeasts developed. The colonies of B. bulgaricus resembled Bacterium 
lactis and the authors believe that it is on this account that other in- 
vestigators have overlooked them. It is concluded that the Bulgarian 
groups occur in sufficiently large numbers, and at a proper stage in 
ensilage fermentation, to play an important role.— C. M. H. 



ABSTRACTS 373 

LABORATORY TECHNIQUE 

An Eye-Shade for Use with the Microscope. E, Kellert. (Jour. 

A. M. A., 1916, 66, 1023-1024.) 

A device for attachment to the draw-tube of the microscope is de- 
scribed. It is so designed as to prevent diffused hght from entering 
the eye above the ocular. — G. H. S. 

An Apparatus for Filling Vaccine Ampoules. R. G. Davis, U. S. Naval 

Med. Bulletin, 1916, 10, 311-313. 

By the use of this apparatus which is briefly described and figured, 
and which can be made in any laboratory it is claimed that ampoules 
may be filled with vaccine without loss of time or vaccine. — E. B. V. 

The Use of the Sand Tube in Isolating the Bacillus typhosus. M. D. 

Levy. (Journ. A. M. A., 1916, 66, 1022-1023.) 

A pipette 33 cm. long and 5 to 6 mm. in diameter is bent in a U shape. 

Sand is placed in one arm to a height of 10 cm. and the other arm 
is filled with hot bouillon. The bouillon is inoculated and the tube 
incubated for 18 hours. 

Motile bacilli, such as Bacillus typhosus or occasionally Bacillus coli, 
penetrate through the sand and may be isolated from the bouillon 
above the sand. — G. H. S. 

On a Rapid Method of Cultivating the Gonococcus. Wm. B. Wherry 

and Wade W. Oliver. (Lancet Clmic, 1916, 115, 306.) 

The authors found that gonococci from the urethral pus of a boy, 

grew best on Martin's pleuritic agar, under partial oxygen tension. 

Tubes similarly inoculated and grown aerobically, yielded no growth. 

The partial oxygen tension was secured by attaching the culture tubes 

inoculated with the pus containing gonococci, to similar tubes inoculated 

with Bacillus subtilis. When isolated in this way the gonococci can 

not be subcultured aerobically, but partial tension subcultures grow 

promptly. — 0. B. 

A New Method of Separating Fungi from Protozoa and Bacteria. N. 

EIopelop'f, H. C. Lint and D. A. Coleman. (Bot. Gaz. 1916, 61, 

247-250.) 

The dilution method followed by the peculiar manner of plating, 
makes it possible to separate fungi from bacteria and protozoa. 

As the result of this separation it has been possible to eliminate 
fungi from experiments involving the effect of protozoa on bacterial 
activity, by making a sub-culture from the fungi-freed solution of 
bacteria and protozoa. 

In view of the fact that fungi are capable of producing ammonia, 
their presence may introduce a factor not accounted for in measuring 
the effect of soil protozoa on soil bacteria. — J. T. E. 



374 ABSTRACTS 

Study of the Blood with a New Stain. B. Lemchen. (Medical Record, 

1916, 89, 607-608.) 

The stain consists of a saturated solution of benzidine in absolute 
alcohol. Blood smears are made on slides and placed in the stain 
for one-half minute. The slide is then placed in hydrogen peroxide 
for one-half minute, washed in water and dried on filter paper. 

In studying blood stained in this way, it is assumed that cells and 
tissues of similar composition react in the same way, as staining is a 
chemical reaction. Red cells, nucleated red cells including both cell 
and nucleus, and fibrin stain blue; white cells and blood platelets do 
not take the stain. From this it may be concluded that red cells and 
white cells are of different origin, that platelets do not have their origin 
in the nucleus of the red cells, and that fibrin has the same composition 
as the red cells. 

These conclusions may throw some light on the processes of coagula- 
tion of the blood and certain phases of hemophilia leading to pernicious 
anemia. According to this line of reasoning, it may be possible that 
the origin of agglutinins in typhoid is in the red blood cells. — M. W. C. 

A Method of Demonstrating Bacteria in Urine by Means of the Centrifuge. 

With Some Observations on the Relative Value of Examinations by 

Culture or Stained Sediment. E. G. Crabtree. (Surg., Gyn., and 

Obstet., 1916, 22, 221-224.) 

The method consists in slow centrifugation to remove the heavier 
sediment, then rapid centrifugation until the urine is clear in order 
to throw the bacteria out of suspension. C. calls attention to the 
danger of mistaking smegma for tubercle bacilli, guinea pig inoculation 
being the final test for infection with tubercle bacilli. The author 
thinks inconsistent results are obtained because of lack of uniformity 
in culture media, etc., while organisms such as B. coli may overgrow 
the others. Microscopical examination assists in determining the 
degree of infection, and predominating organisms, and if there is a 
mixed infection helps to determine cultural method to be used. 

Comment. The author does not mention the use of Petroff 's method 
for direct culturing of tubercle bacilli or the necessity of using the 
antiformin method where T, B. is suspected and other infection already 
exists.— C. P. B. 

A Rapid Method of Counting Living Bacteria in Milk and Other Richly 

Seeded Materials. W. D. Frost. (Journ. A. M. A., 1916, 66, 889- 

890.) 

A detailed account of the procedure is given. The method is essen- 
tially as follows: 

"One-twentieth cubic centimeter of milk is mixed with standard nutri- 
ent agar and spread over a definite area of a sterile glass slide. When the 
agar is hard, this little plate culture is put in the incubator for about six 
hours under conditions which prevent evaporation. It is then dried, 
given a preliminary treatment to prevent the agar from firmly binding 



ABSTRACTS 



375 



the stain, stained, decolorized and cleared. When this dried and 
stained plate culture is viewed under the microscope, the little colonies 
are definitely stained and appear highly colored on a colorless or slightly 
colored background. These colonies can be readily counted and the 
number of bacteria per cubic centimeter calculated." — G. H. S. 

Counting Bacteria by Means of the Microscope. R. S. Breed and J. D. 

Brew. (N. Y. State Sta. Tech. Bui. 49, pp. 31, pis. 2, figs. 5.) 

This bulletin contains the results of tests of this method which have 
been made since those published in an earlier bulletin of the station 
(N. Y. Dept. Agr. Expt. Sta. Bui. 373, pp. 1-38 (1914) ). A general 
description is given of the technique employed in applying this method 
to milk, the various processes involved being discussed with reference 
to possible errors. The result of this investigation are indicated by 
the following quotations from the authors' summary: 

'The results obtained from the examination of samples of milk 
collected in clean test tubes containing preservatives indicate that just 
as accurate counts of the number of bacteria present can be made from 
such samples as can be made if the samples are collected in sterile 
tubes and iced. . . . 

"Capillary pipettes have been found to be more satisfactory for the 
measurement of 0.1 cc. quantities of milk than standardized wire loops. 

"Faulty calibration of pipettes has been found to be a serious cause 
of error. Allowance must be made for the adhesion of a certain quantity 
of milk to the pipette if accuracy of measurement is to be secured. 

"It has been found that sterihzation of pipettes is an unnecessary 
refinement of technique and that a single pipette may be used for mak- 
ing preparations from a long series of samples, provided it is carefully 
cleaned in glass-cleaning solutions after each day's use and also cleaned 
by rinsing in fresh, clean tap water after using in each sample and 
before passing to the next sample. Carelessless in cleaning pipettes 
causes marked errors in counts. 

"Growth of bacteria has been found to take place in the drops of 
milk as they dry so that it is important that these be prepared either 
from samples containing preservatives or that the milk be dried quickly. 
No growth was detected in the dried films even after incubation in a 
moist, 37°C. incubator for one to four days. 

"The claim made by some that bacteria are removed when the fat 
drops are dissolved by solvents does not seem to have any foundation 
in fact. The dried milk-solids-not-fat appear to act as a practically 
perfect fixative, no detectable mechanical loss of bacteria taking place 
when the fat Vops are removed. On the other hand, serious errors 
in count are introduced where the bacteria are stained in the milk 
before the dried films are prepared, because in this way the bacteria 
are not always sufficiently stained to make it possible to detect the 
full number present. Where the fat drops are left in the fibns, even 
though these be spread out so as to be in a very thin laj^er, they tend 
to obscure bacteria and so lower the count. . . . 



376 ABSTRACTS 

"Microscopical methods of examining dried milk-films are of value 
for two purposes: (a) They may be used for the rapid examination of 
milk in order to grade it according to its bacterial quality, both the num- 
ber and the character of the bacteria present being taken into account. 
A microscopical examination permits a fairly accurate guess as to the 
probable plate count which will be secured from a given sample of 
milk, (b) They are also useful as research methods, the microscopical 
method being the only known method which permits a count of the 
number of individual bacteria. Microscopical counts of the number 
of isolated individual bacteria and compact clumps present in milk 
give figures which compare well with those obtained where petri plate 
methods of counting are used." — H. L. L. 

MEDICAL BACTERIOLOGY 

Foot and Mouth Disease in Man. R. L. Sutton and A. O'Donnell. 
(Journ. A. M. A., 1916, 66, 947-949.) 
Report of a case. — G. H. S. 

Early Tuberculosis of the Cervix. T. S. Cullen. (Surg., Gyn,, and 

Obstet. 22, 261.) 

Tuberculosis of endometrium and cervix. Patient 25 years of age. 
Condition rare. — C. P. B. 

Some Fatal Ear Cases in the Writer's Practice. O. D. Stickney, M.D. 
(Jour, of Ophth., Otol., and Laryngol., 21, 189-204.) 
Eight cases reported. Five had meningitis following otitis. Pneu- 

mococci were isolated from spinal fluid in one case, streptococci from 

another.— C. P. B. 

The Choroidal Tubercle in Tuberculous Meningitis. J. F. Bredeck, 

M.D. (Am. Jr. Ophthalmol., 23, 1-8.) 

Choroidal tubercles may be found in 2 per cent of the cases of tuber- 
culous meningitis if careful, daily search is made. — C. P. B. 

The So-C ailed Primary Tuberculosis of the Conjunctivita and the Con- 
junctival Tuberculosis of Lupus Patients. K. K. K. Lundsgaard. 
"(Am. Jr. of Ophthal., 33, 54-59.) 

Of 48 patients 19 had primary conjunctival tuberculosis; 29 lupus 
patients had conjunctival tuberculosis. Former believed to be endog- 
enous and the latter ectogenous. — C. P. B. 

An Unique Lesion of the Heart in Systemic Blastomycosis. T. B. Hur- 
ley. (Jour. Med. Res., 1916, 33, 499-502.) 

A report of an autopsy of a case of systemic blastomycosis in which 
the musculature of the heart was extensively involved. This is, ac- 
cording to the author, the second case of its kind to be reported. No 
cultural studies are reported.^ — H. W. L. 



ABSTRACTS 



377 



Practical Points in the Prevention of Asiatic Cholera. Allen J. 
McLaughlin. (Bost. Med. and Surg. Jour., 1916, 174, 483.) 
The author describes a rapid method of testing for chronic carriers 
of the cholera vibrio among immigrants. His steps are: inoculating 
peptone solution, streaking out on agar, and agglutination, with the 
possible use of Goldberger's enrichment solution. One hundred to 
150 stools a day can be tested by one worker.— E. C. L. M. 

Diphtheria in Manila. A. P. Goff. (Journ. A. M. A., 1916, 66, 941.) 

As a result of a small outbreak of virulent diphtheria the Bureau of 

Science took more than 7000 throat cultures, finding 600 (or 9 per cent) 

positive for diphtheria. . . 

Of the carriers found, 4 per cent developed symptoms of diphtheria. 

G. H. S. 

The Etiology and Treatment of Rat-Bite Fever. W. Tileston. (Journ. 

A. M. A., 1916, 66, 995-998.) 

A case of rat-bite fever is reported. Organisms were found in the 
blood by darkfield examination which closely resembled Streptothrix 
muris-ratti. These organisms were to be found only during the febrile 
paroxysm, examinations made during the intervals being uniformly 
negative. 

The administration of salvarsan was followed by a cessation of the 

paroxysms. — G. H. S. 

Pathogeny of Diabetes and Fecal Disinfection. G. D. Palacios. (Medi- 
cal Record, 1916, 89, 543-551.) 

The pathogeny of diabetes mellitus is a fecal putrefaction and a 
fecal reabsorption of ammoniacal and acid character. Although fast- 
ing and a very restricted diet are the best dietetic treatment of diabetes, 
fecal disinfection is both preventive and curative. 

Intestinal putrefaction may be overcome in some cases by the acido- 
genous Bacillus bulgaricus. In the tropical Atlantic region, an abso- 
lute intestinal disinfection is effected by the ingestion of Micrococcus 
oxycyanogenes. — M. W. C. 

The Etiology of Scarlet Fever. F. B. Mallory, and E. M. Medlar, 

(Jour. Med. Res., 1916, 34, 127-130.) 

In a short communication, the authors describe finding, in the crypts 
of the tonsils, and in erosions of the epithelium of the tonsils, fauces, 
soft palate, uvula, trachea and lung of a child dying of scarlet fever 
on the second day following the eruption, clumps of Gram-positive 
bacilli, together with streptococci. Similar organisms were found in 
four other cases. The organism is best grown anaerobically on 3 per 
cent glycerin, 0.5 per cent glucose serum-agar. The authors beheye that 
the organism dies out rapidly or is overgrown by streptococci which ac- 
counts for previous failures. In view of the lack of animal experiments 



378 ABSTRACTS 

and the small number of cases examined, the work does not appear 
to be conclusive. — H. W. L. 

The Etiology of Rocky Mountain Spotted Fever. S. B, Wolbach, 

(Jour. Med. Res., 1916, 34, 121-127.) 

Guinea-pigs inoculated by ticks infected with the virus of spotted 
fever, show definite pathological changes characteristic of this disease. 
The author finds in the diseased tissue an organism, agreeing in most 
respects with that of Ricketts, which he feels justified in calling a bacil- 
lus. This organism is described as Gram-negative, resembling some- 
what B. influenzae but stained bluish by Giemsa, in contrast to most 
bacteria. All attempts to cultivate the organism have failed. 

H. W. L. 

Studies on Treponema pallidum and Syphilis. II. Spirochaeticidal 
Antibodies against Treponema pallidum. H. Zinsser, and J. G. 
Hopkins. (Jour. Exp. Med., 1916, 23, 323-328.) 
Cultures of the Treponema were grown on a new medium consist- 
ing of inspissated egg in tubes filled with broth serum mixtures. This 
method makes it possible to obtain clean antigen, unmixed with tissues 
detritus, a disadvantage incident to tissue cultures. The authors 
believe that their experiments have shown that the serum of rabbits 
and sheep immunized with cultures of Treponema pallidum acquire 
spirochaeticidal properties for these culture spirochaetes. The normal 
serum of these animals also possesses spirochaeticidal action if used 
in sufficient quantities, and the action of the immune serum repre- 
sents probably an increase of normal antibodies. Both normal and 
immune spirochaeticidal properties are destroyed by heating to 56°C. 
but the serum can be reactivated by the addition of fresh normal serum of 
the same species, insufficient in amount to exert a spirochaeticidal effect 
by itself. The structure of these spirochaeticidal bodies appears to 
be analogous to that of the well-known bactericidal antibodies known 
to exist in antibacterial sera. It is pointed out by the authors that 
these results apply to culture spirochaetes. — G. B. W. 

III. The Individual Fluctuations in Virulence and Comparative Viru- 
lence of Treponema pallidum Strains Passed Through Rabbits. Hans 
Zinsser, J. G. Hopkins, and M. McBurney. (Jour. Exp. Med., 
1916, 23, 329-340.) 

Rabbits were inoculated with strains from human cases with the 
purpose of studying differences in racial and acquired virulence. The 
authors found no difference in pathogenicity between the different 
strains, although they were isolated from various lesions, and, further, 
these strains show no consistent change in rabbit pathogenicity dur- 
ing progressive rabbit passage (21 generations in one case). Variations 
in the lesions produced, and also in the incubation time are probably 
due to variations in technique. — G. B. W. 



ABSTRACTS 



379 



IV. The Difference in Behavior in Immune Serum between Cultivated 
Non-Virulent Treponema pallidum and Virulent Treponemata from 
Lesions. Hans Zinsser, J. G. Hopkins, and M. McBurney. 
(Jour. Exp. Med., 1916, 23, 341-352.) 

Although antibodies can be produced by the immunization of ani- 
mals with cultivated Treponema pallidum, and although these antibodies 
exert specific agglutinative and treponemicidal action upon the culture 
organisms, they possess, at least in the concentration so far obtained 
by the authors in rabbits and sheep, practically no action on virulent 
treponemata obtained directly from lesions. — G. B. W. 

An Experimental Study of Parotitis (Mumps). Martha Wollstein. 

(Jour. Exp. Med., 1916, 23, 353-375.) 

Cats injected in the parotid gland and testicle with a bacterial sterile 
filtrate of the sahvary secretion of children in the active stage of paro- 
titis, or mumps, can be made to develop a pathological condition hav- 
ing several points of resemblance to the condition present in mumps 
in human beings. Definite changes in the temperature, blood leuco- 
cytes, and inoculated organs take place after an incubation stage of 
from 5 to 8 days. These pathological changes are intensified by suc- 
cessive transfers through a small series of cats of the extract and emul- 
sion of the parotid gland and testicle previously inoculated. These 
changes can also be prevented or reduced when the extract or emul- 
sion is previously incubated with blood serum obtained from a cat 
which has survived inoculation. Normal serum, on the other hand 
has no such inhibiting effect. AVhether the filtered sahvary secre- 
tion contains a microorganism and, if so, whether it is the specific mi- 
crobic cause of parotitis, or mumps, remains to be ascertained. 

G. B. W. 

The Etiologij, Mode of Infection, and Specific Therapy of WeiVs Disease 
(Spirochaetosis icterohaemorrhagica) . R. Inda, Y. Ido, R. Hoki, 
R. Kaneko and H. Ito. (Jour. Exp. Med., 1916, 23, 377-402.) 
In the course of their investigations of that endemic disease of por- 
tions of Japan, which agrees clinically with Weil's disease, so called, 
the authors discovered a spirochaetal microorganism which they 
name Spirochaeta icterohaemorrhagiae, and which they believe to be 
the cause of the disease. These spirochaetes live in the blood outside 
the cellular elements and in various organs and tissues. Infection 
is supposed to be by way of the alimentary canal or it may enter through 
the skin. The spirochaetes are excreted through the urine. The 
serum of convalescents possesses bactericidal and bacteriolytic proper- 
ties and recovery from the disease confers a lasting immunity. Treat- 
ment with salvarsan appears to offer promising possibilities, while 
passive immunization with immune serum has already given gratify- 
ing results. Many excellent plates are appended.— G. B. W. 



380 ABSTRACTS 

Bacteria Associated with Certain Types of Abnormal Lymph Glands. 
J. C. ToRREY. (Jour. Med. Res., 1916, 34, 65-81.) 
With a view to substantiating the claims of various authors that a 
diphtheroid bacillus is the causative agent in Hodgkin's disease, the 
author cultured 40 abnormal lymph glands, including 10 cases of 
Hodgkins. Three distinct groups of aerobic diphtheroid bacilli, one 
anaerobic group, and various other types of organisms were found and 
such a diversity of pathological conditions as to preclude the possibility 
of attaching importance to any one type as the cause of Hodgkin's 
disease. Serological reactions and animal inoculations failed to show 
any specificity. 

The finding of the anaerobic diphtheroid type in 100 per cent of 
the cases of Hodgkin's as well as in various other conditions, although 
interesting, the author does not believe is of any importance as throw- 
ing light on the cause of the disease in question, and only emphasizes 
the need of caution in accepting uncontrolled results as conclusive 
evidence. — H, W, L. 

The Diagnosis of Genitourinary Tuberculosis. J. W. Churchman. 

(Medical Record, 1916, 89, 511-513.) 

The mode of entrance of tubercle bacilli into the urine is not definitely 
proved, but it seems probable that the normal kidney is permeable 
for the tubercle bacillus as well as for other organisms. That infection 
does not extend upward from bladder to kidney is a well-established 
fact. 

The most reliable sign in diagnosing renal tuberculosis is the presence 
of tubercle bacilli in the urine, though in rare cases there may be tuber- 
culosis of the kidney with no demonstrable tubercle bacilli in the 
urine. In such cases the granules described by Much may be worthy 
of attention. These granules are interpreted by him to represent 
types of the tubercle bacillus which do not possess the ordinary acid- 
fast character. Although it is impossible to say what the significance 
of the Much granules is, it is true that they were present in the urine 
in a case of tuberculous kidney where tubercle bacilli were not found 
in the urine.— M. W. C. 

Relapsing Fever in Serbia. J. Rudis-Jicinsky. (New York Med. 

Jour., 1916, 103, 643-645.) 

Hundreds of cases of relapsing fever occurred in Serbia during the 
past winter. 

In many cases examination of the blood revealed Spirillum ober- 
meieri. This organism was filamentous, of spiral form, much elon- 
gated, and in motion followed its long axis. It was about four times 
the diameter of a red blood corpuscle. The organism was aerobic. 
It could be stained easily with anilin colors in dry blood, but was not 
found in other fluids or secretions of the body. 

The stage of the disease at which the spirochaete could be found 
in the blood was not always the same. 



ABSTRACTS 



381 



Inoculation of blood containing spirochaetes into rabbits conveyed 
the disease to these animals. After death spirochaetes were present 
in all the organs, but could not be cultivated upon artificial media. 

In nearly every case, the louse could be considered as the carrier 
of the infection, and the prevention of lousiness was a necessary step 
in eradicating the disease. — M. W. C. 

Colon Bacillus Infection of the Bladder. R. T. Morris. (New York 
Med. Jour., 1916, 103, 631-632.) . , . .u 

Many cases of cystitis of obscure origin may be due to the colon 
bacillus, as this bacillus is sometimes found upon examination of the 
urine, and conditions similar to cystitis have been induced m experi- 
mental animals by injecting Bacillus coli into the bladder. 

The differences in the type of infection are probably due to the 
particular strain of colon bacillus causing the condition, for there is 
a wide variation among the members of this group. According to 
the recent work of Rosenow it may even be possible that unusual 
types of cystitis are caused by other bacilli, which have assumed the 
form of B. coli. Uncertain action of vaccines in cases of colon bacillus 
infection is perhaps due to such a variation in the infecting organism. 
Colon bacilli may be responsible for any of the widely different 
manifestations of cystitis. , , , , , ui 

The mode of entrance of the colon bacillus into the bladder probably 
differs with varying conditions.— M. W. C. 

Studies on Diphtheria. II. The Treatment of Diphtheria Carriers by 

Tonsillectomy. H. 0. Run, M. J. Miler and R. G. Perkins. (Journ. 

A. M. A., 1916, 66, 941-943.) ,. . ,^ ^ ,, ,, . , . 

The termination of the carrier condition through the use oi biologi- 
cal or chemical methods did not meet with great success. 

In a series of 19 cases tonsillectomy was performed. The average 
duration of the carrier state before operative treatment was resorted 
to was 31 days. The average duration of release from quarantine 
after the tonsillectomy was 8 days. . . • , , , 

In all cases cultures made from the crypts after excision yielded 
Bacillus diphtheriae in nearly pure culture although surface cultures 
were frequently negative. — G. H, S. 

Bacteriological Work at the American Ajnbulance. Orville F. Rogers 
and George Benet. (Bost. Med. and Surg. Jour., 1916, 174, 418.) 
The authors report on the bacteriological work done in the Harvard 

University Service of the American Ambulance from April 1 to July 

1, 1915. , , . • nf 

From 100 men examined 28 showed gas-producmg organisms. Ut 

these 28, 18 were obtained in pure culture and run through sugar 

media. The authors conclude that sugar media are not suitable for the 

differentiation of the gas-producing bacilli. 

"The majority of cultures showed staphylococci either alone {60) 



382 ABSTRACTS 

or with other organisms (58). Forty cultures showed an anaerobic 
growth of other than gas producers. Other organisms seen were: 
pneumococcus (11), streptococcus (9), pyocyaneus (5), and varieties 
of other than gas producers (25)." — E. C. L. M. 

The Use of Kaolin to Remove Diphtheria Bacilli from the Nose and 

Throat. B. Rappaport. (Journ. A. M. A., 1916, 66, 943-945.) 

A study of the use of kaohn in 100 cases, 96 being diphtheria patients 
and 4 carriers. 

Kaohn, thoroughly dried and finely powered, is distributed over the 
surfaces to be treated. In young children application can best be 
made to the nasal mucous membrane even though the bacilli are in 
the pharynx. Some of the kaolin will work its way into the throat 
but the greater part will remain in the nose. Before a second treat- 
ment the kaohn already applied and now holding organisms should 
be removed by a mild alkaline spray. 

Six treatments per day at two hour intervals are given. With older 
patients the kaolin is swallowed, four half teaspoonful doses at two hour 
intervals six times during the day. 

The action of kaolin appears to be wholly mechanical, no bactericidal 
action being evident. 

The nose may be freed of bacilli much more readily than the throat. 

Compared with 100 consecutive cases dismissed before the use of 
kaolin, the treatment effected a percentage reduction of hospital 
stay of 23.4. 

Various pathological conditions, as adenoids and diseased tonsils, 
interfere with the action of kaolin. In such cases surgical treatment 
is required. — G. H. S. 

The Practical Value of Guinea Pig Tests for the Virulence of Diphtheria 
Bacilli. JoHH A. Kolmer, Samuel S. Woody. Emily L. Moshage. 
(American Jour. Diseases of Children, 1916, 4, 257-268.) 
The paper is based upon the results obtained with the guinea pig 
test for virulence on 1054 diphtheria cultures. The method employed 
consists of isolation of the bacilli upon slants of Loeffler's blood serum 
media, subculturing in 0.2 per cent glucose broth with a reaction of 
plus 0.8, incubation at 37°C., for seventy-two hours and injection sub- 
cutaneously in the median line of a pig weighing from 250 to 300 grams 
with a dose corresponding to 0.5 per cent of the weight of the animal 
expressed in cubic centimeters. The total amount injected is brought 
up to 4 cc. The animal is observed for four days and the development 
of a typical local inflammation with toxemia is regarded as diagnostic 
If in doubt a second pig is inoculated and at the same time is given 
500 units of diphtheria antitoxin. 4 cc. of a good 24 hour culture 
grown upon a tube of Loeffler's blood serum washed off in 10 cc. of 
salt solution can also be used for injection and has the advantage of 
saving 48 hours in time. Granular and barred types were found viru- 
lent in about 70 per cent of cultures from throat, nose and ear, long 



ABSTRACTS 383 

solid forms in about 42 per cent of cultures, while short solid types were 
uniformly found to possess no virulence. The authors, especially 
emphasize the importance of tests for virulence in recovery cases be- 
fore dismissal and in suspected carrier cases. — R. M. T. 

A Preliminary Report on Pneumonia in Children, with Special Reference 
to its Epidemiology. Godfrey R. Pisek and Marshall C. Pease. 
Am. Jour. Med. Sc, 1916, 151, 14. 

In an analysis of 1000 cases of pneumonia, not including those cases 
secondary to other infectious diseases, the authors found a mortality 
of 34.5 per cent in children under six years of age in the Babies' Wards 
of the New York Post-Graduate Hospital. The series contained 445 
cases classed as bronchopneumonia which occurred chiefly during the 
first two years of life, and were relatively uncommon after the third 
year. The lobar form also occurred more frequently during the first 
two years, and was the type usually found after the third year if the 
terminal and secondary infections following other diseases are ex- 
cluded. The highest mortality was found in the first year of life with 
both forms, but relatively less frequently with the lobar. No evidence 
was found of either epidemic or house infection in studying the cases. 
The authors felt that the broncho- and lobar types formed rather dis- 
tinctive groups clinically, their conclusions being based upon both 
pathological and bacteriological differences. For bacteriological study 
the materials were taken from the upper part of the larynx by means 
of a bent applicator. In 23 cases of lobar pneumonia, with sputum 
virulent for mice, all showed Gram-positive diplococci predominating 
in nearly every case with a few streptococci and staphylococci. In 

10 cases of mild bronchopneumonia with sputum which seemed virulent 
to mice, the predominating organism in 5 cases was the streptococcus. 
2 cases each showed staphylococci and the influenza bacillus, and 
in 1 case, tubercle bacilli with other organisms. In 4 cases a few 
pneumococci were present. In 8 cases of bronchopneumonia with 
sputum virulent to mice, smears showed large numbers of pneumococci, 
in addition to large numbers of other organisms, chiefly streptococci 
and staphylococci. Bacteriologically, the authors consider broncho- 
pneumonia as being a mixed infection, or an infection chiefly with one 
type of organism other than the pneumococcus. This differentiates 
this form from the lobar type, which is due chiefly or entirely to the 
pneumococcus. A study was made of the types of pneumococci 
occurring in a group of 48 clinical cases of pneumonia, which gave 
conclusive results. Of these, 28 cases were classified as lobar and 20 
cases as bronchopneumonia. For this purpose the Dochez-Gillespie 
grouping of pneumococci was followed, using specific sera for Groups 
I and II prepared by the Rockefeller Institute, and the methods recom- 
mended by them. The series gave the following results: Group I, 

11 cases; Group II, 14 cases; Group III, 4 cases; and Group IV, 19 
cases. Cases clinically classed as lobar pheumonia showed pneumo- 
cocci Groups I and II, decidedly predominating, while more than 



384 ABSTRACTS 

half of the dinical bronchopneumonias fell in Group IV. The mor- 
tality rate according to groups was as follows: Group I, 9 per cent.; 
Group II, 36 per cent.; Group III, 25 per cent.; and Group IV, 21 per 
cent. Eleven strains of pneumococci taken from the throats of children 
showing no lung involvement fell in Group IV. — L. W. F. 

PALEONTOLOGY 

Mesozoic Pathology and Bacteriology. Roy L. Moodie. (Science, 

1916, 43, 425-426.) 

Attention is called to this rather unusual, though fascinating and 
important branch of bacteriology. The author feels that we have 
convincing proof of the existence of fungi and bacteria in coprolites, 
and of pathologic conditions in various fossil tumors and fractures. 
— C. M. H. 

PLANT PATHOLOGY 

Further Studies in the Role of Insects in the Dissemination of Fire Blight 
Bacteria. V. B. Stewart and M. D. Leonard. (Phytop. 1916, 
152-158.) 

From observations made throughout several seasons the writers 
believe that all the sucking bugs of the nursery bear infection. Experi- 
ments were conducted by caging upon trees insects which had on their 
bodies organisms from a pure culture of Bacillus amylovorus. The 
various species of flies are thought not to be active agents in transmit- 
ing infection though they may be important in carrying the organism 
to blossoms or to wounds. The experiments included the following 
suspected carriers: Pollenia rudis, Empoasca mali, Psylla pyricola, 
Plagiognathus politus, Sapromyza hispina. — F. L. S. 

Citrus Canker. F. A. Wolf. (J. Agr. Res. 6, 69-99.) 

A serious citrus disease has recently been introduced into the Gulf 
States, known as citrus canker. The primary cause is Ps. citri Hasse, 
an organism with a single flagellum, shown by the writer to have the 
group number Ps. 221. 3332513. It attacks both twigs and leaves. 
Fungi of the genera Phoma, Fusarium and Gleosporium have been found 
associated with this organism, although the Phoma is the only one 
found to be notably active in disintegration of the tissues. The only 
method of control recommended is by means of quarantine and thorough 
destruction of diseased trees. — H. J. C. 



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JOURNAL OF BACTERIOLOGY 

OFFICIAL ORGAN OF THE SOCIETY OF AMERICAN BACTERIOLOGISTS 



DEVOTED TO THE ADVANCEMENT AND DIS- 
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CONTENTS 

M. R. Smirnow: Biological Variations of Bacteria. 1 385 

W. Whitridge Williams and Ward Burdick: A New Culture Medium for 

the Tubercle Bacillus 411 

Edward S. Good and Wallace V. Smith: Bacillus Abortus (Bang) as an 

Etiological Factor in Infectious Abortion in Swine 415 

T. L. Hills: The Relation of Protozoa to Certain Groups of Soil Bacteria. 423 

P. G. Heinemann and E. E. Ecker: A Study of the Boas-Oppler Bacillus. . 435 

J. M. Sherman: A Contribution to the Bacteriology of Silage 445 

Ward Giltner. Book Review: Laboratory Manual in General Micro- 
biology 453 

Abstracts of American Bacteriological Literature: 

Bacteriology of Food 455 

Bacteriology of the Mouth 455 

Bacteriology of Soils 456 

Bacteriology of Water and Sewage 457 

Disinfection 459 

Immunology 461 

Industrial Bacteriology 463 

Medical Bacteriology 464 

Number one of volume one of the Journal of Bacteriology, dated January, 
appeared April 22; number two, dated March, appeared May 17. 

The Journal of Bacteriology is issued bimonthly. Each volume will con- 
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of Canada, and correspondence pertaining thereto, should be addressed to Mr. 
C. F. Clay, Manager, Cambridge University Press, Fetter Lane, London, E. C. 



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BIOLOGICAL VARIATIONS OF BACTERIA^ 
I. Induced Variations in the Cultural Characters 

OF B. COLI 

M. R. SMIRNOW 
Yale Medical School, New Haven, Connecticut 

Variations in the biological characteristics of the various mem- 
bers of the Colon-typhoid group have been reported by num- 
erous investigators. These range from mere observations as 
to peculiarities in the fermentative or other biological tests 
up to actual mutations as interpreted by De Vries. 

The most important observations along the latter line are 
reported by Massini (1907), who isolated a type of B. coli he 
called "B. coli-mutahilis." In his observations, he found this 
organism would produce flat colorless colonies on Endo's medium, 
if transplanted every twenty-four hours. When transplanted 
at a later period, however, it would produce nodular shaped 
colonies which became red. The colorless colonies always gave 
rise to colorless colonies when transplanted not later than twenty- 
four hours, whereas the red colonies once obtained, never gave 
rise to any but red colonies irrespective of the time of transfer. 
The knob like colonies appeared only on lactose media though 
other carbohydrate media were used. He observed a single 
reversion from the red to the colorless type, which, however, 
could not be repeated. 

Burk (1908) reports the isolation of a similar mutant and 
records careful observations which were continued over a period 
of five months. 

Of the more interesting reports in literature on modifications 
of B. coli and allied organisms may be mentioned those of Peck- 

^ Read in part, before the meeting of the Society of American Bacteriologists, 
held at Philadelphia, December, 1914. 

385 



386 M. R. SMIRNOW 

ham, Herter, Penfold, Twort, Manfredi and others. The work 
of the first of these investigators will be mentioned below, in 
conjunction with the experiments of the writer. 

Herter (1910) has shown that sodium benzoate in weak glu- 
cose broth considerably inhibits the fermentative activities 
of B. coli, whereas other biological features are but shghtly 
affected. Such action is entirely prevented by the addition 
of calcium carbonate. He has also shown that there are no 
gas producers in food stuffs preserved with sodium benzoate, 
though 22 of 28 samples contained bacteria of some sort. Pen- 
fold (1911) has shown similar action in the case of sodium ace- 
tate on B. coli, B. enteritidis and B. paratyphi with diminishing 
and total disappearance of gas formation in the sugars, though 
the organisms were still capable of producing gas from the cor- 
responding alcohols. This indicated an inhibition or destruc- 
tion of the enzyme, invertase, without effect upon the gas pro- 
ducing power. Twort (1907) has shown that B. typhi, B. para- 
typhi and B. dysenteriae when continuously grown in saccha- 
rose media will ultimately ferment saccharose. Manfredi (1889) 
states that fat-containing media impair the vegetative energy 
of bacteria. 

The observations here reported were undertaken in connection 
with a series of experiments on the biological variations of bac- 
teria, which the writer intends pubhshing in sections whenever 
a sufficient amount of interesting material is accumulated to 
warrant it. Twenty-one different strains of the various bacilli 
of the colon-typhoid group were used, in the study but this 
report is confined only to the B. coli, of which seven different 
strains were experimented on. All of these strains were ob- 
tained from the American Museum of Natural History, New 
York, through the kindness of Prof. C.-E. A. Winslow, and 
were the stock nos. 19, 44, 45, 46, 52, 57, and 95. The bacteria 
were subjected to continuous growth at 37.5°C. in 3 per cent 
glucose, 4 per cent sodium chloride and 1.5 per cent sodium sul- 
phate broth. They were also grown in plain broth and then 
exposed to he action of phenol in the following manner. The 
culture was first inoculated into 9 cc. of plain nutrient broth 



BIOLOGICAL VARIATIONS OF BACTERIA 387 

and incubated for three or four days, at which time 1 cc. of 7.5 
per cent of phenol was added to the culture. The phenol ex- 
posure was limited to two to three minutes at the beginning of 
the experiment and then the time was gradually ncreased with 
each transfer until thirty or more minutes time was attained. 
The exposed culture was then reinoculated into plain broth 
by i:ouring over a small quantity (0.5 to 1 cc.) from it. Con- 
tinuous growth of the B. coli in 0.75 per cent of phenol broth 
was also tried, beginning with 0.25 per cent, with the same gen- 
eral results. The transplanting in all media was carried out 
every three or four days over periods varying from one to three 
months, thus allowing from ten to thirty transfers. The ex- 
periments were repeated two or three times to assure constant 
and uniform findings. 

Control cultures were carried on in plain broth throughout 
the experiment. It might be stated at once that there were 
very sHght variations between the original stocks and these 
control cultures, no more than would be expected as normal 
variations. These were seen as slightly increased or decreased 
amounts of gas or acid formation, differences in time of coagula- 
tion, or slight changes in the growth on potato. At no time, 
however, were the biological characteristics markedly changed 
nor enzyme production completely inhibited simply by continual 
passage through broth. 

The accompanying tables show the results obtained in some 
of the more typical series of experiments. These tabulations 
were all made at seventy-two or ninety-six hours after inocula- 
tion and were verified again, especially those on potato and in 
milk, after a week or ten days growth. The tests for indol were 
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BIOLOGICAL VARIATIONS OF BACTERIA 391 

CHANGES IN MORPHOLOGY 

During the course of experimentation several of the strains 
of B. coli showed a change in morphology, the individual organ- 
isms becoming two and three times the length of those in the 
controls, somewhat wider and more vacuolated. This however 
was not constant, as often on the very next sub-culture they 
would assume their usual morphological appearances. The only 
other thing noted under this head was the decrease in motility, 
which was more marked in the phenol broth than in the other 
media. Very little importance should be attached to this, 
however, since some of the strains were hardly motile to be- 
gin with, and again, too few observations were made to permit 
of absolute statements. 

GROWTH ON POTATO 

Glucose seemed to have a special effect upon the character 
of growth of B. coli on this medium. Five of the seven treated 
strains, showed at best only a very light yellow color or a slight 
brownish growth on ordinary potato with practically no discolora- 
tion of the medium. Very frequently indeed, the glucose affected 
organisms would give the typical "invisible" growth seen with 
the B. typhi. Both the original stock and the broth control 
showed the characteristic colon growth on this medium. This 
change was noted so many times that an explanation based on 
differences in the composition of the potato can be excluded. 
Three of these five strains also showed this change after exposure 
to phenol. One strain of the B. coli, which did not change in 
this respect with either glucose or phenol, showed this same 
variation after growing in either sodium chloride or sodium 
sulphate broth. 

ACTION IN MILK 

Both phenol and glucose diminished the acid production and 
inhibited the formation of lab enzyme in three of the seven 
strains of the B. coli, either entirely or for a period of at least 
two weeks. These results were not seen with the use of the strong 
saline or sodium sulphate broth. 



392 M. R. SMIRNOW 

FERMENTATION OF SUGARS 

The results obtained with these substances on B. coli with 
reference to variations in sugar fermentations can be best seen 
in the accompanying tables. The most striking changes here 
also were seen in those organisms exposed to glucose and phenol. 
The former completely inhibited both acid and gas formation 
in all the sugars tested in three different strains. In two others, 
glucose varied the amount of acid and gas formation, with an 
occasional complete inhibition in some of the sugars. Phenol 
inhibited these fermentations in all of the sugars in only one 
strain, and in four others, diminished this reaction to the point 
of inhibition at times only and in different carbohydrates. Sodium 
chloride and sodium sulphate had less effect than did phenol, 
giving usually slight variations in amount of acid or gas pro- 
duced with an occasional inhibition. 

Inhibition of all the sugar fermentations in any one experi- 
ment was almost always accompanied by inhibition of the 
usual changes in milk, the characteristic growth on potato, and 
the formation of indol. In other words, the most typical varia- 
tions were those in which all acid or ferment production was 
inhibited. 

VARIATIONS IN INDOL PRODUCTION 

The production of indol is held by many bacteriologists to 
be as important a biological characteristic of B. coli as its fer- 
mentations of the sugars, and is even thought to be of greater 
importance in its differentiation. This reaction, however, even 
under normal conditions varies considerably in its quantity and 
time of appearance with most strains, and at times requires 
more deUcate tests than the usual Salkowski method for its 
determination. 

In the experiments here reported it appears that of the varia- 
tions induced in B. coli that of indol production is the first to 
take place, often disappearing in the third or fourth culture in 
glucose broth. This does not hold however when the bacteria 
are grown in the other media, as evidenced below. 



BIOLOGICAL VARIATIONS OF BACTERIA 



393 



Each strain of B. coli was grown in plain broth as control, 
and in glucose, phenol, sodium chloride and sodium sulphate 
broth and on potato. Thirty-five sub-cultures were made in all. 
Indol was tested for after the 10th, 15th, 25th, and 35th transfers. 
The tests for indol were made by inoculating one loop of cul- 
ture from the respective medium to which each strain was sub- 
jected into standard peptone solution, growing for seven days 
at 37°C. and then testing by the Salkowski method. All the 
tests were done at the same time using the same batch of pep- 
tone solution throughout the experiment. 

The results were uniform for all strains and may be readily 
interpreted from the following table: 



TABLE VIII 



NUMBER OP TRANSFER 



Control 

Glucose broth. 
Phenol broth. . 
NaCl broth*.. 
Na2S04 broth* 
Potato 



1 


10 


15 


25 


+++ 


+++ 


+ + + 


+ + + 


+++ 


- 


- 




+++ 


++++ 


+ + + + 


+ + + + 


+++ 


++ 


+ + 


+ + + 


+++ 


++ 


+ + 


+ + + 


+++ 


+++ 


+ + + 


+ + + 



+++ 

++++ 

++ 

++ 
+++ 



* Exposure to these substances gave variable results, at times an increase and 
at others a decrease in indol production. 

All the controls, grown in plain broth, gave good indol tests 
even after the 35th sub-culture. Those grown in glucose broth 
gave none at the 10th sub-cultm-e or thereafter. In phenol 
broth the property of indol production seemed to be somewhat 
increased, judging from the intensity of the reaction. Sodium 
chloride and sodium sulphate, and prolonged cultivation on 
potato practically exerted no influence, or if any, showed a 
shght inhibitory effect. 

Experiments were then carried out to see how soon the prop- 
erty of indol production is interfered with by growth in 3 
per cent glucose broth, and it was found that B. coli lost this 
property usually on the thu'd and at times on the second transfer 
over a period of from seven to ten days. In one experiment 
sub-cultures were made every twenty-four hours with a total 



394 M. R. SMIRNOW 

disappearance of the indol tests in from forty-eight to seventy- 
two hours in all the strains. 

The tests in these latter experiments were made in the cul- 
ture tubes themselves, not transferring to the peptone solution, 
after seven days of growth. In order to exclude the possibility 
of interference with the indol test by the presence of the glucose, 
several cultures in both plain broth, and peptone, were made, 
and grown at 37°C. for seven days. Glucose was added to 
each of the cultures and they were then tested for indol. Posi- 
tive tests were obtained in all cases, excluding any possibility 
of such interference by the presence of the carbohydrate. An 
interesting observation may also be mentioned at this junc- 
ture. Cultures of the organisms in plain broth of seven days 
growth to which phenol or sodium chloride were added showed 
a decided increase in the indol reaction in case of the phenol 
and a diminished reaction in the tubes to which the sodium 
chloride was added. In the interpretation of these tests compari- 
son was made with controls. It may be possible that the 
presence of these substances intensifies or diminishes the color 
produced, the differences not being due to actual variations in 
the amount of indol formed. The different culture media them- 
selves were tested for indol, after incubating for seven days, 
for the purpose of control and they were found negative. 

Experiments were then carried out to determine the per- 
manency of this change. The cultures in glucose broth after 
the 35th transfer were grown in plain broth, transplanting every 
day and tested on the seventh day of incubation. Four of the 
strains of B. coli; nos. 44, 45, 46 and 52 gave sHght indol reactions 
on the third transfer, no. 46 gave a good positive on the fifth 
transfer, but the others took from five to ten more transfers 
before they could be called " + " or " + +" positive. Nos. 57 
and 95 took six transfers before a trace of indol appeared. No. 
19, a very feeble indol producer in the control, remained nega- 
tive up to the fifteenth transfer at which time the experiment 
was discontinued. 

Investigations as to the agglutinabihty of these altered strains 
of B. coli were also made, but the work is too meagre and the 



BIOLOGICAL VARIATIONS OF BACTERIA 



395 



results too indefinite to be reported at the present time. The 
writer intends to continue work along this line, and also with 
respect to pathogenicity, which seems to suggest itself as a 
fruitful subject for investigation. 

In summing up, it can be said that glucose and phenol, par- 
ticularly the former, cause partial inhibition or total disappear- 
ance of acid and enzyme formation in some strains of B. coli. 
These changes together with the suspension of the production 
of indol and the characteristic colon growth on potato, makes 
the B. coli approach the B. typhi type. These changes have 
been noted time and again but in varying degrees, in those strains 

TABLE IX 

Changes produced by glucose and phenol in various strains of B. coli and the complete or 
incomplete reversion towards their previous biological characteristics. The coinplete- 
ness of the change and the incompleteness of the reversion in strain No. 95 is note- 
worthy, as such a change might be regarded in the light of a mutation instead of a 
variation 





POT.^TO 


MILK 


GLUCOSE 


SACCHAROSE 


FERMLNTATION 
TUBE 




Br. Gr. 


Disc. 


Acid 


Coag. 


Acid 


Gas 


Acid 


Gas 


Acid 


Gas 


B. Coli, No. 95 

Control 

Glucose 

Reversion 

B. Coli. No. 19 

Control 

Glucose 

Reversion... . 
B. Coli. No. 44 

Control 

Glucose 

Reversion 

Phenol 

Reversion... . 
B. Coli. No. 45 

Control 

Glucose 

Reversion 

Phenol 

Reversion... . 


+ + + 
+ + + 

+ + + 

+ + + 

+ + + 

+ + 

+ + + 

+ + + 
+ + 


+ + + 
+ + + 

+ + + 

+ 

+ + + 

+ + 

+ + + 

+ + 
+ + 


+++ 
++ 
++ 

+++ 

+++ 

+++ 

++ 

++ 

+++ 

++ 

++ 

++ 


+ + + 
+ + 
+ + 

+ + + 

+ + + 

+ + + 

+ + + 
+ + + 
+ + + 

+ + + 

SI. 
SI. 


+ + 
+ + 

+ + + 

+ + + 

+ + 

+ + + 

+ 

+ + 

+ + 

+ 

+ + 

+ 


+ + + 

+ + + 

+ + 

+ + + 

+ + + 

+ 

+ + + 

+ 

+ + + 

+ 


++ 

++ 

+ 

+++ 

++ 

+++ 

++ 
++ 
++ 

++ 

+ 

+ 


+ + 

+ + + 
+ + + 
+ + + 

SI.* 

++ 
+++ 

+ 

+ 


+ + + + + , + 
+ 1 + 1+ +1 + 1+ +1+ 1 1 + 
+ + + + + + + 


+ + + 

+ + + 

+ + 
+ + + 

SI. 

+ 

+ 

+ 
+ 
+ 



* SI. = slight. 



396 M. R. SMIRNOW 

that are susceptible to variations, but for some unexplained 
reason cannot be regarded as altogether constant. Indol for- 
mation would invariably return when these altered bacteria 
were transplanted into plain broth at frequent intervals. Lab 
enzyme would also return in most of the altered strains but not 
invariably so. The same can be said of the fermentative proper- 
ties. Very often, however, these characteristics appear to be 
entirely done away with, the change being permanent as far 
as could be made evident by sub-culturing into plain broth. 
(See Table IX.) In these cases observations were made up to 
two months after the last exposure to the influencing substance, 
making frequent transfers. There seemed to be no definite 
rule of reversion, and no relation between the reappearance of 
one enzyme and another. The reappearance of the ferment- 
ing enzymes in one sugar was not necessarily accompanied by 
those in other sugars. At times the fermentation of one sugar 
might have returned to nearly normal, while others might show 
httle or no presence of gas with the same strain of B. coli. 

ADDENDA 

The work under this heading was undertaken as supplement- 
ary to the foregoing section for the purpose of verification and 
also in response to comments made upon the report of this 
paper at the meeting of the Society of American Bacteriologists, 
held at Philadelphia, in December, 1914. 

To avoid any objection arising as to the possible existence of 
mixed cultures, or of "weak members" at the start, each strain 
of B. coli was plated, a single colony selected, and replated, and 
from this latter plate, several cultures were selected, inoculated 
on agar slants and after twenty-four hours cultivation, carried 
through on all media. The strain that showed the greatest 
amount of enzyme formation was selected as the "strongest" 
and the one to be subjected to experimentation. 

In general, the technique of these experiments was identical 
with that already described, observations being made with 
special reference to indol production which was taken as the 



BIOLOGICAL VAKIATIONS OF BACTERIA 



397 



index of proteolytic activities. To control the possible effect 
of the acid production of B. coli, upon proteolysis, three of the 
sugars, namely, glucose, lactose, and saccharose, had 1 per cent 
of calcium carbonate added to neutralize any acid formed. An- 
other control was also carried along, a stock strain of Sp. cholerae, 
which produces but a shght amount of acid as compared with 
B. coli, but, on the other hand, gives a marked amount of indol. 
Seven different sugars were used besides the three containing 
calcium carbonate, making a total of ten inoculations for each 
organism. The organisms were cultivated in the respective 
sugar peptone solutions for six days, at which time small amounts 
of the cultures were poured over into fresh media for the con- 
tinuance of the experiment, and then the Salkowski's test was 
apphed to the original culture. After the seventh inoculation, 
when all the strains gave negative indol tests, experiments for 
reversions were begun, by transfering a small quantity of each 
of the cultures into plain peptone, and proceeding with the test 
as before. 



TABLE X 



Showing the effect of various carbohydrates upon the proteolytic activities of seven different 
strains of B. coli, and one of Sp. cholerae, as evidenced by the indol test 



SUBCULTURE 


CONTROL 


GLUCOSE 




1 


2 


3 


1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


B. coli No. 1 

B. coli No. 2 

B. coli No. 3 

B. coli No. 4 

B. coli No. 5 

B. coli No. 6 

B. coli No. 7 

Sp. cholerae 


+ 

++ 

+++ 

++ 

+++ 

+++ 

++ 

+++++ 


tr. 

++ 

++ 

++ 
+++ 
+++ 

++ 
+++ + 


+ 

++ 

+++ 

++ 

+++ 

++ 

++ 

+++++ 


tr. 

tr. 
tr. 

tr. 
tr. 




- 


tr. 
tr. 
tr. 
tr. 
tr. 
tr. 
tr. 


- 


- 


- 


- 


- 


- 



TABLE XL 



Results oj the indol tests in the experiments on reversion. * = Positive test obtained with 
H2S0i only, f = Positive test obtained only after the addition oj nitrite 



B. coli No. 1 








_ 


_ 


_ 


_ 


_ 


_ 


_ 


tr. 


_ 


+ 


B. coli No. 2 








- 


— 


•> 


? 


- 


si. tr. 


si. tr. 


— 


— 


— 


B. coli No. 3 








_ 


— 


? 


— 


? 


- 


- 


? 


- 


— 


B. coli No. 4 








- 


- 


- 


- 


? 


- 


- 


- 


- 


7 


B. coli No. 5 








- 


— 


— 


- 


? 


— 


— 


? 


tr. 


+ 


B. coli No. 6 








— 


— 


— 


- 


? 


- 


- 


— 


? 


? 


B. coli No. 7 








— 


— 


si. tr. 


si. tr. 


— 


- 


— 


si. tr. 


— 


— 


Sp. cholerae 








- 


- 


? 


- 


si. tr. 


tr. 


tr.t 


+ 


+ 


+ 



398 



M. R. SMIRNOW 



TABLE '^—Continued 





CONTROL 


GLUCOSE + CaiCOa 




1 


2 


3 


1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


B. coli No. 1 

B. coli No. 2 

B. coli No. 3 

B. coli No. 4 

B. coli No. 5 

B. coli No. 6 

B. coli No. 7 

B. cholerae 


+ 

++ 

+++ 

++ 

+++ 

+++ 

++ 

+++++ 


tr. 

++ 

++ 

++ 

+++ 

+++ 

++ 

++++ 


+ 
++ 

+++ 
++ 

++ + 
++ 
+ + 


tr. 
tr. 
tr. 
tr. 
tr. 
tr. 
tr. 
+ 


? 
tr. 
tr. 
tr. 

? 
tr. 
tr. 


? 
7 

? 
? 
? 


tr. 
tr. 

tr. 
tr. 
tr. 


- 


- 


- 


- 


- 


- 



B. coli No. 1. 
B. coli No. 2. 
B. coli No. 3, 
B. coli No. 4 
B. coli No. 5 
B. coli No. 6 
B. coli No. 7 
B. cholerae. . 



TABLE m— Continued 
Reversion from above 



si. tr. 

si. tr. 
tr. 



+ + 
+ + + 
++ 
+ + 
++ 



tr. 

tr. 
++ 
++ 
++ 
++ 
+ + 
si. tr. 



tr.* 



tr.' 



TABLE 'K.— Continued 





CONTROL 


LACTOSE 




1 


2 


3 


1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


B. coli No. 1 

B.coli No. 2 

B. coli No. 3 

B. coli No. 4 

B.coli No. 5 

B. coli No. 6 

B. coli No. 7 

B. cholerae 


+ 

++ 

+++ 

++ 

+++ 

+++ 

++ 

+++++ 


tr. 

++ 

+ + 

++ 
++ + 
+++ 

+ + 
+++ + 


+ 

+ + 
+ + + 

++ 
++ + 

+ + 

+ + 
+++++ 


tr. 
+ 
tr. 
tr. 
tr. 
tr. 
++ 


tr. 
tr. 
tr. 
tr. 
tr. 


1 
tr. 

? 
tr. 


tr. 
tr. 

tr. 
tr. 


- 


- 


- 


- 





- 



TABLE 'Xl— Continued 
Reversion from above 



B. coli No. 1 
B. coli No. 2 
B. coli No. 3 
B. coli No. 4 
B. coli No. 5 
B. coli No. 6 
B. coli No. 7 
Sp. cholerae. 



_ 


_ 


_ 


tr. 


si. tr. 


tr. 


+ * 


+ * 


++ 


- 


- 


tr. 


+ 


si. tr. 


tr. 


+ * 


tr.* 


+ 


- 


- 


si. tr. 


+ 


+ 


— 


+ 






- 


- 


si. tr. 


+ 


++ 


+ 


+ 






- 


- 


? 


+ 


+ + 


+ 


+ + 






- 


- 


? 


+ 


+ 


+ 


+ 






- 


— 


si. tr. 


+ 


+ 


+ 


+ 






- 


- 


si. tr. 


+ 


+ 


+ 


+ t 


tr.t 


+ 



BIOLOGICAL VARIATIONS OF BACTERIA 



399 



TABLE X— Continued 





CONTROL 


LACTOSE + CaCOs 




1 


2 


3 


1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


B. coli No. 1 

B. coli No. 2 

B. coli No. 3 

B. coli No. 4 

B. coli No. 5 

B. coli No. 6 

B. coli No. 7 

Sp. cholerae 


+ 
++ 
+++ 
++ 
++ + 
+++ 
++ 


tr. 

++ 

++ 

+ + 

+++ 

+++ 

++ 

++++ 


+ 

+ + 

+++ 

++ 

++ + 

++ 

++ 

+++++ 


tr. 
tr. 
tr. 
tr. 
tr. 
tr. 
+ 


tr. 

tr. 

? 

? 
tr. 
tr. 


? 
? 

? 

+ 


tr. 
tr. 

tr. 
tr. 

tr. 


tr. 


- 


- 


- 


- 


- 



TABLE XI— Continued 
Reversion from above 



B. coli No. 1 
B. coli No. 2 
B. coli No. 3 
B. coli No. 4 
B. coli No. 5 
B. coli No. 6 
B. coli No. 7 
Sp. cholerae. 









_ 


_ 


? 


_ 


_ 


_ 




_ 


_ 








- 


- 


si. tr. 


si. tr. 


si. tr. 


tr. 


+ * 


+ * 


+ 








- 


- 


tr. 


+ 


+ + 


+ 














— 


- 


? 


+ 


++ 


+ 














- 


- 


si. tr. 


+ 


+ 


+ 














- 


- 


— 


+ 


+ 


+ 














— 


— 


si. tr. 


+ 


+ 


+ 














— 


— 


si. tr. 


tr. 


+ 


+ 


tr. 


si. tr. 


tr. 



TABLE X—Continued 





CONTROL 


SACCHAROSE 




1 


2 


3 


1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


11 


12 


13 


14 

+ 

+ 

+ 


15 

+ 

tr. 

tr. 


16 

tr. 

+ 
tr. 


17 


18 


B. coli No. 1. 
B. coli No. 2. 
B. coli No. 3. 
B. coli No. 4. 
B. coli No. 5. 
B. coli No. 6. 
B. coli No. 7. 
Sp. cholerae.. 


• + 

++ 

+ + + 

+ + 

+ + + 

+++ 

++ 

+ + +++ 


tr. 

++ 

+ + 

++ 

+++ 

++ + 

++ 

++++ 


+ 

++ 

+++ 

++ 

+++ 

++ 

+ + 

+++++ 


tr. 
tr. 

+ + 
tr. 
++ 
++ 
tr. 
+ 


tr. 

+ 
tr. 

+ 
tr. 


tr. 

+ 

+ 

+ 


tr. 

+ 
tr. 
+ 
+ 
tr. 
tr. 


+ 

+ 
+ 


+ 

+ 
^ + 


tr. 

tr. 

+ 


+ 

+ 
+ 


+ 

+ 
+ 


+ 

+ 
+ 


+ 

+ 
tr. 


+ 

+ 
+ 


tr. 

+ 

+ 


sl.tr. 

tr. 

+ 


sl.tr. 

+ 
tr. 



TABLE XI— Continued 
Reversion from above 



B. coli No. 1.. 
B. coli No. 2.. 
B.coli No. 3.. 
B. coli No. 4.. 
B.coli No. 5.. 
B. coli No. 6.. 
B. coli No. 7.. 
Sp. cholerae. . 











_ 


_ 


_ 


_ 


_ 


_ 




_ 


tr. 


sl.tr. 
























- 


- 


- 


- 


tr. 


+ * 


tr* 


+ * 


tr. 


tr. 
























=t 










































- 


- 


+ 


++ 


+ + 


+ 
































± 










































=^ 




+ 


++ 


+ 


+ 
































- 


- 


+t 


tr. 


sl.tr. 


sl.tr. 


tr.t 


tr.t 


tr. 


tr. 

















400 



M. R. SMIRNOW 



TABLE X— Continued 







CONTROL 










S.4.CCHAROSE + CaCOj 












































1 


2 


3 


1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


11 


12 


B. coli No. 1.... 


+ 


tr. 


+ 


tr. 


_ 


tr. 


_ 


— 


_ 


_ 


_ 


_ 


_ 


_ 


_ 


B. coli No. 2,... 


++ 


++ 


++ 


- 


tr. 


- 


tr. 


- 


— 


- 


- 


- 


- 


— 


— 


B.coli No. 3.... 


+++ 


++ 


+++ 


+ 


+ 


+ 


+ 


+ 


+ 


tr. 


+ 


+ 


tr. 


+ 


tr. 


B. coli No. 4.... 


++ 


++ 


++ 


tr. 


tr. 


7 


tr. 


+ 


- 


- 


__ 


- 


- 


- 


— 


B.coli No. 5.... 


+++ 


+++ 


+++ 


+ 


+ 


? 


+ 


+ 


+ 


+ 


tr. 


+ 


tr. 


tr. 


tr. 


B.coli No. 6.... 


+++ 


+++ 


++ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


tr. 


+ 


+ 


+ 


B. coli No. 7.... 


++ 


++ 


++ 


tr. 


tr. 


? 


tr. 


— 


— 


— 


— 


— 


— 


— 


— 


Sp. cholerae.. . . 


+++++ 


++++ 


+++++ 


+ 


tr. 


tr. 


tr. 


tr. 


- 


- 


^ 


- 


- 


- 


— 



TABLE XI— Continued 
Reversion from above 



B.coli No. 1... 










_ 


_ 


_ 


_ 


_ 


_ 


_ 


tr.* 


_ 


tr.* 


_ 


B.coli No. 2.... 








- 


- 


sl.tr. 


tr. 


tr. 


tr.* 


tr. 


+ * 


+ * 


+ * 


+ * 


+ 


B.coli No. 3... 








=b 
























B. coli No. 4.... 








+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


tr. 


+ 


+ 


+ 


B.coli No. 5. .. 








- 


- 


- 


? 


tr. 


tr. 


+ 


+ 


tr. 


+ 






B.coli No. 6,... 








=fc 
























B. coli No. 7.... 








— 


— 


tr. 


+++ 


++ 


+ 


4- 












Sp. cholerae 








— 


- 


- 


si. tr. 


tr. 


tr. 


tr.t 


tr. 


+ 


tr. 


+ 


+ 



TABLE X— Continued 





CONTROL 


MALTOSE 




1 


2 


3 


1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


B. coli No. 1 

B. coli No. 2 

B. coli No. 3 

B. coli No. 4 

B. coli No. 5 

B. coli No. 6 

B. coli No. 7 

Sp. cholerae 


+ 

++ 

+++ 

++ 

+++ 

+++ 

++ 

+++++ 


tr. 

++ 

++ 

++ 
+++ 
+++ 

+ + 
++++ 


+ 

++ 

+++ 

+ + 

+++ 

++ 

+ + 

+++++ 


tr. 
tr. 
tr. 

tr. 
tr. 
tr. 

+ 


tr. 
tr. 
tr. 
tr. 
tr. 
tr. 
tr. 


? 

? 
? 
? 
? 
7 
7 


? 

? 
7 
? 

7 

7 


? 

? 


- 


- 


- 


- 


- 



TABLE XI— Continued 
Reversion from above 



B. coli No. 1 








_ 


_ 


si. tr. 


_ 


_ 


si. tr. 


si. tr* 


_ 


tr. 


tr. 


B. coli No. 2 








- 


- 


si. tr. 


tr. 


tr. 


tr. 


tr. 


tr. 


tr. 


tr. 


B. coli No. 3 








tr. 


— 


tr. 


+ 


++ 


+ 










B. coli No. 4 








- 


— 


+ 


+ 


+ 


+ 










B. coli No. 5 








- 


- 


+ 


+ + 


+ 


+ 










B. coli No. 6 








- 


- 


+ 


++ 


+ 


+ 










B. coli No. 7 








— 


tr. 


+ 


+ + 


+ 


+ 










Sp. cholerae 








- 


- 


- 


tr. 


tr. 


tr. 


tr.t 


tr.t 


tr. 


tr. 



BIOLOGICAL VARIATIONS OF BACTERIA 



401 



TABLE X— Continued 





CONTROL 


GALACTOSE 




1 


2 


3 


1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


B. coli No. 1 

B. coli No. 2 

B. coli No. 3 

B. coli No. 4 

B. coli No. !i 

B. coli No. G 

B. coli No. 7 

Sp. cholerae 


+ 

++ 

++ + 

+ + 

+++ 

+++ 

+ + 


tr. 

++ 

++ 

+ + 
+++ 
+++ 

+ + 
+++ + 


+ 

+++ 

++ 
+++ 

++ 

++ 
+++++ 


tr. 

tr. 
tr. 
tr. 
tr. 
tr. 


ti 




- 


- 


- 


_ 


- 


- 


- 


- 



TABLE XI— Continued 
Reversion from above 



B. coli No. 1. 
B. coli No. 2 
B. coli No. 3 
B. coli No. 4 
B. coli No. 5 
B. coli No. 6 
B. coli No. 7 
Sp. cholerae. 



_ 


_ 


si. tr. 


+ 


+ 


+ 


tr. 


tr. 


++ 


— 


— 


— 


— 


— 


tr. 


tr. 


— 


tr. 


- 


- 


si. tr 


+ 


+ 


+ 








— 


- 


si. tr. 


++ 


+ 


+ 








- 


- 


? 


+ 


+ 


+ 








- 


- 


+ 


+ 


+ 


+ 








- 


- 


tr. 


+ 


+ 


+ 
+ 


tr. 


tr. 


tr. 



+ 

si. tr. 



TABLE X— Continued 





CONTROL 


DEXTRIN 




1 


2 


3 


1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


B. coli No. 1 

B. coli No. 2 

B. coli No. 3 

B. coli No. 4 

B. coli No. 5 

B. coli No. 6 

B. coli No. 7 

Sp. cholerae 


+ 
++ 
++ + 
++ 
+++ 
+++ 
++ 


tr. 

++ 

++ 

++ 
+++ 
+++ 

++ 
++++ 


+ 

++ 

+++ 

++ 

+++ 

++ 

++ 

++++ + 


tr. 
tr. 
tr. 
tr. 
tr. 
tr. 
tr. 


tr. 
tr. 
tr. 

tr. 


tr. 


tr. 

tr. 

tr. 
tr. 


tr. 


tr. 


tr. 


tr. 


tr. 


7 



TABLE XI— Continued 
Reversion from above 



B. coli No. 1 
B. coli No. 2 
B. coli No. 3 
B. coli No. 4 
B. coli No. 5 
B. coli No. 6 
B. coli No. 7 
Sp. cholerae. 



_ 


_ 


_ 


_ 


+ * 


++* 


+ * 


+ * 


+ + * 


- 


- 


tr. 


— 


si. tr. 


tr. 


-1-* 


tr.* 


+ 


+ 


+ 


+ 


++ 


+ 


H- 








+ 


+ 


+ 


+ 


+ 


+ 








+ 


+ 


+ 


+ + 


+ 


+ 








+ 


+ 


+ 


+ + 


+ + 


+ 








+ 


+ 


+ 


++ 


+ 


+ 








- 


- 


si. tr. 


si. tr. 


si. tr. 


tr. 


tr. 


tr.t 


+ 



402 



M. R. SMIRNOW 



TABLE X— Continued 





CONTROL 


MANNITE 




1 


2 


3 


1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


B. coli No. 1... 
B. coli No. 2.... 
B. coli No. 3.... 
B. coli No. 4... 
B. coli No. 5... 
B. coli No. 6... 
B. coli No. 7.... 
Sp. cholerae. . . . 


+ 

++ 
+++ 

++ 
+++ 
+++ 

++ 
+++++ 


tr. 

++ 

++ 

++ 
+++ 
+++ 

++ 
++++ 


+ 

++ 

+++ 

+ + 

++ + 

++ 

+ + 

+++++ 


tr. 
tr. 
tr. 
tr. 
tr. 
tr. 
tr. 
+ 


tr. 
tr. 
tr. 
tr. 
tr. 
tr. 
tr. 




} 

} 


- 


- 


- 


- 


- 


- 


- 









TABLE 


XI— Continued 














Reversion from above 








B. coli No. 1.... 








_ 


_ 


_ 


_ 


_ 


_ 


_ 


tr. 


— 


- 


B. coli No. 2.... 








- 


- 


+ 


+ 


+ 


+ 


tr.* 


++ 


+ 


+ 


B. coli No. 3.... 








- 


- 


+ 


+ 


+ 


+ 










B. coli No. 4... 








— 


- 


+ 


+ 


+ 


+ 










B. coli No. 5.... 








- 


- 


sl.tr. 


+ 


+ 


+ 










B. coli No. 6... 








- 


- 


+ 


+ 


+ 


+ 










B. coli No. 7.... 








— 


— 


tr. 


+ 


+ 


+ 










Sp. cholerae 








tt 


tr.t 


+++t 


++t 


+++t 


+ t 


+ 


+ t 


+ t 


+ t 



A study of the accompanying tables will best convey to the 
reader the results obtained. It will be noted that, strain no. 
1 is the "weakest" member of the group, giving but httle 
indol in the controls and but traces in five of the ten sugar 
media used. Strains 2, 4 and 7 are readily changed by all 
sugars, with a rapid disappearance of indol formation. The 
most interesting results, however, were seen in strains 3, 5 and 6. 
These were affected similarly to nos. 2, 4 and 7 by all the sugars 
except saccharose, in which medium the indol reaction con- 
tinued in each case until the 18th transfer, at which time the 
experiment was discontinued. It was suspected that these 
latter were strains of B. coli communis, and since B. communis 
normally does not ferment saccharose with the production of 
gas, possibly not affected by this carbohydrate. Inoculating 
the original agar culture of nos. 3, 5 and 6 into saccharose 
media proved this supposition to be correct. At the time of 
the seventh transfer, the amount of indol produced by these 
three strains was markedly less than at the beginning of the ex- 
periment. This, coupled with the fact that the bacteria were 



BIOLOGICAL VARIATIONS OF BACTERIA 403 

subjected to saccharose for over forty days, suggested the pos- 
sibihty that they had acquired the function of fermenting sac- 
charose, and that the resultant glucose, in its turn, had its effect 
upon the indol production as evidenced in the other experiments. 
Planting these organisms after the seventh subculture into 
saccharose agar proved this to be the case, as each of the strains, 
3, 5 and 6, now fermented saccharose with the production of 
gas to a moderate degree. 

The writer desires to emphasize at this juncture what appears 
to him a certainty, namely, that it is the carbohydrate per se 
that causes the fluctuating biological modification noted in 
those experiments, though a definite modus operandi is not 
known. This is well seen in the action of saccharose which 
had but slight effect upon three of seven strains of B. coli until 
the organisms were capable of splitting it into glucose, which, 
in its turn, effected the amount of indol produced. The fact 
that the inhibition was not complete, does not contradict this 
supposition, for it can be accounted for either by the presence 
of individual bacteria that remained unaltered by virtue of 
their greater resistance, or by the fact that the attack on sac- 
charose was sufficiently slow to permit a small amount of pro- 
teolytic cleavage. Further proof of such effects of carbohydrates 
will be offered below. 

The cholera spirillum, though it produced a large amount of 
indol in the control, readily succumbed to the action of the 
various carbohydrates, and, in several instances, even sooner 
than the B. coli. The fact that this strain of cholera spirillum 
produced but very little acid would indicate that the inhibitory 
action was not due to acid accumulation. The possible effect 
of excess acid is likewise shown to be negligible by the results 
obtamed in the carbohydrate media, wherein, as may be seen 
by consulting the tables, the differences were so slight that they 
cannot be regarded as having any bearing. 

It is of interest to note the differences in the action of the 
various carbohydrates. Saccharose has already been commented 
upon. Galactose, it appears, had the most pronounced effect 
upon all of the organisms, permitting but a single trace of indol 



404 M. R. SMIRNOW 

in five of the eight organisms in the first culture tube, and prac- 
tically none thereafter. Dextrin, on the other hand, showed 
the greatest variation in its effect upon the different strains 
of B. coll. Strain no. 1 gave no indol throughout the experiment; 
both nos. 5 and 6 gave a single trace in the first culture but none 
thereafter, whereas no. 7 gave a trace in each sub-culture up 
to the tenth, at which time the experiment was discontinued. 
Maltose and mannite appear to have a more pronounced in- 
hibitory effect upon indol formation than either glucose, lactose 
or saccharose and would stand intermediate between them and 
galactose. 

Experiments were undertaken to determine the permanency 
of this change in biological activities using the same technique 
as previously described. The bacteria, after being carried 
through the carbohydrate peptone media for at least seven 
transfers, were then inoculated into plain peptone, making suc- 
cessive transfers until three consecutive positive indol tests 
were obtained. 

By referring to Table XI, the most striking difference is seen 
between the action of glucose and dextrin. In the case of glu- 
cose the indol reaction reappeared only in the cases of B. coli nos. 
1, 2, 5 and 7, and the cholera spirillum, the others remaining 
negative up to the tenth transfer in plain peptone, at which 
time the experiment was discontinued. For some unexplained 
reason, the re-appearance of the property of indol formation 
was slower and less marked in this set of experiments than on 
previous occasions. When contrasted with the action of dex- 
trin, a marked difference is seen. This substance permits a 
prompt return of the indol producing property in the first trans- 
fer of the different strains of bacteria used, with the exception 
of B. coli nos. 1 and 2, and the cholera spirillum. Inasmuch 
as this peculiarity was evident in each of the sugars, it would 
appear that it was not due to the carbohydrate, but to a pos- 
sible greater susceptibility on the part of these strains of bac- 
teria. B. coli nos. 3, 4, 5, 6 and 7 gave positive indol tests on 
the third or fourth transfers quite uniformly in all sugars except 
glucose as already mentioned. It may be concluded that dex- 
trin acts entirely unlike the other carbohydrates, having, in 



BIOLOGICAL VARIATIONS OF BACTERIA 405 

the first place, a somewhat selective action on the different 
strains of B. coli, and secondly, permitting an immediate re- 
turn to normal proteolytic activity, under the conditions of 
experimentation here outlined. 

Aside from the peculiar manner in which B. coli nos. 1 and 2 
and the cholera spirillum acted on reversion in contrast to the 
other strains, there is another observation worthy of mention. 
It was noted that frequently in doing the Salkowski test with 
these colon strains, a positive reaction occurred without the 
addition of nitrite. This is rarely found with the B. coli and had 
not been noticed in any of the control cultures nor m the previous 
experiments. This pecuharity of the test was quite irregular 
in its occurrence, it appeared as if at will, and disappeared 
and re-appeared without any apparent reason, since the con- 
ditions of the experiment were unaltered. The reverse was 
often noted with the cholera spirillum, in which case, instead 
of obtainmg a positive mdol test on the addition of acid alone, 
none occurred until nitrite was added. This observation would 
indicate some changes in the power of nitrite formation entirely 
independent of that of indol production, and would well fit 
in with the carbohydrate effect upon the other biological activi- 
ties already mentioned. 

In order further to ascertain the action of the various sugars 
on the biological activities of the different strains of bacteria 
in question, a proteid-free medium was used as a base, to which 
3 per cent of the different sugars was added. Those used were 
glucose, lactose, saccharose, maltose, and dextrin. Inocula- 
tions were made from the same stock cultures of B. coli as in 
the above experiments, a control of each strain being carried 
in the proteid-free medium itself. The medium selected was 
the writer's modification of Naegeli's proteid-free medium and 
had the following composition: 

Ammonium tartrate ^ 

Potassium phosphate 

Magnesium sulphate 

Calcium chloride ^ 

Glycerin ^^-^ 

Water ' 1000 



406 M. R. SMIRNOW 

The medium base was made up in moderate quantities, steril- 
ized in the autoclave and kept until needed. The sugars were 
added to separate small quantities as required, tubed and steril- 
ized by the intermittent method. Inoculations were made 
every fourth or fifth day over a period of fifteen weeks, making 
a total of twenty-six transfers. Tests were made from the last 
culture tube to determine biological variations by planting into 
the various laboratory media, final observations being noted 
after seven days inoculation. 

This series of experiments was not as successful as the writer 
desired. Many of the strains suddenly refused to grow, and 
were lost at different stages of the experiment. This can be 
explained as due either to a lack of proper nutrition, or, possibly, 
to an inhibitory action of the sugars, or both. "Sudden death" 
was also noted by Peckham (1897) in her experiments with 
B. coll grown in glucose peptone, and is accounted for by her 
as due to the accumulation of by-products coupled with the 
complete exhaustion of the bacteria. She believes that on ac- 
count of incomplete proteolytic activities, through the prefer- 
ence for the carbohydrate food, there is a deficiency in plastic 
material of the bacteria sufficient to interfere with reproduction 
and building up of the cells. Besides the loss of a number of 
strains, several were found to be contaminated and had to be 
discarded. 

Though but five cultures were subjected to the carbohydrate 
action, and two controls were carried through to the completion 
of the experiment, the writer beheves the results worthy of 
record, particularly since they verify the results obtained in 
the work above reported. This is also desirable since the in- 
tent of these experiments was to show the ability of the controls 
to retain their various biological characteristics even though 
subjected to carbohydrates.^ 

Table XII indicates the results obtained. Strains 3 and 6, 
subjected to the action of glucose in a proteid-free medium, 
seem to compare well with the results obtained with glucose 

2 These experiments are being repeated, and will again be reported on at a 
later date. 



oq 






,::S S 



fi is 



5l 
?5- e 



s S 



lOONI 


+ 1 1 1 rs I 


•a 

o 

K 
El 




+ 4- + 

+ 1 1 1 1 + 1 
+ + + 


."2 

< 


+ + + 


s 


C3 

o 


+ 1 + 1 1+ 1 




+ + + 1 ++ 1 


X 

» 
Q 




+ 1 + I ++ 1 




+ 1 + 1 ++ 1 


s 


a 


+ 1 + 1 ++ 1 


IS 


±++ 1 +±+ 


o 

< 

\ 




1 1 j; 1 + 1 1 


!2 


+++ 1 ++ 1 


to 
O 


O 


+ 1 + 1 + 1 1 


"2 
'3 

< 


+ 4- + + 
t 1 T 1 ++ 1 

+ + + + 


m 
O 

o 
D 

o 




+ ' + '++' 


-a 
'3 




s 


o 


+ + , + 
+ 1 + 1 1+ 1 
+ + + + 


r2 


+ + , + 
+++ 1 t+ 1 
+ + + + 


o 

H 
O 
Pi 


Q 


+ 1 + 1 ++ 1 
4: ' + ' ++ ' 


a 

M 


+ 1 + 1 ++ 1 
+ ' + ' +J ' 


•ON NIVHXS 


00 CO CO 01 lO 1-H l-H 






; ; • • • 

. to ; ; 

. . . . . • 

' — ^ 0) 0) *- ' — ^ tt> 
CC 03 03 Cj IB 

h; ^ H; 
C 3 3 f3 "TS 



407 



408 M. R. SMIRNOW 

broth media. B. coli no. 3 is markedly changed from its control, 
which itself appears weaker in its cultural characteristics than 
controls carried in broth. The changes in the other strains 
need but little comment. When compared with the glucose 
action as seen in the previous tables, a similar action may be 
noted here. 

Too much stress cannot be placed upon so few experiments 
with proteid-free media, but the writer feels confident that in- 
hibitory action of other sugars than glucose does occur, and 
can readily be demonstrated. It may be well in passing, to 
note that indol production is invariably completely suppressed. 
Next to indol, the greatest amount of inhibition is manifest upon 
gas production, then, on lab enzyme and the characteristic 
growth on potato and finally on acid production. This sequence 
is not always adhered to, but holds good in a general way. All 
of these activities seem to correspond very well indeed with 
the action of glucose in broth media. 

Peckham in a series of experiments along similar lines prefers 
to regard the changes as not. due to any direct carbohydrate, or 
chemical effect, but rather to what she terms exhaustion, sheer 
inability to produce normal biological effects on account of 
previously expended energy. She describes numerous experi- 
ments carried out with a number of strains of B. coli, in which 
she claims that proteolytic activities were suspended when these 
organisms were grown in fresh peptone sugar broth. The index 
she took for determining proteolysis was the amount of indol 
produced by those subjected to carbohydrate as compared to 
control cultures. She concluded that this suppression of pro- 
teolytic activities was due to the preference of the B. coli for 
carbohydrate, a more readily assimilated food, to proteins, 
with a subsequent exhaustion on the part of the bacteria by the 
time they reached the protein material. That this "exhaustion 
of energy" was not due to the amount of lactic acid produced 
was proven by her in another series of tests in which quantities 
of lactic acid were added to similar peptone solutions without 
carbohydrate, and in which indol was produced as in the control 
cultiu*es. 



BIOLOGICAL VARIATIONS OF BACTERIA 409 

This preference for carbohydrate food to the exclusion of 
proteolysis has also been shown by other investigators to occur 
both with B. coli and numerous other bacteria, but in the opinion 
of the writer there is still an open question in regard to the 
modus operandi, namely, whether it is due to sheer exhaustion, 
or to a certain yet unexplained carbohydrate effect upon the 
metaboHc activities of the bacteria. 

Peckham did not return the B. coli into plain peptone to de- 
termine whether the absence of indol was due to a simple sus- 
pension or inhibition of proteolytic activities, or whether this 
inhibition was a pronounced one extending over a period of 
time. Had she done so, she would probably have found, as 
did the writer, that this inhibition was a decided one, not readily 
overcome, that it varied with different strains of B. coli, and 
also varied with the carbohydrate used. In the Hght of the 
experiments here reported the writer feels quite convinced that 
exhaustion, though it may be given a certain amount of credit, 
cannot explain all of his findmgs. This contention is empha- 
sized by: (1) The different effects obtamed on different strains of 
B. coli. (2) Differences in effect by the various sugars, dextrin act- 
ing much more readily, and permitting an immediate return of 
indol formation, whereas in the case of glucose, the carbohydrate 
effect was more gradual and more lasting. (3) The fact that 
in an occasional experiment, the organism would not revert to 
its original type, but remam permanently changed in some or 
even all of its biological characteristics. (4) Differences in 
the quantity, and time of disappearance or reappearance of 
the various enzyme activities, with no special sequence of events. 
(5) Exhaustion, though in spite of the above factors it might 
still be regarded as being the cause in regard to indol, cannot 
explain the more gradual loss of lab enzyme, fermentation of 
carbohydrates with the production of gas, the lack of typical 
growth on potato and finally loss of acid production. If we 
were still to insist that it is a matter of exhaustion, we must 
assume that it was so profound that it could have been handed 
down from one culture to another over certain periods of time 
as evidenced in the experiments for reversion. This would 



410 M. R. SMIRNOW 

be contrary to any existing ideas in respect to the effects of ex- 
haustion and would intimate that exhaustion can be inherited. 
The following conclusions may be drawn from the foregoing 
experiments with B. coli. 

1. Both glucose and phenol give rise to either partial or com- 
plete inhibition of the cultural characteristics of some strains 
of B. coli. Sodium chloride and sodium sulphate also display 
inhibitory action but by no means as marked as either of the 
above compounds. 

2. When complete, the change is more lasting, but there is 
always present a strong tendency for the modified bacteria to 
return to their former status of biological activities. 

3. Occasionally, complete reversion does not take place, in 
which case the organism remains permanently devoid of certain 
enzjrmes or of the power of fermenting one or more of the carbo- 
hydrates. 

4. There is no well defined nor constant sequence of events 
either during the process of modification or reversion, and no 
relationship between the changes produced in the various en- 
zymes, but, it may be stated, in a general way, that indol pro- 
duction is the first to disappear, then, the fermentations of the 
various carbohydrates, the characteristic growth on potato, 
lab enzyme, and finally, acid production. 

In closing, the writer desires to take this opportunity of ex- 
pressing his sincerest thanks to his students, Messrs. Bingaman, 
Braude, Denehey, Nachamofsky, Rubinsky, Russo, and Miss 
Wright, for their kind cooperation in the above work. 

BIBLIOGRAPHY 

BuRK 1908 Arch, fiir Hygiene, 65, 235. 

Herter 1910 Jour. Biol. Chem., 7, 59. 

Manfredi 1889 Ref. by Burton, Macfayden, Royal Soc, 46, 542. 

Massini 1907 Arch, fur Hygiene, 61, p. 250. 

Peckham 1897 Jour. Exp. Med., 2, 549. 

Penfold 1911 Brit. Med. Jour., 1911, Suppl. 2, 363. 

TwoRT 1907 Proc. Royal Soc, Lond., 79, Sec. B, 329. 



A NEW CULTURE MEDIUM FOR THE TUBERCLE 

BACILLUS 

WM. WHITRIDGE WILLIAMS and WARD BURDICK 

(From the Laboratory of the National Jewish Hospital for Consumptives, Denver, 

Colorado 

Contamination is an occurrence which has always troubled 
bacteriologists in the isolation and cultivation of the tubercle 
bacillus. Even the various digestive methods which are used 
at present, such as the antiformin, the sodium carbonate, and 
the sodium hydroxid methods, have failed to yield uniformly sat- 
isfactory results. It is obvious that the ideal culture medium 
for this purpose would be one which included some substance 
that had the property of preventing the growth of other micro- 
organisms while not interfering with the isolation and repro- 
duction of the tubercle bacillus. 

The observation of v. Drigalski and Conradi (1902), that 
crystal violet was able to inhibit the growth of many bacteria 
but had no effect on the cultivation of typhoid and colon baciUi 
was the first step in this direction. Then followed the splendid 
researches of Churchman (1912) who worked with a number 
of common dyes and discovered that gentian violet possessed 
a striking selective power. He found that by making divided 
plates, one-half filled with plain agar and the other filled with 
agar to which 0.001 per cent gentian violet was added, and 
stroking the surface with a mixed culture, he could get rid of 
the contaminating organisms. For instance, by this means he 
purified a culture of B. tuberculosis which had become contami- 
nated with B. suUilis. The former organism is ''gentian-nega- 
tive" while the latter, like almost all air contaminations, is 
''gentian-positive." He states that the effect of gentian violet 
on gentian-positive organisms is better described as bacteriostatic, 
rather than bactericidal, meaning that the dye suspends repro- 

411 



412 W. W. WILLIAMS AND W. BURDICK 

duction without implying that the organisms are necessarily 
killed. He finds that this gentian reaction is much more definite 
and constant than the Gram stain. 

Next came the attempt of Petroff (1915) to devise a simple, 
practical and rehable method for the isolation and cultivation 
of the tubercle bacillus from the sputum and feces. He experi- 
mented with gentian violet, methyl violet, methylene blue, 
crystal violet, and fuchsin added to a meat-juice-glycerin -egg 
medium. He found that gentian violet was the most favorable 
stain on account of its inhibitory action on many organisms and 
reported that he obtained sixty-nine positive cultures from sixty- 
nine specimens of sputum from practically all stages of tuber- 
culosis. Six of these specimens were negative by direct micro- 
scopic examination. 

Shortly after the publication of Petroff's article, we began 
the use of his method. We were soon impressed with the in- 
hibitory effect of the sodium hydroxid and gentian violet which 
he used, but not with the medium as a whole. It did not seem 
to contain enough moisture to prevent rapid drying; it required 
inspissation on three successive days, with the result that fre- 
quently an uneven surface was obtained in spite of the utmost 
care; also, it occasionally became contaminated with spore 
bearing organisms which would usually cause liquefaction of 
the medium with consequent destruction of hopeful cultures. 

It seemed to us that if a gentian violet medium could be made 
which did not require inspissation, gave a smooth surface, con- 
tained sufficient moisture, and could be sterilized in an auto- 
clave, we should have a nearly ideal culture medium. 

While working with the medium devised by Besredka (1913) 
for growing tubercle bacilli to make his tuberculin, which serves 
as a good antigen in tuberculosis complement fixation tests, 
it occurred to us to utilize it in an attempt to overcome the 
defects of Petroff's medium. We believe we have accomplished 
this by making a medium as here described. 



CULTURE MEDIUM FOR TUBERCLE BACILLUS 413 
PREPARATION OF THE MEDIUM 

1. Egg-white solution. This is made by diluting the egg- 
white with ten parts of distilled water and thoroughly shaking. 
The fluid is opalescent and contains numerous whitish flakes. 
To clear it, it is passed through a thin layer of cotton and then 
heated to 100°C. to hasten precipitation. It is then filtered 
through paper. 

2. Egg-yolk solution. The yolks are diluted with ten parts 
of water and well stirred. The very cloudy emulsion is clarified 
by adding normal sodium hydroxid. Too much hydroxid is 
harmful and therefore complete solution of the yolk is not de- 
sirable. The emulsion should be slightly turbid. To attain 
the proper degree of turbidity, one cubic centimeter of normal 
sodium hydroxid is usually added to each one hundred cubic 
centimeters of the emulsion. This is not a constant amount, 
however, because some yolks wifl be completely dissolved by 
less than half this amount of alkali. The solution is heated to 
100°C. and filtered. 

3. Meat infusion. Five hundred grams of finely chopped 
lean veal are covered with one Hter of water containing 15 per 
cent of glycerin, allowed to infuse for twenty-four hours and 
filtered; 5 grams of sodium chlorid are added, and the infusion 
heated to boiHng. It is again filtered and then rendered plus 
1 per cent alkaline. 

With the above solutions, the medium is made as foHows: 
Place 300 cc. of the 10 per cent egg-white solution in a hter 
flask; 300 cc. of the 10 per cent egg-yolk solution in another flask; 
and 400 cc. of the meat infusion, to which is added 15 grams of 
powdered agar-agar, in a third flask. These are then sterihzed 
in the autoclave at 15 pounds pressure for fifteen minutes. They 
are removed from the steriHzer and, while hot, 1 cc. of a 1 per 
cent alcohohc solution of gentian violet is added to the broth- 
agar. The contents of this flask are now poured into that con- 
taining the egg-white and then the egg-yolk is added. The 
whole is poured back and forth from this flask to another so 
as to insure thorough mixing and then it is tubed and slanted. 



414 W. W. WILLIAMS AND W. BURDICK 

The tubes are left in their slanted position for about seventy- 
two hours at room temperature until the contents are well set. 
The cotton plugs are then trimmed and flamed and the tubes 
sealed with corks. This medium presents the same smooth 
inoculating surface as ordinary agar slants, contains as much 
moisture, is quickly made and is rendered absolutely sterile. 

METHOD OF ISOLATING TUBERCLE BACILLI FROM SPUTUM 

About 10 cc. of fresh sputum, which has been thoroughly 
washed in a ininning stream of sterile 0.85 per cent salt solution, 
is placed in a sufficiently large centrifuge tube containing a 
piece of blue litmus paper. An equal amount of 3 per cent 
sodium hydroxid is added and the whole well shaken. It is 
put in the incubator for about one hour or until the sputum is 
fairly well digested. The mixture is neutralized with normal 
hydrochloric acid, then centrifugalized, and after removing the 
supernatant fluid the sediment is planted on several tubes con- 
taining the herewith described medium by means of a large 
platinum loop or a capillary pipette. After from five to four- 
teen days incubation, a good growth appears which is free of 
any contamination. 

This method has given us uniformly good results and the 
medium remains serviceable for at least one month. 

REFERENCES 

Besredka, a. 1913 Compt. rend. Acad. d. sc, 156, 1633. 
Churchman, John W. 1912 Jour. Exper. Med., 16, 221. 
V. Drigalski and Conradi. 1902 Ztschr. f. Hyg., 39. 283. 
Petroff, S. A. 1915 Jour. Exper. Med., 21, 38. 



BACILLUS ABORTUS (BANG) AS AN ETIOLOGICAL 
FACTOR IN INFECTIOUS ABORTION IN SWINE 

EDWIN S. GOOD and WALLACE V. SMITH 

From the Laboratory of the Department of Animal Husbandry, Kentucky Agricul- 
tural Experiment Station, Lexington, Kentucky 

So far as we are able to learn from the literature, the cause 
of infectious abortion in swine has never been determined. Lynch 
in his "Diseases of Swine," says, "Infectious abortion results 
from the infection of the genital passages by some specific germ, 
the true nature of which is as yet undetermined. The disease 
is not nearly so common as in mares and cows, and, while it may 
run through an entire herd, it is not likely to be spread from 
one farm to another except in unusual instances." He fur- 
ther states, "The infectious type of the disease is especially 
mild in its sjonptoms, and unless the animals are carefully 
watched the pigs may be slipped without any notice of the fact 
until several weeks later, when it is found that the sow is no 
longer pregnant." He also says, "The nearer to full term the 
sow is at the time of abortion, the less dangerous the occurrence 
and the more mild the symptoms. Signs of threatening abortion 
are loss of appetite, restlessness, making of the bed, shivering, 
trembling of the muscles, dulness, and in some cases very severe 
labor-pains." 

While considerable work has been done with regard to in- 
fectious abortion in mares and cows in this laboratory, only 
three outbreaks of infectious abortion among sows, have come 
to our investigation. We have found that it is no uncommon 
occurrence for one or two sows in a herd to abort. Numerous 
cases of this kind have been studied by the writers, with no 
etiological results, which would lead to the opinion that such 
abortions were due to some accident. 

415 



416 EDWIN S. GOOD AND WALLACE V. SMITH 

In the lai^e outbreak investigated by the writers early this year, 
some twenty sows aborted in rather quick succession. On \dsit- 
ing the place, it was found that one sow had aborted the night 
before, and two of the fetuses together with the attached after- 
birth were brought to the laboratory for examination. The 
fetuses were fairly well developed, although not far enough along 
to have any hair. On one of the afterbirths there were noted 
numerous brownish, villus-like projections ranging in size from 
1 to 3 mm. in diameter. They were so numerous in some places 
as to become confluent. On opening these, a dark serous fluid 
was noted. In our investigations upon the etiology of infectious 
abortion of animals, streak dilutions on agar or agar serum are 
always made and incubated under aerobic, Nowak, and anaerobic 
conditions. In this instance, streak dilutions were made on a 
series of agar plates, the agar being slightly alkaUne to phenol- 
phthalein, with material from the nodules on the afterbirth, from 
bits of the afterbirth from both pigs, and from the contents of 
the umbilicus, heart, liver, stomach and kidney of each fetus. 
From appearances, the kidneys of the pigs were very much 
enlarged and gorged with blood. As these pigs had lain on straw 
for a few hours, several of the plates showed on incubation in 
the air the growth of a considerable number of contaminating 
organisms, such as B. coli, B. subtilis, etc. This, of course is 
what would be expected on plating the afterbirth as the material 
had lain on straw for several hours before being secured by us. 
Cultures from the internal organs of the fetuses, however, were 
nearly sterile. The clear places on the plates were marked with 
India ink, after which they were subjected to the cultural method 
of Nowak. 1 The material was incubated at 37°C. for four days 
and removed from the jars. On casual examination of the petri 
dishes, we were led to beheve that they contained nothing but the 
growth of bacteria usually encountered in plating material which 
had lain on the ground for some hours. Upon examining one 
of the plates carefully with a Coddington lens, however, there 
were noted in some of the clear spaces of the dishes a few very 

1 E. S. Good, Investigations of the Etiology of Infections Abortion of Cows 
and Mares, Bull. No. 165, Ky. Agri. Exper. Sta., 1912, p. 249. 



INFECTIOUS ABORTION IN SWINE 417 

small dew drop like colonies, which on being examined with the 
microscope resembled in every respect colonies of the B. abortus 
of Bang. Examined by the aid of the hand lens they were nearly 
water clear to direct light and of bluish tint to reflected light. 
They were round and raised, with exceptionally well defined 
borders. Most of these minute dew drop colonies were homo- 
geneous, with the exception that a few of them had a few granules 
in the center, typical of many colonies produced by the B. abortus. 
Viewing the larger colonies under the microscope, it was seen 
that the centers had assumed a granular consistency, while the 
outer portion was homogeneous and transparent. The colonies 
on some of the plates were so large that they might have been 
taken for some other species, measuring If mm. in diameter. 
These conformed, however, to similar colonies of B. abortus 
derived from the tissues of the cow. To reflected light they had 
assumed an amber color, the centers having a whitish appearance. 
On examining stained preparations of these colonies, the mor- 
phology of the organism was identical, so far as we could deter- 
mine, with B. abortus. On examining the plates carefully with 
a Coddington lens and microscope, colonies resembling those 
mentioned and measuring from a pinpoint to 1.5 mm. in diameter 
were seen on streaks made from the blood of the umbihcus, on 
the streaks made from the small nodule-like growths on one of 
the afterbirths, and from the afterbirth and internal organs 
of the fetuses. Eighty-four colonies in all were counted on 
streaks made from one of the small nodules. Some of these colonies 
were so small that they could not have been seen with the naked 
eye. There were one or two colonies on the streaks made from 
the livers. The plates streaked with the amniotic fluid were 
almost completely covered with contaminating bacteria, but, 
in the clear spaces five dew drop colonies were noted. The streaks 
of the contents of the stomachs of both pigs showed numerous 
small dew drop colonies. Streaks from the kidneys were negative. 
Stained preparations had been made from the different organs 
mentioned but were not examined until the cultures had de- 
veloped. Upon examining these preparations, germs identical 
with the Bacillus abortus (Bang) were seen in large numbers in 
those made from the stomach contents of the pigs. 



418 EDWIN S. GOOD AND WALLACE V. SMITH 

Typical colonies were streaked on agar slants, some of which 
were incubated by the Nowak method and others in the air. 
At the end of twenty-four hours, no visible growth was noted 
on the streak cultures incubated in the air. At the end of forty- 
eight hours, however, some little growth could be detected even 
with the naked eye, and at the end of seventy-two hours, quite 
a luxuriant growth of the organism was obtained, in appearance 
identical with streak cultures made with the B. abortus. On 
examining the tubes which had been incubated under the Nowak 
method, we found that they had made but slightly better growth 
than those incubated in the air. Streaks on agar at room tem- 
perature showed no growth in the time mentioned. We came 
to the conclusion that if this organism was the B. abortus and grew 
in the air after the first generation it was different from any we 
had ever isolated. The organism responded to the following 
tests in the following manner: It was found to be non-motile; 
gram negative; did not produce gas in either lactose or glucose; 
did not coagulate milk; grew readily in plain bouillon, showing 
a fair degree of cloudiness at the end of seventy-two hours; 
and did not liquefy gelatin. Serum-agar tubes heavily inoculated 
with this organism and quickly solidified in ice water and in- 
cubated in the air, developed a growth characteristic of the B. 
abortus, as noted by this laboratory, in that a narrow ring of 
growth appeared as a slight haze 3 mm. beneath the surface of 
the medium at the end of sixty hours, and eventually extended 
to the top of the medium. All the above tests conform to the 
biological and cultural characteristics of the Bang bacillus. 

We were not, however, satisfied that the organism isolated 
was the Bacillus abortus on account of its growing in the air 
so readily, so we subjected the culture to the agglutination and 
complement fixation tests, using the serum from a rabbit made 
immune to the Bang bacillus, which agglutinated in high dilu- 
tions. It was found that this serum agglutinated our organism 
in a dilution of 1:1200, which was exactly the same dilution in 
which the serum agglutinated an antigen made of a well known 
strain of the B. abortus, which had been obtained from an abort- 
ing cow. Using as an antigen the organism isolated from the 



INFECTIOUS ABORTION IN SWINE 419 

SOWS and the immune serum mentioned, the complement was 
completely fixed with 0.02 cc. of serum. We were thus convinced 
that this organism was identical with the germ that produces 
abortion in the cow, the only difference being that it grew readily 
in the air after the first generation, while the cultures we have 
derived from the cow usually do not grow in the air until a ter 
being cultured for several generations by the Nowak method. 
An exception to this rule, was discovered by Dr. Frank M. Sur- 
face,2 who accidently inoculated a cow with a culture of the 
Bang bacillus which had repeatedly been transplanted for some 
two years from agar to agar, or from agar to plain peptone broth, 
and then back to agar. He had obtained this particular strain 
while in Denmark. The cow injected, aborted and Surface 
isolated the organism, the first generation of which developed in 
the air. He was able to determine definitely, by using this or- 
ganism as an antigen in the complement fixation test with an 
immune serum, that it was the Bang bacillus. Surface states 
that the growth obtained in a Novy jar (Nowak method) was 
in no respect better than that obtained in the free air. 

We cannot state whether the organism isolated from the abort- 
ing sow would have grown directly from the tissues, as we cultured 
none of the material in that way for any length of time. Upon 
re-culturing the original material, which had been kept in the 
ice box, we found that the contaminating bacteria had become 
so numerous as to make streak dilutions impossible. 

INOCULATION EXPERIMENTS WITH THE ORGANISM OBTAINED 
FROM THE ABORTING SOW 

To test this organism further, a streak culture on an agar 
slant was washed with 5 cc. of physiological salt solution and 
2 cc. of this material was diluted in 3 cc. of normal salt solu- 
tion and injected intravenously into a pregnant sow, no. 1, on 
February 25, 1916. On March 13, seventeen days after the 
inoculation, this sow aborted five fetuses. The only symptom 

2 Surface, F. M., A Note on the Maintenance of Virulence by Bacillus Abor- 
tus Bang, Journal of Infectious Diseases, 1913, 12, p. 359. 



420 EDWIN S. GOOD AND WALLACE V. SMITH 

shown by this sow before aborting was that she did a great 
deal of rooting a day or two before slipping her pigs. As soon as 
she aborted she ceased rooting. The aborted fetuses, while 
quite well developed, were not haired over. Stained slides were 
made from the contents of the different organs. Streak dilutions 
were made of the heart, liver and stomach contents of each pig, as 
well as of the afterbirth, on 2 per cent agar poured in petri dishes 
and solidified. Some of the dishes were incubated in the air, 
while others were cultured according to Nowak. These fetuses 
were numbered 1 to 5. The organism with which this sow was 
injected was obtained from the heart, liver and stomach of pig 
no. 1; from the heart of pig no. 2; from the heart, liver and stom- 
ach of pig no. 3; from the heart and liver of pig no. 4; and from 
the heart and stomach of pig no. 5. Streak dilutions of these 
organs grown in the air showed no growth at the end of twenty- 
four hours. After forty-eight hours, however, the growth was 
distinctly visible to the naked eye, and at the end of seventy- 
two hours it was abundant. The streak dilutions grown under 
diminished oxygen (Nowak method) showed no more growth 
than that obtained in the air. Upon microscopical examination 
of the contents of the stomachs of pigs no. 1 to 5, it was seen that 
these organs harbored the germs in exceedingly large numbers. 
On February 29, 1916, a pregnant sow (no. 2) was fed in ship- 
stuff the growth of the organism, obtained from the aborting 
sow, on two large agar slants washed off with 40 cc. of sterile 
normal salt solution. This sow was kept in an inclosure separate 
from sow no. 1. On March 10, 1916, she received the contents 
of five small agar tubes in a similar feed. On March 17, the 
attendant informed us that this sow was going to abort because 
she was acting like the other sow, previous to aborting, in vigor- 
ously rooting the ground floor of her pen. On March 19, nine- 
teen days after being fed the initial dose of the organism, the 
sow aborted. We obtained three of the pigs. She had eaten 
the afterbirth, and in all probability had also eaten some of the 
pigs, as she had bitten out a large piece from the side of one 
of the pigs secured. After a long series of dilutions, we were 
able to isolate the original organism from the stomach contents 



INFECTIOUS ABOKTION IN. SWINE 421 

of one of these pigs. The bacillus was present in this instance 
in very small numbers. 

On March 27, 1916, the tails of these sows were carefully 
washed, shaved and disinfected, the ends cut off, and 25 cc. of 
blood taken. The blood serum of each of these sows caused 
complete agglutination of a known culture of B. abortus Bang in 
a dilution of 1 :100, with 75 per cent agglutination in a dilution 
of 1:250, and the complement was completely fixed with 0.02 
cc. of the serum. Serum from a normal hog tested at the same 
time did not agglutinate the agglutinating fluid in any dilution, 
nor did it fix the complement. 

The slipping of the pigs produced no after effects upon the 
sows that we could notice. They will be kept under observa- 
tion for some time. 

Taking into consideration all the results mentioned in this 
paper, we may conclude that the Bacillus abortus (Bang) is an 
etiological factor in infectious abortion of sows. Whether or 
not it is the only etiological factor, will have to be determined 
by further investigations. This is the second time, so far as 
we know, that the Bacillus abortus has been associated naturally 
with aborting animals of a species other than the cow. The first 
was discovered by Dr. Surface^ when he found the disease epi- 
zootic among guinea pigs which were being reared in an inclosure 
in which inoculation experiments were being carried on with 
the Bang bacillus. Some of the Utter from the cages containing 
the inoculated pigs had gotten into the pens of breeding pigs 
and caused the spread of the disease. 

SUMMARY 

1. Epizootic infectious abortion occurs occasionally among 
sows, though not so frequently as among cows and mares. 

2. Previous to the time of this investigation, no etiological 
factor connected with the disease in the sow had been discovered. 

3. In this investigation the B. abortus of Bang, the organism 

5 F. M. Surface, Bovine Infectious Abortion Epizootic Among Guinea Pigs, 
Journal of Infectious Diseases, 1912, 11, no. 3, p. 464. 



422 EDWIN S. GOOD AND WALLACE V. SMITH 

causing the disease of infectious abortion in the cow, was isolated 
from the afterbirth of an aborting sow and from the contents of 
the umbihcus, heart, hver and stomach of two aborted fetuses. 

4. The strain of Bacillus abortus isolated from the sow responded 
to all the biological and physiological tests of the strains isolated 
from the uterine exudate of aborting cows by this laboratory, 
with the exception that the original culture grew in the air 
after the first generation. 

5. Pregnant sow no. 1, inoculated intravenously with 2 cc. 
of an agar slant culture of the bacillus secured from the abort- 
ing sow, washed off with 5 cc. of normal salt solution, aborted 
five fetuses seventeen days after the injection, and the organism 
was isolated from the afterbirth and internal organs of the fe- 
tuses. The bacillus in this instance grew directly from the tis- 
sues under strictly aerobic conditions. Pregnant sow no. 2, on 
being fed the organism derived from the aborting sow, aborted 
nineteen days afterwards. The sow ate the afterbirth and pre- 
sumably some of the pigs. The organism was secured from the 
stomach contents of one of the fetuses obtained. 

6. The blood serum of each of these sows, after aborting, com- 
pletely agglutinated a strain of Bacillus abortus (Bang) derived 
from an aborting cow, in a dilution of 1 : 100. The complement 
was fixed in each case with 0.02 cc. of the serum. The serum 
of a normal hog did not agglutinate in any dilution, nor did it 
fix the complement. 



THE RELATION OF PROTOZOA TO CERTAIN 
GROUPS OF SOIL BACTERIA^ 

T. L. HILLS 

From the Laboratory of Agricultural Bacteriology, University of Wisconsin 

INTRODUCTION 

The theory advanced by Russell and Hutchmson (1909; 1913) 
that protozoa are an important factor in limiting bacterial ac- 
tivity and consequent fertility in the soil has aroused no little 
interest. Their work has stimulated much investigation, some 
of the results of which seem to uphold their theory, while others 
do not substantiate it. 

It was thought that results of interest might be obtained by fur- 
ther studies concerning the effect of the protozoa on certain bio- 
logical processes of the soil — ammonification, nitrification and 
free nitrogen fixation. 

For this study soil cultures of Miami silt loam soil from the 
Experiment Station Farm were used. The moisture content was 
maintained at as near one-half saturation as possible. 

AMMONIFICATION 

In this work 300 gram portions of soil were placed in each of 
18 flasks. The flasks were of 500 cc. capacity, Erlenmeyer form, 
and rather narrow so that the soil was in approximately 2 inch 
layers. The proper amount of moisture was added, the flasks 
plugged, and sterihzed at 15 pounds pressure for two hours. 
This was found sufficient to kill all the bacteria. Upon coohng, 
one half of the flasks were inoculated, each with 2 cc. of a suspen- 

1 Presented at Seventeenth Annual Meeting of the Society of American Bac- 
teriologists, Urbana, 111., December 29, 1915. 

423 



424 



T. L. HILLS 



sion of normal soil known to contain protozoa. The remaining 
half were inoculated with the same amount of soil free from 
protozoa. The protozoa-free soil was obtained by sterilizing a 
portion of the Miami soil and inoculating it with as many kinds of 
bacteria as could be isolated by the plate method, using differ- 
ent kinds of media. The flasks were then incubated at room 
temperature (approximately 22° to 25°C) and the ammonia and 
nitrate content determined at the end of four, eight and twelve 
weeks. The ammonia was determined by distilling 100 grams 
of the soil with 10 grams of magnesium oxide and 250 cc, distilled 
water. The distillate was received into N/20 sulphuric acid 
and the excess acid remaining after the distillation was titrated 
with alkali of the same normality. The nitrate was determined 
by the phenolsulphonic acid method. 

The results of this work are given in the following table: 

TABLE 1 
Ammonia and nitrate in soils with and without protozoa 







NITROGEN PER 100 GRAMS DR\ 


SOIL 




TREATMENT 


Ammonia 


Nitrate 




After 
4 weeks 


After 
8 weeks 


After 
12 weeks 


After 
4 weeks 


After 
8 weeks 


After 
12 weeks 




mgm. 


mgm. 


mgm. 


mgm. 


mg m . 


vigm. 


f 


8.12 


4.48 


4.62 


4.50 


7.69 


10.00 


With protozoa ' 


7.70 
8.05 


4.69 
5.60 


4.90 
4.20 


4.50 
4.50 


7.69 
7.69 


10.00 
10.00 


' 


8.19 


8.26 


8.96 


3.60 


3.57 


3.57 


Without protozoa. . • 


8.10 
8.12 


8.19 
8.19 


8.96 

8.85 


3.60 
3.60 


3.57 
3.57 


3.57 
3.57 



From the data of table 1 it will be noted that in the soil contain- 
ing protozoa the ammonia decreased somewhat while in the 
soil free from protozoa it tended to increase to a slight extent. 
This may be explained by the fact that in the soil containing 
protozoa the nitrifying organisms were also present and func- 
tioning, whereas in the soil free from protozoa they were absent 
and thus the ammonia tended to accumulate. In the case of 
the nitrate formation, in the soil with protozoa the nitrate con- 



RELATION OF PROTOZOA TO SOIL BACTERIA 



425 



tent naturally increased since the nitrifying organisms were 
present and the ammonia as it was formed was oxidized to nitrites 
and further to nitrates. Where the soil contained no protozoa, 
the nitrate content remained practically unchanged because of 
the absence of the nitrifying bacteria. 

In order to show the correlation between ammonia formation 
and nitrate formation the following table giving the total ammonia 
and nitrate nitrogen was compiled. 

TABLE 2 
Total ammonia and nitrate nitrogen in soils with and without protozoa 



TREATMENT 



With protozoa 

Without protozoa. 



AMMONIA AND NITRATE NITROGEN PER 100 
GRAMS DRY SOIL 



After 4 wesks 



■nigm. 

12.62 

12.20 

12.55 

11.79 

11.70 

11.72 



After 8 weeks 



mgm. 

12.17 
12.38 
13.29 
11.83 
11.76 
11.76 



After 12 weeks 



mgm. 

14.62 
14.90 
14.20 
12.53 
12.53 
12.42 



From the summary data in table 2 it is very evident that the 
presence or absence of protozoa has very little effect on the sum 
total of ammonia and nitrate nitrogen. 

On account of the absence of the nitrifying bacteria in the 
protozoa-free soil it is hardly fair to draw very definite conclu- 
sions from these results. Therefore, other experiments were 
made, in which three sets of the same soil were used : (1) untreated, 
(2) heated to 90°C. for one hour and (3) heated to 90°C. for one 
hour and later reinoculated with 1 per cent of normal soil, thus 
introducing the nitrifying organisms and also the protozoa. 
These soils were incubated at room temperature and the am- 
monia and nitrate nitrogen determined every ten days for a 
period of thirty days. 

The following results were obtained. 



426 



T. L. HILLS 



TABLE 3 

Ammonia and nitrate in untreated, heated, and heated and reinoculated soils 







NITROGEN PER 100 GR.\MS DRK 


SOIL 




THBATMENT 


Ammonia 


Nitrate 




After 
10 days 


After 
20 days 


After 
30 days 


After 
10 days 


After 
20 days 


After 
30 days 


Untreated 


mgm. 

2.10 
8.96 

8.96 


mgm. 

1.82 
9.80 

10.08 


mgm. 

3.50 
11.75 

6.44 


mgm. 

4.63 
2.50 

2.96 


mgm. 

5.55 
2.51 

6.66 


mgm. 

9.80 


Heated 


3.03 


Heated and reinocu- 
lated 


8.33 







As expected the ammonia content in the untreated soil re- 
mained quite constant while the nitrate increased. In the heated 
soil where the nitrifying bacteria and the protozoa were absent, 
the ammonia increased and the nitrate content remained prac- 
tically the same. A part of the initial increase of ammonia 
in the heated and in the heated and reinoculated soils is undoubt- 
edly due to the heating alone. The bacteria surviving this 
treatment cause some increase in ammonia as seen by the re- 
sults obtained. But in the heated soil which was subsequently 
reinoculated with 1 per cent normal soil the ammonia decreased, 
due no doubt to its oxidation by the nitrifying bacteria, and the 
nitrate nitrogen increased. In the latter instance the protozoa 
were present, a factor which Russell and Hutchinson claim is 
detrimental to bacterial activity. The nitrifying bacteria were 
also present and active. 

The following table compiled in the same manner as table 2 

TABLE 4 

Total ammonia and nitrate nitrogen in the untreated, heated, and heated and 

reinoculated soils 



TREATMENT 


AMMONIA AND NITRATE NITROGEN PER 100 
GRAMS DRY SOIL 




After 10 days 


After 20 days 


After 30 days 




mgm. 

6.73 
11.46 
11.92 


jngm. 

7.37 
12.31 
16.74 


mgm. 

13.30 


Heated. 


14.79 


Heated and reinoculated 


14.77 







RELATION OF PROTOZOA TO SOIL BACTERIA 



427 



shows the total ammonia and nitrate nitrogen present in the 
untreated, heated, and heated and reinoculated soils. 

Here the introduction of the supposedly harmful factor, the 
protozoa, into the soil did not seem to have any depressing effect 
on the bacteria as far as their production of ammonia and sub- 
sequent oxidation of the same was concerned. 



NITRIFICATION 

Flask experiments were carried out somewhat similar to the 
ammonification tests, except that at the time of inoculation with 
soil containing protozoa and soil free from protozoa, the soil was 
also inoculated with cultures of Nitrosomonas and Nitrohacter 
free from ciUates and flagellates. The nitrifying organisms were 
obtained by inoculation and subsequent occasional transfer into 
media suitable to their growth and unfavorable to the growth of 
other organisms. After the inoculation of the sterilized soil as 
previously stated, ammonium sulphate was added in quantities 
equal to 20 mgm. of nitrogen as ammonia per 100 grams (dry 
weight) of the soil. The ammonia and nitrate content was de- 
termined at the end of fourteen and twenty-eight days. 

The figures in tables 5 and 6 show the results of these deter- 
minations. 

In the case of the soil with protozoa at fourteen days the aver- 
age ammonia content was approximately 18.6 mgm. and at 
twenty-eight days, 16.0 mgm. Where the protozoa were ab- 

TABLE 5 
Rate of conversion of ammonia to nitrate in soil with and without protozoa 



ANALYSES 



TBEATMENT 



With protozoa. . . 
With protozoa ... 
With protozoa. . . 
Without protozoa 
Without protozoa 
Without protozoa 



NITROGEN AS AMMONIA PER 100 
GRAMS DRY SOIL 



After 14 days After 28 days 



mgm. 

20.58 
19.02 
16.10 
21.14 
23.24 
20.16 



mgm. 

15.82 
15.96 
16.24 
19.32 
19.18 
19.32 



428 



T. L. HILLS 



TABLE 6 
Rate of nitrate formation in soil with and without protozoa 



ANALYSES 



TREATMENT 



With protozoa. . . 
With protozoa. . . 
With protozoa. . . 
Without protozoa 
Without protozoa 
Without protozoa 



NITROGEN AS NITRATE PER 100 
GRAMS DRY SOIL 



After 14 days After 28 daya 



mgm. 

9.90 
11.90 
11.76 
7.52 
6.57 
8.20 



mgm. 

13.88 

13.88 

13.51 

7.81 

8.06 

8.06 



sent the relative decrease in the amount of ammonia was about 
the same, 21.5 mgm. at fourteen days to 19.3 mgm. at twenty- 
eight days. There seemed to be practically no difference in 
the rate of conversion of ammonia into nitrate in the two soils. 

Where the rate of nitrate formation was determined the in- 
crease in nitrate formation seemed to be slightly in favor of 
the soil which contained the protozoa. 

From the small amount of work done on the effect of protozoa 
on nitrification in soil, it seems that their presence is at 
least not detrimental to the process as determined by these 
experiments. 

FREE NITROGEN FIXATION 

Some very interesting results were obtained in this part of 
the work. Here both soil and liquid cultures were employed. 
The liquid medium gave the protozoa an environment better 
adapted to their growth and activity than did the soil cultures. 

Soil cultures. Four hundred grams of soil were weighed out 
and spread in approximately one inch layers on six flat porcelain 
plates. These were then carefully covered with parchment 
paper and tied and then steriUzed at 15 pounds for one hour. 
Upon cooling each culture was inoculated with a suspension 
of Azotohacter in sterile distilled water. Then one-half were 
inoculated with a suspension of soil containing protozoa and the 
remaining half with the same amount of protozoa-free soil. Af- 



RELATION OF PROTOZOA TO SOIL BACTERIA 



429 



ter incubating at room temperature for one week, the soils in 
the plates were treated with 1 per cent of mannite. The 
mannite was thoroughly mixed with the soil by means of a 
sterile spatula. The plates were then incubated for three 
weeks at 25°C. The moisture content was kept as near one- 
half saturation as possible by the addition of sterile distilled 
water. At the expiration of the incubation period, the soils 
were placed in a 30°C. incubator until air dry. They were then 
ground in a mortar and thoroughly mixed and finally sieved. 
Duplicate total nitrogen analyses were made according to the 
modified Gunning method. 

The results of the analyses are given in the following table. 

TABLE 7 
Rate of fixation of free nitrogen in soil with and without protozoa 



TREATMENT 



With protozoa. . . . 
With protozoa. . . , 
With protozoa. . . 
Without protozoa 
Without protozoa 
Without protozoa 



TOTAL NITROGEN PER 100 
GRAMS OP DRY SOIL 



After 21 days 



mgm. 

148.40 
145.60 
150.50 
144.20 
149.10 
144.90 
147.00 

144.20 
144.90 
140.00 
147.00 



> 



Average 



147.45 



145.02 



From the data above it will be seen that there is a difference 
in total nitrogen in favor of the soil containing the protozoa. 
However, it is probably not marked enough to cause any dif- 
ference in the fertility of the soil. 

From these results it seems probable that the protozoa do 
not have any particularly harmful effect on the fixation of free 
nitrogen in the soil. 

Liquid cultures. One hundred cubic centimeters of mannite 



430 



T. L. HILLS 



solution (Asliby's) were placed in each of ten liter Erlenmeyer 
flasks. To each of these flasks 10 grams of soil were added and 
the flasks and contents sterilized at 10 pounds for thirty min- 
utes. After cooling each flask was inoculated with a suspension 
of Azotohacter in sterile distilled water. Finally, one half of 
the flasks were inoculated with 10 cc. of a suspension of 40 
grams of normal soil in 400 cc. of sterile distilled water and the 
remaining half were similarly treated using protozoa-free soil. 
The flasks were incubated at 25°C. for three weeks. Previous 
to determining the total nitrogen the flasks were examined in 
order to ascertain whether or not protozoa were present. In 
those flasks inoculated with soil containing the protozoa they 
were present and in a very active state and in those inoculated 
with soil free from protozoa they were not found. 

The results of the total nitrogen analyses are given below. 

TABLE 8 
Rate of fixation of free nitrogen in solution with and without protozoa 



ANALYSES 



TREATMENT 



With protozoa. . . 
With protozoa. . . 
With protozoa. . . 
With protozoa. . . 
Without protozoa 
Without protozoa 
Without protozoa 
Without protozoa 



TOTAL NITROGEN PER 100 CC. 
OF SOLUTION 



After 21 days 




Average 



31.74 



33.79 



The results of the total nitrogen determinations revealed a 
difference of 2.05 mgm. of nitrogen in favor of the cultures with- 
out the protozoa. Apparently the protozoa had a slight detri- 
mental effect on nitrogen fixation in solution. The protozoa- 
free cultures contained 33.79 mgm. of nitrogen and the cultures 
with protozoa contained 31.74 mgm. of nitrogen. 

Thus it appears that in hquid cultures where the protozoa 
are in an actively motile state they seem to exert a harmful 



RELATION OF PROTOZOA TO SOIL BACTERIA 431 

influence on the process of free nitrogen fixation. It is probable 
that the larger protozoa made use of the Azotobacter as food. 
In certain cases upon staining a small amount of the film from 
a liquid culture with Gram's iodine solution Clostridium cells 
could be very readily distinguished within the protozoan cell. 
The probable presence of Azotobacter cells within the protozoa 
cells was also observed but by no means as definitely as in the 
case of the Clostridium. 

It may be concluded that the protozoa have a slight detri- 
mental effect on free nitrogen fixation in solution because the 
individual determinations seem to check closely and to be quite 
outside the limit of experimental error considering the small 
amount of nitrogen in the cultures. In the case of the soil cul- 
tures such a slight difference is not so important because the 
total nitrogen content here is approximately five times that of 
the liquid cultures. 

DISCUSSION 

From the results of this study of the influence of the protozoa 
on ammonification, nitrification and free nitrogen fixation in 
soil it would seem that their effect can not be considered detri- 
mental. This is in accord with the work of other investigators, 
even with that of Cunningham (1915) who claims that his re- 
sults uphold the theory put forth by Russell and Hutchinson. 
In the work referred to he states that soil protozoa in solution 
exercise a decided limitmg effect on the numbers of bacteria and 
that in an ammonifying solution they show their activity by 
causing a decrease in the amount of ammonia produced as com- 
pared with cultures free from protozoa. These results are in 
accord with those already presented in this paper, in regard to 
free nitrogen fixation in solution. The protozoa seemed to 
have a detrimental effect on this process but when experiments 
were carried out on free nitrogen fixation in soil, the protozoa 
did not appear to influence the amount of nitrogen fixed. Cun- 
ningham may well conclude that protozoa have a limiting effect 
on the number of bacteria in solution, for here an environment 



432 T. L. HILLS 

is furnished for the protozoa which is never met with in soils 
under normal conditions. 

The results of the work at the New Jersey Experiment Station 
are not in accord with Russell and Hutchinson's theory. Lip- 
man et al. (1910) found that the protozoa do not play any im- 
portant part in depressing the activity of the soil bacteria. This 
was shown by a series of experiments performed relative to a 
possible influence which the protozoa might have on the im- 
portant soil process of ammonification. 

Concordant results have been obtained by Sherman (1916) 
who worked with six species of protozoa, namely, the two ciliates 
Colpoda cucullus and Balantiophorus elongatus, which are not 
active in soil and four flagellates, which by test were shown to 
be active in soil. The ciliates had a very marked detrimental 
effect upon the number of bacteria in soil extract but had no 
effect upon them in soil. Three of the flagellates had no effect 
upon the number of bacteria either in soil extract or soil. The 
fourth flagellate had a very marked detrimental effect in soil 
extract but none in soil. These experiments were performed 
many times and always with the same results. 

In an earlier work Sherman (1914) showed conclusively 
that some protozoa can increase in numbers in the soil under 
ordinary conditions but from the results of his later work it 
is probably doubtful if they have any appreciable effect on limit- 
ing the numbers of the soil bacteria. 

Grieg-Smith (1912), drawing conclusions from his own work, 
thinks that the protozoa have but little effect on the bacteria 
of the soil. He tested the action of the soil phagocytes (the 
protozoa) in the same manner as Russell and Hutchinson did 
and from his experiments he concluded "that Russell's conten- 
tion cannot be sustained; the protozoa have little or no action 
in limiting the number of soil bacteria." 

Goodey (1911), working with the ciliates only, thinks that 
these protozoa do not exist in the soil in an active state, but 
that they are present in an encysted condition. He made a 
careful study of recently excysted Colpoda cucullus obtained 
from soil which had been added to a suitable medium but a few 



RELATION OF PROTOZOA TO SOIL BACTERIA 433 

hours before. He concluded that if these organisms had been 
in the soil in an active state they would have possessed food 
vacuoles, as these develop soon after the protozoan begins to 
ingest its food. 

CONCLUSIONS 

In conclusion it maj^ be said that in the soil cultures the 
presence of protozoa under the conditions of the experiments 
did not have any noticeable effect, detrimental or otherwise, 
on the processes of ammonification, nitrification and free nitro- 
gen fixation. In the case of the liquid cultures employed in 
the study of free nitrogen fixation the conditions w^ere at an 
optimum for the development of the protozoa and under these 
circumstances they limited bacterial activity as evidenced by 
the harmful effect on the fixation of free nitrogen. Undoubtedly 
under these conditions the protozoa were active in destroying 
the Azotobacter cells. But in the soil cultures conditions were 
evidently not favorable for the activity of the protozoa as these 
organisms did not appear to exert any harmful influence on the 
three soil processes studied. 

REFERENCES 

Cunningham, A. 1915 Studies on soil protozoa. Centbl. f. Bakt. (etc.) 
Abt. II. 42, 8-27. 

GooDEY, T. 1911 A contribution to our knowledge of the protozoa of the 
soil. Proc. Roy. Soc. (London), Ser. B., 84, No. B570, 165-180. 

Grieg-Smith, R. 1912 The inactivity of soil protozoa. Proc. Linn. Soc. 
N. S. Wales. (1912), 655-672. 

LiPMAN, J. G., Blair, A. W., Owen, I. L., and McLean, H. C. 1910 Experi- 
ments relating to the possible influence of protozoa on ammonification 
in the soil. N. J. Exp. Sta. Bull. 248. 

Russell, E. J. and Hutchinson, H. B. 1909 The effect of partial steriliza- 
tion of soil on the production of plant food. Jour. Agr. Sci. 3, pt. 2, 
111-144. 

1913 The effect of partial sterilization of soil on the production of 
plant food. Jour. Agr. Sci., 5, pt. 2. 152-221. 

Sherman, J. M. 1914 The number and growth of protozoa in soil. Centbl. 
f. Bakt. (etc.) Abt. II, 41, 625-630. 

1916 Studies on soil protozoa and their relation to the bacterial flora. 
Jour. Bact., 1, 35-66; 165-185. 



A STUDY OF THE BOAS-OPPLER BACILLUS 

p. G. HEINEMANN and E. E. ECKER 
From the Department of Hygiene and Bacteriology, the University of Chicago 

In 1895 Boas and Oppler (1895) published a paper in which 
they reported observations on a large bacillus occurring in 
the gastric juice of patients afflicted with carcinoma of the 
stomach. In the same year Schlesinger and Kaufmann (1895) 
found a similar bacillus in 19 cases out of 20 cases of gastric 
carcinoma examined. These findings were further confirmed 
by several investigators and the presence of large numbers of 
these bacilh in the stomach was taken to indicate carcinoma. 
Strauss (1895) reported finding similar organisms in normal gastric 
juice although in small numbers. Kuntze (1908) was the first 
to suggest that the Boas-Oppler bacillus was related to the 
lacto-bacilli. Rodella (1908) has also^ shown the similarity 
of the Boas-Oppler bacillus to the so-called acidophile or aciduric 
bacilli and the B. bifidus of Tissier. These suggestions were 
given further experimental support by the work of Heinemann 
and Hefferan (1908). The authors found in an extensive in- 
vestigation of that group of bacteria, now commonly known as 
the B. bulgaricus group, that similar bacilli were present in two 
samples of normal gastric juice and in two cases of gastric car- 
cinoma. These organisms are difficult to cultivate as they do 
not grow on ordinary laboratory media to an appreciable extent ; 
but they grow well in milk or on media prepared from milk. 
The presence of glucose or some other carbohydrate favors 
growth. For a detailed description of the organism and its 
cultural characteristics the reader is referred to the pubhcation 
of Heinemann and Hefferan (1908). 

In 1914 Gait and lies (1914) reported the finding of the same 
organism in three cases of gastric carcinoma. They thought 
that mahgnant cases of carcinoma could be distinguished from 

435 



436 P. G. HEINEMANN AND E. E. ECKER 

benign ones by the presence of these bacilli. This conclusion 
was reached because in malignant cases the hydrochloric acid 
disappears, while lactic acid is frequently present. 

Bacilli of the B. bulgaricus group are widely distributed in 
nature as shown by several authors. Heinemann and Hefferan 
have found them in the feces of man, horses and cows, in soil, 
in fodder for cattle (bran, silage, dry brewer's grains), in corn- 
meal, sauerkraut, olive juice, dill pickles, pepper mango, mar- 
ket milk and in human saUva. Hastings and Hammer (1909) 
reported the presence of these bacilU in milk, butter and cheese 
and recently Hunter and Bushnell (1916) ascribed the fermenta- 
tion of silage to the activity of the B. bulgaricus group. That 
they are active in final stages of the ripening of Emmenthaler 
cheese has been shown by Eldredge and Rogers (1914) and they 
have been reported by Evans, Hastings and Hart (1914) in 
Cheddar cheese. Dotterrer and Breed (1915) during a tour of 
inspection of cheese factories in New York State have found that 
the pasteurized whey undergoes a practically pure lactic acid 
fermentation due to B. bulgaricus and that in unpasteurized 
whey the organisms are present in enormous numbers. The 
authors state also that the pasteurization temperature applied 
to these cases (66°-71°C.) was not sufficient to destroy the or- 
ganism, although it destroyed most other bacteria present. 

Since lacto-bacilU have been found in saliva and feces under 
normal conditions, it would be surprising if they did not exist 
normally in the stomach. Their presence in the digestive tract 
is the natural result of their frequency in foods, especially milk 
and milk products. Furthermore, this group of bacilli is able 
to resist a relatively high degree of acidity and survive where 
other bacteria are largely destroyed. Consequently the hydro- 
chloric acid of the gastric fluid is not destructive to them, although 
it undoubtedly restrains their growth. 

That lacto-bacilU actually exist in normal gastric juice has 
been shown by Strauss and Heinemann and Hefferan as stated 
before. If the hydrochloric acid restrains multipUcation, it 
seems logical to assume that reduction in the amount of hy- 
drochloric acid or its absence will permit growth and this assump- 



A STUDY OF THE BOAS-OPPLER BACILLUS 437 

tion may explain the finding of lacto-bacilli in large numbers 
in cases of gastric carcinoma. 

However, if it be true that the presence of lacto-bacilli in 
large numbers in the stomach is due to a reduced quantity of 
hydrochloric acid, it may be assumed that they ought to be 
easily found in any pathological condition which reduces the 
normal acidity of the gastric juice. This reasoning led us to 
carry on a study of the problem the results of which are pre- 
sented in this paper. We were aided by the kindness of Mr. 
A. G. Bower who furnished samples of gastric juice from a 
variety of sources. 

The method of isolation was the same as the one employed 
by Heinemann and Hefferan. About half a cubic centimeter 
of gastric fluid was inoculated into acetic acid broth and incu- 
bated at 44°C. After twenty-four hours incubation several loop- 
fuls were transferred to htmus milk and this was also incubated 
at 44°C. After a further twenty-four hours, transfers were 
made from the litmus milk tubes to other similar tubes and this 
proceeding was repeated every twenty-four hours until the 
characteristic reaction in Htmus milk was observed. The coagu- 
lum should be smooth and compact with the appearance of 
httle or no whey; the major part of the milk should be decolor- 
ized and a surface layer of intense red appears. Stains with 
methylene blue were prepared to ascertain the presence of long 
bacilli in pure culture. In order to study colony formation, 
plates were prepared in whey agar. Maltose broth was inocu- 
lated with five strains (P, S, F, H and the stram from infant 
feces) to test their reaction on this carbohydrate and finally 
500 cc. of sterihzed milk were inoculated with the same five 
strains. The evolution of acid was determined by titrating 
5 cc. of the undiluted milk with N/20 NaOH and the acidity 
was calculated as lactic acid. The flask inoculated with strain H 
(from carcinoma) became contaminated and the results had 
to be excluded. The optical rotation of the lactic acid produced 
was determined after preparing the zinc salts in the usual manner. 

Since lacto-bacilli are present in saliva it was suggested by 
Heinemann and Hefferan that they might be identical with 



438 



p. G. HEINEMANN AND E. E. ECKER 



Leptothrix huccalis, the organism thought to be the cause of 
caries of teeth. We examined the decayed portion of four 
teeth after having carefully cleaned the teeth with sterile NaCl 
solution to avoid the chance of lacto-bacilli being present in 
the dried saliva on the outside of the teeth. After cleansing 
the teeth the inside decayed part was scraped out with a sterile 
knife and placed in acetic acid broth. Otherwise the same 
technic was employed as with the other material. 

Material for examination was obtained from the following 
sources : 



SAMPLE NUMBER 


CONDITION 


KIND OF MATERIAL 


STAIN FROM MATERIAL 


1. P 


Gastric ulcer 


Gastric juice 


Many large granular bacilli 


2. M 


Gastric ulcer 


Gastric juice 


Many large granular bacilli 


3. F 


Gastritis 


Gastric juice 


Many large granular bacilli 


4. S 


Pernicious 


Gastric juice 


Many large granular bacilli 




anemia 




5. H 


Carcinoma 


Gastric juice 


Many large granular bacilli 


6. P 


No diagnosis 


Gastric juice 


Some large granular bacilli 


7. J 


Normal 


Gastric juice 


Few large granular bacilli 


8. Bottle-fed 




infant 




Feces 


Many large granular bacilli 


9. Breast-fed 








infant 




Feces 


Few large granular bacilli 


10. Tooth 


Abscess 


Decayed part 


No stain made 


11. Tooth 


Pyorrhea 
Pyorrhea 


Decayed part 
Decayed part 


No stain made 


12. Tooth 


No stain made 


13. Tooth 


Ulceration 


Decayed part 


No stain made 



From all these cases typical lacto-bacilli were isolated. After 
four to six transfers in litmus milk the typical appearance of 
the milk was observed. Stains were prepared from all cultures 
and the bacilli appeared as large, rather slender or fairly thick 
organisms with blue granules. They were Gram positive. The 
colonies were all of the compact type. Sandberg (1904) first 
called attention to the two kinds of colonies formed by the 
Boas-Oppler bacillus. One of these has woolly edges, the other 
is solid. Similar observations have been reported by several 
authors. 



A STUDY OF THE BOAS-OPPLER BACILLUS 



439 



The progressive amount of acid formed is shown in the follow- 
ing table : 





ORIGINAL 
MILK 


AFTER DAYS 




1 


2 


3 


4 


6 


9 


13 


17 


20 


I. p 

4. S 


0.14 
0.14 
0.14 

0.14 


0.22 

0.20 
0.25 

0.25 


0.66 
0.43 
0.90 

0.39 


1.12 
0.93 

1.27 

0.57 


1.34 
1.21 
1.29 

0.71 


1.56 
1.38 
1.32 

0.95 


1.56 
1.41 
1.32 

0.98 


1.56 
1.42 
1.33 

1.03 


1.56 
1.48 
1.35 

1.05 


1.57 
1.49 


3. F 


1.36 


8. Infant 
feces 


1.05 



The amount of acid formed by the different strains is remark- 
ably uniform and the rate of acid formation nearly the same 
in the first three samples. The strain from infant's feces is 
somewhat slower in acid formation than those from pathological 
conditions. 

In maltose broth the strains P, S and F produced no change 
in reaction, while the strain from infant feces produced 4 per 
cent normal acid or 0.36 per cent lactic acid in five days at 37°C. 
and the same amount in two days at 44°C. 

The strain from carcinoma lost its power to coagulate milk 
after seven transfers. We are unable to give an explanation 
of this phenomenon, unless it was due to enfeebhng of the 
organism. 

We foimd typical lacto-bacilU in the decayed contents of the 
four teeth. In one of the teeth (sample 10) long bacilli forming 
filaments and showdng granular staining were found in large 
numbers. A few streptococci were also present. In one of 
the pyorrhea cases (sample 11) the bacilh were somewhat shorter 
than in the previous case. There were some that stained solidly, 
while others showed distinct granular staining. In the second 
pyorrhea case (sample 12) the long form was prevalent and 
filament formation was common. The fourth tooth (sample 13) 
was decayed and an ulceration was present at the root. The 
lacto-bacilH present were rather slender, the granular staining 
form being prevalent. Streptococci were also numerous. In 
the cultures obtained from two of the teeth (samples 10 and 13) 



440 P. G. HEINEMANN AND E. E. ECKER 

branching forms resembling the letter Y were observed. In all 
cases of material obtained from teeth the typical milk reaction 
resulted after three transfers in litmus milk. On account of 
the granular staining we assume that the lacto-bacilli from dis- 
eased teeth belong to the low-acid type. 

This investigation confirms the results of previous work in- 
dicating that the Boas-Oppler bacillus is a member of the group 
of lacto-bacilli. Its cultural characteristics are in harmony with 
the descriptions of the group given by various authors and it 
appears established that members of the B. hulgaricus group 
are present in saliva, diseased teeth, gastric juice and the in- 
testinal contents. The source can undoubtedly be looked for 
in certain foods, especially milk and milk products. 

The finding of lacto-bacilU in large numbers in carious teeth 
is perhaps not conclusive evidence that they are actually the 
cause of decay. Experimental evidence to prove this would 
of course be difficult to obtain. However, since that kind of 
decay of teeth is usually ascribed to the presence of relatively 
large quantities of acid and since normal saliva is of an alkaline 
reaction the assumption is not difficult to arrive at that lacto- 
bacilli may be the cause. This is further supported by the 
fact that Heinemann and Hefferan found large numbers of 
lacto-bacilli in a sample of saliva of acid reaction. 

White and Avery (1909) have attempted to separate the 
group of lacto-bacilli into two types as follows: Type A stains 
homogeneously with Loffler's methylene blue and Neisser's 
stain, produces 2.7 to 3.7 per cent lactic acid in milk and the 
lactic acid formed is of the inactive variety. Type B, stained 
with Loffler's methylene blue or Neisser's stain shows intensely 
stained granules; the bacilh of type B produce 1.2 to 1.6 per cent 
lactic acid in inilk and the lactic acid formed is always levo- 
rotatory. According to this classification the strains examined 
by us belong to type B. 

Rahe (1914) has classified aciduric bacteria according to their 
ability to clot milk and produce acid from maltose. He dis- 
tinguished three varieties, namely: Variety A which clots milk, 
but has no action on maltose; variety B which clots milk and 



A STUDY OF THE BOAS-OPPLER BACILLUS ' 441 

ferments maltose; and variety C which ferments maltose, but 
does not clot milk. Strains F, P and S of our series belong 
to variety A of Rahe and the strain from mfant feces belongs to 
variety B. 

The presence of lacto-bacilli throughout the digestive tract 
has some bearing on the hypothesis of Metchnikof that life 
can be prolonged by estabUshing bulgarian bacilli permanently 
in the digestive tube. Rahe (1915) studied the problem of 
implanting B. hulgaricus in the alimentary tract and came 
to the conclusion that it cannot be adapted to the human lower 
intestine and that in monkeys the B. hulgaricus is capable only 
of an apparently limited survival in the upper intestine. The 
author further claims that B. hulgaricus can be readily distin- 
guished from intestinal aciduric bacteria by its lack of ability 
to ferment maltose. 

With these facts before us it seems clear that there is no 
support for the theory that lactic acid in statu nascendi is of 
value in suppressing intestinal putrefaction. The chief differ- 
ence between the lacto-bacilli normally present in the alimentary 
tract and typical B. hulgaricus is the smaller amount of lactic 
acid produced by the former. However, the actual quantity 
produced is about 1.5 per cent, an amount which is greater than 
can be assumed actually to exist in the digestive tube. If we 
consider further that earher findings of B. hulgaricus in feces, 
even after ingestion of bulgarian milk, were not entirely trust- 
worthy on account of imperfect technic and that no attempt was 
made to distinguish between the high-acid and low-acid types, 
it becomes evident that the existence of an appreciable quan- 
tity of lactic acid in the digestive tract as a result of bacterial 
action is at least questionable. There can be no reasonable 
doubt about the actual formation of lactic acid by bacteria m 
the digestive tract, but the acid is promptly decomposed and 
utilized by the system. 

The question naturally presents itself whether the types A 
and B of White and Avery are pennanent or the result of en- 
vironmental conditions and changeable. Type A is represented 
chiefly by typical B. hulgaricus. It forms about twice as much 



442 P. G. HEINEMANN AND E. E. ECKER 

lactic acid of the inactive modification as type B which produces 
only levo-rotatory acid. Granular staining is frequently a 
characteristic of enfeebled forms of bacteria and it is possible 
that type B is an enfeebled strain of type A that has lost the 
power to form dextro-rotatory lactic acid. If equal amounts of 
both modifications of active lactic acids are formed they unite 
to form inactive acid and it might be possible to convert type 
B into type A if suitable conditions of growth were offered. 
Type B is the one that is found active in cheese ripening and 
this fact suggests that type B grows at lower temperatures 
than type A whose optimum temperature is about 45° C. It 
is usually stated that type A does not grow below 30°C. It is 
possible therefore that type B is a modification of type A accus- 
tomed to lower temperature. We hope to study this problem 
at some future period. 

SUMMARY AND CONCLUSIONS 

1. Boas-Oppler baciUi occur in normal gastric juice in moderate 
numbers and in gastric juice containing either no hydrochloric 
acid or materially less than the normal amount in large numbers. 

2. The presence of Boas-Oppler bacilli in large numbers in 
gastric juice is an indication of reduction of the normal hydro- 
chloric acid content, whether this is due to gastric ulcer, gastritis, 
pernicious anemia or gastric carcinoma and possibly other 
pathological conditions. 

3. The Boas-Oppler bacillus belongs to the group of lacto- 
bacilli which occur frequently in foods, chiefly milk and milk 
products. It gains access to the saliva, the stomach and in- 
testinal contents with food. 

4. The Boas-Oppler bacillus is the lactic acid producing or- 
ganism that occurs in saliva and in the contents of the digestive 
tube. 

5. The Boas-Oppler bacillus is common in feces of bottle- 
fed infants, but relatively scarce in the feces of breast-fed infants. 

6. Four strains of Boas-Oppler bacilli studied by us produced 
in milk from 1.05 to 1.57 per cent acid, consisting chiefly of 



A STUDY OF THE BOAS-OPPLER BACILLUS 443 

lactic acid. No acid was produced in maltose broth by strains 
from pathological conditions. 

7. The lactic acid produced by the strains of Boas-Oppler 
bacilli studied by us is of the levo-rotatory modification. 

8. A stram isolated from the intestinal contents of a bottle- 
fed infant coagulated milk and produced 1.05 per cent (lactic) 
acid in twenty days. In maltose broth 0.36 per cent (lactic) 
acid was produced by this strain. 

9. Lacto-bacilli were found in material from decayed teeth 
from which desiccated mucus had been removed. This fact 
suggests that Streptothrix huccalis is perhaps a lacto-bacillus 
of the low-acid forming type. 

REFERENCES 

Boas and Oppler 1895 Zur Kenntniss des Mageninhalts beim Carcinoma 

ventriculi. Deutsch. Med. Wchnsch., 21, 73-75. 
DoTTERER AND Breed 1915 The pasteurization of dairy by-products. New 

York Agri. Exp. Sta. Bull. 412, December 1915, 581-610. 
Eldredge and Rogers 1914 The bacteriology of cheese of the Emmenthal 

type. Cent. f. Bakt., Abt. 2, 40, 5-21. 
Evans, Hastings and Hart. 1914 Bacteria concerned in the production of 
the characteristic flavor in cheese of the Cheddar type. Jour, of 
Agricultural Res., 2, 167-192. 
Galt and Iles 1914 A study of the Boas-Oppler bacillus. Jour, of Path. 

and Bact., 19, 239-244. 
Hastings and Hammer 1909 The occurrence and distribution of organisms 
similar to the B. bulgaricus of Yoghurt. Cent. f. Bakt., Abt. 2, 25, 
419-426. 
Heinemann and Hefferan 1908 A study of B. bulgaricus. Jour, of Inf. 

Diseases, 6, .304-318. 
Hunter and Bushnell 1916 The importance of Bacterium bulgaricus group 

in ensilage. Science, 43, 318-320. 
KuNTZE 1908 Studien liber fermentierte Milch (1), Yoghurt und Mazun. Cent. 

f. Bakt., Abt. 2, 21, 737-768. 
Rahe 1914 An investigation into the fermentation activities of the aciduric 

bacteria. Jour, of Inf. Diseases, 15, 141-150. 
Rahe 1915 A study of the so-called implantation of the Bacillus bulgaricus. 

Jour, of Inf. Diseases, 16, 210-220. 
Rodella 1908 Magen Carcinoma und Milchsaure Bacillen. (B. Oppler, 
B. gastri, bacterium gastrophilus, Lehmann-Neumann, B. acidophilus, 
B. bifidus communis.) Cent. f. Bakt., Abt. 1, 47, 445-466. 



444 P. G. HEINEMANN AND E. E. ECKER 

Sandberg 1904 Ein Beitrag zur Bakteriologie der Milchsaure Gahrung im 

Magen, mit besonderer Beriicksichtigung der langen Bacillen. Ztschr. 

f. klin. Med., 51, 80-94. In this article a complete bibliography of 

the Boas-Oppler bacillus is published. 
ScHLESiNGER AND Kaufmann 1895 tjber einen Milchsaure bildenden Bacillus 

und sein Vorkommen im Magensaft. Wien. Klin. Rundschau, 15, 

225-229. 
Strauss 1895 tJber die Abhiingigkeit der Milchsaure Gahrung vom HCl Gehalt 

des Magensafts. Zeitschr. f. klin. Med., 28, 567-578. 
White and Avery 1909 Observations on certain lactic acid bacteria of the 

so-called Bulgaricus Type. Cent. f. Bakt., Abt. 2, 25, 161-178. 



A CONTRIBUTION TO THE BACTERIOLOGY OF 

SILAGEi 

JAMES M. SHERMAN 

From the Bacteriological Laboratories of the Pennsxjlvania State College and Agri- 
cultural Experiment Station 

The fermentation which ensilage undergoes during its curing 
process was looked upon a few decades ago as being entirely 
of microbic origin, and we find, in the older hterature on the 
subject, frequent reference to the alcohohc, acetic acid and 
lactic acid ferments which were supposed to exist in the ensilage. 
The evidence upon which such statements were based was, as 
far as can be ascertained, merely the occurrence in silage of the 
products characteristic of the action of such organisms. At 
the present time, due chiefly to the work of Babcock and Rus- 
sell (1900, 1901), opinion has swung in the opposite direction 
to such an extent that microorganisms are now generally con- 
sidered of httle if any significance in the normal fermentation 
of silage. 

That most microbiologists in this country do not consider 
bacteria of significance in the formation of silage is indicated 
by a review of the recent textbooks on the subjects of general 
and agricultural bacteriology. Marshall (1911) and Jordan 
(1914) make no mention of silage, although other related fer- 
mented products are discussed. Russell and Hastings (1915) 
state that the fermentation is beheved to be due to the respira- 
tion of the Hving plant cells. Lipman (1911) outlines the res- 
piration theory of Babcock and Russell and states that silage 
may be made under conditions which exclude bacterial action. 

1 This paper, originally entitled "The Occurrence of Aciduric Bacilli in Corn 
Silage," was prepared for publication when a paper appeared by Hunter and 
Bushnell bearing a somewhat similar title. This article, slightly changed so 
as to contain reference to their work, is published only to confirm the observations 
of Hunter and Bushnell. 

445 



446 JAMES M. SHERMAN 

He further states that the question as to whether bacteria have 
any beneficial function can not be answered at the present time. 
Conn (1901) gives the old ideas concerning the supposed roles 
of microorganisms, and then reviews the work of Babcock and 
Russell (1900, 1901). His views on the subject may be well exem- 
plified by the following quotation: 

From all these facts it becomes clear that while this method of pre- 
paring food is due to a fermentation, it can not be attributed to the 
growth of microorganisms. It certainly involves other factors, and it 
is uncertain whether bacteria, or other microorganisms, have anything 
to do with the process as normally carried out. 

Since the work of Babcock and Russell fifteen years ago, 
little has been pubUshed on the processes involved in the cur- 
ing of ensilage. E. J. Russell's work (1908) was in substantial 
agreement with that of Babcock and Russell, though he thought 
it possible that bacteria play a minor part. Esten and Mason 
(1912) considered the process entirely bacteriological. Three 
chief fermentations were thought to take place: the lactic acid, 
alcoholic and acetic acid fermentation. The lactic acid fermen- 
tation was thought to be due to organisms similar to those con- 
cerned in the sourmg of milk. It was also beheved by these 
workers that yeasts cause an alcoholic fermentation and that 
acetic acid bacteria then oxidize the alcohol so formed to acetic 
acid. Samarani (1913) concludes that the acetic acid fermenta- 
tion in silage is due to the respiration of the plant cells, while 
the lactic acid fermentation is due to bacterial action. The 
organisms responsible for the latter process were identified by 
Samarani as a bacillus and a coccus which occurred in about 
equal proportions. The former he designated as the B. acidi- 
lactici of Hueppe, and the latter was considered identical with 
the common streptococcus of milk. 

The role of acid producing bacteria of the Bulgarian type 
in such fermented products as sauerkraut, milk beverages, 
cheese, etc., is well known. That they should function in silage 
would appear probable, but until very recently no data were at 
hand which gave any evidence for such a belief. Although 



BACTERIOLOGY OF SILAGE 447 

suggestions that these organisms may be of importance in the 
ensiling of foods are found in the hterature (Heinze, 1913), 
such suppositions have not been based upon estabhshed facts. 
The lactic acid bacteria mentioned by Esten (1910) as impor- 
tant in silage were inhibited by the presence of only 0.45 per 
cent of lactic acid when grown in corn juice. Gorini (1906) 
made a detailed study of the bacteria of silage and mentioned 
Streptococcus lactis-acidi and B. lactis-acidi as the most impor- 
tant of the acid-forming organisms. None of the organisms 
which he described had the property of forming a high degree 
of acidity. Lohnis (1907) in his classification of the lactic acid 
bacteria described a number of strains of the aciduric bacteria 
but made no mention of a group derived from silage. Steven- 
son (1911) studied the distribution of the high acid bacteria 
but did not report silage as one of the sources from which they 
were obtained. Heinemann and Hefferan (1909) noted silage 
as one of the substances from which they had isolated cultures 
of B. bulgaricus. The recent paper by Hunter and Bushnell 
(1916) however is the first report, so far as the writer is aware, 
of the constant occurrence and probable importance of organisms 
of the B. bulgaricus group in silage. 

OBSERVATIONS 

The notes recorded in this paper are those which have been 
made on ensilage, more or less incidentally, during the past 
year. It was first noted on April 26, 1915, that sterilized milk 
inoculated with silage developed a high acidity. After incuba- 
tion for ten days at 37°C. the milk was found to contain an acid- 
ity of 2.3 per cent calculated as lactic acid. This observation 
indicated the presence of organisms belonging to the group of 
aciduric bacilh, and these bacteria were isolated from the milk 
culture by plating on ordinary lactose agar. Their occurrence 
has been repeatedly verified in samples of corn ensilage from 
four different silos taken at various stages during the feeding 
season. 

That the high acid-producing organisms not only exist in 



448 JAMES M. SHERMAN 

silage but that they occur in large numbers is shown by the 
fact that sterile milk usually develops a high acidity when 
inoculated with dilutions of silage representing only 1/1,000,000 
of a gram of the moist material, or when inoculated with 1/1,000,- 
000 of a cubic centimeter of the juice expressed therefrom. The 
occurrence of the high-acid organisms in such large numbers 
has been observed in silage over nine months old. 

The aciduric bacilli may also be readily isolated by the direct 
plating of the silage on lactose agar on which this type can be 
grown. In fact these organisms constitute a majority of the 
bacteria found in ensilage during the latter part of the curing 
process. Unfortunately, it was not possible to make a study 
of the silage during the first two months when the most impor- 
tant bacterial processes undoubtedly take place. Plate counts 
made on lactose agar of the juice expressed from silage between 
three and six months of age have given counts ranging from a 
few thousand to over 1,000,000 bacteria per cubic centimeter. 
As is well known, most types of the aciduric bacilli do not grow 
well on agar plates, and it would seem very probable that the 
actual number of such organisms is considerably greater than 
is indicated by the plate count. That this supposition is cor- 
rect is shown by the observation that these organisms may be 
present in numbers approximating 1,000,000 per cubic center- 
meter of juice,as revealed by the dilution method, when the 
plate Count shows only 15,000. 

Counts made on silage juice by the direct microscopic method 
of enumeration have shown the presence of from 1,500,000,000 
to 4,800,000,000 bacterial cells per cubic centimeter, most of 
which were slender rods. Many of the organisms observed 
under the microscope were, in all probability, dead, since autoly- 
sis would undoubtedly be greatly retarded in such an acid me- 
dium. However, these observations indicate that immense 
bacterial activity had taken place. 

The morphological and cultural characteristics of the acid 
producing bacilli which have been isolated agree with those 
reported by Hunter and Bushnell (1916). The rods were of 
variable size, but the growth characters of the cultures thus 



BACTERIOLOGY OF SILAGE 



449 



far collected are very similar in so far as the cultural observations 
have been made. The colonies on agar appear exactly like those 
of the B. lactis-acidi group and, in the presence of a ferment- 
able carbohydrate, they are surrounded by the characteristic 
haze. The development of colonies is not so rapid as with 
organisms of the B. lactis-acidi type, but on prolonged incubation 
they usually develop to a greater size. The readiness with which 
this group of bacteria grows on ordinary laboratory media differ- 
entiates it quite sharply from the typical B. bulgaricus of milk. 
Not only do these organisms cause a high acid fermentation 
in milk but they have a similar action in corn juice in which 
they grow very rapidly. In the table given below are the data 

TABLE I 
Acidity produced by silage organisms in milk and in corn juice 





PER CENT ACID AS LACTIC ACID 


CULTURE NUMBER 








Milk 


Corn Juice 


1 


1.36 


1.36 


2 


2.25 


1.67 


3 


1.38 


1.21 


4 


1.53 


1.67 


5 


2.34 


1.35 


6 


1.36 


1.53 


7 


1.51 


1.69 


8 


2.28 


1.25 


9 


1.39 


1.44 


10 


1.44 


1.69 


11 


2.25 


1.24 


12 


1.34 


1.55 


13 


1.39 


1.64 


14 


1.53 


1.51 



obtained with fourteen cultures grown in milk and in corn juice. 
The corn juice used was obtained from green plants at about 
the tasselling stage. The juice was expressed from the stalks 
by pressure, heated for a few minutes in the autoclave, filtered 
through filter paper, tubed and sterilized. The cultures were 
incubated twelve days at 37°C. 



450 JAMES M. SHERMAN 

The observations reported in this paper would appear to 
indicate that acid producing bacteria might play a part in the 
fermentation of silage. How much of a factor they are in 
ordinary silage can not be answered from the meager data 
which have been collected. From the evidence presented by 
various workers, there can hardly be any question but that 
cell respiration is of vital importance in the fermentation of 
normal silage, but that this may be supplemented greatly by 
the action of bacteria certainly appears reasonable. It would 
seem that microorganisms might be responsible for the fermen- 
tation which takes place in silage made from shocked corn. 
The ensiling of shocked corn and corn stover, a practice which 
has been in vogue to a limited extent in some localities for years, 
in which we would expect the plant cells to be inactive, must 
be largely dependent, it would seem, upon the action of bacteria. 

A laboratory test on this point was made by ensiling some 
corn stover with double the amount, by weight, of water in a 
glass jar. The stover used had been shredded and baled and 
was about fifteen months old. After one month at laboratory 
temperature the jar was opened and the ensilage examined. 
The material had a clean acid odor quite typical of ordinary 
silage, but on comparison of the two it was found to lack a 
certain richness in aroma so characteristic of silage put up in 
the usual way The juice expressed from the stover silage had 
an acidity of 1.35 per cent, calculated as lactic acid, and a bac- 
terial count on lactose agar of 1,700,000,000 organisms per cc. 
of which 600,000,000 were of the high acid producing type. A 
direct microscopic examination of the juice revealed a count of 
11,000,000,000 bacteria per cc. 

The subject of the fermentation in stover silage is under 
f