^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
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is offered at a price which is intrinsically
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quality of this pepton for all bacteriological
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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
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C. M. Hilliard
I. J. Kligler
j. a. kolmer
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H. W. Lyall
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E. C. L. Miller
E. H. Nollau
L. Pearse
E. B. Phelps
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W. Sadler
G. H. Smith
F. L. Stevens
F. W. Tanner
R. M. Taylor
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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-
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Manuscripts should be sent to Prof. C.-E. A. Winslow, Yale Medical School,
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to A, P. Hitchens, Glenolden, Pa.
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Length, mm 100 100 120 120 120 150 150 150
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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
0
0
0
0
0
0
0
B. coli ■
0.5 per cent peptone. . .
0
0
0
0
0
0
0
0
0.25 per cent peptone . .
0
0
0
0
0
0
0
0
'
0.25 per cent peptone. .
0
0
0
0
0
0
0
0
0.25 per cent peptone
0.5 per cent beef ex- >
0
0
X
X
X?
0
tract
0
0
xo
0
0
B. coli (H). ■
0.25 per cent peptone 1
0
1.0 per cent glucose . . ^
0.25 per cent peptone
0.25 per cent ammo- '
0
0
0
X
X
X
nium sulphate
0.25 per cent peptone . .
0
0
X
xo
0
0
0
0
0.25 per cent peptone ]
0.5 per cent beef ex- ■
0
X
?
0
X
•?
tract
0
0
0
0
0
B. coli (U). -
0.25 per cent peptone 1
0
1.0 per cent glucose . . J
0.25 per cent peptone.
0.25 per cent ammo- -
0
0
xo
0
0
xo
nium sulphate ,
0.25 per cent peptone
0.5 per cent beef ex- >
0
X
0
X
tract
B. coli (A). ^
0.25 per cent peptone 1
1.0 per cent glucose . . J
0.25 per cent peptone
0
0
X
0
0
X
0.25 per cent ammo- •
0
0
0
0
0
X
x
nium sulphate
0.25 per cent peptone . .
0
0
0
0
X
0
0
0.25 per cent peptone
0.5 per cent beef ex- •
0
X
X
0
0
X
X
tract
0
0
0
0
0
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- •
0
0
0
0
0
0
nium sulphate
B para- 1
typhi B. . J
0.25 per cent peptone .
0
0
0
0
0
0
0
0
B. pullorum.
0.25 per cent peptone .
0
0
X
0
0
X
B. aerogenes
0.25 per cent peptone .
0
0
0
0
0
0
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§
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UTILIZATION OF PROTEID AND NON-PROTEID NITROGEN 25
S
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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)
0 ^
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)
0
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)
0
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
0
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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^ • S
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
0
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
0 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 0 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
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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.
De Khotinsky
Electrically Heated and Regulated
Constant Temperature
Heating Appliances
We are pleased to announce that we have taken over the manufacture and sale of
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Four electric heating units of 45 watts each are used, which may be added one by one, by
means of the switch which constitutes a part of the bath, and the desired temperature thus secured.
The bath has a constant water level attachment, so that the water level may be kept constant
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ICOO cc.
As the heating units are inserted in brass tubes placed permanently inside of the baths, 20 mm.
from the bottom, their heating value comes to 99% of the theoretical expenditure of energy. Placed
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VOLUME I NUMBER 2
JOURNAL
OF
BACTERIOLOGY
OFFICIAL ORGAN OF THE SOCIETY OF AMERICAN
BACTERIOLOGISTS
MARCH, 1916
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open new fields to our vision, — Pastetjr
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Bacteriological Pepton
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FAIRCHILD BROS. & FOSTER
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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WE HAVE ON HAND FOR IMMEDIATE SHIPMENT IN ANY QUANTITIES
PETRI DISHES AND TEST TUBES
'^ *" '
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OF AMERICAN MAKE
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Size, mm 100x10 100x15 150x10
Outside diameter of bottom, mm 95 95 145
Outside diameter of top, mm 100 100 150
Each .20 ^20 M
Per gross 25.00 25.00 70.00
25606. Porous Earthenware Covers for Petri Dishes, unglazed throughout; the smaller size
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Dishes 100 mm in diameter.
Inside diameter, inches 31 4|
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Per 100 3.50 3.50
25607. Earthenware Covers for Petri Dishes, unglazed inside but glazed on top and sides;
reducing evaporations of water from the medium and preventing possibility of contami-
nation due to absorption in the porous cover. As suggested by C. C. Young of the
Kansas State Water Survey.
Each 08
Per 100 7.00
47948A. Test Tubes, thick wall, without lip, for use as culture tubes in bacteriology. These
tubes are guaranteed not to corrode or give off alkali after repeated sterilization in the
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strength and will stand an unusual amount of mechanical stress without breaking.
Length, mm 100 100 120 120 120 150 150 150
Outside diameter, mm 12 15 13 16 18 16 18 20
Per 100 1.60 1.65 1.75 1.85 1.90 1.95 2.00 2.10
47952A. Test Tubes, medium weight, with flat well formed lip, for Wassermann and other
serological work. Of resistance glass showing a minimum amount of color and in sizes
selected especially for serological work.
Length, mm 65 75 100 150
Outside diameter, mm 12 10 10 10
Per 100 95 .95 1.00 1.05
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ARTHUR H. THOMAS COMPANY
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WEST WASHINGTON SQUARE PHILADELPHIA, U. S. A.
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
0
5
0
100.0
90.9
95.5
68.2
95.5
36.4
59.1
00.0
22.0
00.0
0
0
17
5
1
0
0
0
0
0
00.0
00.0
85.0
25.0
5.0
00.0
00.0
00.0
00.0
00.0
0
1
2
0
1
1
0
0
0
0
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.
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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.
ScHREiNER, O. AND Reed, H. S. 1907 Some factors influencing soil fertility.
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.
1912 Uber Bodenprotozoen. Centbl. Bakt. (etc.), Abt. 2, 33, 314^
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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SPORE-FORMING BACTERIA
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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
0
/
/
/
/
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.
De Khotinsky
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VOLUAIE I
NUMBER 3
JOURNAL
OF
BACTERIOLOGY
OFFICIAT. ORGAN OF THE SOCIETY OF AMERICAN
BACTERIOLOGISTS
MAY, 1916
It is characteristic of Science and Progress that they continually
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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. HITCHENS
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. Williams
H. Zinsser
S. H. Ayers
F. Bachmann
D. H. Bergey
O. Berghausen
C. P. Brown
P. E. Brown
V. Birckner
H. J. Conn
M. M. Cook
J. T. Emerson
L. W. Famulener
C. P. Fitch
D. Greenberg
Abstract Editors
P. B. Hadley
I. C. Hall
C. M. Hilliard
J. G. Hopkins
T. G. Hull
A. Itano
I. J. Kligler
J. A. KOLMER
H. L. Lang
H. W. Lyall
W. J. MacNeal
E. C. L. Miller
E. H. NOLLAU
Zae Northrup
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
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.
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.
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No claims for copies lost in the mails can be allowed unless such claims are
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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
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with proof.
Manuscripts should be sent to Prof. C.-E. A. Winslow, Yale Medical School,
New Haven, Conn.
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to A. P. Hitchens, Glenolden, Pa.
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BACTERIOLOGICAL INCUBAT
No. 20548 C. S. &. E. Anhydric Electric Incubator
C. S. Si. E.
lie ELECTRIC
MCUBATORS
The walls of these Incubators consist
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and transite. No water jacket is used
and the C. S. & E. Electric Thermostat
maintains a constant temperature within
^° C with no attention after adjustment
is once made. No special switch or
wiring is necessary as the Incubators
operate on any 110 volt alternating or
direct circuit by screwing plug into
ordinary lamp socket, and will operate
on 220 volt direct or alternating circuit
by simply changing the voltage of the
lamp heaters.
These are supplied in six different sizes,
the smallest with inside dimensions
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as shown in illustration — with inside
dimensions 30 x 36 x 18 inches at S125.00,
all of which are carried in stock in
Philadelphia for immediate shipment.
COMPLETE DESCRIPTIVE PAMPHLET
SENT UPON REQUEST
HEARSON INCUBATORS
WITH PATENT CAPSULE TEMPERATURE
CONTROL FOR GAS, OIL AND
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We have supplied over two hundred Hearson
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Hospital bacteriological laboratories through-
out the U. S. Their performance has been
universally satisfactory and in view of this
experience we are willing to recommend the
Hearson patent capsule temperature control
as being the most accurate and simple device
for the uniform control of incubator tempera-
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We supply them in nine sizes for gas heating,
six sizes for oil heating, six sizes for electric
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and keep a stock on hand of the more popular
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COMPLETE DESCRIPTIVE PAMPHLET SENT
UPON REQUEST
No. 20740 Hearson Gas Heating Incubator
ARTHUR H. THO
IMPORTERS— DEALERS— EXPORTERS
LABORATORY APPARATUS AND REAGENTS
WEST W/ASHINGTON SQUARE
PHILADELPHIA, U. S. A.
Thomas Jonathan Burrill
[1839-1916]
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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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
0 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
0 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
0 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
0 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 0 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 0 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
0
+ 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
0
+ 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.
De Khotinsky
Fixing and Hot Staining Apparatus
No. 8049A
Electrically Heated
This apparatus meets the need for a clean and neat apparatus for hot staining. By
its use it is possible to fix the smears of sputum, etc., without danger of burning, to stain
them without danger of boiling the carbolfuchsin solution and also to avoid dropping carbol-
fuchsin over the laboratory tables and floors. It consists of an asbestos wood frame with
base which will stand red heat without destruction. In the bottom is a sliding metal shelf,
to be filled with sand for catching any drippings and to aid in reflecting all the heat upward
from the heating units, of which there is one for every two slides. These units, which are
easily replaceable, are made of lavite, bound with nichrome wire, which is insoluble in boil-
ing carbolfuchsin. The slide carriers are nickel-plated brass frames made to fit in the as-
bestos frame at a suitable height above the electrical units. The base is provided with
leveling screws. A set of fuses is placed on the apparatus, so that there is no danger from
accidental short circuit. The apparatus is made in two sizes, as listed below, and is in-
tended for standard 3 x 1 in. slides. It consists of heating box, slide carrier, tray, cross test
level and five feet of cord with two plugs. For 110 volts A. C. or D. C. and furnished with
a rheostaf for close regulation of heat.
8049A. STAINING APPARATUS, for 20 slides Net $45.00
8049B. STAINING APPARATUS, for 10 slides Net 35.03
Electrically Heated and Regulated
This apparatus is constructed for the same purpose as described above, with the ad-
dition of the de Khotinsky Automatic Thermo-Regulating system to facilitate maintaining
a constant temperature, which is especially convenient in research work.
8049K. STAINING APPARATUS, Electrically Heated and Regulated, for 20
standard slides Net $75.00
CENTRAL SCIENTIFIC COMPANY
460 East Ohio Street
(Lake Shore Drive, Ohio and Ontario Streets)
CHICAGO
U. S. A.
Thelco "LOWTEMP"
Electric Incubator
Although primarily
designed for use at
20° C. the **LOWTEMP"
Incubator can be used
just as satisfactorily
at any temperature
from about 10°to45° C.
^The Thelco "all metal" ther-
mostat is quickly and easily set
for any temperature within the
range of the incubator and will
maintain indefinitely constant
temperature to one degree
Centigrade.
^ Adjustable cold air circulation
around the inside jacket of incu-
bator and splendid construction
reduces the ice consumption to
a minimum and secures highest
possible uniformity.
^Exterior of incubator and the
working chamber are of glazed
porcelain fused on steel at 2000'^
F. The most perfect sanitary
construction.
Made in Two Sizes
Dimensions of Working Chamber
18 by 15 by 12 inches, $195.00 Net
30 by 23 by 14 inches, $240.00 Net
Descriptive pamphlet on
request
EIMER & AMEND
FOUNDED 1851
Laboratory Supplies
NEW YORK and PITTSBURGH, PA.
VOLUME I
NUMBER 4
JOURNAL
OF
BACTERIOLOGY
OFFICIAL ORGAN OF THE SOCIETY OF AMERICAN
BACTERIOLOGISTS
JULY, 1916
It is characteristic of Science and Progress that they continually
open new fields to our vision. — Pasteur
PUBLISHED BI-MONTHLY
WILLIAMS & WILKINS COMPANY
BALTIMORE. U. S. A.
THE CAMBRIDGE UNIVERSITY PRESS
FETTER LANE, LONDON; E. C.
Entered as second-class matter April 17, 1916, at the Post Office at Baltimore, Maryland, under the
Act of March 3, 1879
Bacteriological Pepton
Fairchild Building
Washington and Laight Sts.
New York
Fairchild Bros. & Foster
Offer to the bacteriologist a Pepton
which is perfectly serviceable for the for-
mulas and in all the technic of the bac-
teriological and antitoxin laboratory. It is
employed in the usual proportions and for
whatever purposes pepton of this most
desirable quality is required.
It may be mentioned that this product
is offered at a price which is intrinsically
low, in view of *the completely serviceable
quality of this pepton for all bacteriological
purposes.
Pepton, Fairchild
Pepton, Fairchild, is put up in 30 gram
vials; in bottles — quarter, half, and one
pound.
We shall be pleased to send a 30 gram
vial for trial.
FAIRCHILD BROS. & FOSTER
COPE EXTENSION SCOOP
DESIGNED BY
W. G. COPE, Chemist
U. S. Bureau of Mines
MANUFACTURED BY
SCIENTIFIC MATERIALS CO.
PITTSBURGH, PA.
Used for introducing solids into Kjeldahl or other long-
necked flasks. The apparatus consists of an aluminum
weighing scoop attached to an extension by means of a
bayonet joint. The weighing scoop is detachable and of
such a size that it can be placed on a balance pan.
This permits the sample to be weighed and introduced into the flask
without transferring and without getting any oj the material on flask
neck walls.
Permits most accurate results in Kjeldahl determinations
on food products, fertilizers and feed stuffs, also in the
determination of nitrogen in explosives.
FRY RESISTANCE GLASS
MADE BY
H. C. FRY GLASS CO.
IN THE PITTSBURGH DISTRICT
DISTRIBUTED BY
SCIENTIFIC MATERIALS GO.
PITTSBURGH, PA.
This is a real resistance glass as it resists the action of
both alkalies and acids and also temperature changes and
the effects of shocks incident to handling.
Fry Resistance Glass is one that is extremely heat
resisting but not brittle, a very important feature as it is
essential that such glassware should withstand ordinar}'
handling under conditions as they exist in the laboratory.
Complete line oj Flasks, Beakers, and Petri Dishes.
Style K. Flasks, Kjeldahl, with round bottom and long
neck.
Size No.
Capacity, oz.
Capacity, cc.
Stopper No.
1
2
3
10
16
27
300
500
300
6
6
7
Net, Per Doz. $3.50 4.50
Gross lots 10% discount; 5 gross, 15%.
5.50
SCIENTIFIC MATERIALS CO., Pittsburgh, Pa.
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. PARKER 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. Williams
H. Zinsser
S. H. Ayers
F. Bachmann
D. H. Bergey
O. Berghatjsen
C. P. Brown
P. E. Brown
V. Birckner
H. J. Conn
M. M. Cook
J. T. Emerson
L. W. Famtjlener
C. P. Fitch
D. Greenberg
Abstract Editors
P. B. Hadley
I. C. Hall
C. M. Hilliard
J. G. Hopkins
T. G. Hull
A. Itano
I. J. Kligler
J. A. Kolmer
H. L. Lang
H. W. Lyall
W. J. MacNeal
E. C. L. Miller
E. H. Nollau
Zae Northrup
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
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-
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 .S5.50 (23s).
No claims for copies lost in the mails can be allowed unless such claims are
received within 30 iays 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.
BACTERIOLOGICAL INCUBATORS
No. 20548 C. S. &. E. Anhydric Electric Incubator
HEARSON INCUBATORS
WITH PATENT CAPSULE TEMPERATURE
CONTROL FOR GAS, OIL AND
ELECTRIC HEATING
We have supplied over two hundred Hearson
Incubators to important U. 8., State, City and
Hospital bacteriological laboratories through-
out the U. S. Their perforniance has been
universally satisfactory and in view of this
experience we are willing to recommend the
Hearson patent capsule temperature control
as being the most accurate and simple device
for the uniform control of incubator tempera-
tures now on the market.
We supply them in nine sizes for gas heating,
six sizes for oil heating, six sizes for electric
heating and eight sizes for low temperatures,
and keep a stock on hand of the more popular
sizes for immediate delivery.
COMPLETE DESCRIPTIVE PAMPHLET SENT
UPON REQUEST
C. S. &, E.
ANHYDRIC ELECTRIC
INCUBATORS
The walls of these Incubators consist
of five-ply wood, tar paper, animal hair
and transite. No water jacket is used
and the C. S. & E. Electric Thermostat
maintains a constant temperature within
i° C with no attention after adjustment
is once made. No special switch or
wiring is necessary as the Incubators
operate on any 110 volt alternating or
direct circuit by screwing plug into
ordinary lamp socket, and will operate
on 220 volt direct or alternating circuit
by simply changing the voltage of the
lamp heaters.
These are supplied in sixdifferent sizes,
the smallest with inside dimensions
9x7x7 inches at $15.00 and the largest —
as shown in illustration — with inside
dimensions 30 x 36 x 18 inches at $125.00,
all of which are carried in stock in
Philadelphia for immediate shipment.
COMPLETE DESCRIPTIVE PAMPHLET
SENT UPON REQUEST
No. 20740 Hearson Gas Heating Incubator
ARTHUR H. THOMAS COMPANY
IMPORTERS-DEALERS— EXPORTERS
LABORATORY APPARATUS AND REAGENTS
WEST WASHINGTON SQUARE
PHILADELPHIA, U. S. A.
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
made as described below after seven days growth.
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03 02
390
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 0
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
; ; • 0 • •
. to ; ;
. . . . 0 . •
' — ^ 0) 0) 0 *- ' — ^ tt>
0 CC 03 03 Cj 0 IB
h; 0 0 0 ^ H; 0
C 3 3 0 0 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
further investigation at this station.
The constant occurrence of a group of organisms in silage
with characteristics which differentiate its members from
other related groups is of interest from a biological as well as
from the practical viewpoint. The question naturally arises
as to how they gain entrance to the ensilage, or what is their
habitat in nature. Observations made in this connection indi-
BACTERIOLOGY OF SILAGE 451
cate an intimate relationship between the corn plant and this
group of aciduric bacilli. Juice expressed from corn cut a
few weeks prior to the time it was ready for the silo underwent
an acid fermentation, when kept in the laboratory, with the
production of a high degree of acidity and an aroma resembling
that of good silage. From this were then isolated cultures of
organisms similar to those obtained from silage. Sterile milk
inoculated with bits of corn stover, either from the leaves or
pith, always develops large amounts of acid and from it the
typical organisms may be obtained. On a sample of shredded
corn stover nearly a year and a half old the acid forming
bacilli were found in numbers approximating 1,000 per gram
as determined by dilutions in sterile milk.
SUMMARY
The data presented in this paper suggest the probable impor-
tance of a group of acid-tolerant, acid-producing bacilli in the
curing of corn silage.
The organism concerned, while closely related to the B. hul-
garicus group of milk and the B. acidophilus group of the intes-
tines, appears to differ somewhat from the typical members
of these groups, notably by its comparatively abundant growth
on ordinary laboratory media.
The microscopic examination of silage juice deraonstrates
the presence of immense numbers of bacterial cells (always
over one billion per cubic centimeter), most of which are bacilli
which resemble morphologically the high acid producing bacilli
described above.
The aciduric bacilli of silage are constantly found in quite
large numbers on corn fodder, so that silage made from corn
is always amply seeded with these organisms.
452 JAMES M. SHERMAN
BIBLIOGRAPHY
Babcock, S. M. and Russell, H. L. 1900 Causes operative in the production
of silage. Ann. Rpt. Wis. Agr. Expt. Sta., 123-141.
Babcock, S. M. and Russell, H. L. 1901 Causes operative in the production
of silage. Ann. Rpt. Wis. Agr. Expt. Sta., 177-184.
Conn, H. W. 1901 Agricultural bacteriology. Philadephia: P. Blakiston's
Son and Company.
EsTEN, W. M. 1910 Some observations on the fermentation of silage. Science,
n. s., 31, 547-548.
EsTEN, W. M. and Mason, C. J. 1912 Silage fermentation. Conn. (Storrs)
Agr. Expt. Sta. Bull. 70.
Gorini, C. 1906 Recerche batteriologiche sui foraggi conservati nei silos.
Ann. 1st. Agr. (Milano). 6 (1901-1905) lOS-122.
Heinemann, p. G. and Hefferan, M. 1909 A study of B. bulgaricus. Jour.
Infectious Diseases, 6, 304^318.
Heinze, B. 1913 Uber die Einsaurung von Futterstoffen unter BeriJcksichti-
gung von Impfungen mit geeigneten Milchsiiurebakterien-zuchten.
Jahresber. Ver. Angew. Bot., 11, 142-167.
Hunter, O. W. and Bushnell, L. D. 1916 The importance of Bacterium bul-
garicus group in ensilage. Science, n. s., 43, 318-320.
Jordan, E. O. 1914 General bacteriology. Philadelphia: W. B. Saunders
Company.
LiPMAN, J. G. 1911 Bacteria in relation to country life. New York: Macmillan
Company.
LoHNis, F. 1907 Versuch einer Gruppierung der Milchsaurebakterien. Centbl.
Bakt. (etc.), Abt. 2, 18, 97-149.
Marshall, C. E. 1911 Microbiology. Philadelphia: P. Blakiston's Son and
Company.
Russell, E. J. 1908 The chemical changes taking place during the ensiling
of maize. Jour. Agr. Science, 2, 395-410.
Russell, H. L. and Hastings, E. G. 1915 Agricultural bacteriology. Madi-
son, Wisconsin: H. L. Russell.
Samarani, F. 1913 Studi intorno alia conservazione dei foraggi alio stato
verde. Bol. Min. Agr., Indus, e Com. (Rome), Ser. C, 13, 87-103.
Stevenson, W. 1911 The distribution of "long lactic bacteria" — Lactobacilli.
Centbl. Bakt. (etc.), Abt. 2, 30, 345-348.
BOOK REVIEW
Laboratory Manual in General Microbiology. Ward Giltner. Pp.
418. John Wiley and Sons. 1916.
It is refreshing to have a laboratory guide for the study of micro-
organisms with a new title. This Manual is the result of the teaching
experience of the author and co-workers at the Michigan Agricultural
College and aims to be a guide for the teaching of general microbiology.
I suspect that many teachers experience the same difficulty the
reviewer has felt in adapting another's laboratory directions to his
own classroom work. The present Manual will be found extremely
helpful and suggestive in the hands of the teacher but its wide accept-
ance as a class book seems doubtful, partly because of its length, and
partly because of its lack of emphasis on any particular field or group.
The Manual is divided into three parts: Part I, General Morphologi-
cal and Cultural Methods with 53 exercises; Part II, Physiology of
Microorganisms with 33 exercises; Part III, Applied Microbiology,
including 1 exercise on Air, 4 exercises on Water and Sewage, 10 exer-
cises on Soil, 10 exercises on the Dairy, 1 exercise on Plant Micro-
biology, and 14 exercises on Animal Diseases and Immunity. The
Appendix is of great value, including directions for the preparation
of special media, stains, various tables and other data.
The illustrations are admirable and an excellent selection of perti-
nent references is given at the end of each exercise.
The book may be highly commended to teachers, who will cull
from it many valuable suggestions, and when sufficiently extensive
courses in general microbiology are offered it may well be adopted in the
classroom.
C. M. HiLLIARD.
453
ABSTRACTS OF AMERICAN BACTERIOLOGICAL
LITERATURE
BACTERIOLOGY OF FOOD
Feeding Experiments with Bacterium Pullorum. The Toxicity of In-
fected Eggs. Leo F. Rettger, T. G. Hull and W. S. Sturges.
Jour. Exp. Med., 1916, 23, 475^89.
This investigation is divided into two phases: first, a study of the
toxicity or disease-producing properties of Bacterium pullorum when
administered orally; and, second, an investigation of the heat toler-
ance of this organism in infected eggs when prepared for the table
by the usual processes of cooking. It was found that eggs which har-
bor Bacterium 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. Many market eggs
are infected with this organism and since such methods of cooking as
soft boiling, coddling and frying on one side only do not render the yolks
free from viable bacteria, the feeding of eggs thus prepared may be
the cause of serious disturbances in persons who are particularly sus-
ceptible to such influences, and especially in infants.. Inasmuch as
the wide distribution of ovarian infection in the domestic fowl has
come about only in the last few years, its possible danger to man is
one of recent development. — B. W.
BACTERIOLOGY OF THE MOUTH
Observations upon the Endamoebae of the mouth; I. Endamoeba gingivalis
(buccalis). C. F. Craig. Jour. Infect. Diseases, 1916, 18, 220-237.
A general discussion of this protozoan, its historical significance,
geographical distribution, morphological features, relation to Enda-
moeba hystolytica, and its pathogenic role. The author concludes that
"it is more than doubtful that Endamoeba gingivalis is the cause of
pyorrhea alveolaris and that it is yet too early to make positive state-
ments regarding the relation of this parasite to disease." — P. B. H.
Pyorrhea Alveolaris and Some Modern Discoveries Regarding Its Causes
and Treatment. F. E. Stewart. New York State Jour. Med.,
1916, 16, 71-79. . . , ,.
In a comprehensive discussion of pyorrhea alveolaris or Rigg s dis-
ease, the author calls attention to the prevalence of the condition, and
its importance as an etiological factor in many systemic diseases.
Microorganisms, either of virulent types or those ordinarily saprophytic
in the mouth, may invade the living tissues and cause the infection.
455
456 ABSTRACTS
Associated with pyorrhea alveolaris, various types of bacteria have
been recognized for some years, and recently certain protozoa. A re-
view of the more important contributions on the bacteriology and
protozoology of these cases is given. Under treatment the use of
ipecac and emetine as an amebicide against Entameba buccalis is dis-
cussed, likewise the use of ammonium bifluoride, as recommended
by Head, as a bactericide. The use of bacterial vaccines (bacterins)
is indicated, especially when the condition does not respond to local
treatment, or when it becomes systemic. The technique of collecting
the material for, and the . preparation of, bacterial vaccines is out-
hned. In considering the use of autogenous and stock vaccines the
author holds that in certain cases a polyvalent stock vaccine is suffi-
cient. A discussion of the theoretical principles of immunity in rela-
tion to vaccine therapy is presented. — L. W. F.
BACTERIOLOGY OF SOILS
Variation in Nodule Formation. L. T. Leonard. Jour. Amer. Soc.
Agronomy, 1916, 8, 116.
Varying degrees of susceptibility between varieties of the soy beans
to inoculation regarding the production of nodules were investigated.
Nineteen varieties of soy beans were taken and inoculated with 1 cc.
of a broth culture of the soy bean organism. From the results obtained,
the author draws the conclusion that a soy bean culture isolated from
a single strain of soy beans will be successful in inoculating any of
the varieties studied. — A. I.
Nitro-cultures and their Commercial Application. F. C. Harrison.
Trans. Roy. Socy. Can., 1915, Ser. Ill, 9, Sec. IV, 219-223.
The writer refers to the paper presented to the Royal Society in
1906 by himself and Barlow on "The Nodule Organism of Leguminosae —
its Isolation, Cultivation, Identification and Commercial Application."
He reviews the commercial application of the methods then described,
and notes that several firms in the United States have manufactured
and distributed nitro-cultures according to these methods. The On-
tario Agricultural College and the Macdonald College, Quebec, have
sent out some 32,000 cultures with very favorable results, particularly
with Alfalfa and Red Clover. Further experiments with media are
reported, and the following revised medium which has been used suc-
cessfully for the past two years is recommended.
Three solutions are prepared:
A. 75 grams of agar are dissolved in 3000 cc. of water, by placing
in the autoclave at 10-15 pounds pressure.
B. 25 grams of hard wood ashes are boiled in 1000 cc. of water and
filtered.
C. 0.5 gram of acid potassium phosphate, 0.5 gram of magnesium
sulphate, 0.5 gram sodium chloride, 0.25 gram calcium sulphate, 6.25
ABSTRACTS
457
grams of calcium carbonate are dissolved in the order named in 1000
cc. of hot water (about 80°C.). ,
The three solutions, A, B and C are mixed and 87.5 grams saccharose
and 12.5 grams of mannite are added. The resulting 5 litres of medium
are filled into ounce and a half wide mouth Blake bottles, plugged with
cotton. These bottles when filled are sterilised in the autoclave at a
pressure not exceeding 10 pounds. On removal, the bottles are sloped,
and inoculated by means of a pipette. About 2 cc. of a suspension
of the desired organism is run into each bottle. The bottles are kept
in a sloped position, and incubated at 25°C. for about a week, when
they are ready for distribution. — W. S.
BACTERIOLOGY OF WATER AND SEWAGE
Experiences in the Application of the Activated Sludge Process to Chicago
Stockyard Sewage. Arthur Ledbrer. Eng. Contrg., 45, 388.
The temperature of the hquid affects the action m winter. The
turbidity removal is an index of degree of treatment, and with the rela-
tive stabiUty test aids in control. — L. P.
Winter Experience with the Activated Sludge Process at Milwaukee.
W. R. CoPELAND. Eng. Contrg., 45, 386-387.
In winter with a sewage averaging 10.5°C. (minimum 4.5 C.) the
oxidation was retarded. Two and one-fourth cubic feet air per gallon
removed 90 per cent of bacteria and reduced the suspended matter to
15 parts per miUion. The sludge contained 5 per cent N as NH3 and
could be readily pressed. Analytical details are given.— L. P.
Grease and Fertilizer Base from Boston Sewage. R. S. Weston. Jour.
Am. Pub. Health Assn., 1916; 6, 334-343; Eng. News, 1916, 75, 913
The use of SO2 or H2SO4 on Boston sewage disinfects the liquid and
precipitates most of the grease with the greater part of the suspended
matter. Experiments showed a recovery of 1738 pounds dry sludge
per million gallons containing 21.7 per cent grease. The estimates
show an apparent profit of $6 per million gallons. Further investiga-
tion is recommended. — L. P.
The Sterilization of Chicago's Water Supply. Eng. Contrg., 1916,
45, 18. , . „ , n • . ,
After 5 years trial, hquid CI plants are to be installed on all intake
Imes. With less danger of tastes and odors, liquid CI is cheaper, more
flexible and capable of more precise control and reUabihty than other
disinfectants. — L. P.
Manjland Experiences in the Disinfection of Water Supplies. R. B.
Morse. Eng. Contrg., 45, 396-397. ., , , ^, ^ ^ ,
From 0 2 to 3.0 parts per million of available CI have been used
in hypochlorite plants and 0.25 to 0.67 in liquid CI plants. Fewer
458 ABSTRACTS
complaints have been noted with Hquid CI. With intelUgent opera-
tion the plants afford a cheap method of treatment for supplies not
requiring filtration, and cause a marked decrease in typhoid. — L. P.
Copper Sulfate Treatment of St. Paul, Minnesota Water Supplies. N. L.
Huff and G. 0. House. J. Am. W. W. Assoc, 1916, 3, 581-621.
One part per 12,000,000 parts of water was found to be adequate
to eliminate Spirogyra, Cyclotella and most Cyanophyceae. One part
per 10,000,000 remained effective for five weeks after which time
treatment was repeated. Charts accompany the paper. — F. W. T.
The Water Supplies of Interstate Common Carriers on the Great Lakes.
H. P. Letton. J. Am. W. W. Assoc, 1916, 3, 364-384.
Letton discusses mechanical features of the subject together with
various methods for treatment of such water. Bacteriological analyses
were made according to the method promulgated by the Secretary
of the Treasury (Public Health Reports, November 6, 1914, p. 2960).
From these bacterial examinations Letton considers it an impossibility
to obtain a drinking water for boats directly from the lakes that will
at all times conform to the Treasury Department standard. — F. W. T.
The Use of Ammonia in the Chlorination of Water. Joseph Race.
Can Engr., 1916, 30, 345-346.
Ammonium hypochlorite proved to have a very rapid action, 20
to 30 times as rapid as CaOCla. The ammonium salt is not absorbed
readily, preventing after-growths. The use of ammonia (aqua 16°B.)
with CaOCl2 may be economical when the CaOCl2 costs over $2.08
per 100 pounds. Difficulties in application require quick mixing with
large dilution after ammonia is added to bleach solution to avoid loss.
L. P.
Some Aspects of Chlorination. Joseph Race. J. Am. W. W. Assoc,
1916, 3, 439-449.
Among other subjects Race discusses the questions of aftergrowths
and types of B. coli which survive chlorination. Much difference of
opinion exists with regard to the question of aftergrowths. The after-
growths, under usual working conditions, vary according to the dosage
of chlorine employed. A small amount .^f chlorine exerts a selective
action. With large amounts a flora of spore formers survives which
is unlike the original flora of the water. A study to determine whether
the B. coli found after chlorine treatment were more resistant, indicated
that there was little difference in the resistance of various types to
chlorine.— F. W. T.
A Study of Some Organisms Which Produce Black Fields in Aesculin —
Bile-Salt-Media. F. C. Harrison and J. Vanderleck. With 4
plates. Trans. Roy. Socy. Can., 1915, Ser. Ill, 9, Sec IV, 207-217.
Six hundred pure cultures were isolated from aesculin bile-salt-agar,
and lactose htmus agar plates made during analyses of 1000 milk sam-
ABSTRACTS 459
pies from dealers or farmers in the Province of Quebec. These cul-
tm-es were used to test the reliability of aesculin bile-salt-agar for the
identification of the colon-aerogenes group. Of the 600 colonies many
were selected because they seemed to be slightly atypical. There
were only 10 exceptions; a percentage of 1.5 of all colonies tested; and
a percentage of 0.03 of the colonies on the plates from which the 600
were isolated. Seventeen of the sub-cultures were subjected to the
usual classification tests, and the results together with information
as to the source of the milk are given in tabular form. Drawings
showing the appearance on aesculin bile-salt-agar plates of surface
and deep colonies respectively are included on plates I-IV. — W. S.
DISINFECTION
Further Investigation into the Precipitation of the Typhoid Bacillus by
Means of Definite Hydrogen-ion Concentration. C. F. Kemper.
Jour. Infect. Diseases, 1916, 18, 209-215.
The aim of the present work was to ascertain whether there exists
a specific hydrogen-ion concentration for the precipitation of all strains
of B. typhi. None was found; the effective range of hydrogen-ion con-
centration was wide, most strains being precipitated in the presence
of a 3.6 by 10^ hydrogen-ion concentration derived from the dissocia-
tion of acetic acid in the presence of sodium acetate. — P. B. H.
Effect of Pasteurization on Mold Spores. Charles Thom. J. Agr.
Res., 1916, 6, 153-166.
The object of this investigation was to see whether mold spores
could resist the heat applied in pasteurization. Several species of
Aspergillus and Mucor, and a large number of species of Penicillium
together with a few other kinds of molds were tested. It was found
that nearly all the mold spores were killed either by the holder process
(30 minutes at 63°C.) or by the flash process (30 seconds at 74° or 80°C).
The flash process at 74°C. proved the most efficient, only occasional
spores surviving.
The effect of dry heat was also investigated. Dried preparations
of the spores were submitted to various degrees of temperature. The
resistance to dry heat was found to be much greater than to moist heat,
considerable numbers of the spores surviving unless temperatures of
120°C. (for 30 seconds) were used.— H. J. C.
The Physical Chemistry of Disinfection. J. F. Norton and Paul H.
Hsu. Jour. Infect. Diseases, 1916, 18, 180-194.
This contribution attempts to apply physico-chemical methods
to the problem of the mechanism of disinfection; and in the first place
to demonstrate the roles which undissociated acids, hydrogen ions and
anions play in the process. The first test was made with formic acid,
using B. typhi as the test organism. The results seemed to justify the
following conclusions : that acids act as disinfectants through the agency
of the hj^drogen ions, and that the disinfecting power is proportional
460 ABSTRACTS
to the H ion concentration. The addition of a salt possessing a com-
mon anion diminishes the power through decrease in the H ion con-
centration and increase in the concentration of the undissociated acid
molecules. Salts which do not appreciably affect the dissociation of
an acid greatly increase the disinfecting properties. In acid disinfec-
tion acid anions are positive catalyzers and undissociated acid mole-
cules are negative catalyzers. — P. B. H.
The Bactericidal Properties of the Quarternary Salts of Hexamethylene-
tetramine. I. The Problem of the Chemotherapy of Experimental
Bacterial Infections. W. A. Jacobs. Jour. Exp. Med., 1916, 23,
563-568.
Introductory to subsequent papers. A discussion of the factors to
be considered in formulating a program for systematic studies in
chemotherapy.
Ibidem. II. The Relation between Constitution and Bactericidal Action
in the Substituted BenzylhexametMjlenetetraminium Salts. W. A.
Jacobs, M. Heidelberger and H. L. Amoss. Jour. Exp. Med.,
1916, 23, 569-576.
By the addition of substituted benzyl halides to hexamethylene-
tetramine, a series of quarternary salts of this base was obtained. These
salts represent a new group of organic bactericides. The results ob-
tained in the tests with these substances upon Bacillus typhi have
demonstrated the existence of direct relationships between chemical
constitution and bactericidal action within the series. The bactericidal
character is directly attributable to the presence of the hexamethylene-
tetramine nucleus. The degree of the bactericidal action, however,
is determined by the position, character, and number of the groups
substituted in the benzene nucleus. By the introduction of the methyl,
chlorine, bromine, iodine, cyano, and nitro groups into the benzene
nucleus of the parent benzyl hexamethylenetetraminium salt, the
bactericidal power of this compound was notably enhanced. The sub-
stitution of these groups in the ortho position almost invariably resulted
in substances which were more active than their meta or para isomers.
The introduction of the methoxy group was without marked effect.
Several substances in which two hexamethylenetetraminium side-
chains occurred were found to be the most active of the substances of
this series when tested against Bacillus typhi. Comparative tests
with other bacterial types demonstrated that these compounds pos-
sessed a marked degree of specificity for Bacillus typhi.
Ibidem. III. The Relation between Constitution and Bactericidal Action
in the Quarternary Salts Obtained from Halogenacetyl Compounds.
W. A. Jacobs, M. Heidelberger and C. G. Bull. Jour. Exp,
Med., 1916, 23, 577-599.
The extension of the study of the quarternary salts of hexamethylene-
tetramine to those obtained by the addition of this base to the most
ABSTRACTS 461
varied types of substances containing aliphatically bound halogen
has demonstrated that the introduction of the hexamethylenetetramine
nucleus in this manner results in the production of bactericidal sub-
stances or enhances the bactericidal action if already present.
In particular it was found possible by the use of the halogenacetyl
group, XCHoCO, as a connecting link, to furnish primary and secondary
ahphatic and aromatic amines, alcohols, and hydrocarbons of the
most varied character with the hexamethylenetetramine molecule and
to study the relation between chemical constitution and bactericidal
action in the series of substances so prepared. Because of the variety
of chemical types studied, the results are too involved for a detailed
summary here. Many of the substances were found to be very power-
ful bactericides, and in a number of instances derivatives of purely
aliphatic nature were found to possess an unusual bactericidal power.
Bacillus typhi, streptococci, meningococci, and gonococci were the
microorganisms used for the tests, and striking instances of partial
specificity were observed. This specificity was found to favor not one
species alone, but instances were found in which each of the types of
bacilli was shown to be especially susceptible to one or another of the
particular types of compound employed. The source of this partial
specificity is to be sought not in the hexamethylenetetramine nucleus
itself but in the molecule to which it is attached. The action of some
of the substances was tested in the presence of serum or protein and
was found to be not at all or only slightly i ihibited. In other cases
marked inhibition occurred. The factors controlling the serum —
or protein — compatibility of these substances are likewise to be sought
in that portion of the molecule other than the hexamethylenetetramine.
B. W.
IMMUNOLOGY
Anti-Typhoid Inoculation. Eng. News, 1916, 75, 530.
The Canadian Pacific Railway has used inoculation with success,
reducing cases in two years to 3, as compared with 290 for two years
among non-inoculated. — L. P.
Development of Immune Reactions in Serum Disease. W. T. Longcopb
AND F. M. Rackemann. Proc. Soc. Exp. Biol, and Med., 1916,
13, 101-102.
Eleven patients, who had received horse serum for therapeutic
purposes, were studied by two methods. Skin sensitiveness to horse
serum was tested by intracutaneous injections of 0.02 cc. of horse
serum, both undiluted and diluted ten or one hundred times with salt
solution. Second, anaphylactic antibody was tested for by injecting
the serum of the patient into guinea pigs and testing these for passive
sensitization. The results show that anaphylactic antibodies for horse
serum appear in the blood serum in maximmn concentration towards
the close of serum sickness and suggest that their presence determines
recovery from this disease. — W. J. M.
462 ABSTRACTS
Immunization with Sensitized Bacteria. Homer F. Swift and Ralph
A. KiNSELLA. Proc. Soc. Exp. Biol, and Med., 1916, 13, 103.
Two strains of green-forming streptococci were used. Rabbits were
injected with sensitized vaccines and compared with other rabbits in-
jected with unsensitized vaccines. In the latter group of animals there
was strong formation of antibodies in from twelve to sixteen days. The
animals injected with sensitized vaccines, on the other hand, showed
only weak agglutinins or complement-fixing bodies and the serum was
without protective value for mice. — W. J. M.
Complement Fixation in Acne Vulgaris. Albert Strickler, John
A. Kolmer and Jay F. Schamberg. Jour. Cutan. Dis., 1916, 34,
166-178.
The authors call attention to the indefinite bacteriology of acne
vulgaris and the probable etiological role of colon bacilli in this dis-
ease. They attempted a study of the relationship to the disease of
the bacillus of acne, the cocci from acne lesions and of B. communis
and B. communior from the feces of persons suffering with this disease,
by means of complement fixation tests using the same antigens with
the sera of normal persons and persons suffering with non-acneiform
diseases and controlling the results in acne by testing the serum of their
patients with polyvalent antigens prepared with cocci from furuncles
and with colon bacilli from the feces of healthy persons.
Of 57 cases of acne vulgaris, 84.2 per cent reacted positively with an
antigen of B. acne; 64 per cent reacted positively with the antigen of
staphylococci from acne lesions and practically the same results were
observed with the control antigen of staphylococci; 63.1 per cent
reacted positively with the antigen of B. coli from the feces of acne pa-
tients and 32 per cent reacted positively with the antigen of B. coli
from the feces of normal and healthy persons.
The sera of normal and syphilitic persons reacted uniformly negatively
with all antigens; the sera of persons suffering with various skin dis-
eases likewise reacted negatively in the majority of instances except
those with acne rosacea and seborrholic dermatitis.
From these studies the authors conclude that B. acne may be an
etiological factor in skin diseases other than acne vulgaris; that the
cocci found in these lesions possess no peculiar serological characteris-
tics such as would differentiate them from other staphylococci found
in furunculosis and that B. coli appears to exert an etiological influence
in some diseases of the skin and particularly acne vulgaris. — J. A. K.
The Variations in Reaction of the Blood of Different Species as Indicated
by Hemolysis of the Red Blood Cells ivhen Treated with Acids or Alkalies.
J. G. Gumming. Jour. Infect. Diseases, 1916, 18, 151-179.
It had been shown in earlier work that the sign of the Wassermann
reaction might be reversed by the influence of salts, acids and alkalies
on the hemolytic system. The aim of the present work was to estab-
lish a chemical standardization for definite hemolytic time indices of
ABSTRACTS 463
different animal species, as a preliminary to determining the percent-
age of ammonia, sodium hydroxide, hydrochloric acid and other mor-
ganic and organic compounds and salts, necessary to cause complete
hemolysis under certain arbitrary conditions. For a fifteen minute
hemolytic system there was a marked difference in the requirements
for NHg, NaOH, and HCl, for some species but not for others. Va-
rious hemolytic time indices for the substances mentioned were worked
out, and by this means it was found possible to identify blood cell sus-
pensions of different species with considerable accuracy. The follow-
ing conclusions were drawn: Alkaline hemolysis may be considered
due to the hydroxy 1 group, while acid hemolysis is due to the H-ion.
"The hemolysis of the red blood cell may be used an as indicator
to ascertain the degree of acidity or alkalinity of certain solutions."
"AlkaUne hemolysis can be influenced by acids and acid hemolysis
by alkalies. Both can be influenced by the neutral salt content of
the suspension." It was shown that there was a distinct variation
between the normal and the pathologic blood of the same species,
since the time indices of the latter specimens were increased or de-
creased. It is suggested that this may be due to increased alkalinity
or decreased acidity, or to variation in the natural salt content.
P. B. H.
INDUSTRIAL BACTERIOLOGY
The Removal of the Natural Impurities of Cotton Cloth hy the Action
of Bacteria. B. S. Levene, Journ. Ind. and Eng. Chem., 1916,
8, 298.
Levene investigated the possibility of removing the nitrogenous and
fatty impurities of cotton fiber by means of bacteria in place of the
vigorous chemical treatment now employed. After preliminary ex-
perimentation the following organisms were found most suitable :
B. amylolyticus, B. fimi, B. bihulus, B. carotovorus, B. suhtilis.
These bacteria are capable of hydrolyzing starch, and decomposing
cellulose or pectin or both.
Coarse cotton cloth was sterilized in nutrient broth inoculated with
the above named bacteria and incubated at 37.5°C. Tests were
made after one, two, and three. months respectively. Slight changes
were observed after one month, more marked effects after two months,
and decided alterations after three months. Cloth washed and bleached
was perfectly white and was not yellowed by steaming. Chemical
tests showed complete removal of nitrogenous impurities, about 80
to 90 per cent of the ether soluble impurities, and from 2.5 to 40 per
cent of the alcohol soluble substances. The effect on the last men-
tioned substances varied with the different types, B. caratovorus being
the most effective. Two forms— B. bihulus and B. fimi— caused weak-
ening of cloth; the others apparently had no such effect.
By using different combinations of organisms and different media
the incubation period could be reduced to 24 to 72 hours.— I. J. K.
464 ABSTRACTS
MEDICAL BACTERIOLOGY
The Pure Cultivation of Spirochaeta Icterohaemorrhagiae (Inada) . T. Ito
AND H. Matsuzaki. Jour. Exp. Med., 1916, 23, 557-562.
This organism, claimed to be the causative agent of Weil's disease,
was obtained by the authors in a solid, a semi-solid, and a fluid medium,
enriched with blood. The spirochaete thus isolated remains patho-
genic for guinea pigs for many generations. The characteristics of
three strains are described. — B. W.
A Milk-Borne Paratyphoid Outbreak in Ames, Iowa. Max Levine
AND Frederick Eberson. Jour. Infect. Diseases, 1916, 18, 143-150.
The authors explain a slight epidemic on the grounds of a paratyphoid
B. infection originating either from a carrier or from a convalescent.
The paratyphoid cultures isolated, it is stated, produced gas in glu-
cose broth.— P. B. H.
A Leptothrix Associated with Chronic Hemorrhagic Nephritis. G. R,
Dick, G. F. Dick, and B. Rappaport. Jour. Infect. Diseases,
1916, 18, 216-219.
The authors report a case of chronic nephritis characterized by recur-
ring attacks of hematuria probably caused by a Leptothrix which was
present in the urine. The causal relation was suggested by the con-
stant occurrence of the organism, its virulence for animals and the
improvement in the case which took place under vaccine treatment.
P. B. H.
Technique of Cultivating Human Tissues in Vitro. R. A. Lambert.
Proc. Soc. Exp. Biol, and Med., 1916, 13, 100-101.
Chick plasma with the addition of an equal quantity of human serum
furnishes a satisfactory medium, in which the fibrin network resists
digestion. Human tissue may be preserved in viable condition for
five to ten days, by immersion in salt solution in a cool place. The de-
struction of bacteria in infected tissues by means of chemical disinfec-
tants is being investigated. — W. J. M.
The Cytology of the Exudate in the Early Stages of Experimental Pneu-
monia. Frank A. Evans. Proc. Soc. Exp. Biol, and Med., 1916,
13, 99-100.
Pneumonia was induced in rabbits by intrabronchial injection of
pneumococci and streptococci and by the injection of egg yolk. The
exudate in each instance contained many polynuclear cells but more
often the predominant cells were mononuclear. — W. J. M.
Pneumotyphoid, with Report of a Case. F. Billings. Am. Jour. Med.
Sc, 1916, 151, 36-39.
Billings reports a case which "seems to present an example of typhoid
fever presenting its primary manifestations in the lung in the form of
ABSTRACTS 465
a frank, outspoken croupous pneumonia." Cultures from the blood,
and from the sputum yielded B. typhi, but the organism was not found
in the stools or the urine. — L. W. F.
The Mode of Infection in Pulmonary Distomiasis. Koan Nakagawa.
Jour. Infect. Diseases, 1916, 18, 131-141, Pis. II-IV.
The author reports on the distribution, origin and means of infec-
tion in the case of the distomiasis observed in Formosa. It is shown
that at least two species of crabs found in Formosa and Japan proper
contain in the liver or gills large numbers of encysted larvae, and that
the number of infested crabs in any district is roughly proportional
to the number of cases of distoma infection. Dogs were successfully
infected as a result of eating liver or lungs of infested crabs. In the
final host it was shown that the encysted larvae, after entering the
intestines, reach the abdominal cavity by perforating the intestinal
wall near the jejunum. They then penetrate the diaphragm and
pleura and finally pierce the lung parenchyma. Here they develop
and lay eggs which are discharged with other degenerative tissue prod-
ucts through the trachea. — P. B. H.
The Reactions between Bacteria and Animal Tissues under Conditions
of Artificial Cultivation. II. Bactericidal Action in Tissue Cultures.
H. F. Smyth. Jour. Exp. Med., 1916, 3, 265-274.
In a previous paper the author described an original method for
studying the action of tissue cultures in vitro when inoculated with
living pathogenic bacteria. In the present experiments chick embryo
tissues were grown in a mixture of equal parts of plasma and Ringer
solution. It was found that chicken plasma exerts a marked bacteri-
cidal action on B. typhi and on B. diphtheriae but is less marked with
B. dysenteriae, and sKght, if present at all, with B. coli. Chick tissues,
particularly splenic tissue, counteract this action. The migrating
white cells from splenic cultures have a distinctly bactericidal influence
on all organisms tested except B. coli.
Ibidem. III. The Action of Bacterial Vaccines on Tissue Cidtures
in Vitro. H. F. Smyth. Jour. Exp. Med., 1916, 3, 275-291.
In plasma tissue cultures in vitro with tissue containing lymphatic
elements the changes characteristic of early tubercle formation may
be seen when such cultures contain masses of tubercle bacilli. — B. W.
Experiments with Poliomyelitis in the Rabbit. M. J. Rosenau and
L. C. Havens. Jour. Exp. Med., 1916, 23, 461^74.
The poliomyelitic virus obtained from an experimental monkey
was passed through eight generations in rabbits with no apparent
change in virulence. It is filterable and is virulent only for young
rabbits. Even in these only about 40 per cent succumb. Inocula-
tions were made intracranially, intravenously, into the sheath of the
sciatic nerve and by placing the virus upon the uninjured nasal mucosa.
466 ABSTRACTS
The incubation period varied from two to forty-one days with an aver-
age of twelve days. The lesions produced while dej&nite and consist-
ent lack the distinctive features of the pathologic picture of polio-
myelitis in man and the monkey. The symptoms differ in individual
rabbits and show variations from those seen in the monkey and in
man. — B. W.
The Protection of Pathogenic Microorganisms by Laving Tissue Cells.
P. Rous AND F. S. Jones. Jour. Exp. Med., 1916, 23, 601-612.
One series of experiments was carried out to determine whether
phagocytes protect ingested bacteria against the bactericidal action
of sermn and of potassium cyanide. While the conditions of the
tests are scarcely comparable with conditions in the body, yet they
point to a protecting action on the part of the phagocyte. When
erythrocytes and a hemolytic system were substituted for bacteria
and bactericidal substances the results were sharp and conclusive.
Suspensions of dog leukocytes were incubated with rat erythrocytes
and dog serum was added for its opsonic action. After an hour's
incubation anti-rat erythrocj^te serum was added. It was then found
that while all extra cellular rat erythrocytes were dissolved, the pha-
gocyted red cells remained unaffected. From their experiments the
authors conclude that living phagocytes are able to protect ingested
organisms from the action of destructive substances in the surrounding
fluid, and even from a strong homologous antiserum, and that this
protection by phagocytes is largely, if not entirely, conditioned on their
being alive. These findings should be taken into consideration in
the study of diseases caused by infectious agents capable of living within
tissue cells. — B. W.
Chemical versus Serum Treatment of Epidemic Meningitis. Simon
Flexner and H. L. Amoss. Jour. Exp. Med., 1916, 23, 683-701.
The authors studied the therapeutic effect of lysol and protargol
in experimental meningococcus infections. Neither substance proved
to have any curative action on the experimental infection in guinea
pigs and protargol failed to influence favorably the infection follow-
ing sub-arachnoid inoculation in monkeys. It was found that both
lysol and protargol exert antileukotactic and antiphagocytic effects,
and are also potent protoplasmic poisons, and the leukocytes with
which they come in contact are injured and made to degenerate. The
mixture of antiserum with lysol and with portargol reduces to a certain
extent the antileukotactic and antiphagocytic effect of the chemicals;
but this action is insufficient wholly to set aside the injurious effects
which they produce. Any theoretical advantages they may possess
are more than offset by the harmful effects which they cause, hence
specific antiserum seems to provide the logical therapeutic agent with
which to combat epidemic meningitis, since it is itself innocuous and
promotes those processes essential to recovery from the disease.
B. W.
ABSTRACTS 467
Further Experimentation in Animals with a Monilia Commonly Found
in Sprue. B. K. Ashford. Am. Jour. Med. Sc, 1916, 151, 520-
528.
This report is a continuation of the author's studies in experimental
monihasis in animals by means of a new moniha found in sprue. He
feels that sufficient evidence has been gained to justify Bahr's opinion
that sprue is due to a monilia, but not Monilia albicans, since he has
found in Porto Rico a distinct, undescribed species in nearly one hun-
dred cases of true sprue, and in only a small percentage of carriers.
This organism is designated as Monilia X, and is ordinarily of low
virulence. The vu-ulence on long cultivation is partially or completely
lost, but may be recovered by passage through susceptible animals.
When promptly injected, after recovery from patient with sprue,
deaths generally result from mycotic septicaemia. Feeding tests
with the freshly isolated organism from the patient ordinarily failed
to kill animals, but when the virulence was raised by passage, it killed
by this method of administration. Some of the animals died rapidly
of a monilia septicaemia, others more slowly, probably from a toxin
developed by a localization of the organisms in the intestinal tract;
stomatitis has been observed, and also long continued severe diarrhoea
following the feeding tests. Most of the tests were carried out on
guinea pigs, although rabbits, monkeys, and the white rat were also
used. The gross and microscopical findings of the autopsied animals
are recorded. Noteworthy is the fact that the monilia, if attacking
an internal organ, were seen as large colonies having the appearance
of emboli. The intervening tissue spaces were generally free from
the organism, thus radically differing from a bacterial septicaemia.
L. W. F.
Analysis of One Hundred and Thirty-Four Cases of Baderiemia. M.
Warren and W. W. Herrick. Am. Jour. Med. Sc, 1916, 151,
556-577.
The author's report, as the title states, is an analysis of one hundred
and thirty-four cases of bacteriemia observed during the past five
years in the various services of a general hospital. An outline of the
laboratory methods employed is given; also the bacteriological classi-
fication of the streptococcus group is considered. The cases are re-
corded under hospital case number, with age of the patient, the diag-
nosis, the maximum temperature, the leucocyte count and polyniiclear
percentage, the bacteriological blood findings, the treatment, and the
result. Tables are given summarizing the authors' findings. Of
especial interest to the bacteriologist is the table giving the General
Summary, which follows:
468
ABSTRACTS
General Summary
ORGANISM
Str. hemolyticus
Str. viridans
Str. mucosus
Staph, aureus
Staph, albus
Pneumococcus
B. coli
B. influenzae
Anaerobic streptococci
B. mucosus
B. mallei
B. alkaligenes
Mixed infection
Str, hemolyticus and Staph, aureus.
Str. hemolyticus and B. typhi
Str. viridans and Staph, aureus
Str. viridans and B. coli
Staph, aureus and B. coli
B. proteus and B. coli
B. influenzae and Staph, albus
All cases
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134
83
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86.0
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100.0
0
100.0
100.0
100.0
100.0
61.1
In the table giving the results of treatment are found twenty-
five cases treated with vaccines with a mortality of 81 per cent, and
four cases treated with serum, with a mortality of 75 per cent, which
according to the authors shows "the futility of present measures of
specific therapy of generalized infections."— L. W. F.
Tonsillar Endamehiasis and Thyroid Disturbances. J. S. Evans, W. S.
MiDDLETON AND A. J. Smith. Am. Jour. Med. Sc, 1916, 151,
210-222.
The authors discuss the various theories of the causation of endemic
goitre, in particular, those more recently advanced, based on the view
that the disease is of an infectious nature. From data obtained from
the physical examination of a large number (1328) of men (University
of Wisconsin) it was found that 27.2 per cent had thyroid involvement.
Of these, 22.8 per cent showed infective cryptic tonsillar lesions; this
was increased to 90 per cent when those having nasal lesions were
included. These observations suggest a connection between the nasal
and throat affections, and the thyroid involvement. Since Entameba
gingivalis (Gros) apparently plays a role in the etiology of pyorrhea,
ABSTRACTS 469
certain cases of chronic tonsilitis and systemic complications, the
writer's attention was directed toward the organism. In thirty-four
cases showing typically diseased tonsils, 97 per cent showed entamebae
in the crypts. Of this group, sixteen individuals were treated by
means of emetin hydrochloride with a disappearance of the amebae
from the crypts in thirteen cases (81 per cent). Emetin was adminis-
tered to twenty-three individuals, sixteen of whom showed an appre-
ciable reduction in the bulk of the thyroid. The group included seven
dysthyroid cases ; six were benefited in varying degrees. The improve-
ment under the treatment with emetin led the writers to believe that
an indirect relationship existed, and they concluded that "A symbiosis
of entamebae with appropriate bacteria, leading to the elaboration and
absorption into the thyroid of selective thyrotoxic poisons, is at least
conceivable in explanation of such relation." But they do not consider
this to be an exclusive explanation of all goitres. No entamebae were
found in the thyroid gland. — L. W. F.
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VOLUME I
NUMBER 5
JOURNAL
OF
BACTERIOLOGY
OFFICIAL ORGAN OF THE SOCIETY OF AMERICAN
BACTERIOLOGISTS
SEPTEMBER, 1916
It is characteristic of Science and Progress that they continually
open new fields to our vision. — Pasteur
PUBLISHED BI-MONTHLY
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Act of March 3, 1879.
Bacteriological Pepton
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New York
Fairchild Bros. & Foster
Offer to the bacteriologist a Pepton
which is perfectly serviceable for the for-
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teriological and antitoxin laboratory. It is
employed in the usual proportions and for
whatever purposes pepton of this most
desirable quality is required.
It may be mentioned that this product
is offered at a price which is intrinsically
low, in view of the completely serviceable
quality of this pepton for all bacteriological
purposes.
Pepton, Fairchild
Pepton, Fairchild, is put up in 30 gram
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pound.
We shall be pleased to send a 30 gram
vial for trial.
FAIRCHILD BROS. & FOSTER
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Used for introducing solids into Kjeldahl or other long-
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This permits the sample to be weighed and introduced into the flask
•without transferring and without getting any of the material on flask
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Price, $1.50 each.
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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
Managing Editor
C.-E. A.
WINSLOW
A. PARKER HITCHENS
Yale Medical School,
New Haven, Conn.
Glenolden, Pa.
Advisory
Editors
C. C. Bass
F. C. Harrison
V. A. Moore
W. T. Sedgwick
R. E. Buchanan
H. W. Hill
M. E. Pennington F. L. Stevens
P. F. Clark
E. 0. Jordan
E. B. Phelps
A. W. Williams
H. \V. Conn
A. I. Kendall
L. F. Rettger
H. Zinsser
F. P. Gay
C. B. LiPMAN
L. A. Rogers
F. P. GORHAM
C. E. Marshall
M. J. ROSENAU
Abstract Editors
S. H. Ayers
L. W. Famulener
J. A. Kolmer
W. Sadler
F. Bachmann
C. P. Fitch
H. L. Lang
G. H. Smith
D. H. Bergey
D. Greenberg
H. W. Lyall
F. L. Stevens
0. Berghausen
P. B. Hadley
W. J. MacNeal
F. W. Tanner
C. P. Brown
I. C. Hall
E. C. L. Miller
R. M. Taylor
P. E. Brown
C. M. HiLLIARD
E. H. Nollau
E. B. Vedder
V. Birckner
J. G. Hopkins
Zae Northrup
A. R. \\ ARD
H. J. Conn
T. G. Hull
L. Pearse
B . W HITE
M. M. Cook
A. Itano
E. B. Phelps
J. T. Emerson
I. J. Kligler
G. H. Robinson
CONTENTS
Dorothy F. Pettibone, Franklin P. Bogart and Paul F. Clark: The Bacteriology
of the Bubble Fountain 471
James M. Sherman: The Advantages of a Carbohydrate Medium in the Routine
Bacterial Examination of Milk 481
Hans Zinsser and J. G. Hopkins: On a Species of Treponema Found in Rabbits. . . . 489
W. W. Ford and Others: Studies on Spore-Bearing Non-Pathogenic Bacteiia. Part II 493
C. A. Laubach : Spore-Bearing Bacteria in Dust 493
C. A. Laubach : Spore-Bearing Bacteria in Water 505
C. A. Laubach and J. L. Rice: Spore-Bearing Bacteria in Soil 513
W. W. Ford: Miscellaneous Cultures 518
W. W. Ford: Classification 527
Paul R. Cannon: A Rapid and Simple Indol Test 535
Nathan Berman and Leo F. Rettger: Bacterial Nutrition, a Brief Note on the Produc-
tion of Erepsin by Bacteria 537
A. Parker HiTCHENs: A Practical Method for the Identification of Guinea-pigs under
Treatment 541
C. L. Williams and H. P. Letton: A Note on the Preparation of Agar Agar Culture
Media 547
Book Reviews: McFarland's Pathogenic Bacteria and Protozoa; D. Greenburg. 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
Immunology 568
Laboratory Technique 576
Plant Pathology 577
Public Health Bacteriology 578
Medical Bacteriology 579
INFORMATION FOR CONTRIBUTORS AND SUBSCRIBERS
Number one of volume one of the Journal of Bacteriology, dated January, appeared
Apri' 22; number two, dated March, appeared May 17.
The Journal of Bacteriology is issued bi-monthly at present.
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 non-receipt.
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 c'ontribtitprs 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 Dr. A
Parker Kitchens, Glenolden, Pa.
Correspondence concerning business matters should be addressed to the Williams and
Wilkins Company, 2419-2421 Greenmount Avenue, Baltimore, U. S. A.
Subscriptions are received — For the British Empire, except Canada: Mr. C. F. Clay,
Manager, The Cambridge University Press, Fetter Lane, London, E.C. Subscription price,
23s. net, post paid.
For Japan and Korea: Maruzen Company, Ltd. (Maruzen-Kabushiki-Kaisha, 11 to 16
Nihonbashi Tori-Sanchome, Tokyo; Osaka, Kyoto, Fukuoka, and Sendai, Japan. Sub-
scription price, $5.50 net, post paid.
For the United States, Canada, and all other countries, except as above: The Williams
and Wilkins Company, 2419-2421 Greenmount Avenue, Baltimore, U. S. A. Subscription
price. North America, and U. S. possessions, $5.00 net, post paid; other countries, $5.50 net,
post paid.
For Switzerland: Georg & Cie, Freiestrasse 10, Basel, Switzerland. Subscription price
$5.50, post paid.
Subscriptions to the Journal are taken for the volume only. A volume consists of 600
to 650 pages. As soon as this number of pages is reached a new volume is started and a sub-
scription bill for the new volume will be rendered.
HEARSON
LOW TEMPERATURE INCUBATORS
No. 20848— Model C
No. 20854— Model G
LOW TEMPERATURE INCUBATOR, HEARSON MODEL C, for operation with a constant flow of water. Most
of the low temperature incubators can only be worked successfully as long as the external air is 10° b".low the tempera-
ture required in the incubating chamber, and the expedient sometimes resorted to of running cold water through them
to keep the temperature down can, in summer time, be adopted with advantage, but the results are not altogether
satisfactory, cultures being frequently spoiled by an unexpectedly vyarm day or night.
ice,
other predete — ^ , = . , , , a . j ■ »i. • * •
even though the external air may be 30 or 40 degrees above the temperature desired in the interior.
With capsule adjusted to 20° C unless otherwise ordered, with thermometer, ice funnel, gas fittings and 4 ft. of
flexible metalic tubing (or, if for oil lamp, 2 chimneys and one yd. wick) and packing. With suitable burner for either
oil or gas heating. Please specify when ordering. , .j ,, , n .„ c,»» ci.-i,
^ Inside Measurements Duly Free Slock
2')844. Low Temperature Incubator Model C 9 x 9 x 12 inches S78.30 S94.00
~n8i8 " " " " " 12 X 12 X 14 111.80 134.15
inlT^- .. " " " " 15x15x18 " 140.75 158.90
^llll] .. .. «' " " ;■. 20x20x24 " 198.75 238.45
LOW TEMPERATURE ELECTRIC INCUBATOR, HEARSON MODEL G, for ice and electricity, working
independently of any constunt water supply as required in model C. This apparatus is identical with Model C except
that an electric heater is provided which automatically operates when the room temperature is lower than the tempera-
ture required in the chamber and an electric motor which automatically circulates the water from melting ice to the
water jacket when the temperature of the room is higher than the temperature required in the chamber. No connection
with a water supply is required and the apparatus may be set to operate at any temperature from 10° to 37 C. and can
be operated in any climate and in any place where water and electricity are available. Control is by the Hearson
capsule and full operating directions are supplied with each utensil. Voltage must be specified in ordering.
Inside Measuremenls Duly Free Stock
20850. Low Temperature Electric Incubator Model G 9 x 9 x 12 inches $113.40 $136.10
TnsAi' " " " " " " 12 X 12 X 14 " 143.10 171.75
inlll' .. " " " " " 15x15x18 " 199.80 229.00
2Qg72. " " " " " " 20x20x24 " 252.00 302.40
These Low Temperature Incubators have been supplied by us to leading Universities, City and State
Boards of Health, U. S. Government laboratories, etc., all of whom are
unanimous in reporting satisfactory service
ARTHUR H. THOMAS COMPANY
IMPORTERS— DEALERS— EXPORTERS
LABORATORY APPARATUS AND REAGENTS
WEST WASHINGTON SQUARE PHILADELPHIA, U. S. A.
THE BACTERIOLOGY OF THE BUBBLE FOUNTAIN^
DOROTHY F. PETTIBONE, FRANKLIN B. BOGART and PAUL F. CLARK
From the Laboratory of Medical Bacteriology, The University of Wisconsin
The public conscience has been aroused to the dangers of the
common drinking cup as a possible means of spreading disease.
Many state legislatures have passed laws prohibiting its use;
other states have accomplished the same object by rulings of
their state boards of health ; and interstate passenger trains have
been compelled to discard the common cup.
To meet this situation, three substitutes have been recom-
mended; first, the individual cup carried and used by a single
person, second, the single service paraffin cup, and third, the
bubbling fountain. Expense and inconvenience have been large
factors in preventing the general adoption of the first two meth-
ods. The bubble fountain, however, has met with cordial ap-
preciation both on the part of the public and also in the hearts of
health officials, and has been largely adopted in this country as
the modern hygienic method of publicly providing for all comers
drinking water free from possible contamination.
But is the bubble fountain as constructed at present free from
danger? Have we not seized too readily upon an obvious im-
provement over the common drinking cup without adequate
experimental basis?
Our attention was drawn to the bubble fountain as a possible
source of danger during an epidemic of streptococcus tonsilitis
which occurred in the fall of 1914 in one of the women's dormi-
tories at the University of Wisconsin. Within a week's time,
fifty cases were reported in this building. In studying the situa-
tion, it was noted that in this building the water pressure was so
low that it was scarcely possible to drink from the bubble foun-
^ Presented at seventeenth annual meeting of the Society of American Bac-
teriologists, Urbana, 111., December 29, 1915.
471
472 DOROTHY F. PETTIBONE, F. B. BOGART AND P. F, CLARK
tains in the hall without touching the metal portions with the
lips.
An examination of these fountains showed them to be heavily
contaminated with streptococci. Positive results were obtained
from the surface of the fountain, from the inside, and from sam-
ples of the water. In fact, the swab from the inside of the bub-
ble fountain on the third floor, where the greatest number of
cases were located, gave an almost pure culture of streptococci.
Several examinations of the city water supplying the fountains
showed, on the other hand, no evidence of these organisms. The
sanitary character of the city water supply is excellent, the
water being obtained from deep wells in the Potsdam sandstone
which underlies all southern Wisconsin.
Owing to a laboratory accident the identity of the organisms
found in the bubble fountain and those isolated from the cases
of tonsilitis was not established; and there were other cases of
streptococcus tonsilitis in the town and also scattered cases
throughout the University at the time. It seems a fair presump-
tion, nevertheless, that these heavily contaminated fountains
were a factor in spreading the infection within the dormitory.
A subsequent investigation of all the other bubble fountains
in the University yielded surprising results. In examining the
fountains, the following observations were made : type of foun-
tain, height of column, bacterial count of the water on litmus
lactose agar and action in glucose broth fermentation tubes
which were subsequently examined for streptococci by the hang-
ing drop method and by Gram stained smears. In addition,
sterile throat swabs were rubbed thoroughly over the top of the
fountain and as far into the bulb as possible. These swabs were
placed in glucose broth which also was examined for streptococci
by the methods mentioned above. The swabbings proved to be
the most fruitful source of positive results. Only in a few in-
stances did the amount of water tested prove to contain strep-
tococci. Forty-three out of seventy-seven bubble fountains
(55.8 per cent) were found contaminated with Gram positive
streptococci which frequently occurred in long chains.
Many different types of bubble fountains are in use in the
THE HYGIENE OF THE BUBBLE FOUNTAIN 473
University ranging from a plain rusty half-inch iron pipe with
constant flow, in some remote corners of the campus, to the lat-
est porcelain device with automatic shutoff in more prominent
situations. In general the simpler fountains such as the rusty
pipe were not infected, whereas the complex fountains with inter-
mittent flow and those with crevices and crannies between the
inner and outer portions were the worst offenders, although pre-
sumably the location of the fountain was a factor in this result.
In fact it is worthy of note in connection with the epidemic in
the women's dormitory that streptococci were found most often
in those buildings frequented by the women students while the
Law Building, Engineering Building, Armory, etc., were quite as
uniformly free from such contamination.
To control these rather surprising results a second survey of
the fountains was made about a year later, at a time when there
was no unusual number of upper respiratory infections, but fol-
lowing a period of unusual incidence of "grippe" both in the
University and throughout the country. Samples of water and
swabbings were taken from fifty fountains, excluding many of
those less commonly used. The earlier results were completely
confirmed, as Gram positive streptococci were found in 35 out
of the 50, 70 per cent of the total.
The streptococci found are somewhat difficult to isolate, and
readily overgrown by the common water organisms, developing
well in glucose broth but not on other media. They are only
slightly virulent to experimental animals. Further studies of
these strains are being carried on.
As a further control a Berkefeld filter was attached to a city
water faucet and the water allowed to flow for one week. During
this period it was estimated that about 3300 liters passed through
the filter and yet smears and cultures made from the deposit on
the filter showed not only no streptococci but no Gram positive
organisms present. This control was repeated.
In order to study the factors involved in the contamination of
the fountains, a series of tests were performed on an experi-
mental bubbler in the laboratory. This particular fountain is
one of an ordinary continuous flow type, the bubble being fed
474 DOROTHY F. PETTIBONE, F. B. BOGART AND P. F. CLARK
through four perforations in the top of a cylinder which screws
into the center of a hollow metal bulb. Uniform suspensions of
B. prodigiosus were made by emulsifying each of two twenty-four
hour cultures of the organism in 1 cc. of salt solution. This emul-
sion (2 cc.) was then dropped slowly from a pipette into the top
of the bubble, care being observed not to touch the metal por-
tions of the fountain. Ten cubic centimeter samples of the
water were then taken at frequent intervals and plated in tripli-
cate on agar, 1 cc. to each plate. After incubation the plates
were allowed to stand at room temperature for several days in
order to allow the B. prodigiosus colonies to develop their char-
acteristic red pigment. Red colonies and total colonies were
then counted. In all cases control samples of the water were
taken before introducing the organisms. In a total of over 100
control plates, only one red pigment producer was found. This
formed colonies quite different from B. prodigiosus colonies
macroscopically and could never be mistaken for that organism.
Between each two experiments the bubble fountain was removed,
washed and sterilized.
The experiments were repeated several times with the column
of water at different heights, varying from about 3 to 4 mm.
(approximately the height of column prevailing in the women's
dormitory at the time of the epidemic) to 10 cm., a column much
higher than we have ever observed in use. The results obtained
with the column at each level were somewhat variable. So
many factors are involved that this is to be expected. The rela-
tively small amount of water sampled as compared with the
volume flowing during the period of sampling, the variation in
the column caused by variation in water pressure, chance con-
ditions such as the position of the pipettes both in introducing the
organisms and in withdrawing the samples, together with un-
known factors, must all play a part in the results observed.
In brief, when the bubble is extremely low (3 to 4 mm.) some
of the introduced organisms are found in repeated experiments
for as long as 135 minutes. With a column 2 to 3 cm. high, the
average height of the jets in the University, B. prodigiosus or-
ganisms are retained frequently for 10 to 20 minutes and have
THE HYGIENE OF THE BUBBLE FOUNTAIN
475
been found for as long as 90 minutes. A column 7.5 cm. in
height has shown the presence of B. prodigiosus for 20 min-
utes although commonly no organisms have been found for
more than 2 minutes. Even with the column 10 cm. high, B.
prodigiosus has always been found in the plates poured one or
two minutes after the introduction of the suspension.
Another type of experiment was tried more nearly approxi-
mating the ordinary use of the bubble fountain. The writers
washed out their mouths with suspensions of B. prodigiosus and
while the lips were still moist drank from a column 2 to 3 cm. in
height, exercising due care not to touch the metal portions of
the fountain. Samples of water were taken as before and plated.
In one instance the organisms were retained for 135 minutes,
and the average was 25 minutes.
The following tables show the results of sample tests taken at
random from those made with the experimental fountain:
TABLE 1
Column 2 cm. high. Suspension of B.
■prodigiosus introduced by pipette,
two twenty-Jour hours cultures 1 cc.
salt solution to each culture
TIME AFTER
BACTERIAL
B. PRODIGIOSUS
INTRODUCING
COUNT
COUNT
ORGANISMS
IN MINUTES
58
0
^
52
0
■ Controls
56
0
58
0
5
106
0
10
52
9
15
72
0
20
44
7
25
64
3
30
104
0
40
48
0
50
39
0
60
104
7
70
58
0
80
59
0
90
62
0
100
69
0
110
TABLE 2
Column 2 cm. high. Suspension of B.
prodigiosus introduced as in table 1
TIME AFTER
BACTERIAL
B. PRODIGIOSUS
INTRODUCING
COUNT
COUNT
ORGANISMS
IN MINUTES
160
0
130
0
Controls
142
0
58
13
5
136
4
10
52
1
15
72
13
20
64
2
25
149
21
30
104
35
45
104
5
60
61
1
75
95
2
90
95
0
105
476 DOROTHY F. PETTIBONE, F. B. BOGART AND P. F. CLARK
TABLE 3
Column 2 cm. high Suspension of B.
prodigiosus introduced as in table 1
TABLE 4
Column 3 cm. high. Suspension of B.
prodigiosus introduced as in table 1
TIME AFTER
BACTERIAL
B. PRODIGIOSUS
INTRODUCING
COUNT
COUNT
ORGANISMS
IN MINUTES
Not made
0
^
0
• Controls
0
5
1
1
2
3
3
0
4
1
5
1
7
1
10
0
15
0
20
0
25
0
30
0
40
0
50
0
60
0
70
0
80
0
90
TIME AFTER
BACTERIAL
B. PRODIGIOSUS
INTRODUCING
COUNT
COUNT
ORGANISMS
IN MINUTES
Not made
0
0
' Controls
0
7
1
1
2
1
3
1
4
0
5
0
7
193
10
0
15
■
1
20
0
25
0
30
0
40
0
50
0
60
These tables show that the efficiency of a bubble fountain de-
pends partly upon the height of the jet thrown. But even at the
maximum height used, 4 inches, which is higher than practicable,
some organisms are still retained in the column for about 2
minutes. Considering also the fact that when the organisms
were introduced on the moist lips, a very small number were
added as compared with the milUons introduced in the turbid
suspension by means of the pipette, it is possible that the saliva
of the mouth may be a factor in causing the bacteria to remain a
longer time than would otherwise be the case.
How shall we explain these facts and how shall the bubble
fountain be made safe for public use?
Our conception of the problem is that the organisms dance in
the column of water much as the toy ball dances on top of the
fountain in the garden. Most of the organisms introduced are
THE HYGIENE OF THE BUBBLE FOUNTAIN
477
TABLE 5
Column 10 cm. high. Suspension of B.
prodigiosus introduced as in table 1
BACTERIAL
COUNT
B. PRODIGIOSUS
COUNT
TIME AFTEH
INTRODUCING
ORGANISMS
IN MINUTES
12
0
^
20
0
18
20
0
0
> Controls
14
0
18
0
15
2
1
12
1
2
8
0
3
10
0
4
6
0
6
12
0
8
9
0
10
15
0
15
18
0
20
8
0
30
TABLE 6
Column 2 cm. high. B. prodigiosus
introduced by rinsing m,outh with
suspension and then drinking while
lips were still moist.
TIME AFTER
BACTERIAL
B. PRODIGIOSUS
INTRODUCING
COUNT
COUNT
ORGANISMS
IN MINUTES
Not made
0
0
[ Controls
0
Drank from
fountain
0
1
1
2
1
3
2
4
1
5
0
7
0
10
0
15
1
20
0
25
0
30
0
40
0
50
0
60
0
70
0
80
flushed off at once, whereas others ''dance" for varying periods
of time until they finally fall outside the column and disappear
in the waste pipe. Doubtless other factors are concerned, such
as variation in pressure due to excessive use elsewhere on the
same line and subsequent cessation of this unusual consumption
of water. Indeed a control series of plates of water samples taken
at five minute intervals from a bubble fountain shows wide vari-
ation in bacterial count due partly to variation in pressure.
When the pressure is suddenly increased the pipes are vigorously
flushed and the bacterial count is higher. Keith^ (2) has sug-
gested a modified form of fountain which automatically regu-
2 Keith, S. C, Jr. Amer. Jour, of Public Hygiene, 1910, 20, 163.
478 DOROTHY F. PETTIBONE, F. B. BOGART AND P. F. CLARK
lates the flow so that regardless of pressure changes, the height
of the jet will remain constant.
But why retain a vertical column of water when it is obviously
the upward direction of flow which more than any other factor
renders the bubble fountain unsafe? A fountain (see fig. 1) was
constructed with a simple tube, having no crevices or complica-
tions placed at an angle of about 55 degrees from the vertical.^
'\ 1 V t
Fig. 1. Simple Type of Fountain Which Should Be Generally Adopted.
No Contaminating Organisms Are Retained in the Jet or Water
Even with a minimal stream flowing from this type of fountain,
the introduced organisms were all immediately washed away and
never did we find a single B. prodigiosus colony even on the plates
poured immediately after the introduction of the organisms.
This simple type of fountain with an adequate collar guard to
prevent possible contact with the orifice would, we believe, solve
the problem and furnish water uncontaminated by human lij>s
even to a constant succession of thirsty persons.
s Later experiments have shown that a tube erected at an angle of 15 degrees
from the vertical is adequate to prevent organisms from "dancing" on the
column of water. This makes the construction of a safe bubble fountain much
more practicable.
THE HYGIENE OF THE BUBBLE FOUNTAIN
479
TABLE 7
New model fountain with a small stream of water issuing from a simple tube at
an angle of 50 degrees from the vertical. Suspension B. prodigiosus introduced
as in table 1
BACTERIAL COUNT
B. PRODIGIOSUS COUNT
TIME AFTER INTRODUCING
ORGANISMS
Not made
0
1
0
[ Controls
0
seconds
0
2
0
5
0
10
0
15
0
20
0
25
0
30
0
35
0
40
0
45
0
50
0
65
0
60
minutes
0
1
0
2
0
3
0
4
0
5
0
7
0
10
0
15
0
20
0
25
0
30
0
40
0
50
0
60
CONCLUSIONS
1. During an epidemic of streptococcus tonsilitis in a woman's
dormitory at the University of Wisconsin, streptococci were
found in the bubble fountains in this building and in the water
issuing from these fountains.
480 DOROTHY F. PETTIBONE, F. B. BOGART AND P. F. CLARK
2. The city water supply was at the time, and has been, excel-
lent in its sanitary character. It is obtained from the underlying
Potsdam sandstone. No streptococci were found on a Berkefeld
filter through which water had been flowing continuously for one
week.
3. Presumably the bubble fountains were a factor in trans-
mitting the disease.
4. A survey of all the fountains of the University showed the
presence of streptococci in over 50 per cent of the total number.
The streptococci varied in abundance from a few chains to an
almost pure culture obtained by swabbings from the fountains
in the women's dormitory.
5. In an experimental bubble fountain, B. prodigiosus when
introduced either by means of a pipette or by the moistened lips
remained in the water from 2 to 135 minutes depending partly
on the height of the "bubble."
6. Most of the organisms are flushed away but some remain
dancing in the column much as a ball dances on the garden foun-
tain even though the bubble be increased to the impractical
height of 4 inches.
7. To avoid the difficulty always present in the vertical col-
umn, a simple fountain with a tube at an angle of 50 degrees from
the vertical was constructed. B. prodigiosus was never found
in the plates from this type of fountain even when samples were
taken immediately after the introduction of the organisms.
8. We believe that this type of fountain should be generally
adopted. Its simplicity, low cost of construction and freedom
from lurking danger should recommend it to all.
THE ADVANTAGES OF A CARBOHYDRATE MEDIUM
IN THE ROUTINE BACTERIAL EXAMINATION
OF MILK
JAMES M. SHERMAN
From the Bacteriological Laboratories of the Pennsylvania State College and
Agricultural Experiment Station
INTRODUCTION
The object of this paper is to direct the attention of labora-
tory workers to the advantages over plain agar of an agar con-
taining a fermentable carbohydrate, for use in the bacteriological
control of market milk. Some laboratories are probably aware
of these facts, but since the standard methods for the bacterial
analysis of milk prepared by the committee of the American
Public Health Association^ call for the use of plain agar, and since
this medium is used in many of the largest laboratories making
routine analyses at the present time,^ it seems pertinent to sub-
mit a few data on these points.
METHODS
The bacterial counts reported herein were made on plain agar
prepared according to the standard formula, and on lactose agar
with the same ingredients plus 1 per cent of lactose. Liebig's
beef extract and Witte's peptone were used. The media were
adjusted to a reaction of 0.5 per cent normal acid to phenol-
phthalein, which reaction is, according to the results obtained in
this laboratory,^ better than a higher acidity.
In the determination of bacterial num.bers triplicate plates
were made of each dilution, and the counts were made after
1 Meetings of Am. Pub. Health Assn., Rochester, N. Y., September, 1915.
' U. S. Pub. Health Service, Public Health Reports, 30 : 2349-2394.
•'Penn. Agr. Expt. Sta., Report for 1914-1915.
481
482
JAMES M. SHERMAN
forty-eight hours incubation at 37°C. A hand glass with a
magnification of three and one-half diameters was used in count-
ing the plates made with plain agar, but the lactose agar plates
were counted with the naked eye.
For the comparison of the size of colonies which developed on
plain and lactose agars the plates were incubated forty-eight
hours at 37 °C. and measurements made by means of a com-
pound microscope and eyepiece micrometer which gave a mag-
nification of fifty diameters.
QUANTITATIVE BACTERIAL ANALYSIS
In table 1 are given the results obtained from eighteen samples
of raw market milk when plated on plain and on lactose agars.
Among these milks were samples representing all kinds of milk
from a very good to a very low-grade type.
TABLE 1
Number of bacteria on plain and lactose agars
Raw Milk
NUMBER OP BACTERIA
PER CUBIC CENTIMETER
SAMPLE NUMBER
INCREASE IN FAVOR
OF LACTOSE AGAR
Plain agar
Lactose agar
pel cent
1
817
1,017
25
2
1,340
2,070
55
3
1,630
2,000
23
4
3,230
3,530
9
5
7,330
7,330
0
6
8,000
11,300
41
7
8,900
12,100
36
8
9,030
8,970
-1
9
11,800
11,170
-5
10
186,000
610,000
228
11
192,000
278,000
45
12
260,000
361,000
39
13
369,000
463,000
26
14
53,700,000
109,000,000
103
15
216,300,000
346,600,000
60
16
421,300,000
640,000,000
52
17
576,000,000
1,216,000,000
111
18
1,036,000,000
1,312,000,000
27
ADVANTAGES OF A CARBOHYDRATE MEDIUM
483
The data given in this table indicate that lactose agar is of
considerably more value than plain agar for the quantitative
bacterial analysis of milk. Of the eighteen samples examined,
fifteen gave higher counts on lactose agar; with one sample
there was no difference, while two samples gave counts which
showed a negligible advantage in favor of plain agar. If we con-
sider the experimental error as about 20 per cent, and discard
all differences of less than that amount, it will be seen that four-
teen, or 78 per cent, gave increased counts on the lactose agar,
while none of the samples showed a similar increase in favor of
plain agar. Taking all eighteen samples we find an average in-
crease of 43 per cent in the counts obtained with lactose agar.
The results reported in table 1 are all from samples of raw
milk. In table 2 will be found the data obtained from six sam-
TABLE 2
Number of bacteria on plain and lactose agars
Pasteurized Milk
SAMPLE NUMBER
NUMBER OP BACTERIA PER CUBIC CENTIMETER
INCREASE IN FAVOR
Plain agar
Lactose agar
per cent
1
7,600
25,300
233
2
6,500
42,500
554
3
6,000
69,000
1050
4
18,100
43,600
141
5
23,800
58,000
144
6
72,000
177,000
146
pies of pasteurized milk. Three of the samples were pasteur-
ized by the holder method at 140°C. for twenty minutes, while
the other three were treated by the flash method at 180°C. The
milks were plated within an hour after pasteurization. The re-
sults are very striking and demonstrate the superiority of lac-
tose agar over plain agar for the examination of freshly pas-
teurized milk.
THE SIZE OF COLONIES ON PLAIN AND LACTOSE AGARS
Although the difference in the counts obtained is sufficient
reason for advocating the use of a carbohydrate medium for the
484 JAMES M. SHERMAN
bacterial examination of milk, probably the strongest point in
its favor is the difference in size of colonies which develop on the
two media. The colonies on lactose agar are always consider-
ably larger than those on plain agar, this being especially true
of the acid-forming organisms. Photographs were taken of the
petri dish cultures obtained and reproductions of some typical
plates are appended to this paper. As was stated before, the
plain agar plates were counted with the aid of a hand lens as
prescribed by the standard methods for the bacterial analysis of
milk, whereas the plates containing lactose agar were counted
with the naked eye. This practice was followed because the
colonies on lactose agar were of sufficient size to make the use of
a lens unnecessary. Many trials have failed to reveal colonies
on lactose agar under a hand lens which were not visible to the
unaided eye. This, however, might not be true in the case of a
worker with defective eyesight.
In order to get some definite information on the difference in
size of colonies on the two media, pure cultures of B. lactis-acidi —
which is usually the predominating organism in ordinary market
milk — and a streptoccus — which is usually the predominating
organism in milk of a certified grade — were plated out on plain
and lactose agars, and the colonies which developed were meas-
ured after incubation at 37°C. for forty-eight hours. Only plates
which contained less than one hundred colonies were used so as
to eliminate as far as possible the factor of inhibition. The ten
largest colonies on each plate were measured. These data are
given in table 3.
These differences in size of colonies are presented graphically
in figures 1,2, and 3.
DISCUSSION
It should be understood that the data here presented are in-
tended to apply only to the routine examination of milk for which
a short period of incubation is used. Although the few data at
hand indicate that quite similar results are obtained with lower
temperatures and longer periods of incubation, it is not within
the province of this paper to draw such conclusions.
TABLE 3
Size of colonies on plain and lactose agars
DIAMETER OF COLONIES IN MICRONS
NUMBER OF COLONY
B. lactis-acidi
(Culture A)
B. lactis-acidi
(Culture B)
Streptococcus
Plain
Lactose
Plain
Lactose
Plain
Lactose
1
2
3
4
5
6
7
8
9
10
50
80
60
50
60
70
80
60
60
70
660
720
540
520
500
640
820
540
620
580
340
280
320
260
240
220
300
300
300
280
560
600
700
1300
600
640
590
1140
1080
1120
220
230
220
220
240
210
240
220
260
220
460
440
500
480
510
580
600
570
580
640
Average
64
614
284
833
228
536
15 ACia 1
(culture R")
Fig. 1. The relative size of colonies of B. lactis-acidi grown on plain and lac-
tose agars. Plates were incubated at 37°C. for forty-eight hours. Graphs are
plotted from the average of ten colonies from each medium.
485
486
JAMES M. SHEKMAN
Lactose agar was used in all of the tests herein reported, but
from the results of a number of comparisons it may be said that
glucose is apparently just as efficient as is lactose, both as to
number and size of colonies. According to the work of Heine-
mann and Glenn" glucose and lactose agars are of equal value
(CultureB'i)
Fig. 2. Same as figure 1 except a different strain of B. lactis-acidi was used.
for the quantitative bacterial analysis of milk, and since glu-
cose is cheaper it should, perhaps, be recommended as the stand-
ard for routine work. A limited number of trials have also
* Jour. Infect. Dis., 5: 412-420.
ADVANTAGES OF A CARBOHYDEATE MEDIUM
487
indicated that the amount of carbohydrate may be reduced to
0.1 per cent without impairing the value of the medium.
Another advantage of a medium containing a carbohydrate
over plain agar is that the former is of considerable value in
differentiating the types of organisms on the plates. The colo-
nies of acid-producing bacteria on sugar agar are surrounded by
^1 YepiOCOCCU5
Fig. 3. Same as figure 1 except a culture of a streptococcus was used.
a precipitate of protein matter, thus giving the typical hazy
edge which is so characteristic of organisms of the B. lactis-
acidi group. On agar made without clarification with egg albu-
men, and which is consequently more opaque, the peptonizing
bacteria may be quite readily identified by means of the clear
488 JAMES M. SHERMAN
halo which surrounds their colonies. This differentiation is
much clearer on plates containing lactose or glucose agar than on
those with plain agar, as the opacity of the medium is very much
increased in the presence of a fermentable carbohydrate, due to
the growth of acid-producing organisms. In fact, unclarified
lactose agar gives, in our hands, nearly as clear a differentiation
of bacterial types as does the special casein agar devised by
Ayers.^ These points are well illustrated by the following
photographs.
SUMMARY
Data are presented which it is thought warrant the recommen-
dation that agar containing glucose or lactose be adopted as the
standard medium for the routine bacteriological analysis of milk.
The advantages of a carbohydrate medium over plain agar
may be summed up in the following points :
1. A higher count is obtained.
2. The colonies grow larger and more rapidly which greatly
facilitates the counting of plates.
3. It is of some value as a differential medium.
* U. S. Bureau of Animal Industry, Report for 1911.
JOURNAL OF BACTERIOLOGY VOL. I
PLATE 1
Plain agar
Lactose agar
Milk "a." Dilution of 1/100 on plain and lactose agars. Incubated at 37°C. for
forty-eight hours.
Plain agar
Lactose agar
Milk "b." Dilution of 1/1000 on plain and lactose agars. Incubated at 37°C. for
forty-eight hours.
(Sherman: Advantages of a Carbohydrate Medium)
JOURNAL OF BACTERIOLOGY VOL. I
PLATE 2
Plain agar
Lactose agar
Milk "c." Dilution of 1 100 on plain and lactose agars. Incubated at 30°C. for
six days.
Plain aoar
Lactose agar
Milk "d." Dilution of 1/10,000 on plain and lactose agars. Incubated at 37°C. for
twentv-four hours.
(Sherman: Advantages of a Carbohydrate Medium.)
ON A SPECIES OF TREPONEMA FOUND IN RABBITS
HANS ZINSSER and J. G. HOPKINS
From the Department of Bacteriology, College of Physicians and Surgeons,
Columbia University, New York
In the course of many attempts to isolate new strains of Tre-
ponema pallidum from luetic rabbits, the writers have encoun-
tered a spiral microorganism which has appeared on six different
occasions in tubes prepared from the tissues of five different
strains of rabbit syphilis. On first observation of this micro-
organism it seemed difficult to decide whether it represented a
distinct form, or a morphological mutation of Treponema palli-
dum. Subsequent studies, however, have led us to feel reasonably
sure that the organism has no relation to T. pallidum, but repre-
sents a distinct species present in the rabbit tissue, which grows
in the tubes as prepared by the Noguchi method. It is a curious
fact that although innumerable tubes have been prepared by the
same method, in the course of the last three years, with normal
rabbit kidney for transplantation of pure cultures of Treponema
pallidum (both Noguchi's strains and our own), the microorgan-
ism concerning which we wish to report has never appeared in
such cultures but has been seen only in those implanted with the
syphilitic testicular tissue of diseased rabbits. We therefore be-
lieve it at least very unlikely that the organism had its origin in
the normal rabbit kidney tissue which was used in both kinds of
culture. Since we have not made more than a few dozen plants
with normal rabbit testis, we can not decide whether the micro-
organism we are describing may be a parasite in normal rabbit
testes, or whether its presence is incidental only to the abnormal
conditions occurring in the luetic lesions. To determine this
would probably require a great deal of experimentation and time
(to say nothing of luck), since we have encountered the organism
but six times in the course of three years during which many
hundreds of tubes were planted and examined.
489
490 HANS ZINSSER AND J. G. HOPKINS
It was startling to find this organism growing only when syphi-
litic material had been planted, and then multiplying with sparse-
ness and cultivated with difficulties much greater than those at-
tending the eventual cultivation of the limited number of strains
of T. -pallidum successfully grown by various workers. However,
the obvious suspicions as to its connection with the syphihtic
lesions aroused by these, facts seemed easily refuted by the morph-
ology of this treponema which is very different from that of both
the virulent treponemata and of the cultivated T. pallidum de-
scribed by Miihlens, Noguchi, and others, and studied for several
years by us.
The microorganism is a very fine spiral with curves having the
absolute regularity of a corkscrew in most of the individuals,
with finely tapering ends, and varying in length from 2 to 10 or
20 rather shallow curves. Both short and long forms are from
two to three times as thick as the Treponema pallidum, and in
most individuals a definite double contour is visible. The aver-
age length varies from about one-half to three or four times that of
the Treponema pallidum and occasional long forms are seen which
extend completely across the dark field of a one-twelfth oil im-
mersion lens. The curves are long and shallow.
What is most noticeable about these treponemata is their ab-
solute rigidity and lack of any kind of motility. At first we took
H for granted that the organisms were dead. However, when
we found that subsequent generations in culture were equally
immobile, it became evident that this was characteristic of the
species.
The organism stains with great difficulty. Its contours ap-
pear faint and are often less distinct than those of the Treponema
pallidum after 12 hours staining in dilute Giemsa. It can be dem-
onstrated with the Loeffler flagella stain and by the Fontana
method. We have so far failed to stain it with the ordinary dyes.
Cultivation is extremely difficult. As stated above, multi-
plication has appeared in ascitic agar tubes prepared with rab-
bits' kidney as in the Noguchi method. We have had three or
four strains which have proceeded to the third or fourth genera-
tion only to be lost. At present we have a strain still growing
TREPONEMA FOUND IN RABBITS 491
in the fourth generation. The cultures in which it multiphed
were sealed with oil, and at first we found the organism near the
bottom of the tubes, about the kidney tissue. Subsequent study,
however, revealed that the most likely place to find them was
about one inch from the top under the oil. In such tubes the
growth was sometimes indicated by a very faint haziness not seen
without careful examination and usually not larger than the head
of a pin.
In the material fished from such an area, small clumps of
three, four, or five spirals may be seen under the dark field as
depicted in the photographs. We have not yet been able to
purify this organism, a task of great difficulty because of its im-
mobility. We have not had a sufficiently profuse growth to at-
tain success by the dilution methods, and the first appearance of
the microorganism has so far occurred only in contaminated
tubes together with motile bacilli. The continued cultivation
of the microorganism has, as it were, hung on a thread, the growth
being very sparse and taking about a month or two to develop
at all.
We have withheld the report on this organism for many
months because we have been unable to obtain it in pure cul-
ture. Unless chance favors our efforts more than heretofore, it
may take a year or two to achieve this, and perhaps we may lose
and recover it several times before this result is obtained. We
venture to make this brief report now, therefore, since many
investigators are studying the cultivation of Treponema pallidum
from rabbits and we have not seen any mention of a microor-
ganism of this kind, so easily differentiated morphologically from
T. pallidum, yet appearing in rabbits in the same location
and under the same conditions.
Knowledge of its occurrence and its limitation, so far in our
experience, to cultures from syphilitic testis should be of value
to others working in this field. Should it eventually prove to be
a hitherto undescribed species, we would suggest as a suitable
name for it ^^ Treponema rigidum.'^
Dark Field Photo-micrographs of Non-motile Treponema found
IN Testes of Enetic Rabbits
492
STUDIES ON AEROBIC SPORE-BEARING NON-
PATHOGENIC BACTERIA
Part II
From the Laboratory of Hygiene and Bacteriology, Johns Hopkins University
SPORE-BEARING BACTERIA IN DUST
BY C. A. LAUBACH
Spore-bearing organisms from dust were obtained by rubbing
moist sterile swabs over various dust-laden surfaces, transfer-
ring the material thus obtained to melted agar and then heating
to 90°C. for fifteen minutes to destroy all non-sporulating bac-
teria. Plates were then poured in the usual way and different
colonies selected for study and identification. In many in-
stances the cultures had to be replated a number of times before
the purity of the strain was established, so closely do the spores
adhere to each other. In general the most prolific source of
the spore-bearing organisms was dust which had lain undisturbed
for long periods of time as in closets or on high shelves. Dust
particles circulating in the air seemed relatively free from spore-
bearing bacteria but an increase of these species was always
noted with an increased velocity of the wind. Dust from moist
surfaces allowed to dry down and from surfaces exposed to di-
rect sunhght contained few spore-bearers. Numerous strains
were obtained from the dust found on books. Some 312 cul-
tures were studied and the types (as established in accord with
results of the previous work on milk and on miscellaneous cul-
tures) were found to be distributed as follows.
QO
Bacillus cereus Frankland
Bacillus subtilis (Ehrenberg) Cohn '1
Bacillus vulgatus (Fliigge) Trevisan 46
(Bacillus mesentericus vulgatus Fltjgge.)
Bacillus megatherium De Bary
493
494 C. A. LAUBACH, J. L. KICE AND W. W. FORD
Bacillus petasites Gottheil 21
Bacillus mesentericus (Flligge) Migula 17
{Bacillus mesentericus fuscus Flugge.)
Bacillus aterrimus Lehmann & Neumann 8
{Bacillus mesentericus niger Lunt.)
Bacillus fusiformis Gottheil 4
Bacillus brevis Migula 2
Bacillus alholactus Migula 1
Bacillus terminalis Migula 1
In addition to these previously established types, on four oc-
casions an organism was encountered giving the same reactions
as the species discovered by Prausnitz in Flligge' s laboratory
and described by Flugge (1886) as Bacillus ramosus liquefaciens.
This organism was correctly named Bacillus prausnitzii by Tre-
visan (1889).' It is distinct from Bacillus mycoides of Flugge
but the use of the term/'Wiirzelbacillus" and the name ''Ramo-
sus" for this latter organism by both Eisenberg (1891) and the
Franklands (1894) with a coincident description of Bacillus my-
coides as a distinct species by the latter has led to hopeless con-
fusion. Our investigations show that the majority of organ-
isms of this group, producing felted growths in the depths of
agar, correspond in all particulars to Bacillus mycoides of Fliigge
which is probably identical with the ''Wiirzelbacillus" and also
with the Bacillus ramosus of both Eisenberg and the Frank-
lands. The Bacillus ramosus liquefaciens of Flugge is a distinct
species which we shall describe under its correct name Bacillus
prausnitzii Trevisan (syn. Bacillus ramosus liquefaciens Flugge) .
Two cultures were isolated which exhibited the morphology
and general cultural characters of the members of the "mesen-
tericus" group but produced an abundant yellow pigment. At
first we were inclined to regard this organism as identical with
the species described by Sternberg (1892) as Bacillus suUilis
similis, but the morphology was so clearly that of the mesenteri-
cus type that it was deemed best to describe it as a new variety
of Bacillus mesentericus to which the varietal name flavus is
given. On one occasion a culture was obtained which seems to
represent Bacillus ruminatus of Gottheil. It shows the peculiar
porcelain-white growth on agar said by Gottheil to be the prin-
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 495
cipal feature distinguishing B. ruminatus from Bacillus mega-
therium, which it most closely resembles. The species described
originally by Vogel (1897) as Bacillus mesentericus panis viscosi I,
now known as Bacillus panis Migula was found once; and one
isolation proved to be a new species belonging to the mycoides
group to which we have given the name Bacillus adhaerens.
The above list of organisms found in dust must therefore be
supplemented by the following types whose descriptions are
given below in full.
Bacillus prausnitzii Trevisan 4
{Bacillus ramosus liquefaciens Flijgge.)
Bacillus mesentericus variety fiavus nov. var 2
Bacillus ruminatus Gottheil 1
Bacillus panis Migula 1
(Bacillus mesentericus panis viscosi 1 Vogel.)
Bacillus adhaerens, nov. sp *
^■^'^Bacillus prausnitzii Trevisan
This organism was originally described as Bacillus ramosus
liquefaciens by Flugge. It is sometimes regarded as identical
with Bacillus mycoides but a culture obtained from the Krai
collection in Vienna shows different reactions from those of
Bacillus mycoides. Several cultures corresponding closely to
the Krai culture were isolated from dust. The following de-
scription applies to the Krai culture and to our own isolations,
as well.
Morphology. In young cultures 6 hours old on plain agar the
organisms are homogeneous, have round ends when free and
flattened ends when in juxtaposition. They generally occur in
chains of 2 to 4 elements and resemble Bacillus mycoides in mor-
phology. The single cells measure 0.625 to 0.75 by 3 to 5 mi-
crons. On glucose agar they are thicker measuring 0.75 to 1
by 3 to 5 microns. In older cultures, 24 hours, on both plain
and glucose agar the protoplasm is converted into globular
bodies which take the stain badly. These are especially abun-
dant on glucose agar which also shows long and thick vegetative
rods measuring 0.75 to 1 by 4 to 6 microns and peculiar washed-
496 C. A. LAUBACH, J. L. RICE AND W. W. FORD
out organisms which seem to be made up of a fine network or
skein of filaments. (Figures 53 and 54.)
Motility. Active motiUty in young cultures.
Staining properties. Gram-positive.
Spore formation. Spores begin to form early appearing in 24
hours on plain and on glucose agar. They are usually central,
one to a cell, and are slightly wider than the vegetative rods.
The organisms with spores retain their chain formation and
later the free spores may also remain attached in chains. The
free spores are cylindrical and measure 0.75 to 1 by 1.5 to 2
microns. As they lose their protoplasm they become oval and
measure 0.625 to 0.75 by 1 to 1.25 microns.
Agar slant. Profuse spreading dull growth consisting of fine
interlacing filaments developing from the central line of inocu-
lation as a rhizoid mass. The early growth is extremely tena-
cious and extends deeply into the underlying agar. Later the
growth becomes finely granular and friable and can be scraped
from the medium. In general the appearance on agar is like
that of a culture of Bacillus mycoides.
Agar stab. Abundant growth along line of inoculation and
spreading surface growth.
Agar colonies. Colonies consist of profusely interlacing fila-
ments spreading from opaque centers. They are dull grayish
and penetrate the agar, under the surface of which they grow in
the medium.
Glucose litmus agar slant. Scanty growth on the surface with
a pronounced acid reaction which remains permanent.
Glucose litmus agar colonies. Colonies much the same as
those on plain agar but somewhat more profuse, with the fila-
mentous character more pronounced.
Gelatin stab. Progressive funnel-like liquefaction often com-
plete within three days.
Gelatin colonies. Colonies consist of profusely interlacing fila-
ments spreading from opaque centers. They are dull greyish
and penetrate the gelatin, under the surface of which they grow
as in agar. Each colony is soon surrounded by a zone of liquid
gelatin.
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 497
Broth. Granular scum ^nd flocculent growth which soon set-
tles to the bottom.
Peptone. Similar granular scum and flocculent sediment.
Potato. Viscous yellowish-gray growth spreading profusely
and rapidly over the whole medium.
Litmus milk. Reaction highly acid within 24 hours and a firm
coagulation within 48 hours. Peptonization soon begins and
proceeds very slowly eventually converting the coagulum to an
amber-colored fluid. Coagulation is more rapid in freshly iso-
lated strains.
Blood serum. Profuse moist dull interlacing or mycelioid
growth. No peptonization.
Fermentation tubes. Glucose : a flocculent growth in the bowl
and a granular scum. Turbidity in closed arm. Reaction acid.
Saccharose: a flocculent growth in the bowl with an acid reac-
tion. Arm usually clear.
Lactose: a similar flocculent growth with an acid reaction.
Arm usually clear.
Thermal death point. Spores survive 12 pounds in the auto-
clave but are killed by 15 pounds. In the Arnold they survive
45 minutes but are destroyed by an hour's exposure.
Bacillus mesentericus variety flavus nov. var.
This is a new variety of Bacillus mesentericus to which the
name flavus is given because of the abundant yellow pigment it
produces. We have encountered it repeatedly in dust and in
soil.
Morphology. Thin homogeneous rods with round ends, in
young cultures on plain agar measuring 0.375 to 0.5 by 1.5 to
4 microns. On glucose agar the organisms are a little thicker
and longer, measuring 0.5 to 0.75 by 3 to 5 microns. They of-
ten grow in long threads measuring 9 to 12 microns in length.
Shadow forms are formed early both on plain and on glucose agar.
In old cultures especially on glucose agar the long forms tend to
curve. (Figures 55, 56 and 57.)
Motility. Actively motile in young cultures.
498 C. A. LAUBACH, J. L. RICE AND W. W. FORD
Staining properties. Gram-positive.
Spore formation. Spores begin to form within 24 hours. By
the end of the 2nd day the spores are abundant. They arise in
the center or towards one end of the organism and bulge the rod
but Uttle. The free spores retain rims of protoplasm often
greater in quantity at one end than at the other. They resemble
the mesentericus spores and measure usually 0.625 by 0.75 to 1.5
microns. As they mature they lose their rims of protoplasm,
become more oval and measure 0.5 to 0.625 by 0.75 to 0.875
microns.
Agar slant. Moist, smooth, non-spreading lemon-yellow
growth. The lemon-yellow color becomes more pronounced with
age.
Agar stab. Profuse granular growth along line of stab with
slight irregular outgrowths.
Agar colonies. The colonies are smooth, moist, round, with no
tendency to spread, glistening and raised, lemon-yellow in color.
There is a definite opaque yellow center while the periphery is
translucent and shell-like. Deep colonies have a tendency to
spread and become faintly iridescent.
Glucose litmus agar slant. A very scant, moist, granular and
sHghtly yellow growth is produced with a slight acid reaction.
Glucose litmus agar colonies. Colonies similar to those on
agar but less profuse. Reaction of medium acid.
Gelatin stab. A very slow liquefaction is produced along the
line of stab and a slight yellow tinge is imparted to the medium .
Gelatin colonies. The colonies rest in slight depressions caused
by slow liquefaction. They are dark yellow in color, moist, round
and smooth. Under the low power of the microscope each
colony is seen to be composed of concentric circles of varying
densities with lobate edges.
Broth. A slight turbidity appears after a considerable pe-
riod. The medium clears by sedimentation and the sediment has
a sHght yellow tinge.
Peptone. A slight turbidity, somewhat less than in broth
occurs. This soon settles to the bottom.
Potato. No visible growth.
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 499
Litmus milk. No change in reaction even after a long period
of time.
Blood serum. A scant, moist, smooth, glistening and yellow-
ish growth is produced. No solution of the medium occurs.
Fermentation tubes. Glucose : a slight turbid growth occurs in
the bowl with the production of a slight acidity.
Saccharose : the same appearance. No acid.
Lactose: shght turbidity. No acid.
Thermal death point. Spores survive 18 pounds pressure in
the autoclave but are destroyed by 20 pounds. They survive one
hours steaming in the Arnold.
Bacillus ruminatus Gottheil 1901
The type ''ruminatus" was first described by Gottheil (1901).
In morphology and cultural characters it closely resembles Bacil-
lus megatherium from which it is distinguished by its porcelain-
white growth, particularly in young cultures. The present de-
scription applies to an organism found in dust and subsequently
in water which corresponds to Gottheil's original description
but not however to that given by Chester. We believe that it
may properly be called Bacillus ruminatus.
Morphology. Homogeneous rods with rounded ends measur-
ing 0.625 to 0.75 by 2.5 to 4 microns in young cultures on plain
agar. On glucose agar they are distinctly thicker and longer
measuring 0.75 to 1.125 by 2.25 to 5 microns. Rarely, long
forms are found on this medium measuring 8 to 10 microns in
length. Shadow or washed out forms are common on both plain
and glucose agar measuring 1.025 to 1.5 by 3 to 5 microns. Or-
ganisms often appear in short chains. (Figures 58 and 59.)
Motility. Active motility in young cultures.
Staining properties. Gram-positive.
Spore formation. Spores are formed early, often in 48 hours,
especially when the strains are first isolated. They are central or
sHghtly ex-centric and on sporulation swell or bulge the organ-
isms from which they spring. They measure usually 0.625 by
1.25 to 1.5 microns. Some spores are however somewhat
smaller and more oval.
500 C. A. LAUBACH, J. L. RICE AND W. W. FORD
Agar slant. Moderate growth along line of inoculation form-
ing a definite ridge. There is a tendency for the growth to
spread to either side and the opacity of the growth decreases
towards the periphery which is translucent. The growth is glis-
tening, raised, moist and has a pure white color which becomes
more apparent with age.
Agar stab. Growth along the line of inoculation distinctly
granular.
Agar colonies. The colonies are very characteristic. Some are
round and regular while others show a considerable tendency to
spread. They are moist, opaque, raised, glistening and white.
The majority show dense centers surrounded by thin areas which
are in turn surrounded by translucent shell-like peripheries.
Glucose litmus agar slant. The growth is similar to that on
agar although somewhat variable as to amount. The distinct
white coloration is also evident. A definite and permanent
acidity is produced.
Glucose litmus agar colonies. The colonies correspond closely
to those on agar but are less profuse.
Gelatin stab. A fairly rapid progressive cone-like liquefaction
occurs.
Gelatin colonies. The colonies rest in a cup-like excavation
caused by liquefaction. They are definitely circumscribed and
have an opaque center which is surrounded by a less dense gray-
ish area. This in turn is enclosed by a more dense grayish ring.
Outside this ring the opacity decreases towards the periphery.
Broth. A fine fragile pellicle is formed with some turbidity.
The medium clears by sedimentation.
Peptone. Similar scum but less marked turbidity. "*
Potato. Cream-white moist profuse growth developing in 24
to 48 hours.
Litmus milk. Within 24 hours a slight acid reaction occurs
and the milk often shows a distinct thickening at the bottom.
The coagulation is not definite however and peptonization begins
usually within 48 hours. As it advances a clear zone of amber-
colored fluid is found at the upper part of the milk tube. This
gradually increases until all the milk is peptonized.
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 501
Blood serum. Fairly profuse moist glistening smooth whitish
growth. Some softening of the serum but no definite liquefac-
tion.
Fermentation tubes. Glucose: a sUght turbidity in the bowl
and neck with a reduction of the Htmus in the closed arm. Re-
action acid.
Saccharose : a similar growth with an acid reaction.
Lactose: only a slight turbidity develops with an alkaline
reaction.
Thermal death point. The spores survive 20 pounds pressure
in the autoclave but are destroyed by 22 pounds. They sur-
vive one hours steaming in the Arnold.
Bacillus panis Migula 1900
This organism was originally described by Vogel (1897) as
Bacillus mesentericus panis viscosi I. It has been found in Bal-
timore, but once, in dust.
Morphology. When first isolated this organism showed only
encapsulated forms, the capsules staining readily with gentian
violet. As the organism was cultivated on artificial media in
the laboratory it lost its capacity of forming easily-stained
capsules but continued to manufacture a quantity of viscous ma-
terial which gave a characteristic appearance to the cultures.
In young cultures on plain agar, 6 to 24 hours old, the organisms
are small and homogeneous with round to flattened ends, meas-
uring 0.375 to 0.5 by 1.5 to 3 microns. They show no appreci-
able difference in thickness on glucose agar, but tend to show
long forms measuring 5 to 6 microns in length. Occasional
shadow forms are seen measuring 0.75 to 1 by 1.5 to 4 microns.
(Figures 60, 61, and 62.)
Motility. No motihty has thus far been demonstrated.
Staining properties. Gram-positive.
Spore formation. When first isolated, spores were formed early,
often in 24 to 48 hours. After long cultivation in the laboratory,
they appear in the cultures only after 6 to 8 days growth. They
are formed in the centers or towards one end of the rods and are
502 C. A. LAUBACH, J. L. RICE AND W. W. FORD
typical mesentericus spores. They retain definite rims of proto-
plasm at times concentrated at one end of the spore and measure
0.375 to 0.5 by 1 to 1.25 microns. As they lose their rims of
protoplasm they become more oval and measure about 0.75 by
1 micron.
Agar slant. Growth scanty in 24 hours, then becomes slightly
raised, finely wrinkled, translucent, non-spreading and viscous.
When older the growth has a tendency to become somewhat
dry and gray and is easily scraped off. On highly acid agar the
growth is more profuse within 24 hours but is not viscous.
Agar stab. Slight granular growth along the line of inocula-
tion with occasionally a slight budding out from the stab.
Agar colonies. Colonies small, drop-like, slightly irregular,
showing little or no tendency to spread, glistening, elevated and
viscous. In some cases a scum-like covering which enclosed
clear gelatinous material was produced about each colony.
Glucose litmus agar slant. Moderate non-spreading growth
within 24 hours. The surface of the growth has a sort of honey-
combed appearance caused by fine interlacing wrinkles. It is
also viscous and is somewhat bluish-gray in color. The reac-
tion is definitely acid within 24 hours. This acidity is followed
by a reduction of the litmus and a gradual return to alkalinity.
Glucose litmus agar colonies. The colonies correspond closely
to those on plain agar but usually attain somewhat greater
dimensions.
Gelatin stab. Rapid funnel-like and progressive liquefaction.
Complete liquefaction results with the formation of a tenacious
grayish scum.
Gelatin colonies. The colonies on gelatin plates rest in cup-
like excavations caused by rapid liquefaction. A definite brown
center with a surrounding grayish granular area is evident in
each colony under the low power of the microscope.
Broth. A slight turbidity is produced within 24 hours with
the beginning formation of a scum. The scum later is finely
granular and is formed of discrete colonies. The medium clears
itself by sedimentation.
Peptone. Reaction the same as that in broth.
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 503
Potato. Growth finely wrinkled, grayish and viscous. The
wrinkles appear wave-like. When older the growth loses its
viscosity and becomes dry and granular.
Litmus milk. A gradual clearing from the top due to progres-
sive peptonization occurs within 24 hours. Within 48 hours
peptonization is generally completed. The remaining fluid has
a port-wine color but becomes amber-colored after a variable
period.
Blood serum. Smooth, moist, glistening and viscous growth.
Within 24 hours some solution of the medium occurs along the
line of inoculation (a trough-like excavation). The medium
may be entirely dissolved within 2 weeks, and a tenacious scum
may be formed.
Fermentation tubes. Glucose: finely granular scum is formed
which generally breaks up into large flakes. A flocculent growth
is present in the bowl. The reaction is definitely acid.
Saccharose : the growth is the same and the reaction also acid.
Lactose: the growth is also abundant with more pronounced
scum-formation. Reaction alkaline.
Thermal death point. The spores survive 10 pounds in the
autoclave but are destroyed by 15 pounds pressure. They sur-
vive 30 minutes steaming in the Arnold but are destroyed by
one hours exposure.
Bacillus adhaerens nov. sp.
This organism has been encountered but once, in dust. It is
apparently a new species.
Morphology. Slender long rods with homogeneous prot oplasm
and flat ends, growing usually in long curved chains made up of
18 to 20 elements. In young cultures on plain agar the indi-
vidual cells measure 0.375 to 0.5 by 1.5 to 4 microns. Some
longer forms, 6 microns in length, may also be found. On glu-
cose agar the organisms are homogeneous, measure 0.625 to 0.75
by 3 to 5 microns, but are often longer measuring 6 to 8 microns.
In older cultures (4 days) many globular bodies occur on glu-
cose agar. They resemble the globular bodies seen in Bacillus
cereus. (Figures 63 and 64.)
504 C. A. LAUBACH, J. L. RICE AND W. W. FORD
Motility. No motility has ever been observed, even in very
young cultures.
Staining properties. Gram-positive.
Spore formation. When first isolated this species formed
spores in 24 hours. After long artificial cultivation spores are
formed only after 4 to 5 days growth on both plain and glucose
agar. They are usually subterminal but may be central. The
rods swell appreciably before sporulation, sometimes in the cen-
ter and sometimes at the ends. The free spores are oval and
measure 0.625 to 0.75 by 0.875 to 1 micron. They often remain
fastened to each other in long chains. Frequently a bit of proto-
plasm remains attached to the spore which then resembles a ten-
nis racket with its handle.
Agar slant. In early growth (18 hours) this species slightly
resembles Bacillus mycoides. The line of inoculation shows a dis-
tinct ridge from which shoot out fine interlacing filaments. These
adhere closely to and grow into the agar. Considerable puck-
ering of these interlacing filaments causes a roughened leathery
appearance on the surface. The early growth is moist and
slightly glistening but these properties are soon lost. The edges
of the growth are serrated, with little or no tendency to spread.
A brownish color is found in old cultures throughout the entire
medium.
Agar slant. Profuse growth along the line of inoculation and
out into the medium. This has the appearance of an inverted
fir tree.
Agar colonies. The colony is very characteristic. It first ap-
pears like a small colony of Bacillus mycoides, but within 24
hours the filaments seem to swell and produce a somewhat cor-
rugated surface with a very definite, elevated and yellow-brown
center. The entire colony adheres closely to the agar and gradu-
ally grows into it.
Glucose litmus agar slant. The growth is similar to that on
plain agar but is very scanty. A definite and permanent acidity
is produced.
Glucose litmus agar colonies. Colonies in this medium are
considerably smaller than those on plain agar and have the same
general appearance.
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 505
Gelatin stab. In gelatin, growth is slow and a very slow fun-
nel-like liquefaction is produced.
Gelatin colonies. They appear coarsely granular, slightly
raised mth definite yellow-brown centers. The centers are
coarsely flocculent under the low power of the microscope. The
colonies are surrounded by a slight area of liquefaction.
Broth. A slight turbidity is produced and a definite scum is
formed which settles to the bottom.
Peptone. Growth similar to that in broth.
Potato. A fairly profuse grayish-white moist growth. When
the medium is dry the growth is scale-like.
Litmus milk. No change is noticed within 24 hours. After
48 hours a slowly progressive peptonization occurs. The med-
ium becomes amber-colored.
Blood serum. A fine-grained leather-like growth occurs, dull
gray and adherent to the medium. Later this is easily scraped
off. No solution of the serum occurs.
Fermentation tubes. Glucose : a flocculent growth occurs in the
bowl and extends into the closed arm. Reaction acid.
Saccharose: turbidity in bowl. Arm clear. Reaction neu-
tral or slightly acid.
Lactose : turbidity in bowl. Arm clear. Reaction not changed.
Thermal death point. The spores resist 18 pounds pressure in
the autoclave but are destroyed by 20 pounds. They survive
one hour's steaming in the Arnold.
SPORE-BEARING ORGANISMS IN WATER
BY C. A. LAUBACH
The spore-bearing organisms in water were obtained by pass-
ing the tap water in the laboratory through Berkefeld filters
under pressure for a period of three days, washing the filters in
sterile salt solution, heating the washings to 80°C. for 15 min-
utes and then plating. Ten samples were obtained by this
method and 313 organisms studied. The species previously es-
tabUshed from studies of milk and dust were found as follows:
506 C. A. LAUBACH, J. L. RICE AND W. W. FORD
Bacillus cereus Frankland 93
Bacillus subtilis (Ehrenberg) Cohn 84
Bacillus vulgalus (Flijgge) Migula 45
(Bacillus mesentericus vulgatus FliJgge.)
Bacillus mycoides FliJgge 41
Bacillus megatherium De Bary 14
Bacillus mesentericus (Fliigge) Migula 12
{Bacillus mesentericus fuscus Fliigge).
Bacillus petasites Gottheil 8
Bacillus aterrimus Lehmann & Neumann 7
(Bacillus mesentericus niger Lunt.)
Bacillus fusiformis Gottheil 2
Bacillus brevis Migula 2
Bacillus prausnitzii Trevisan 1
(Bacillus ramosus liquefaciens Prausnitz.)
Bacillus ruminatus Gottheil 1
In addition to the above types one organism was found which
had not thus far been isolated in our laboratory but which was
described originally by Chester as a viscous variety of Bacillus
subtilis. Another sporulating organism new to us, producing a
green pigment, was found on one occasion. An attempt was
made to identify this as one of the numerous fluorescent sporu-
lating organisms already described in water but the morphology
and cultural reactions were quite the same as those of Bacillus
cereus and it was therefore decided to regard it as a fluorescent
variety of this species. It has subsequently been isolated on a
number of occasions. An organism was encountered on one in-
stance which had such peculiar properties as to merit description
as a new species. Therefore the following may be added to the
above list.
Bacillus subtilis var viscosus Chester 1
Bacillus cereus var fluorescens nov. var 1
BACILLUS LATEROSPORUS nOV. Sp 1
Bacillus subtilis variety viscosus Chester 1903
Attention was first called to this organism by Chester (1903).
We have encountered it but once, in water.
Morphology. Homogeneous rods with round and flat ends
measuring 0.5 to 0.625 by 1.5 to 4 microns in young cultures on
plain agar. No change in morphology on glucose agar. Trans-
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 507
parent or shadow forms abundant on plain and on glucose agar.
These measure usually 0.75 to 1.125 by 2.5 to 4 microns. In old
cultures an abundance of viscous substance appears staining
slightly with gentian violet. (Figures 65, 66 and 67.)
Motility. Active motility in young cultures.
Staining properties. Gram-positive.
Spore formation. When first isolated, spores were formed
within 48 hours. After long cultivation on artificial media, the
spore fomiation has become considerably delayed. Spores are
now formed only in ten to fourteen days. They are central or
slightly ex-centric and resemble the spores of Bacillus subtilis.
They measure usually 0.5 to 0.625 by 1 to 1.125 microns and are
thus oval to cylindrical. They do not bulge the organisms vfhen
formed.
Agar slant. Within 48 hours there is a fair growth which is
moist, glistening, non-spreading, glairy and transparent. Its
characteristic quality is its viscosity. Later the growth becomes
dull and grayish in its appearance and loses its viscosity. A slight
wrinkling appears along the outer edge of the growth and forms
a narrow border surrounding the entire line of growth. After a
few days the medium acquires a brownish color.
Agar stab. A faint granular growth along the line of
inoculation.
Agar colonies. Colonies round, definitely circumscribed, with
but little tendency to spread, raised, dull and viscous. There is
a scum-like covering which encloses gelatinous material about
each colony which is very characteristic.
Glucose litmus agar slant. The growth is bluish-gray, viscous,
wrinkled and dull. A sHght reduction of the Htmus occurs with
a definite acidity. There is a later return to alkalinity usually
within ten days.
Litmus glucose agar colonies. The colonies are similar to those
on plain agar but are more profuse and have a definite bluish-
gray color.
Gelatin stab. Rapid progressive surface liquefaction. Com-
plete liquefaction occurs within 96 hours with the formation of a
definite flocculent sediment.
508 C. A. LAUBACH, J. L. RICE AND W. W. FORD
Gelatin colonies. The colonies are definitely circumscribed
and rest in a cup-like excavation due to liquefaction. They are
translucent, grayish, viscous with opaque centers.
Broth. A granular, lace-like scum is formed which settles to
the bottom. A flocculent growth occurs throughout the medium.
Peptone. Growth similar to that in broth.
Potato. A profuse, grayish, slightly glistening and viscous
growth is produced. More characteristic is the appearance of
numerous blebs filled with gelatinous material on the entire
medium within 24 hours. Later the growth becomes dry and
scale-like.
Litmus milk. Within 24 hours a slight reduction of the lit-
mus. Within 24 hours a thickening occurs at the bottom and
peptonization begins. This progresses rapidly and within four
days the process is generally completed. There remains first a
port-wine, then an amber-colored fluid.
Blood serum. The growth is scant, glistening and bluish-gray.
There is occasionally a fine wrinkling. A gutter-like excavation
along the line of inoculation is often visible. Within 96 hours
a pinkish tinge is produced in a wrinkled scum if water of con-
densation be present at the bottom of the tube.
Fermentation tubes. Glucose: a turbid growth occurs in the
bowl and neck with the formation of a thin smooth bluish-gray
scum. Some acidity is produced.
Saccharose: a similar growth is apparent but the scum is
finely granular. Here also a definite acidity is produced.
Lactose: the growth is identical with that in saccharose but
the reaction remains neutral or becomes alkaline.
Thermal death point. Spores survive 15 pounds in the auto-
clave but are killed by 20 pounds pressure. They are killed
within an hour in the Arnold sterilizer.
/
Bacillus cereus variety fluorescens. no v. var.
An organism producing a greenish fluorescence has been en-
countered frequently in water and in soil. It does not agree
with any previously described organisms in its cultural reactions,
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 509
but has the same morphology and m many respects the same
reactions on media, as Bacillus cereus. It has therefore been
described as a fluorescent variety of this organism.
Morphology. In young cultures on plain agar (6 to 20 hours)
the vegetative rods have rounded ends, slightly granular proto-
plasm and measure 0.5 to 0.75 by 1.5 to 4 microns. They fre-
quently grow in short chains. On glucose agar they are slightly
thicker and longer measuring 0.75 to 0.875 by 2 to 5 microns.
As the organisms mature they show many globular bodies in
their protoplasm which take the stain with difficulty. These are
particularly abundant on glucose agar. (Figures 68, 69, and 70.)
Motility. Active motility in young cultures.
Staining properties. Gram-positive.
Spore formation. Spores begin to form early appearing in 24
hours on plain and on glucose agar. They are located in the
center or towards one end of the vegetative rods which swell
noticeably on sporulation. As the spores mature the protoplasm
disintegrates but remains attached to the spore, sometimes in
equal quantities at the ends and sometimes in greater amount at
one end than at the other. The spores thus resemble mesenteri-
cus spores but are much larger. The free spores measure 0.5
to 0.75 by 0.75 to 1.5 microns. They frequently remain attached
to each other in long chains.
Agar slant. The growth is profuse, smooth, moist, and dull.
There is a definite ridge along the line of inoculation and the
edges are thin and sHghtly serrated. A yellow-green pigment is
diffused throughout the entire medium. This remains perma-
nently and increases with age. The pigment is especially pro-
nounced in an acid medium. Rarely there is considerable
wrinkhng and the formation of rather coarse folds.
Agar stab. Shght granular growth along the fine of stab with
the gradual appearance of the characteristic pigment.
Agar colonies. Irregular colonies which have a great tend-
ency to spread in amoeboid fashion. They are ghstening,
slightly translucent and shghtly raised. They become more
dense and dull as growth progresses and assume a greenish-yel-
low coloration. The colonies can be scraped off and are of a
510 C. A. LAUBACH, J. L. RICE AND W. W. FORD
mealy consistency. The same pigment appears in the medimn.
Glucose litmus agar slant. The growth is fairly profuse and
similar to that on agar. The reaction is first acid with a return
to alkalinity after a considerable period.
Glucose litmus agar colonies. The colonies are similar to those
on agar but more profuse. Reaction of medium acid.
Gelatin stab. Very rapid liquefaction along the line of inocu-
lation. The medium is entirely liquefied within 48 hours and
acquires an intense yellow-green color. A scum of the same color
is formed.
Gelatin colonies. The colonies rest in cup-like excavations
caused by liquefaction. They have a definite greenish center
surrounded by a net-work of fine interlacing, clear, thread-like
filaments. The medium assumes the characteristic fluorescence.
Broth. Profuse, flocculent growth with the production of a
slight green-yellow coloration.
Peptone. Growth more pronounced than in broth and the
coloration more intense. A scum is also formed.
Potato. Profuse, moist, mealy, yellow-green growth with a
coloration of the medium.
Litmus milk. Slight reduction of the litmus with a suggestion
of peptonization within 24 hours. A precipitation of the pro-
teins occurs within 48 hours and peptonization progresses rap-
idly. A scum is formed and the fluid remaining is deeply col-
ored with the characteristic pigment.
Blood serum. Moist, profuse, dull and slightly greenish-yel-
low growth. No liquefaction.
Fermentation tubes. Glucose: profuse flocculent growth with
the formation of yellow-green scum in the bowl and a turbid
growth in the closed arm. The usual fluorescence is present. A
definite acidity is produced.
Saccharose: the growth is identical with that in glucose but
no acidity is produced.
Lactose: the growth is the same. Reaction unchanged.
Thermal death point. The spores survive 12 pounds pressure
in the autoclave but are destroyed by 15 pounds. They are
also destroyed by one hour's exposure to steam in the Arnold
sterilizer.
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 511
Bacillus laterosporus nov. sp.
This organism was obtained but once, from water. It is ap-
parently a new species.
Morphology. The typical vegetative rods are found only m
very young cultures 6 to 8 hours old. They are homogeneous or
slightly granular with round ends, measuring 0.375 to 0.5 by
1.125 to 4 microns on plain agar. On glucose agar they are
slightly thicker and about the same length measuring 0.5 to 0.75
by 1.125 to 4 microns. The rods rapidly lose their characteristic
appearance and assume a fusiform or spindle shape with a swol-
len middle and pointed ends. Such forms take the stain much
less intensely. (Figures 71 and 72.)
Motility. Active motility in young cultures.
Staining properties. Gram-positive.
Spore formation. As the organisms swell and assume the fusi-
form shape the spores appear at one side as slightly staining
globules. The protoplasm soon disintegrates but more on one
side of the spore than on the other so that the free spores which
are almost egg-shaped retain a rim of protoplasm on one side and
present a characteristic appearance. Such spores measure 0.75
to 0.875 by 1.5 microns. As the spores mature they lose their
rims of protoplasm, become more oval and measure 0.75 to 1 by
1 to 1.125 microns. The spores begin to appear in about 48 hours
and are abundant in 72 hours. Rarely small tags of protoplasm
remain attached at the ends of the spores.
Agar slant. The growth is very characteristic. It is profuse,
rapidly spreading, smooth, moist, and has a silvery metallic-hke
lustre. Within 72 hours the growth becomes sUghtly wrinkled.
With age the growth loses its metalhc lustre and becomes dull
and scrapes off in scales.
Agar stab. A slight grayish granular growth along the line of
stab.
Agar colonies. The colonies are flat, transparent, irregular,
moist, and have the same pecuhar metallic lustre.
Glucose litmus agar slant. Growth shght, granular, moist and
gray. Shght reduction of the htmus within 24 hours and a per-
manent acidity within 96 hours.
512 C. A. LAUBACH, J. L. RICE AND W. W. FORD
Glucose litmus agar colonies. Colony formation is very slow
and the colonies only appear within 48 hours. They are similar
to those on agar but less profuse. At times a slight wrinkling
occurs.
Gelatin stab Very slow surface liquefaction.
Gelatin colonies. The colonies are definitely circumscribed,
gray and moist, under the low power of the microscope showing
finely granular centers surrounded by small gray rings. Beyond
the rings there are other granular areas the outer borders of
which consist of dense gray rings. A very peculiar odor like that
of semen plus some aromatic radical occurs in gelatin.
Broth. Peculiar pellicle made up of discrete colonies which
sink to the bottom. There is also a slight turbidity.
Peptone. Growth similar to that in broth.
Potato. The growth is dull, moist, gray, granular and finely
wrinkled. A slight lustre appears which becomes more pro-
nounced as the medium becomes drier.
Litmus milk. Within 24 hours there is a complete reduction of
the litmus with an alkaline ring at the surface. A soft coagulum
is formed within 24 hours which is somewhat firmer than the
€oagulum formed by other spore-bearing organisms. Peptoniza-
tion begins after 48 hours and requires a considerable period be-
fore it is completed. The fluid remaining is straw-colored.
Blood serum. The growth is grayish, moist, non-spreading and
not profuse. Within 96 hours it becomes finely wrinkled.
Fermentation tubes. Glucose : there is a heavy pellicle formed
and a turbid growth in the bowl and neck of tube. If litmus is
present it is reduced in the closed arm. Also a turbid growth is
found in this arm. Reaction acid.
Saccharose: a similar condition is apparent with little or no
acidity.
Lactose : there is the same reaction with a slight alkalinity.
Thermal death point. The spores survive 12 pounds pressure in
the autoclave but are destroyed by 15 pounds pressure. They
are destroyed by one hour's steaming in the Arnold sterilizer.
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 513
SPORE-BEARING BACTERIA IN SOIL
BY C. A. LAUBACH AND J. L. RICE
In a preliminary investigation of the germ content of soils ob-
tained in the neighborhood of Baltimore the attempt was made
to verify the results already obtained by the work on milk, dust
and water and to determine whether the types previously iso-
lated from the other sources could also be found in this environ-
ment. Altogether some 63 cultures were obtained and found to
consist of the following types :
Baltimore soil
Bacillus cereus Frankland 28
Bacillus mesentericus (Flijgge) Migula 15
{Bacillus mesentericus fuscus FliJgge.)
Bacillus subtilis (Ehrenberg) Cohn 8
Bacillus vulgatus (Fliigge) Trevisan 6
(Bacillus mesentericus vulgatus Flugge.)
Bacillus brevis Migula 3
Bacillus megatherium De Bary 1
Bacillus mycoides Flugge 1
Bacillus petasites Gottheil 1
It is interesting to note the great predominance of Bacillus ce-
reus which makes up nearly half the isolations, the relative in-
frequency of Bacillus subtilis and the rarity of Bacillus megather-
ium, Bacillus petasites, and Bacillus mycoides. Bacillus mesen-
tericus appears also as a more frequent isolation than Bacillus
vulgatus.
Eight different samples of raw and heated soil were selected for
this examination, some samples being plated immediately, others
being incubated at 37°C. for 48 hours before plating. It was
hoped that a great diversity of organisms would be obtained by
this method and no special attempt was made to estimate with
any great accuracy the germ content of any particular sample.
In general it was noted however, that heated soil furnished a
greater variety of types than unheated soil and that the incuba-
tion of the sample was distinctly an unfavorable factor since one
type tended to overgrow the other types present. It was thought
that the prevalence of Bacillus cereus might be due to its power of
514 C. A. LAUBACH, J. L. RICE AND W. W. FORD
crowding out the other microorganisms with which it was asso-
ciated in soil, rather than to its original presence in greater
numbers.
In a subsequent investigation 520 cultures were obtained from
8 soils, 5 of them from the vicinity of Baltimore and 3 from Naza-
reth, Pennsylvania. The heating of the soil to temperatures of
60° and 80°C., and the incubation of the sample was abandoned
owing to the inconsistency of the results obtained and the over-
growth by Bacillus cereus. All samples were boiled for 20 min-
utes and plated immediately. Of the 520 cultures 306 were
from Baltimore and 214 from Nazareth. The cultures included
the following previously described types:
Baltimore Nazereth
Soil Soil
B. petasites Gottheil 73 116
B. cereus Frankland 134 41
B. megatherium De Bary 29 13
B. subtilis (Ehrenberg) Cohn 24 9
B. mesentericus (Fliigge) Migula 9 11
{Bacillus mesentericus Juscus Fltigge.)
B. vulgatus (Flugge) Trevisan 12 6
(Bacillus mesentericus vulgatus Flugge.)
B. mycoides Fltigge 15 2-
B. mesentericus var. flavus 9
B. cereus var. fluorescens 3
B. fusiformis Gottheil 3 2
B. brevis 3
B. simplex Gottheil 1
B. cohaerens Gottheil 1 2
304 214
One new species (Bacillus agri) belonging to the Cohaerens-
simplex group was isolated from the Baltimore soil on two
occasions.
In the results obtained from Baltimore it is interesting to note
the predominance of Bacillus cereus, the large number of isola-
tions of members of the Megatherium-petasites group and the
small number of Bacillus mycoides. Bacillus mycoides indeed is
a rare organism in Baltimore and soil conditions here apparently
differ markedly from those in other locahties. Bacillus subtilis
and the members of the mesentericus group are also uncommon
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 515
while the other organisms mentioned are extremely rare. The
conclusions reached by the study of the total number of organ-
isms are borne out by the analysis of the individual samples
which may be tabulated as follows:
B. cereus
B. petasites
B. mycoides
B. megatherium
B. subtilis
B. vulgatus
B. mesentericus
B. cohaerens
B. fusiformis
B. agri
B. simplex
B. cereus var. fluorescens .
B. mesentericus var. flavus
B. brevis
Total
BALTIMORE SERIES
16
10
5
4
2
1
39
20
18
6
6
4
57
23
15
3
8
3
4
2
60
17
21
1
8
7
4
6
1
66
58
9
84
NAZARETH SERIES
20
3
1
2
35
10
30
4
4
67
23
66
2
6
4
2
3
1
2
112
Source of samples: 1, Paving sand, Baltimore City; 2, surface soil, lawn of
Johns Hopkins Hospital ; 3, soil from fertilized flower bed, Baltimore City, under
cultivation, 20 years; 4, soil from cultivated garden, Baltimore; 5, soil from
field, suburb of Baltimore; 6, clay from depth of 5 feet, Nazareth; 7, soil from
highly fertilized garden, under cultivation more than 25 years; 8, richly fertilized
field soil.
In the Nazareth series the predominant organism was Bacillus
petasites which made up more than half the isolations. Bacillus
cereus stood next while Bacillus mycoides was obtained on but
two occasions. The analysis of the individual samples shows the
same predominance of Bacillus -petasites and is chiefly interesting
in demonstrating the small number of organisms isolated from
soil collected at the depth of five feet as contrasted with the
more diversified flora of highly fertiUzed top garden and field
soil.
In general it may be seen that the study of soil confirms the
conclusions reached by the study of milk, dust and water. The
516 C. A. LAUBACH, J. L. RICE AND W. W. FORD
predominating types isolated from these latter sources were also
found predominating in soil, since the 520 cultures revealed only-
one new species.
^ Bacillus agri nov. sp.
This organism was isolated on two occasions from Baltimore
soil and cannot be identified with any previously-described spe-
cies. It evidently belongs in the Simplex-cohaerens group and
because of its different cultural reactions must be described as a
new species.
Morphology. Large uniform homogeneous rods with round
ends measuring 0.625 to 0.75 by 2.5 to 5 microns in young cultures
on plain agar. Occasionally chains with a number of elements
are found and shadow or transparent forms measuring 12-15
microns in length. On glucose agar there is no special change in
morphology, but the proportion of shadow forms is greater. The
morphology resembles that of Bacillus simplex. (Figures 73 and
74.)
Motility. Active motility in young cultures.
Staining properties. Gram-positive.
Spore formation. Spores begin to form within 24 hours but
are not abundant till after the lapse of three or four days. They
are central or slightly ex-centric, no wider than the rods from
which they spring. The free spores retain rims of protoplasm
for some time. They measure 0.625 to 1.125 to 1.25 microns.
As they lose their protoplasm they become more oval and
measure 0.5 to 0.625 by 0.75 to 1.125 microns. The spore-for-
mation resembles that of Bacillus simplex.
Agar slant. Fairly profuse gray moist smooth glistening
opaque growth along the line of inoculation with a slight tendency
to spread.
Agar stab. Faint granular growth along line of inoculation.
Agar colonies. Small gray moist glistening elevated colonies
with opaque centers. No tendency to spread.
Glucose litmus agar. Fairly profuse gray moist smooth glis-
tening opaque growth with a slight tendency to spread. The lit-
mus is completely reduced within 48 hours with a very slight
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 517
initial acidity which is rapidly replaced by a permanent alka-
linity. The growth wrinkles slightly.
Glucose litmus agar colonies. Grayish, moist, opaque glisten-
ing and slightly wrinkled colonies. The reaction is the same as
that on the glucose litmus agar slant.
Gelatin stab. A definite granular growth along the line of in-
oculation followed by complete liquefaction. The liquefaction
is funnel-like.
Gelatin colonies. Definitely circumscribed grayish, moist,
glistening colonies with opaque centers and thin lace-like pe-
ripheries. The colonies rest in excavations caused by Hquefaction.
On examination with the low power, they appear to be composed
of large flocculi.
Broth. A very turbid growth which clears by sedimentation.
No pellicle is formed.
Peptone. Growth similar to that in broth.
Potato. Fairly abundant, moist, heaped up, glistening, vis-
cous and grayish growth.
Litmus milk. No change is noticed within 96 hours, after
which period a slight reduction becomes evident. The reduction
is completed within about 7 days and then the milk is slowly
peptonized. Peptonization is generally completed within 14
days. The remaining liquid is at first grayish in color but after
a variable period becomes amber-colored.
Blood-serum. A fairly abundant, thin, smooth, slowly spread-
ing, slightly glistening, brownish growth occurs. No liquefac-
tion of the medium.
Fermentation tubes. Glucose: a turbid growth occurs in the
bowl and neck but does not extend up the closed arm. No scum
is formed and a very slight initial acidity is produced which
gives way to a slight alkalinity.
Saccharose : the growth corresponds closely to that in glucose.
There is a slight alkalinity.
Lactose : the growth is similar with the production of a slight
alkalinity.
Thermal death point. The spores survive 10 pounds in the auto-
518 C. A. LAUBACH, J. L. RICE AND W. W. FORD
clave but are destroyed by 12 pounds pressure. In the Arnold
they survive 30 minutes steaming but are destroyed by one
hour's exposure.
MISCELLANEOUS CULTURES
BY WILLIAM W. FORD
On a number of occasions miscellaneous cultures from a va-
riety of sources have been studied with care and identified.
Such cultures, about 100 in number, include spore-bearing organ-
isms found at times as laboratory contaminations, in the normal
and abnormal dejecta of children and adults, in milk products, etc.
The majority of such cultures were easily identified and found
to correspond to the well-known types already described. The
most frequent isolation was Bacillus cereus which is our common
laboratory contamination. Next to this organism Bacillus suh-
tilis was most frequent, then Bacillus vulgatus or Bacillus mesen-
tericus. Members of the Megatherium-petasites group were
also not uncommon. In addition to such isolations as these
Bacillus circulans of Jordan was found on one occasion. It is
recognized by its cylindrical terminal spores and by its cultural
reactions. Dr. Jordan was kind enough to examine the culture
and confirmed our identification. Bacillus circulans is evidently
one of the rarer sporulating bacteria. At another time a round-
spored organism resembling Bacillus tetani in morphology was
encountered as a contamination on an old agar plate. This spe-
cies was apparently first described by Kruse (Fliigge, 1896) as
an aerobic variety of the tetanus bacillus. On three occasions an
organism was isolated which was originally obtained by Fliigge
(1894) and named later Bacillus brevis by Migula (1900). The same
species was found by us in Montreal. Finally on one instance a
culture was found in the normal dejecta which could not be iden-
tified with any previously described organisms. The morphology
and spore formation were so characteristic and so different from
that seen with other spore-bearing bacteria that it was decided to
create a new species for which the name Bacillus centrosporus is
selected. A detailed description of these organisms follows.
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 519
bacillus circulans Jordan 1890
This organism was described originally by Jordan in 1890. The
present culture was obtained from normal human dejecta and cor-
responds closely with Jordan's description.
Morphology. Rather long and thin bacilU with rounded or
square ends measuring about 0.5 by 2.5 to 4 microns in 24 hour
agar cultures. Occurs usually as single cells. Protoplasm homo-
geneous. On glucose agar the organisms are thicker and longer
measuring 0.75 by 5 to 8 microns. (Figure 75.)
Motility, Active progressive and rotatory motihty in 24 hour
agar cultures.
Staining properties. Gram-positive.
Spore formation. Spores form early on plain agar often in 24 hours.
Abundant in 48 horns. Form more slowly on glucose agar. They
are cyhndrical, wider than the rods from which they spring and
terminal in position. The free spores may retain rims of proto-
plasm at one end or may be naked. They measure usually about
0.75 by 1.125 microns.
Agar slant. Thin shghtly moist non-spreading growth, pale
white in color.
Agar colonies. Round regular non-spreading colonies, under low
power pale yellow, finely granular, with entire edges. Older colo-
nies become irregular and amoeboid showing under low power cen-
tral nuclei and thin peripheries.
Agar stah. Faint growth along hne of inoculation spreading at
point of puncture.
Glucose agar. Faint thin non-spreading growth producing an
acid reaction beginning in 24 hours and quite marked at the end of
5 to 6 days.
Glucose agar colonies. Small fine colonies, under low power pale
yellow, granular with entire or serrated edges. Reaction acid.
Gelatin siah. Faint growth along hne of inoculation. Little or
no surface growth. No hquefaction.
Gelatin colonies. Small fine colonies with hazy outlines, under
low power dark opaque with fuzzy margins. Older colonies quite
amoeboid. No hquefaction.
520 C. A. LAUBACH, J. L. RICE AND W. W. FORD
Broth. Slight turbidity, no scum, no sediment.
Peptone. Slight turbidity, no scum, no sediment.
Potato. No visible growth.
Milk. Complete decolorization within 24 to 48 hours. Gradual
production of acidity and late coagulation, which may appear
spontaneously and always occurs if the milk be heated.
Blood serum. Faint thin white non-spreading growth. No
hquefaction.
Fermentation tubes. Glucose : turbidity in bowl and closed arm.
No scum. Reaction acid, appearing in 3 to 4 days.
Saccharose : appearance the same. Reaction acid.
Lactose : appearance the same. Reaction acid.
Thermal death point. Spores destroyed by steaming 15 minutes
in the Arnold. Survive 18 pounds pressure in the autoclave.
Destroyed by 20 pounds pressure.
Bacillus pseudotetanicus (Kruse) Migula 1900
This organism was first described by Kruse in the 3rd edition
of Flugge's "Die Mikroorganismen" under the name Bacillus pseu-
dotetanicus var. aerohius. It resembled Bacillus tetani in mor-
phology and spore-formation but was distinguished from it by its
aerobic development, its failure to liquefy and its lack of patho-
genicity. Apparently the same organism has been described by
Neide (1904) as Bacillus sphaericus. The species here described
has the morphological and cultural features given by both Kruse
and Neide and under the rules of nomenclature the name given by
Migula should be adopted. It was found but once as a contami-
nation on an agar plate.
Morphology. Short thick bacilU with rounded ends occurring as
single cells or as two cells end to end. In 24 agar cultures they
measure 0.75 by 1.5 to 3 microns. Protoplasm homogeneous.
Many of the cells are fusiform, distinctly swollen toward the mid-
dle while others are swollen at the ends. On glucose agar the or-
ganisms are longer and thicker measuring 1.125 by 1.5 to 4 microns.
They show no change in the character of the protoplasm. In older
cultiu-es the organisms often form long threads measuring 8 to 15
microns in length. (Figures 76 and 77.)
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 521
MoUlity. Active and progressive rotatory motiUty in 24 hour
agar cultures.
Staining properties. Gram-negative.
Spore formation. Spores are formed early often withm 24 hours
on both plain and glucose agar. They are round, and usually ap-
pear at the ends of the organisms m a sub-termmal position, but
may be distmctly terminal. The spores are of greater diameter
than the vegetative rods from which they spring and thus give a
clavate or club-shaped appearance to the organisms. The organ-
isms taken from glucose agar resemble markedly the tetanus baci -
lus cultivated on this medium. The free spores retam fauly thick
waUs of protoplasm for some time. They vary m size from 1 to
1.125 microns in diameter.
Agar slant. Moist rather thin translucent growth m 24 hours,
becoming pale yellowish white in old cultures. No tendency to
spread except when the medium is very moist.
Agar stah. Faint growth along Ime of inoculation with shght
spreadmg on the surface. In older cultures the surface growth is
thicker and more abundant but does not usuaUy spread far from
the line of puncture. Easily scraped from medium.
Agar colonies. Colonies round and regular or amoeboid m 24
hours Under low power they show dark central areas with Ughter
peripheries. As they age they become thicker and denser with
more pronounced central nuclei.
Gliu^ose agar. Pale white moist rather thin growth in 24 hours,
becoming pale yellow or cream yellow in older cultures.
Glucose agar colonies. In 24 hour plates the colomes are nregu-
lar, amoeboid, thick, sho^ving under low power darker central nu-
clei with lighter peripheries but with entire edges. As the colomes
become older they become denser and thicker with heaped-up
edges They give a pecuUar bizarre appearance to the plate as
though it was covered with drops of moisture. Reaction of agar
remains neutral or becomes alkahne. .
Gelatin stab. Faint Une growth appearing after 48 to 72 hours
with a sUghtly spreading growth on the surface. In older cultures
the surface growth spreads considerably from the point of puncture
on top of the gelatin which remains soUd. Rarely the Une growth
becomes arborescent.
522 C. A. LAUBACH, J. L, RICE AND W. W. FORD
Gelatin colonies. Small fine colonies visible only after 48 hours.
Under low power they are round, regular and pale yellow with en-
tire margins. As they grow older they become denser and more
opaque and under low power show central nuclei with pale mar-
gins. Colonies two weeks old are thick brownish non-spreading.
No liquefaction.
Broth. Turbidity and rather faint sediment. No scum.
Peptone. Turbidity with shght sediment. No scum.
Potato. Rather scanty yellowish moist growth becoming brown
in old cultures.
Litmus milk. No change in appearance of the milk in the first
few days then a gradual reduction of the Htmus, completed in 15
to 18 days. No peptonization even in milk cultures kept under
observation four weeks. No coagulation.
Blood serum. Dry thin yellowish growth becoming pale yellow-
brown in old cultures. No Uquefaction.
Fermentation tubes. Glucose: turbidity in bowl, no scum, arm
clear. Reaction alkaUne.
Saccharose: reaction the same.
Lactose: reaction the same.
Thermal death point. The spores resist steaming in the Arnold
sterihzer 15 minutes but are destroyed in 30 minutes. They resist
a pressure of 14 pounds in the autoclave but are destroyed by 16
pounds. /
Bacillus brevis Migula 1900
This organism was first described by Fliigge (1894) as Bacillus
No. I. According to Neide (1904) who has given an accm^ate ac-
count of its morphology and cultural reactions under the name
Bacillus lactis Fliigge, it is probably identical with Bacillus
cylindrosporus Burchard (1898). The correct name is that of
Migula. Three different strains have been isolated from milk,
from soil, and from dust. They agree in their cultural reactions
with the description given by one of us (W. W. F.) of a culture iso-
lated in Montreal.
Morphology. Bacilh with pointed ends and sUghtly granular pro-
toplasm, sometimes fusiform in shape . In 24 hour cultures on plain
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 523
agar they measure 0.375 to 0.5 by 2 to 3 microns. On glucose agar
they are more uniform and shghtly thicker and longer, measuring
usually 0.5 by 3 to 4 microns. (Figures 78, 79 and 80.)
Motility. Active progressive motility in 24 hour agar cultures.
Staining properties. Gram-negative.
Spore formation. Spores are formed early appearing often within
24 hours on plain and glucose agar. They are cyUndrical, usually
subterminal in position, thicker than the organisms from which
they spring, and present a characteristic appearance. They may
retain spurs of protoplasm at each end or may be entirely naked.
They measure usually 0.75 by 1.125 to 1.5 microns. Rarely they
may be distinctly terminal in position.
Agar slant. Thick moist spreading growth with a silvery sheen
abundant in 24 hours and becoming thick white and glistening in
older cultures. Easily scraped from medium.
Agar stab. Fihform growth along line of inoculation with an
abundant surface growth spreading to the tube wall.
Agar colonies. Surface colonies in 24 hours are round aiid regu-
lar or thin spreading translucent, under low power slightly granu-
lar with entire edges. Deep colonies small fine, under low power
irregular and pale yellow. As the superficial colonies get older
they become thicker and heaped up sUghtly. The deep colonies
may show irregular fuzzy margins under the low power.
Glucose agar. Heaped up white moist non-spreading growth in
24 hours, becoming thick and raised in old cultures. Reaction
alkahne.
Glucose agar colonies. Superficial colonies are shghtly thicker
than plain agar colonies in 24 hours and show a greater tendency
to spread. Under the low power they are granular with entire
edges. Deep colonies small and fine, under low power irregular and
pale yellow. Older superficial colonies are thick, irregular and
heaped up. Reaction of agar alkahne.
Gelatin stab. Faint growth along hne of inoculation and at the
point of puncture in 3 to 4 days, with beginning hquefaction. Dis-
tinct cup-shaped or funnel liquefaction in two weeks. Occasionally
a fragile scum appears on the hquid gelatin.
Gelatin colonies. Colonies begin to appear in 48 hours and show
524 C. A. LAUBACH, J. L. RICE AND W. W. FORD
dark central areas with hazy peripheries. Under the low power
they show dark opaque centers with wavy branching peripheries.
Deep colonies frequently show peculiar prickly margins. Gelatin
completely liquefied at the end of about two weeks and may show
a thick scum on the surface.
Broth. Turbidity and a flocculent sediment in 24 to 48 hours
with a gradual production of a heavy scum which appears often
only after 4 days. In old cultures the precipitate is quite
heavy.
Peptone. Turbidity and heavy flocculent sediment. Scum may
be thick and heavy or entirely lacking.
Potato. Usually a rather scanty moist yellow or yellow-brown
growth.
Milk. No change in reaction. Litmus begins to decolorize in
48 hours and by the end of two weeks is completely decolorized.
At the same time the proteins are digested and the resulting fluid
is a pale rather thin colorless solution. No coagulation.
Blood serum. Thin cream-white non-spreading growth becom-
ing yellowish-brown in old cultures. No Hquefaction.
Fermentation tubes. Glucose : thick scum, turbidity in bowl, arm
clear. Reaction alkaline.
Saccharose : reactions the same.
Lactose : reactions the same.
Thermal death point. The spores survive steaming 30 minutes
but are destroyed by 45 minutes exposure. They may survive a
pressure of 16 pounds in the autoclave but are destroyed by 18
pounds pressm^e. ■
V Bacillus centrosporus nov. sp.
This is a new species obtained but once, from the normal de-
jecta of a child from the Harriet Lane Home.
Morphology. In 24 hour cultures on plain agar at 37°C. or
in 36 hour cultures at 22°C. long thin bacilli with pointed ends
fusiform or Clostridium in shape with slightly granular proto-
plasm (grassy), occurring usually as single cells measuring 0.375
to 0.5 by 3 to 4.5 microns in dimensions. The majority of the
AEKOBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 525
organisms measure about 3 to 4 microns in length. On glu-
cose agar the organisms measure about 0.375 by 2.25 to 3 mi-
crons and the ends are rounder but there is no change in the
character of the cytoplasm. (Figures 81 and 82.)
Motility. Active progressive and rotatory motility in 24 hour
agar culture.
Staining properties. Organisms stain readily by the ordinary
dyes. Gram-negative.
Spore formation. Spores are formed early appearing in 24
hours on plain agar in the thermostat, somewhat later on glu-
cose agar. They are usually formed in the central portion of the
rods causing a distinct sweUing so that the bacilli become spindle-
shaped. Rarely the spores appear toward the end of the rods
in a subterminal position or they may even be terminal. They
are much wider than the vegetative rods and when free are dis-
tinctly cylindrical with pointed spurs. They measure 0.625 by
1.125 to 1.5 microns. By the end of 48 hours both at 37° and
at 22° the spores are abundant.
Agar slant. Thick moist rather dark non-spreading growth in
24 hours, becoming thicker and raised in 48 hours and in older
cultures. Easily scraped off from medium.
Agar stab. Growth along hne of inoculation in 24 hours,
gradually spreading on the surface of the agar at the point of
puncture.
Agar colonies. Round regular rather translucent colonies in
24 hours, granular with smooth margins under the low power.
Older colonies show pale central areas with heaped up peripheries
but remain small regular and slightly raised, resembling those of
Bacillus coli.
Glucose agar slant. Thick moist cream-yellow non-spreading
growth in 24 hours, becoming thick heavy with an irregular
surface of a reddish brown color in old cultures. Reaction
alkaline.
Glucose agar colonies. Irregular heaped up colonies in 24
hours tending to become slimy or viscid-looking. Under low
power they are irregular, heaped up or swollen, with entire mar-
gins. At times they have a dew-drop appearance. On old cul-
526 C. A. LAUBACH, J. L. RICE AND W. W. FORD
tures (10 days) they are thicker and shghtly brownish. Reac-
tion of medium alkahne.
Gelatin stab. Fihform growth along Hne of inoculation and
gradual liquefaction in the form of a sphere below the surface.
Liquefaction is slow, taking place only after 7 to 8 days. Lique-
faction gradually reaches the walls of the tube. Growth not
arborescent.
Gelatin colonies. Colonies appear only after 5 to 6 days.
They are small fine and surrounded by a hazy zone. Gelatin
slowly liquefied. Under low power colonies are round regular
finely granular with entire margins or may show irregular out-
lying strands.
Broth. Turbidity within 24 to 48 hours with a faint friable
scum appearing in about 10 days.
Peptone. Turbidity within 24 to 48 hours with faint friable
scum in 10 days.
Potato. Moist yellow growth within 24 hours gradually be-
coming heaped up and irregular and assuming a brown or red-
dish brown color.
Litmus milk. No change in reaction except for an occasional
partial reduction of the litmus. No peptonization. No coagu-
lation.
Blood serum. Pale yellow growth in "24 hours gradually be-
coming thick and moist in old cultures. No liquefaction.
Fermentation tubes. Glucose: turbidity in bowl, no scum,
arm clear. Reaction alkaline.
Saccharose: reactions the same.
Lactose : reactions the same.
Thermal death point. The spores stand boiling 15 minutes but
are destroyed by boiling for 30 minutes. They resist 15 minutes'
exposure in the Arnold but are destroyed by 30 minutes' exposure.
They resist autoclaving at a pressure of 12| pounds but are
destroyed by 14 pounds pressure.
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 527
CLASSIFICATION
BY W. W. FORD
From a study of spore-bearing organisms lasting over a period
of four years, during which over 1700 cultures were worked out
from a variety of sources including milk, dust, water, soil, in-
testinal contents, and laboratory stock cultures 28 species or
varieties of species were isolated and established as distinct
types by the usual moi-phological, developmental, tinctorial and
cultural features available for systematic investigation. The ma-
jority of these types were encountered on many separate occa-
sions and our descriptions of them are based upon those fixed and
permanent characters which we regard as establishing bacterial
species. These types thus represent the organisms met with in
our work quite regardless of their identification as previously
established species or varieties. Of the total number, 28, we
were able to identify 22 as species already described in the lit-
erature. Whether these species are correctly named, is a mat-
ter about which of course opinions may vary. We feel however
that our identification of these 22 species is as satisfactory as
the confusion in the literature and the uncertainty of stock cul-
tures permit. In addition to the 22 types described by previous
workers two distinct varieties of old species were found while
four organisms were isolated which are so different from any de-
scribed in the hterature as to merit estabhshment as new species.
To these six organisms new names have been given. With these
28 types in mind it now becomes possible to classify the spore-
bearing organisms from a preliminary standpoint. The basis
for the classification rests primarily upon morphology, spore-
formation and motility, and secondarily upon tinctorial and cul-
tural features. All the characters are easily demonstrated m any
properly equipped bacteriological laboratory. Both the mi-
crochemical properties of the bacterial cells and the methods of
spore-germination while studied when possible have been dis-
regarded in arranging the species in groups. The extent to
which this classification is of permanent value will be shown, we
believe, by the approval or disapproval with which it is received
by American bacteriologists.
528 C. A. LAUBACH, J. L. RICE AND W. W. FORD
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA
Group I. Suhtilis group
Small homogeneous sluggishly motile organisms measuring
0.375 by 1.5 to 2.5 microns. No threads on glucose agar. Cen-
tral or ex-centric spores, oval, measuring 0.5 by 0.75 to 0.875
microns, often retaining terminal tags of protoplasm. Growth
on soh'd media hard and penetrating, with tenacious scums on
fluid media.
Represented by
Bacillus suhtilis Cohn.
Bacillus suhtilis-viscosus Chester.
(Characterized by viscosity.)
Group II. Mesentericus group
Small homogeneous actively-motile organisms measuring 0.5
by 2 to 4 microns. Often produce long threads on glucose agar.
Spores measure 0.5 by 1 to 1.125 microns, oval, retaining termi-
nal tags of protoplasm. Growth on hard media as soft pulta-
ceous mass with tendency to wrinkle, on fluid media as friable
easily-broken scums.
Represented by
Bacillus vulgatus (Fltigge) Trevisan.
{Bacillus mesentericus vulgatus Fltigge.)
Bacillus mesentericus (Fltigge) Migula.
(Bacillus mesentericus fuscus Fltigge.)
Bacillus aterrimus Lehmann & Neumann.
(Bacillus mesentericus niger Lunt.)
Bacillus glohigii Migula.
(Bacillus mesentericus ruber Globig.)
Bacillus niger Migula.
(Bacillus lactis niger Gorini.)
Bacillus mesentericus var. fiavus.
Bacillus panis Migula.
(Bacillus mesentericus panis viscosi I Vogel.)
(Motility lost by capsule formation.)
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 529
Group III. Cohaerens-simplex group
Motile organisms somewhat larger than either Bacillus sub-
tilis or Bacillus mesentericus, measuring 0.375 to 0.75 by 0.75 to
3 microns. Thicker and longer forms on glucose agar. Invo-
lution and shadow forms common and appear early. Spores
cylindrical, measuring 0.5625 to 0.75 by 1 to 1.5 microns.
Growth as a soft mass on hard media, as turbidity with little
or no scum on fluids.
Represented by
Bacillus cohaerens Gottheil.
Bacillus simplex Gottheil.
Bacillus agri nov. sp.
Group IV. Mycoides group
Large organisms with square ends growing in long chains.
Single cells measure 0.5 by 3 to 6 microns. On glucose agar
organisms are thicker and longer and made up of globular bod-
ies. Tendency for organisms to grow in curves or spirals.
Spores central or ex-centric, round or oval to cylindrical, meas-
uring 0.75 to 1 by 1 to 2 microns. Spores vary greatly in size
and often appear in chains. Growth on hard media dry and
penetrating, on fluid media as firm tenacious scum.
Represented by
Bacillus mycoides Fliigge.
Bacillus prausniizii Trevisan.
{Bacillus ramosus liquefaciens Prausnitz.)
Bacillus adhaerens nov. sp.
(No motility.)
Group V. Cereus group
Large motile organisms with round ends measuring 0.75 by
2.25 to 4 microns. Tend to grow in short chains. Thicker and
longer on glucose agar where protoplasm is converted into glob-
ular bodies. Central or excentric spores, cylindrical, measur-
ing 0.5 to 0.75 by 1.125 to 1.5 microns. Spores retain proto-
530 C. A. LAUBACH, J. L. RICE AND W. W. FORD
plasm at one or both ends often resembling enlarged subtilis or
mesentericus spores. Growth on hard media as soft pultaceous
mass with tendency to fold or wrinkle, on fluid media as thick
friable scum.
Represented by
Bacillus cereus Frankland.
Bacillus albolactus Migula.
Bacillus cereus var. fluorescens nov. var.
Group VI. Megatherium group
•Very large actively motile organisms measuring 0.75 to 1.25
by 3 to 9 microns. Often in long forms which spread out, lose
their cytoplasm and show peculiar aggregations of protoplasm
at the periphery. Protoplasm rapidly converted into peculiar
globular highly refractile bodies, particularly on glucose agar.
Shadow and transparent forms appear early. Spores central,
excentric or sub-terminal, oval to cylindrical, measuring usu-
ally 0.75 to 1.125 by 1.5 to 2 microns. Spores vary greatly in
shape, sometimes round, sometimes rectangular, often reniform.
Growth on solid media as thick pultaceous mass, on fluid media
as turbidity with little or no scum formation.
Represented by
Bacillus megatherium De Bary.
Bacillus petasites Gottheil.
Bacillus ruminatus Gottheil.
Group VII. Round terminal spored group
Small actively motile organisms measuring 0.5 to 0.75 by 1.5
to 3 microns, often forming long threads in old cultures. Proto-
plasm homogeneous. Spores subterminal or terminal, round,
thicker than the organisms from which they spring, measuring
1 to 1.5 microns in diameter.
Represented by
Bacillus pseudotetanicus (Kruse) Migula.
(Bacillus pseudotetanicus var, aerobius Kruse.)
Bacillus fusiformis Gottheil.
AEROBIC SPORE-BEARING NON- PATHOGENIC BACTERIA 531
Group VIII. Cylindrical terminal spared group
Small thin actively motile organisms measuring 0.375 to 0.5
by 2.5 to 4 microns. Slightly larger on glucose agar but no
change in character of protoplasm. Spores terminal, cylindri-
cal, measuring usually 0.75 by 1.125 to 1.5 microns.
Represented by
Bacillus circulans Jordan.
Bacillus hrevis Migula.
Bacillus terminalis Migula.
Group IX. Central spored group
Long, actively motile organisms with pointed ends measuring
0.375 to 0.5 by 1.125 to 4 microns. Slightly larger on glucose
agar, but no change in character of protoplasm. Spores de-
velop in the middle of the rods which become spindle-shaped.
Spores large cylindrical measuring 0.625 to 0.875 by 1.125 to
1.5 microns.
Bacillus centrosporus nov. sp.
Bacillus laterosporus nov. sp.
bibliography
BuKCHABD (1898) Arb. a. d. bakt. Inst. d. teen. Hochsch. zu Karlsruhe, 2,
Heft., 1, 31.
Chester (1903) Fifteenth Annual Report of the Delaware College Agricul-
tural Experiment Station, 42.
EiSENBERG (1891) Bakteriologische Diagnostik, 126.
FLtJGGE (1886) Die Mikroorganismen. 2 aufl., 402. Watson Cheyne trans-
lation.
FLiJGGE (1894) Zeitschr. f. Hyg., 17, 294.
FLtJGGE (1896) Die Mikroorganismen. 3 Aufl., 267.
Frankland, Grace and Percy (1894) Micro-organisms in Water, 419.
GoTTHEiL (1901) Centralbl. f. Bakt. 2 Abt., 7, 485.
Jordan (1890) Experimental Investigations by the State Board of Health of
Massachusetts. Part U, 831.
Migula (1900) System der Bakterien, 2, 583.
Neide (1904) Centralbl. f. BaKt., 2 Abt., 12, 337, 350.
Sternberg (1892) Manual of Bacteriology, 679.
Trevisan (1889) Genera, 20.
VoGEL (1897) Zeitschr. f. Hyg., 26, Heft 3, 404.
532 C. A. LAUBACH, J. L. RICE AND W. W. FORD
LIST OF ILLUSTRATIONS
Plate 1
Fig. 53. Bacillus prausnitzii from dust. {Bacillus ramosus liquefaciens.)
Plain agar, 6 hours
Fig. 54. Bacillus praursnitzii from dust. {Bacillus ramosus liquefaciens.)
Plain agar, 48 hours
Plate 2
Fig. 55. Bacillus mesentericus var. flavus from dust. Plain agar, 6 hours.
Fig. 56. Bacillus mesentericus var. flavus from dust. Glucose agar, 24 hours
Plate 3
Fig. 57. Bacillus mesentericus var. flavus from dust. Plain agar, 12 days
Fig. 58. Bacillus ruminatus from dust. Plain agar, 6 hours
Plate 4
Fig. 59. Bacillus ruminatus from dust. Plain agar, 16 days
Fig. 60. Bacillus panis from dust. {Bacillus mesentericus panis viscosi I.)
Plain agar, 6 hours
Plate 5
Fig. 61. Bacillus panis from dust. {Bacillus mesentericus panis viscosi I.)
Glucose agar, 48 hours
Fig. 62. Bacillus panis from dust. {Bacillus mesentericus panis viscosi I)
Plain agar, 8 days
Plate 6
Fig. 63. Bacillus adhaerens from dust. Plain agar, 6 hours
Fig. 64. Bacillus adhaerens from dust. Plain agar, 5 days
Plate 7
Fig. 65. Bacillus suhtilis var. viscosus from water. Plain agar, 6 hours
Fig. 66. Bacillus suhtilis var. viscosus from water. Glucose agar, 3 days
Plate 8
Fig. 67. Bacillus suhtilis var, viscosus from water. Plain agar, 13 days
Fig. 68. Bacillus cereus var, fluorescens from water. Plain agar, 6 hours
Plate 9
Fig. 69. Bacillus cereus var. fluorescens from water. Plain agar, 24 hours
Fig. 70. Bacillus cereus var. fluorescens from water. Glucose agar, 24 hours
JOURNAL OF BACTERIOLOGY VOL. I PLATE 1
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Fig. 54
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JOURNAL OF BACTERIOLOGY VOL. I
PLATE 2
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JOURNAL OF BACTERIOLOGY VOL. I
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JOURNAL OF BACTERIOLOGY VOL. I
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JOURNAL OF BACTERIOLOGY VOL. I
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JOURNAL OF BACTERIOLOGY VOL.
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JOURNAL OF BACTERIOLOGY VOL. I PLATE 7
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JOURNAL OF BACTERIOLOGY VOL. I
PLATE 8
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JOURNAL OF BACTERIOLOGY VOL. I PLATE 9
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(Laubach, Rice and Ford: Aerobic Spore-bearing Non-pathogenic Bacteria)
JOURNAL OF BACTERIOLOGY VOL. I
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JOURNAL OF BACTERIOLOGY VOL. I PLATE 11
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JOURNAL OF BACTERIOLOGY VOL. I PLATE 12
Fig. 75
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JOURNAL OF BACfERIOLOGY VOL. I PLATE 13
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JOURNAL OF BACTERIOLOGY VOL. I
PLATE 14
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0
.v>^
^: b
0
Fig. 79
L? 0
^ ^/ b ^ J?
o
Fig. 80
fLaubach. Rice and Ford: Aerobic Spore-bearing Non-pathogenic Bacteria)
JOURNAL OF BACTERIOLOGY VOL I
PLATE 15
\
^..
y
A
\^ K
\
/
Fig. 81
0 . ^ ^ ^ ^ ^
0 0
o
<^
0 0
^
^ ^ '^ « . ^ .c^ 0
^
Fig. 82
(Laubach, Rice and Ford: Aerobic Spore-bearing Non-pathogenic Bacteria)
AEROBIC SPORE-BEARING NON-PATHOGENIC BACTERIA 533
Plate 10
Fig. 71. Bacillus laterosporus from water. Plain agar, 6 hours
Fig. 72. Bacillus laterosporus from water. Plain agar, 3 days
Plate 11
Fig. 73. Bacillus agri from soil. Plain agar, 24 hours.
Fig. 74. Bacillus agri from soil. Plain agar, 3 days.
Plate 12
Fig. 75. Bacillus circulans from human dejecta. Plain agar, 24 hours.
Fig. 76. Bacillus pseudotetanicus from contaminated plate. Plain agar, 20
hours.
Plate 13
Fig. 77. Bacillus pseudotetanicus from contaminated plate. Glucose agar, 18
hours.
Fig. 78. Bacillus brevis from dust. Plain agar, 24 hours.
Plate 14
Fig. 79. Bacillus brevis from soil. Plain agar, 48 hours.
Fig. 80. Bacillus brevis from milk. Plain agar, 3 days.
Plate 15
Fig. 81. Bacillus centrosporus from human dejecta. Plain agar plates, 30
hours.
Fig. 82. Bacillus centrosporus from human dejecta. Plain agar, 24 hours.
A RAPID AND SIMPLE INDOL TEST
PAUL R. CANNON
Fro7n the Department of Hygiene and Bacteriology , The University of Chicago
It is well known that tryptophane in an inorganic solution fur-
nishes an excellent medium for the demonstration of indol pro-
duction by bacteria. Zipfel (1) in 1912 found that indol could
be demonstrated by this method at the end of twenty-four hours,
giving the best reaction with p-dimethyl-amido-benzaldehyde.
The test is made by adding to the culture to be tested one cubic
centimeter of a solution consisting of p-dimethyl-amido-benzal-
dehyde, 4 parts; absolute alcohol, 380 parts; and concentrated
hydrochloric acid, 80 parts.
The expense of preparing tryptophane and the great difficulty
in obtaining it at all in the United States at the present time are
drawbacks to the introduction of the tryptophane method. As
a substitute, I have found that hydrolyzed casein can be used.
Cow casein contains about 1.5 per cent tryptophane, which may
be obtained in its amino-acid form by hydrolyzing the casein.
The method is as follows: 10 grams of casein are hydrolyzed
by 200 cc. of 10 per cent sulphuric acid, the mixture being kept
on the water bath for twenty-four hours. At the end of this
time, the casein is completely dissolved and the solution is a
dark brown. Next, the solution is neutralized by the addition
of saturated barium hydrate, thus precipitating out the sulphate.
The resulting solution is then evaporated until the amino-acids
crystallize. Half of the crystalline mass is dissolved in 500 cc.
of Zipfel's inorganic solution consisting of Asparagin and Ammo-
nium lactate, 5 grams each ; Potassium acid phosphate, 2 grams ;
Magnesium sulphate, 0.2 grams; and distilled water, 1000 grams.
The medium is tubed and sterihzed. Assuming that there are
0.15 grams of tryptophane in the cow casein, tryptophane should
be present in the mixture to the extent of about 0.03 per cent.
535
536 PAUL R. CANNON
Tubes of media prepared in this way were inoculated with
known indol-forming bacteria, and, at the end of eighteen hours,
the indol test was made by using p-dimethyl-amido-benzaldehyde.
A pronounced red color almost instantly appeared, showing the
presence of indol. The control and non-indol-formers remained a
straw color after the addition of the aldehyde. It proved un-
necessary in my tests to use amyl alcohol to dissolve out the
color, although this may be done in case indol formation is
doubtful.
The brief time — eighteen to twenty-four hours — necessary for
the test by the above method, is a great improvement over the
old standard peptone test, which required five days. Further-
more, hydrolyzed casein can easily be obtained or prepared, and
the constituents of the inorganic solution are available in most
laboratories.
REFERENCE
(1) Zipfel: Centralblatt f. Bakt., etc., Orig. Abt. 1, 64, 65, 1912.
BACTERIAL NUTRITION: A BRIEF NOTE ON THE
PRODUCTION OF EREPSIN (PEPTOLYTIC
ENZYME) BY BACTERIA
NATHAN BERMAN and LEO F. RETTGER
From the Sheffield Laboratory of Bacteriology and Hygiene, Yale University
In a recent publication (1) the authors stated that gelatin-
non-liquefying bacteria of the Bacillus coli type are able to exert
but little, if any, proteolytic action on Witte's peptone and on
partially purified proteoses. The biuret test was employed in
the earlier experiments, and the results were not expressed in
mathematical terms. In later studies the changes in the pro-
tein content of the media have been recorded in definite figures.
Furthermore, the Sorensen method for the determination of
amino acids has been used along with the biuret test.
The published results in a few instances gave unmistakable evi-
dence of a reduction in the amount of biuret-giving substances
in the media containing Witte's peptone, when B. coli, B. typhi
and B. pullorum were employed. These reductions were only
slight, however, and were noticeable only after two weeks' in-
cubation. While the changes in the protein content of the media
suggested the possibility of ereptic enzyme action, no definite
conclusion was warranted. Subsequent investigation has, in a
measure at least, clarified the situation.
In a medium containing 1 per cent peptone, 0.25 per cent
Liebig's meat extract and 0.5 per cent of sodium chloride B. coli,
B. typhi, and B. para^?/7?/l^ A, under optimum cultural conditions
have slowly acted upon the biuret-positive substances in the
peptone. While at least two weeks were usually required to
produce any appreciable decrease in the ''protein," the loss
amounted to from 10 per cent to 40 per cent of the original when
the digestion was allowed to continue for at least three weeks.
It was impossible to bring about a decomposition of more than
537
538 NATHAN BERMAN AND LEO F. RETTGER
40 per cent of the original ''protein" in the peptone solution,
even under the most favorable conditions, as for example the
addition of ammonium sulphate, asparagine, glycerine, phos-
phates, etc. In some instances 10 to 20 per cent marked the
maximum reduction. When other peptones were employed,
however, namely five different American brands, reductions con-
siderably in excess of 40 per cent of the protein were frequently
obtained. In all of these experiments the Sorensen method gave
corroborative results.
Media containing Witte's peptone which had been dialyzed
for at least two days underwent less change in their protein con-
tent than the corresponding solutions of untreated peptone. Fur-
thermore, the so-called ''proteose fraction" obtained from Witte's
peptone by precipitation with ammonium sulphate, repeated pre-
cipitation with sodium sulphate, and prolonged dialysis, was
apparently unattacked when used in place of the original com-
mercial peptone. The results obtained thus far indicate that
the gelatin-non-liquefying organisms of the Coli-typhi-paraty-
phi group are unable to utilize or in anyway affect the proteose
portion of commercial peptones.
In the light of the researches of Emil Fischer and his pupils
(2) on polypeptides, the deportment of the Bacilus coli type
of gelatin-non-liquefying bacteria towards commercial peptone
may be explained as follows. These peptones, far from being
relatively simple, are mixtures of many organic substances of
varying composition and complexity, particularly amino acids
and polypeptides. Some of these polypeptides are in all prob-
ability quite simple, as for example the condensation product
of two simple amino acids, and if we accept the conclusions drawn
by different investigators, others are of a much more complex
type, and are tied up with the peptone and proteose molecules.
Some of the polypeptides give a positive biuret reaction, even
the relatively simple ones, while others are negative to this test.
All may on decomposition yield ammonia, amines and amino
acids.
The proteose fraction of commercial peptone, it may be as-
sumed, contains the relatively complex polypeptides, and hence
BACTERIAL NUTRITION 539
is less readily attacked and utilized as food by bacteria than the
remainder of the mixture. On the other hand, the real peptone
fraction is undoubtedly a more heterogeneous mixture of poly-
peptides, some of which are indeed quite simple, and are easily
broken up by bacteria or their enzymes. In the proteolysis
which takes place slowly under most favorable conditions, the
simpler polypeptides are attacked. When these have been ex-
hausted so-called "peptolysis" ceases.
The above explanation appears all the more probable from the
fact that as bacterial decomposition continues there is a change in
the biuret-giving ingredients of the peptone which is indicated
by a difference in the color obtained. While the original pep-
tone medium gives a pink color, the reaction changes if there is
appreciable peptolysis, and the color becomes distinctly violet
and indistinguishable from the biuret color obtained with pro-
teoses and some of the higher proteins.
The peptolysis which is brought about by Bacillus coli and
its close allies is undoubtedly the result of an ereptic enzyme
(erepsin) . This enzyme differs, however, from erepsin of animal
origin in that it does not attack casein. In comparison with
intestinal erepsin. and with the proteolytic enzymes of B. sub-
tilis, B. prodigiosus and other strong gelatin-liquefjdng bacteria,
in so far as peptolytic action is concerned, the erepsin elaborated
by the Coli-typhi-paratyphi group of organisms is decidedly
weak.
REFERENCES
(1) Rettger, L. F., Berman, N. and Sturges, W. S.: This Journal, The
utilization of proteid and non-proteid nitrogen. 1, 15-33, 1916.
(2) Hammarsten and Hedin: Textbook of physiological chemistry. English
translation by Mandel, pp. 86-91, 1914.
A PRACTICAL METHOD FOR THE IDENTIFICATION
OF GUINEA-PIGS UNDER TREATMENT
A. PARKER KITCHENS
From the Mulford Biological Laboratories, Glenolden, Pennsylvania
The problem of identifying guinea-pigs under treatment is a
simple one if only a few animals are in use; the popular method of
recording their colors may be adequate; or, separate cages or
pens may be provided for each animal. But in laboratories
where ten or more animals are inoculated every day a more highly
systematized method is required.
Celluloid or aluminum ear tags are both convenient and satis-
factory for a small number of animals ; they are not easily lost un-
less two or more full grown male pigs are kept in the same pen.
The ear tag method, however, has the disadvantage of consuming
considerable time. Furthermore it involves an extra item of
stock to look after; if the tags happen to be all used, there may
be some difficulty and annoyance in finding a substitute.
Another method in common use is a pictorial description of the
animal. On the record sheet is printed the outline of a guinea-
pig with the regions of the body indicated. The colors are then
noted on the diagram according to their regional distribution.
In some laboratories, a rubber stamp is used as the basis for the
pictorial description, an arrangement which may be desirable
when the same record sheet is to be used for other animals.
Many laboratory workers merely draw an elongated oval with
little marks at one end to represent eyes and ears and record on
this simple diagram the color distribution. All these pictorial
methods are reliable, but so much space is required for the esquisse
that the record of a large amount of work becomes extremely
bulky.
541
542 A. PARKER KITCHENS
The most valuable method for practical purposes would seem
to be one whereby (1) ears tags and other equipment are rendered
unnecessary, (2) the description is recorded on a single line, on
ordinary paper without special ruhng or diagram. Although
systems similar to the one about to be described have probably
been in use for some time, none has appeared to the writer so
satisfactory as the elaboration of what was originally merely a
makeshift series of abbreviations invented at the time for a par-
ticular purpose. The fundamental idea of the plan in question
was conceived by Dr. Joseph McFarland more than fifteen years
ago ; and with a few modifications by the writer, the same method
has been used in these laboratories ever since that time. The
readiness with which an assistant of no training can learn to de-
scribe and recognize guinea-pigs accurately seems to be suf-
ficient recommendation for a more widespread use of this system
of abbreviations.
The rules according to which guinea-pigs are described and
identified in the Glenolden laboratories are as follows:
IDENTIFICATION OF GUINEA-PIGS
1. Not more than 5 to 8 guinea-pigs are kept in one pen.
2. The assistant in describing the animals mentions first the
weight, and then the sex, before calling out the description.
3. The sex is denoted thus:
Male cf
Female 9
4. The sides (Right and Left) and the colors are represented
in abbreviations by the capitalized initial letter.
The sides of the animal are written thus :
Right ^
Left ^•
The colors are recorded as follows:
1 The difference between the abbreviations for red, "/t** and right, "(Z.'', are
to be noted.
METHOD FOR IDENTIFICATION OF GUINEA-PIGS 543
White ^
Cream q^
Yellow ij^
Silver (silver agouti) ^2
Silver and yellow ^tU
Red ^
Red and Gold RJLt
Gold (golden agouti) Ai
Fawn (also chocolate) ^g
Yellow and Slate ^
Slate...- ^
Red and Black ^[^
Black ^
5. The varieties of coat are described thus:
Hair Clipped ^
Rough ^
Angora ^
6. The regions of the body are represented by small letters as
follows (see fig. 1) : .. , p, ,
^ Singular Plural
Nose '^^
Face
Head
.bU-
Eye -^ -^^^
Ear ^^ 4*^
Root (of ear) -f \f
Shoulder "4-' ,4^
Pleura -^ 4^
Flank (pleura and hip) "^ 'IT
Sacrum
7. The pigs are described in the order of the depth of their
coloring, a white pig, for instance, is mentioned first, then a pig
with few markings over only a small part of the body. Pigs of
sohd colors come last in the order given above. White is men-
tioned only when the entire pig is white.
8. In describing an animal, the assistant starts with the fore
part of the body on the right side (R) ; he mentions the color-
say, of the right eye— and proceeding backward enumerates the
2 Note the difference between 6" (silver) and •^' (slate).
544
A. PARKER KITCHENS
parts having this color, first on the right side then on the left
(provided, of course, this color is distributed on both sides); he
then mentions the next color in the same order from the head
backward.
9. In the case of two pigs with mixed colors over the entire
body it is convenient to note the two colors and follow this by a
characteristic difference. Occasionally there may be several pigs
T;<.ure I.
Fa.ce ■ /
X
Nose
H e A. J '' !/^\
/^.
..Eye
..Root
jC__
LJ\
...EJLr
/
\
.. 5Kou.Uer
Fl^hK-.-l :
w
exactly or almost exactly ahke — white, for instance, or red and
black. In such cases, it may be necessary to chp the hair on
the nose or some other part of the body.
10. The description of 8 guinea-pigs in a certain pen might be
written as follows:
ttf White
^^ White with red about the right eye
MJLU^^*^ Slate right ear and left eye and hip, yellow right shoul-
der and pleura
/3;i»A>y Black eyes, ears and red pleura
^A«y^ Red and black mixed but with red on nose
-ftiill*/. Red and black mixed but with black on nose
/\A,fe^ Red and black mixed with the hair on the nose clipped
^ Black
METHOD FOR IDENTIFICATION OF GUINEA-PIGS 545
It will be noted that the entire description of the animal is
recorded without raising the pen or pencil from the paper.
Since this system is based upon abbreviations, the question
may be asked, why not go still further and use shorthand charac-
ters? It is true that shorthand once learned would consume less
time, but it would require considerable training for a new assist-
ant to become sufficiently famihar with shorthand characters to
write them rapidly and accurately. In the author's experience,
on the other hand, it has not required more than two or three
fifteen-minute periods to instruct any new assistant in the satis-
factory use of the above method.
A NOTE ON THE PREPARATION OF AGAR AGAR
CULTURE MEDIA
C. L. WILLIAMS AND H. P. LETTON
United States Public Health Service
One of the greatest troubles in preparing agar agar is clearing
the medium. Filtering through paper is very slow and requires
a special water jacket for the filter in order to keep the agar hot
enough to prevent sohdification. Nor does a paper filter entirely
clear the agar. The more usual method of filtering through
cotton requires repeated filtration, and even then seldom gives a
perfectly clear product.
The principal cause of failure is apparently the presence of
finely divided insoluble particles derived from the agar agar,
which become evenly distributed through the medium after this
substance has been dissolved. These particles are often so fine
that a great part of them will pass even through filter paper.
In the laboratory of the Sanitary District of the Great Lakes
various methods were tried to facilitate filtration while securing
a clear product. The use of egg albumin as a coagulant in some
media suggested coagulation as probably successful, and the use
of the Hygienic Laboratory method of preparing Endo's medium
gave us the clue to the present procedure.
In the Hygienic Laboratory method for Endo, the medium is
partially cleared by pouring into large beakers while hot, where
it is allowed to cool and harden. On inverting the beakers the
solid mass of agar slips out, and the bottom where most of the
suspended matter has settled is cut off and wasted. This proc-
ess was modified by us in the case of ordinary agar media by
placing the container in a large water bath (a large saucepan usu-
ally served for this) which was kept over a low flame for several
hours, usually over night. This procedure caused the collection
of the suspended matter into flocculent masses which settled to
547
548 C. L. WILLIAMS AND H. P. LETTON
the bottom. The result was more than a settling of the solid
particles, it was rather the ensnaring of these in a dehcate coagu-
lum, much like the coagulum of aluminum hydrate and like
that substance in requiring some time for its action. After the
formation of this flocculent precipitate a single filtration through
cotton produces a beautifully clear filtrate.
The following points should be remembered. Any stirring be-
fore filtration will quickly break up the coagulum, which is very
dehcate, a cloudy filtrate resulting. For this reason, the reac-
tion should be adjusted before placing on the water bath, as we
have found that this changes very little during its stay there.
After sterihzation in the autoclave the medium remains clear if
soon cooled. If remelted for pouring plates it is clear at first,
but after standing some time in the water bath a second flocculent
precipitate forms, which however settles to the bottom so that
with careful handling the clear supernatant medium maybe
readily poured off.
No attempt has been made by us to review the literature deal-
ing with this subject, so that we do not know if this procedure
has been described before. We have never seen it mentioned
however. Any one who has had to make any considerable
amount of agar will readily understand the value of so simple a
method of obtaining a clear product. We prefer this method
even to clearing with egg albumin, since the result is a clearer
medium although it takes more time to prepare.
BOOK REVIEWS
A Text-book upon the Pathogenic Bacteria and Protozoa. For Stu-
dents of Medicine and Physicians. By Joseph McFarland, M. D.
Eighth edition, thoroughly revised. Octavo of 807 pages with 323
illustrations, a number of them in colors. Philadelphia: W. B.
Saunders Company. 1915. Cloth $4.00 net.
The author opens with an excellent historical introduction, tracing
the evolution of bacteriology through three stages — biologic contri-
butions, chemical contributions and the modern medical and surgical
contributions. The first part of the book, headed " General," is devoted
to a discussion of the biology of microorganisms, methods of studying
them, and infection and immunity. Chapter I — on Structure and
Classification of Microorganisms — includes bacteria, higher bacteria,
yeasts, molds and protozoa. No mention is made of the chemical com-
position of bacteria. The nature of the cell substance is passed over
in one sentence. The author does not take up the bioligoc significance
of spores, the conditions under which sporulation takes place, and con-
ditions under which germination occurs. The classification of bacteria
is treated very briefly, Migula's tables being given, while Jensen's sys-
tem does not appear. Only ten pages are given over to a discussion of
these subjects, about one-third of space allotted in other books.
Chapter II (pageo 50-65), on Biology of Microorganisms is too com-
pact and synoptic in its introductory discussion of biologica relations of
bacteria to temperature. Mere definitions of minimum, optimum and
maximum temperatures ove no significance without some discussion of
the thermal death points of various organisms, range of temperature at
which growth takes palace among different types, etc. Similarly more
than one page should be devoted to a discussion of the food supply of
bacteria, and the synthesis affected by nitrifying bacteria should be
included under such a topic
Chapters III and IV (pages 66-143) on Infection and Immunity are
clearly presented. The Lateral Chain Theory of Ehrlich is explained
by a lengthy quotation from his Croonian Lecture before the Royal
Society of London. A section in this chapter is devoted to Defensive
G^erments with a description of the Abderhalden Reaction and the
technique involved in making the test. Staining and observation of
protozoa, and bacterio-vaccines and their preparation are some of the
new topics treated.
Chapters XI, XII and XIII (pages 234-248) devoted to Bacteri-
ology of Water, Air and Foods are necessarily brief and incomplete.
549
550 BOOK REVIEWS
In Chapter XVII (pages 251-262) on Determination of the Value of
Antiseptics, Germicides and Disinfectants, a detailed description of the
technique of the Anderson-McClintic method is given. The important
theoretical work of Chick is not mentioned in this chapter.
Part II is devoted to a discussion of the infectious diseases and the
specific microorganisms — each disease being treated in a separate chap-
ter,— and this Part is very fully and well treated.
It is curious that the importance of septic sore throat as a new dis-
ease should not find a place in a discussion of streptococci. Cole's
work on the Pneumococci has not yet been included in the chapter
on Pneumonia.
Chapters XIX (pages 471-493) on Malaria and Chapter XX (pages
494-505) on Sleeping Sickness treat these subjects in much detail.
There is no mention of Texas Tick fever or Rocky Mountain Spotted
Fever. Such diseases as measles, scarlet fever, foot and mouth disease
and mumps should find a place in a chapter on filterable viruses, which
the book does not contain.
On the whole the text presents the subject very clearly, and the
illustrations are exceedingly good. The author in the preface says that
the book is a medical work intended for medical students and practi-
tioners; and its purely medical aspects are excellently handled. It is
unfortunate however, that medical students should not master the un-
delying fundamental biological principles of bacteriology in their wid-
est aspects for these are of the utmost importance in their application
to modern preventive medicine.
D. Greenberg.
The Principles of Pathologic Histology. By Frank B. Mallory, M.D.,
Associate Professor of Pathology, Harvard Medical School and Pa-
thologist to the Boston City Hospital. Octavo of 677 pages, with
497 figures containing 683 illustrations, 124 in colors. Philadelphia
and London: W. B. Saunders Company, 1914. Cloth $5.50 net.
This interesting and useful book on pathologic histology reflects
admirably the experience of its well-known author. It is confessedly
individualistic and contains practically no direct reference to the work
and opinions of others. The author is undoubtedly right in insisting
that the study of the lesions themselves is the only proper source of a
balanced judgment in reference to the terminal aspects of disease. It
seems a pity, however, that on controversial points he has not seen fit
to state both sides of the question and then give his own interpretation.
The book may be reviewed from the standpoint of pathology in the
larger sense, and again from the standpoint of the morphological aspects
of disease. From the latter viewpoint it is to be praised for the order-
liness of presentation and the remarkable clearness and beauty of the
numerous original illustrations. In the first part, dealing with Gen-
eral Pathologic Histology, the subjects of inflammation and the retro-
BOOK REVIEWS 551
grade processes are treated in a masterly and deservedly extended
fashion. The section on Tumors, comprising approximately one-third
of the book, is a remarkable monograph which presents a summary of the
author's own extensive material basis on which his mature and deserv-
edly respected opinion is based. The treatment of this phase, impor-
tant as it is from a diagnostic standpoint, seems disproportionate for
the needs of beginning students of disease processes however valuable
it may be for the specialist. The second part dealing with Special
Pathologic Histology is on the other hand somewhat short for general
needs, and lays unusual emphasis on those rarer lesions which have
happened to attract the author.
From the larger standpoint of pathology as a study of the natural
history of disease the book represents an unfortunately restricted atti-
tude. In studying disease we are of course interested in studying the
cause and progress as well as the result produced. In the infectious
diseases of known causation the entire course of the process can be fol-
lowed, particularly with the aid of animal experimentation. Much can
be learned concerning the progress of a disease if an approximately com-
plete set of lesions is gathered by the diligent collector at the autopsy
table, provided always that the successive stages be subsequently fitted
into their proper places. Much more rapidly and certainly can a
chronological series be prepared by injecting the specific microoorganism
into a suitable animal. To Mallory "recourse to animal experimen-
tation has often served to confuse a subject rather than to simplify and
clear it up." Experimentation unfortunately requires a certain type of
ingenuity and the power of inductive reasoning in addition to the de-
ductive reasoning required by the collector. The author has correctly
stated that from a study of lesions alone "we are in a position to read
the process (of disease) backward with some degree of certainty." It
is profitable that the life work of a group of individuals should center
in pathology as viewed from this essentially Chinese angle, but it is
inexcusable that the work of the growing majority who choose to follow
pathology forward rather than attempt to read it backward should not
at least be recognized.
This failure to recognize the essentially dynamic functional view-
point of disease has led Mallory into numerous errors in treating of the
infectious diseases. Bacterial causation he recognizes in so far as bac-
teria may be fixed and stained, but failure to utilize or even absorb the
experimental viewpoint has led to numerous unlikely hypotheses. Prac-
tically all bacteria are stated to act by the production of a "toxin" of
greater or less strength as judged by the response of fixed cells; anti-
toxins are asserted to be formed in typhoid fever and indeed they are
said to be formed in the so-called "endothelial leucocytes." It is need-
less to state that there is no experimental evidence for a statement of
this sort. The very name of "endothelial leucocytes," for the de-
scription of which Mallory is properly appreciated, is probably inexact
if we are to accept their origin from connective tissue as shown by
Evans in his work with vital stains. For his insistence on the objec-
tivity of lesions we have only to thank Mallory, but it is unfortunate
that he has not employed his imagination in experunental verification
of his working hypotheses.
F. P. Gay,
ABSTRACTS OF AMERICAN BACTERIOLOGICAL
LITERATURE
ANIMAL PATHOLOGY
Notes on the Histo-Pathology of the Intestines in Young Chicks Infected
with Bacterium pullorum. G. Edward Gage and James F. Martin.
(Jour. Med. Res., 1916, 34, 149-155.)
Typical strains of Bacterium -pullorum, when injected into young
chicks, produced death with characteristic symptoms of bacillary white
diarrhea in 49 per cent of the animals. Chicks which survived the in-
jection showed retarded growth and general weakness. The chief patho-
logic changes on section were marked injury to the mucosa, associated
with hyperemia, hemorrhagic exudation and leucocytic infiltration.
H. W. L.
The Diptheroid Bacillus of Preisd-Nocard from Equine, Bovine and
Ovine Abscesses. Ulcerative Lymphangitis and Caseous Lymphadeni-
tis. I. C. Hall and R. V. Stone. (Jour. Infect. Diseases, 1916, 18,
195-208.)
The authors call attention to the presence in the United States of
the bacilkis of Preisz-Nocard, which they isolated from horses and
from a calf. They point out the uncertain significance of experimental
orchitis in guinea-pigs as a test for glanders, and emphasize the neces-
sity of microscopic and cultural examination of pus for diagnostic pur-
poses.— P. B. H.
BACTERIOLOGY OF SOILS
Studies in Sulfojication. P. E. Brown and H. W. Johnson. (Soil
Science, 1916, 1, 339-362.)
It is suggested that applications of sulfur-containing commercial
fertihzers to soils will prove profitable unless different methods of soil
treatment are employed than those in use at present, because at some
future time sulfur will be lacking. In other words, for permanent soil
fertihty, the sulfur supply for crops must be considered. Chemical
tests were made for determining sulfofication produced in a sulfur-rich
soil to which various chemicals were added. — Z. N.
Preliminary Experiments on Some Effects of Leaching on the Soil Flora.
C. B. Lipman and L. W. Fowler. (Soil Science, 1916, 1, 291-297.)
It seems that leaching affects the bacterial flora of soils profoundly.
Nitrification, nitrogen-fixation and cellulose decomposition are wholly
553
554 ABSTRACTS
or almost wholly checked by this process, especially if salts are present
prior to leaching. Investigations are being carried on to determine if
the injury done is or is not an ephemeral one which may entirely disap-
pear in a few months under field conditions. This and other experi-
ments in progress may lead to the discovery of the cause or causes of
damage done to relatively new soils by irrigation flooding. — Z. N.
Quantitative Method for the Estimation of Bacteria in Soils. R. C. Cook.
(Soil Science, 1912, 1, 153-163.)
A synthetic medium is most desirable so that results may be compar-
able at different times and places, and for quantitative work the me-
dium which will permit the development of the maximum number of
colonies is usually most satisfactory.
Twelve media were at first compared on four soils, Lipman and
Brown's modified synthetic agar being taken as a basis for comparison,
the only difference being in the methods of sterilization. In the third
series of experiments seven media were eliminated and in the last two
series, Lipman and Brown's agar was eliminated. Results indicate
that sodium asparaginate agar, albumen agar, and urea ammonium
nitrate agar will in most cases give a greater colony development for
soil bacteria than other media in common use in bacteriological work.
A five-day count gives much higher bacterial counts than a three-day
count. — Z. N.
Stimulating Influence of Arsenic upon the Nitrogen-fixing Organisms of
the Soil. J. E. Greaves. (J. Agr. Res., 1916, 6, 389^16.)
The author has previously shown that arsenic stimulates the nitri-
fying and ammonifying powers of soil. The present investigation is
to show whether it has a similar effect upon nitrogen-fixation. Results
show that all forms of arsenic tested, except par is green, stimulate the
nitrogen-fixing power of soil, but not so greatly as the nitrifying power.
These results are obtained only when the tests are made in soil (either
untreated soil or sterilized soil reinoculated) ; for when solutions adapted
to nitrogen-fixation are inoculated with soil, the addition of arsenic
always proves toxic to the organisms concerned. One culture of Azoto-
bacter was obtained that was directly stimulated by the arsenic; but in
general, increase in nitrogen-fixation is observed only when a mixed
flora is used. If the soil infusion used for inoculating sterile soil is pre-
viously filtered or heated as high as 55°C., no stimulation is brought
about by the arsenic.
The author does not favor any one theory to account for these results;
but considers the suppression of harmful microorganisms to be at least
a partial explanation. He points out that the results are almost exactly
what might be expected on the assumption that these harmful organ-
isms were protozoa. — H. J. C,
ABSTRACTS 555
The Occurrence of Bacteria in Frozen Soil. E. C. Harder. (Bot.
Gaz., 1916, 61, 507-517, two graphs.)
Investigations were begun during the latter part of October and the
samples were examined every week until the latter part of February.
A dark, medium rich, slightly sandy garden soil was studied, obtained
from the University campus near the College of Agriculture at Madi-
son, Wisconsin. The soil was sampled to a depth of 6 inches each time.
The entire sample was thawed when necessary and thoroughly mixed
in a mortar previously washed out with 95 per cent alcohol. After
proper dilution, plates were poured and counted after an incubation
period of eight days at 28°C. Hey den Nahrstoff agar was used as a
culture medium.
In order to determine whether the high bacterial content of frozen
soil was due to an actual increase or whether other factors brought
about this phenomenon, two duplicate sets of potted soils were pre-
pared. One set was kept at room temperature and the other set was
placed outside, subject to atmospheric temperatures.
Summary of results. 1. It was found that the number of bacteria in
surface soil increased markedly after heavy frost, and in general main-
tained a high average during the winter months. The increase and
decrease, however, were found to bear a distinct relation to the mois-
ture content.
2. The potted soils failed to show such marked increase in the bac-
terial count after frosts. On the contrary the enriched cultures showed
a distinct retardation of bacterial growth when in a frozen condition.
3. The bacterial flora was much the same during the fall, winter and
spring, with the exception that after heavy frosts the small transparent
colonies characteristic of water and of deeper soils, formed a larger pro-
portion of the growth on the plates.
From these results it seems reasonable to conclude that ordinary soil
bacteria undoubtedly withstand cold to a marked degree, even tem-
peratures as low as 40°C. or more below zero. The increase in num-
bers, however, seems to be due to mechanical transpiration of moisture
coming up from below during heavy frost, and where such transpira-
tion is not possible there is an actual retardation in growth as compared
with that in unfrozen soils. — J. T. E.
Incubation Studies with Soil Fungi. S. A. Waksman. (Soil Science,
1916, 1, 275-285.)
Too little attention has been directed to the possible influence of
fungus forms as one of the important factors in soil fertility.
The longer and more complex life cycle of the fungi no doubt makes
their relation to the fertihty of the soil different at successive stages
of growth.
Bacteriological methods were pursued in the study of three organ-
isms which represent three very important groups of soil organisms:
Mucor plumbeus, Penicillium sp., and, Monilia sitophila. ^
At first the moisture relationship and incubation period were deter-
556 ABSTRACTS
mined for these three molds after 6, 12 and 18 days upon a culture
medium composed of gravelly loam plus dried blood and cotton-seed
meal as ammoniates. From results of this preliminary experiment at-
tempt was made to determine how the biological stage affected ammoni-
fication. The periods of most active ammonification correspond to those
of active spore formation for the respective organisms and the small-
est amount to the time preparatory to actual spore formation. The
Monilia shows the largest ammonia accumulation within the first 3 or
4 days; the Penicillium, between 10 and 15 days and the Mucor be-
tween 6 and 10 days.
An analogy is found in the growth of legumes like the clovers. Nitro-
gen is fixed during the period of active growth of the plant, the fixation
ceasing almost entirely when seed formation begins. — Z. N.
Diastase Activity and Invertase Activity of Bacteria. George P.
Koch. (Soil Science, 1916, 1, 179-196.)
This paper is limited to the study of diastases and invertases pro-
duced by bacteria which are concerned in the production of nitrogen
compounds for consumption by plants and in the decomposition of
carbohydrates in the soil.
The author determined that sufficient diastases and invertases were
secreted by bacteria so that they could be quantitatively determined
and also that there was considerable variation in the enzyme (diastase
and invertase) secretion by organisms developed in culture solutions of
different composition. The enzyme secretion by bacteria at different
periods varies from day to day under conditions otherwise the same
and there seems to be no direct correlation between hydrolytic enzyme
secretion by bacteria and their property of decomposing proteins.
The enzyme activity of various organisms and their ability to decom-
pose proteins vary greatly; there is also a variation in enzyme activity
of different cultures of the same species. No correlation was found be-
tween the secretion of enzyme and the decomposition of proteins by
bacteria, the property of the cultural solution to rotate the plane of
polarized light, the percentages of reducing compounds, the formation
of acid or the numbers of organisms.
The rotatory power of a solution may be increased as well as de-
creased by bacteria; they do not produce a surplus of reducing com-
pounds. Bacteria also seem to have the property of causing a condi-
tion which will prevent starch hydrolysis and sucrose inversion. There
may be a possible correlation between the protein decomposition de-
termined as ammonia and the formation of acid. — Z. N.
Bacterial Numbers in Soils at Different Depths, and in Different Seasons
of the Year, Selman A. Waksman. (Soil Science, 1916, 1, 363-380.)
Four soils were studied. Meadow soil gave the largest bacterial
counts at a depth of 1 inch, the 1-inch layer of this soil being richer
also in organic matter and nitrogen content than that of the garden
and orchard soils. The forest soil, though showing a high carbon and
ABSTRACTS 557
nitrogen content gave the lowest bacteria counts probably because of
the high acidity and large amount of undecomposed matter. The num-
bers of bacteria in the soils studied were not governed either by the
moisture content of the different soils, or by the nitrogen and carbon
contents.
The greatest number of bacteria were found at a depth of 1 inch in
the soils that are under shade the year around. The garden soil gave
on the average the largest numbers 4 inches from the surface. The
numbers of organisms decreased regularly from a depth of 1 inch down
to a depth of 30 inches.
Three soils showed a regular decrease in the lime requirement down
to 30 inches and in all soils this was accompanied with a more or less
gradual decrease in the nitrogen and carbon content.
Frozen soil, though showing a high bacterial content, did not give
the largest bacterial numbers found through the year. — Z. N.
The Inoculation and Incubation of Soil Fungi. Nicholas Kopeloff-
(Soil Science, 1916, 1, 381-403.)
The fungi employed in these studies were isolated from soil on the
College Farm. They were identified as Rhizopus oryzae (Wendt),
Zygorrhyncus vuilleminii (Namyslowski) , Rhizopus nigricans (Ehren-
berg), Penicillium sp. 10. Spores of these fungi were obtained for use
by growing them on Cook's No. II fungi medium. Suspensions of a
known number of spores of each fungus were added to 100 gm. portions
of soil of an optimum moisture content to which dried blood and cotton-
seed meal were also added. Ammonia determinations were made at
the end of 7 days.
An increase in the number of fungus spores inoculated into the soil
was found to be related to a proportional increase in ammonia accumu-
lation but this increase was not proportional beyond a certain limit.
Under the conditions of moisture and temperature employed, cot-
ton-seed meal appears to be the more acceptable source of food for the
organisms studied. A 7-day incubation period may be recommended as
most desirable for the study of soil fungi other than those belonging to
the Penicillium group.
A striking increase in ammonia production was observed to take
place every other day (after the first five days). This leads to the con-
clusion that the production of ammonia is dependent on the meta-
bolic processes of the fungus rather than the biological stage of spore
production and germination — Z. N.
Studies on the Decomposition of Cellulose in Soils. I. G. McBeth.
(Soil Science, 1916, 1, 437-486.)
Detailed directions are given for the preparation of cellulose for cel-
lulose agar. The action of cellulose-dissolving organisms was studied
not only on typical cellulose such as is found in filter paper or in cotton
fiber but on the cellulose of plant tissues freed from encrusting sub-
stances. The method for preparation of this plant cellulose is given
in detail.
558 ABSTRACTS
The cellulose-dissolving bacteria isolated from soils by means of the
cellulose agar plate method, have the power of dissolving the cellulose
of alfalfa. Twenty-five species of cellulose-dissolving bacteria dis-
solved alfalfa cellulose as readily as that prepared from filter paper.
Thirty-six species of this type of bacteria have been isolated and
their cultural and morphological characteristics are given in detail.
Fifteen new species are included.
A provisional key for identifying and comparing species of bacteria
which dissolve cellulose is appended.
Many of the data called for by the card of the Society of American
Bacteriologists seem to have little significance in the separation of mem-
bers of this group.
Filamentous fungi and actinomyces also unquestionably play an im-
portant role in the destruction of cellulose in nature. — Z. N.
Actinomyces of the Soil. Selman A. Waksman and Roland E. Curtis.
(Soil Science, 1916, 1, 99-135, 3 plates.)
Seven different types of soils from different localities and under dif-
fering chmatic and cultural conditions were used. Brown's albumen
agar, slightly modified, was used for isolation of the actinomyces from
the soil and for counts. Each organism isolated was studied on Cza-
peck's solution, agar, potato, and on 15 per cent gelatin in distilled water.
All the actinomyces studied liquefy gelatin and they may be divided
into two groups depending on their differences in color production, one
browning the liquefied portion, the other remaining colorless. A char-
acteristic of some species is the production of an aerial mycelium on
gelatin.
The numbers of actinomyces decrease with soil depth, but their num-
bers, relative to those of bacteria and fungi, increase.
Thirty species of actinomyces are classified, and their morphology
and cultural characteristics on the above media are considered in detail.
A key is included facilitating their identification.
The average optimum, maximum and minimum temperatures are
30°C., 50°C. and 15°C. respectively. The actinomyces do not play any
appreciable role in the soil as ammonifiers; they readily assimilate NO2,
NO3, NH3 and organic compounds of nitrogen and characteristically
reduce nitrate to nitrite, but not to free nitrogen or NH3.
Actinomyces are strong cellulose decomposers and this fact in com-
bination with their weak ammonia production leads the author to think
that the probable role of the organism in the fertility of the soil lies in
the formation of humus. Therefore, in arid soils where cellulose destruc-
tion has been found to be extremely rapid, actinomyces should be ex-
pected in abundance. — Z. N.
Some Factors that Influence Nitrate Formation in Acid Soils. E. B.
Fred and E. J. Graul. (Soil Science, 1916, 1, 317-338, 1 ph)
Acid soils do not possess a strain of nitrifying bacteria especially re-
sistant to soil acidity. The nature of the compound to be nitrified
ABSTRACTS 559
plays an important part. For example, in acid soils organic nitrogen
nitrifies much more rapidly than nitrogen from ammonium sulphate;
the reverse is true in non-acid soils.
In soil treated with calcium carbonate there is an enormous multi-
plication of the nitrate bacteria. At first, one or two weeks after treat-
ing, calcium carbonate stimulates nitrate formation, later the reverse
is true. In the presence of organic nitrogenous substances as casein
and gelatin, calcium carbonate did not permanently increase the accumu-
lation of nitrates.
All soils stored under conditions that prevent leaching showed a gain
in nitrate nitrogen.
Considering the data given, as a whole, it seems that under laboratory
conditions the beneficial effect of CaCOa on plant growth must be ac-
counted for by some processes other than the direct effect on nitrifica-
tion as this effect of CaCOs on nitrification takes place before higher
plants begin to draw heavily on the nitrogen of nitrates. Moreover,
the period of rapid accumulation from liming may result in a loss of ni-
trogen from leaching of the nitrates. These questions can be deter-
mined only by field tests. — Z. N.
The Influence of Some Common Humus-Forming Materials of Narrow
and of Wide Niti-og en-Carbon Ratio on Bacterial Activities. P. E.
Brown and F. E. Allison. (Soil Science, 1916, 1, 49-75.)
Ammonification, nitrification and azofication were found to be in-
creased to a considerable extent by application of the common humus-
forming materials (dry) in maximum amounts for farm conditions;
horse-, cow- and rotted manures, oat straw, timothy-, cowpea- and
clover hays and corn stover. In general the manures favored and the
legume hays depressed ammonification. Increases in ammonification
were independent of the N-C ratio of the materials added, and were
probably dependent on the chemical composition of the substances.
If the humus-forming materials especially the manures, had been ap-
plied undried as under field conditions, ammonification would have
been accentuated.
In contrast, nitrification was increased by leguminous green manures,
and retarded somewhat by animal manures. These increases were ap-
parently independent of the N-C ratio in the substances.
Azofication was favored by manure, straw stover and non-leguminous
hays, and leguminous hays in order, the N-C ratio of these materials
being of little or no significance as indicated by their effects on
azofication.
Indications were, however, that non-legumes and straws might in-
crease azofication on soils to a large enough extent to make their use
more profitable than that of legumes which add nitrogen to the soil, but
are somewhat more expensive to use.
The substances with wide N-C ratio decreased the crop yield while
those of narrow ratios gave increases. The N-C ratio was found to be
of more importance in determining the effect on the second crop of oats
than on the first crop.
560 ABSTRACTS
To increase azofication, non-leguminous manures must remain longer
in the soil than leguminous in order to give time for considerable de-
composition to occur before a crop is grown to test the effects. — Z. N.
Can Soil Be Sterilized without Radical Alteration? David A, Coleman,
H. Clay Lint, and Nicholas Kopeloff. (Soil Science, 1916, 1, 259-
274.)
An effort was made to devise some method whereby soil might be
rendered sterile with a minimum amount of alteration. Four different
lines of experimentation were carried on: 1. The intermittent steri-
lization of soil by dry heat; 2. Various chemical substances used as soil
antiseptics; 3. Volatile antiseptics apphed in partial vacuum; and 4.
Volatile antiseptics applied under pressure at 80°C.
Both moist and dry soils were sterilized at 82°C. for 1 hour on 1 to 5
successive days. Ammonia determinations were made after each heat-
ing, also bacterial counts on Lipman and Brown's synthetic agar. All
protozoa were killed at the first heating; species of PenicilUum and
Mucor persisted throughout. The numbers of bacteria in the moist
soil decreased from 47,750,000 per gram on the first day to 1,500 on the
last day while although there was an initial depression in numbers of
bacteria in the dry soil, an increase occurred on the first day, then a
gradual decrease only. This method of soil sterilization is decidedly
more efficacious in the sterilization of moist than of air-dry soil. It in-
creases the total solids in the soil about 46 per cent, which is only one
sixteenth as much as by the common method of steam sterilization.
Where the time element is of considerable importance this method is
undesirable.
As soil antiseptics, were used 1 per cent (on the basis of 100 gm. of
air-dry soil) of ethyl alcohol, ethyl ether, toluene, carbon bisulfid, chloro-
form and hydrogen peroxid. Chloroform caused a decrease of 86 per
cent of the original bacterial content and caused the least alteration in
the chemical constitution of the soil; carbon bisulfid and toluene were
next in order.
In addition to the first four volatile antiseptics named above, osmic
acid was used in the third method of sterilization. The antiseptic
vapor was allowed to remain in intimate contact with the soil for 1^
hours for 3 successive days. Carbon bisulfid, toluene and ethyl alco-
hol, in order, caused a decrease of 99+ per cent of the original soil
flora, although their action is more efficient in air-dry soil.
Carbon tetrachloride, carbon bisulfid, ethyl ether and chloroform
were used in the heat plus pressure method. They were used at a tem-
perature of 80°C. for 3 successive days upon moist and air-dry soil.
The decrease in bacterial flora, in general, approximated 98 per cent.
Carbon bisulfid was the only chemical which proved superior to the
check treatment; it developed a pressure of 20 pounds. Ethyl ether
which was least efficient developed only 6 pounds. A possible corre-
lation might be obtained between the pressure developed during treat-
ment and the effectiveness of the sterilizing agent. Carbon tetrachlor-
ide, which developed 5 pounds pressure is an exception. — Z. N.
ABSTRACTS 561
Studies on Soil Protozoa. Selman A. Waksman. (Soil Science, 1916,
1, 135-153.)
The author found that no flagellates could be found in a living condi-
tion before sixteen minutes and no ciliates until after sixty-two minutes
in a soil which was covered with water during these periods. The
longer time allows a closer observation among the soil particles and a
more thorough examination of the field. There is no doubt that there
is a minimum moisture content for each soil below which protozoa can-
not be found in a living condition.
Four soils were selected which varied in humus content and were
under crops or orchard. Examinations were made every day for ten
days in succession during which period several rains occurred. The
moisture content proved to be a limiting factor but not the only one as
the structure and humus content of the soil hkewise play an important
part. The protozoa found were all flagellates.
Another experiment with three different soils under different mois-
ture conditions, part sterilized and part unsterilized, the latter plus dried
blood, to each of which cultures of protozoa were added, brings out the
fact that the sterilization of soil and addition of easily soluble organic
matter will make the conditions optimum for protozoan activities at a
lower moisture content than in corresponding unsterilized or untreated
soils.
Protozoa (flagellates) found in the first inch of three cultivated soils
varied in numbers from 1000 to 10,000 per gram of soil; at four inches,
from 100 to 5000; at eight inches from 100 to 5000; at twelve inches
from 10 to 100. None were found at a depth of twenty or thirty inches.
The fourth soil, an acid forest soil, contained from 10 to 100 flagellates
at a depth of one inch and at four inches; at eight inches only 1 to 10
were found and below that none. Ciliates and amoebae were not re-
corded as they occurred only occasionally.
Flagellates are present in greatest numbers at a depth of 1 inch where
conditions favor microorganic activities, ciliates and large flagellates in
largest numbers at a depth of 4 inches where the moisture conditions
may be more favorable for their development. A hst of the common
types is given.
The flagellates are at once the most common and the largest group of
soil protozoa.
Ammonification by bacteria is not influenced appreciably by soil
protozoa although their presence acts detrimentally upon bacterial
numbers.
This might be explained by one of the following assumptions: (1) if
the protozoa destroy bacteria, they destroy non-ammonifying organ-
isms ; 2) the protozoa themselves take part in the process of ammonifica-
tion; (3) the disintegration of the bacterial cells results in decomposi-
tion products which might be responsible for high ammonia produc-
tion.—Z. N.
562 ABSTEACTS
A Detailed Study of Effects of Climate on Important Properties of Soils.
C. B. LiPMAN and D. D. Waynick. (Soil Science, 1916, 1, 5^8, 5
plates.)
A soil block, 5 feet square and 3 feet deep from the fields of each of
three state experiment stations (Maryland, Kansas and California) was
moved to the two other experiment stations and placed in position as
nearly as possible in the original order of layers. A similar block of soil
was dug up and replaced in its position at every station. Strips 5 feet
wide of untouched field soil surrounded the board frame of every plot
which was placed in position. It was then possible to study at Mary-
land, at Kansas and at California four soil blocks as follows: First, nat-
ural field soil undisturbed; second, natural field soil disturbed and re-
placed; third and fourth, soil blocks obtained respectively from each of
the other two stations. The chief aim was to show how any given soil
in its natural location compares, after seven years, with the same soil
under foreign conditions. Unfortunately no studies except brief chemi-
cal studies, were made at the initiation of the soil exchange experiment.
Studies of the hygroscopic coefficient, the moisture equivalent, the
wilting point, changes in color and colloidal nature were among the
physical studies made. Among chemical studies, complete chemical
analyses were made in accordance with the official method, also humus
and humus nitrogen determinations, total nitrogen and soil water-ex-
tract studies. Among bacteriological studies were: Counts on albu-
men agar, ammonifying power for dried blood, nitrifying power for the
soil's own nitrogen, for dried blood, for cotton-seed meal, and for sul-
fate of ammonia, nitrogen fixing power in mannit solution, and qual-
itative tests for cellulose destruction.
The soils were described as follows: The California soil as "Sacra-
mento silt loam," the Kansas soil as a "dark heavy loam," and the Mary-
land soil as a "light yellow clay."
It was found that soils change markedly in color in a period of seven
years, and perhaps less, when moved to other climates. The differ-
ences are so great that samples of any one original soil from the three
different stations today show no outward resemblance among them-
selves, but appear to represent three very distinct soil types.
In general the hygroscopic coefficient, the moisture equivalent, and
the wilting point of any of the soils increased when the soil was placed
at California. Some exceptions to this rule are noted.
Generally speaking bacterial numbers increase in arid soils placed
under humid conditions. In general, also, the opposite is true for
humid soils.
Ammonification, nitrification and nitrogen fixation follow the general
trend of bacterial counts. In the case of nitrification, however, this
applies to certain forms of nitrogen only. In the case of other forms
of nitrogen very pecuhar conditions exist which are fully explained in
the text.
Cellulose destruction by soils proceeds with greater rapidity under
arid than under humid conditions with any given soil type. Cellulose
ABSTRACTS 563
destruction therefore appears to follow in general an opposite course
to those of other microorganic activities in soils as affected by climate.
Marked changes in the acid soluble constituents of soils are wrought
by chmatic effects. The general tendency is for soils to increase in
iron and decrease in alumina when placed under arid conditions, and
vice versa.
Phenonienal losses in certain constituents in five years seem to have
occurred in some soils even when the latter were not moved. Thus
for example the Maryland soil lost in the period named enormous
quantities of magnesia.
Large increases occur in the total water soluble constituents of Cal-
ifornia soil when it is moved to the Kansas or Maryland stations. On
the other hand, the Maryland soil gains in water soluble matter when
moved to Kansas or to California. — Z. N.
BACTERIOLOGY OF THE MOUTH
The Treatment of Pyorrhea Alveolaris and its Secondary Systemic In-
fections by Deep Muscular Injections of Mercruy. B. D. Wright.
(Medical Record, 1916, 89, 807-811.)
Seventy-five cases of pyorrhea were treated with mercuric succin-
imide administered intramuscularly. In conjunction with the injec-
tions of mercury, surgical treatment of the gums and teeth was carried
out.
In all cases, the pyorrhea was entirely cured. Secondary systemic
infections, which were present in 54 per cent of the patients, were
also cured in every case.
The author is convinced that mercury, because of its parasitotropic
action, promises to be successful in treating various infections pro-
duced by the vegetable parasites. — M. W. C.
Constitutional Conditions Caused by Oral Sepsis. J. Daland. (New
York Med. Jour., 1916, 103, 817-820.)
Foci of infection in the mouth may give rise to serious constitutional
disturbances, such as endocarditis, acute parenchymatous nephritis,
furunculosis, and particularly septic polyarthritis. Many other patho-
logic conditions may be traced to a septic mouth as the cause. The
offending organism, in such cases, is usually a streptococcus. This
bacterium, according to the work of Rosenow, may develop greater
virulence than it possesses as it exists in the mouth, if it is transferred
to other parts of the body where different conditions for growth are
present.
As a direct causal relationship exists between oral sepsis and consti-
tutional disease, the dentist should take particular pains to remove
all manifestations of oral sepsis, and it should become a matter of rou-
tine procedure that any septic conditions of a patient's mouth should
be reported to the physician.— M. W. C.
564 ABSTRACTS
A Study of Endameba Buccalis in Alveolodental Pyorrhoea. F. M.
Johns. (Am. Jour. Trop. Dis. and Prevent. Med., 1916, 7, 372-
376.)
The first part of this paper deals with a microscopic study of the
lesion — demonstrating the presence of the Endameba buccalis in great-
est numbers penetrating the healthy peridental tissue beyond the super-
ficial zone of suppuration, and not living in the mass of bacteria, pus
and semi-necrotic material filling the pocket.
Phagocytosis of bacteria is only noted in the few atypical forms that
are swept into the pus of the pocket. From the depths of the lesion
only a large nucleated cell is found to be constantly ingested.
Reproduction in the lesion is apparently only by bilateral fission.
Cyst formation was not observed.
A significant fact was in the adhesion of numbers of bacteria to the
ameba when observed in fresh and wet fixed preparations.
Endamebae following the injection of emetin are gradually decreased
in numbers without any visible morphological or biological changes
being produced. Forms in active reproduction are found up to the
complete disappearance, the inference being drawn that the action of
emetin upon pathogenic amebae is an indirect one in the dosage chn-
ically possible. — F. M. J.
BACTERIOLOGY OF WATER AND SEWAGE
Bacterial Counts in Water Examination. J. Race. (Amer. Jour, of
Pub. Health., 1916, 6, 488-496.)
The conclusions as given by the author are:
1. Not one of the media used at any one of the incubation periods
or temperatures chosen gives a bacterial count that bears a constant
ratio to the organisms of excremental origin as estimated by the B.
coll test.
2. The blood heat count is the nearest to the B. coli content.
3. The largest count is obtained by the use of nutrient gelatin.
4. The bacterial count varies directly with the incubation period and
inversely with the incubation temperature.
5. The largest count in the shortest incubation period is obtained
at 27°C.
6. The ratio of the count at a given temperature to the count at a
higher temperature decreases as the temperature of the water increases.
D. G.
Bacteria in Commercial Bottled Waters. Maud Mason Obst. (Bul-
letin No. 369, U. S. Dept. of Agr.)
The paper is based on results obtained from the examinations in the
Bureau of Chemistry during the last six years of from 1 to 17 samples
of bottled waters from each of 110 American springs and from 57 sources
in foreign countries.
ABSTRACTS 565
The author concludes that the data as summarized show the need
of improvement in the bacteriological condition of many of the brands
of bottled water to be found in the market. There are some springs
used for the production of commercial bottled waters which should not
be used. In other cases, the contaminations found are clearly those of
manipulation.
The results clearly show that bottled water can be made to conform
to the requirements of the United States Public Health Service for
drinking water furnished upon trains; that is, that not more than one
10 cc, sample out of five should show the presence of B. coli. — S. H. A.
Confirmatory Tests for B. coli in Routine Water Examinations. W. H.
Frost. (Amer. Jour, of Pub. Health, 1916, 6, 585-588.)
If no typical colonies develop within 24 hours on Endo plates made
from fermentation tubes showing gas, further effort is made to recover
B. coli as follows: (1) One or more colonies are transferred to lactose
broth fermentation tubes. The formation of gas demonstrates pres-
ence of B. coli. (2) Plates are again made from the original fermenta-
tion tube. (3) A transplant is made from the original fermentation
tube directly to another lactose broth tube. If steps (1) and (2) have
both failed to recover B. coli, plates are are now made from this trans-
planted culture. (4) At the same time a transfer is made from this
tube directly to a third fermentation tube.
If all of the above procedures fail to recover B. coli and gas is still
formed in this fermentation tube, the inference is that gas in the pre-
liminarj'- test was due to an anaerobe.
The presumptive test for B. coli whether in lactose bile or lactose
broth is subject to considerable variations in its specificity as indicat-
ing the demonstrable presence of B. coli. Careful confirmation of the
presumptive test is especially important in the examination of treated
waters, where the error in this test is greater than in raw waters. — D. G.
CLASSIFICATION OF BACTERIA
Studies on the Classification of the Colon-Typhoid Group. J. B. Thomas
and E, A. Sandman. (Amer. Jour, of Pub. Health, 1916, 6, 579-
584.)
The conclusions as given by the authors are: Glycerine is of value in
separating B. cloacae from B. communior and B. aerogenes, but is fer-
mented by both of the latter types, while B. communis isolated from
water frequently fails to ferment it.
Dulcite and adonite are of httle value in separating B. cloacae from
B. aerogenes and B. communior while a majority of B. communis iso-
lated from water fail to ferment these substances.
Sahcin is frequently fermented by all the lactose-positive organisms.
Raflanose is frequently fermented and dextrin usually fermented by
all of the lactose-positive organisms. — D. G.
566 ABSTRACTS
A Study on the Grouping of Meningococcus Strains. Miriam P. Olm-
STEAD, Phoebe L. DuBois, Josephine B. Neal, and Rose Schweit-
zer. (Journal of Immunology, 1916, 1, 307.)
Twenty-nine strains of meningococci were studied as to their im-
munity reactions by the complement fixation method. Immune sera
were prepared in rabbits with each of these strains, and cross fixations
were carried out with extracts of each strain of cocci. Fourteen strains
gave cross fixation and fell into one group. Eight other strains gave
cross fixation and so fell into a second group. Three strains gave cross
fixation with certain members of group 1 but not with all, and two
strains were entirely heterogeneous. The organisms of the second group
gave cross fixation with one strain of Dopter's parameningococcus but
not with a second representative of his organisms. — J. G. H.
The Correlation of the Voges-Proskauer and Methyl Red Reaction in the
CoU-Aerogenes Group of Bacteria. Max Levine. (Jour. Infect.
Diseases, 1916, 18, 358-367.)
The author studied the reactions of 167 colon-like organisms from
the horse, cow, pig, sheep, man, raw sewage and septic sewage. It was
shown that the two reactions studied were correlated; further that (1)
there was a better correlation between saccharose fermentation and
source than between saccharose-dulcite fermentation and source; (2)
cultures which formed acetyl-methyl-carbinol from glucose usually fer-
mented salicin and glycerin, but dulcite only occasionally; (3) organisms
that give the Voges-Proskauer reaction are rarely found in feces; (4) in
this test the glucose-peptone-dipotassium phosphate medium of Clark
and Lubs gives a more distinct test than glucose broth. Since this re-
action is characteristic for non-fecal strains, the author concludes that
it may be of considerable sanitary significance. — P. B. H.
DAIRY BACTERIOLOGY
Pasteurization as a Public Health Measure. C. M. Hilliard. (Jour.
Home Economics, 1916, 8, 307-312.)
The article gives a comprehensive statement of the principles and
methods of milk pasteurization, and discusses the relation of the prac-
tice to the public health, especially the health of infants. Bacterial
reductions obtained, both quantitative and qualitative, are consid-
ered.—C. M. H.
Bacterial Testing Versus Dairy Inspection. C. E. North. (Amer.
Jour, of Pub. Health, 1916, 6, 569-578.)
The author discusses the value of intelligent cooperation of labora-
tory worker and inspector. The function of the laboratory is to exam-
ine results and to locate insanitary milk. The inspector's function is
to ascertain the causes and to apply the remedies. Simple laboratory
equipment and methods within the reach of the small village are dis-
cussed.— D. G.
ABSTRACTS 567
Some Observations on Causes of High Bacterial Counts in Milk. H. D.
Pease. (Amer. Jour, of Pub. Health, 1916, 6, 563-568.)
In parts of the country where adequate field and laboratory inves-
tigations of milk suppHes have been in operation high bacterial counts
are most generally caused through inefficiently cleaned apparatus, or
as a result of inefficient refrigeration. In locations where no super-
vision has been in operation and where the producers are more or less
slovenly, high bacterial counts may be attributed to definite dirty con-
ditions and are found even where the milk can be shown to have been
delivered promptly and with the application of a reasonable degree of
refrigeration. — D. G.
Colo7i Bacteria and Streptococci and Their Significance in Milk. L. A.
Rogers, W. M. Clark, and A. C. Evans. (Amer. Jour. PubHc
Health, 1916, 6, 374-380.)
The authors find that cultures (streptococci) isolated from infected
udders are characterized by weak fermentation of the simpler sugars
and an inability to ferment higher polysaccharides and alcohols, while
cultures from bovine feces may be distinguished by ability to ferment
polysaccharides and inability to attack alcohols. They believe that
the presence in lactose bile inoculated with milk and incubated at
37°C. of streptococci forming distinct chains is good presumptive evi-
dence of milk from infected udders.
Bacteria of the colon group occurring in market milk may be di-
vided into two very distinct groups (1) that which agrees closely
with the characteristic colon bacillus of the bovine intestine, yielding
a low CO2 : H2 ratio ; (2) that which yields a high ratio ; numerous in
milk, but occurring very rarely in bovine intestine (1 out of 150 cultures).
The surface of dried grains is found to be source of a number of types
of colon. That type occurring with greatest frequency probably cor-
responds to conception of B. laciis aerogenes, so that the ordinary pre-
sumptive tests and even the usual confirmatory tests are not necessarily
proof of the contamination of the milk with fecal matter. — D. G.
DISINFECTION
The Antiseptic Action of Ether in Peritoneal Infections. J. Saliba.
(Jour. A. M. A., 1916, 66, 1295-1297.)
The instillation of ether into the peritoneal cavity in infections by
streptococci and B. coli served as a safe and beneficial antiseptic. — G.
H. S.
The Bactericidal and Fungicidal Action of Copper Salts. L. M. DeWitt
and Hope Sherman. (Jour. Infect. Diseases, 1916, 18, 368-382.)
The present study was initiated to ascertain (1) the killing power of
the sulphate and chloride of copper and (2) their inhibiting action, on
B. coli, B. typhi, B. prodigiosus, B. tuberculosis and Staph, aureus;
also on species of yeast, Aspergillus and Penicillium. The results indi-
568 ABSTRACTS
cated that copper is unreliable as a bactericide and as a fungicide, al-
though some organisms are more susceptible than others. Long time
experiments were more satisfactory than short time tests. It is said
that solutions representing one part of the metal (2.5 parts of the
chloride or 4 parts of the sulfate) to the million kill the ordinary water
organisms B. coli and B. typhi, but do not injure the health. One
part to 100,000 was found to inhibit the growth of B. tuberculosis in
vitro. The therapeutic value of copper salts is discussed. — P. G. H.
IMMUNOLOGY
Immunity, Natural and Acquired. W. H. Porter. (Medical Record,
1916, 89, 983-987.)
A theory of immunity which bases the formation and activities of
antibodies upon the metabolic processes of the body proteins. — M. W.
C.
The Production of a Hyperimmune Serum for Infectious Abortion in
Mares. E. S. Good and Wallace V. Smith. (Jour. Infectious
Diseases, 1916, 18, 347-401.)
The authors report on the production of a serum which protected
rabbits from the lethal dose, but did not protect a mare from artificial
infection. In a guinea pig the time for abortion was lengthened. — P.
B. H.
Complement Fixation in Vaccinia and Variola. John A. Kolmer.
(Journal of Immunology, 1916, 1, 59.)
The sera of vaccinated rabbits and of a few recently vaccinated hu-
man beings gave complement fixation with salt solution extracts of
variola virus and vaccine virus. Positive reactions were also obtained
in 9 out of 17 smallpox patients. Alcoholic extracts of the virus could
not be used as antigens as they gave no reactions with these sera. — J.
G. H.
Complement Fixation in Varicella. John A. Kolmer. (Journal of
Immunology, 1916, 1, 51.)
Kolmer obtained weak complement fixations in 7 out of 24 cases of
chicken pox, the antigen used being a saline solution of the contents of
the varicella vesicles. — J. G. H.
The Agglutinability of Blood and Agar Strains of Typhoid Bacilli. C.
G. Bull and I. W. Pritchett. (Jour. Exp. Med., 1916, 24, 35-
40.)
Cultivation on 10 per cent rabbit blood agar did not affect the agglu-
tinability of fifty-seven strains of typhoid bacilli. The authors were
unable to confirm the observations of Gay and Claypole on the varia-
tion in agglutinability caused by cultivating the typhoid bacillus on
blood agar. A typhoid bacillus showing irregularity in fermentation,
agglutination, and indol production is described. — B. W.
ABSTRACTS 569
A Simplijied Method of Producing a Potent Precipitin Serum. Wallace
V. Smith. (Jour. Med. Res., 1916, 34. 169-175.)
The author finds that the serum proteins precipitated from blood
by means of one-third saturation with ammonium sulphate consti-
tute a potent antigen for the preparation of precipitin serum in rab-
bits. The dried precipitate can be kept on hand over a long period
without impairment. — H. W. L.
The Agglutination Reaction with Sera Derived from Human Cases of
Leprosy and from the Experimental Animal upon Various Members of
the Acid-Fast Group. W. H. Harris and J. A. Lanford. (Jour.
Med. Res., 1916, 34, 157-167.)
Sera from 20 human cases of leprosy tested against several strains
of B. leprae, human, bovine, and avian tubercle baciUi, and several of
the non-pathogenic acid-fasts, failed to show any regularity or speci-
ficity. The same was true of experimental sera produced by injections
of the various strains of leprosy bacilU into rabbits. — H. W. L.
Observations on the Typhoid Reaction. C. R. Austrian and A. L.
Bloomfield. (Archives of Internal Medicine, 1916, 17, 663-669.)
By the typhoidin reaction individuals who had suffered from typhoid
fever or received prophylactic treatment could not be differentiated
from those who had never had the disease nor received vaccine treat-
ment. The reaction was positive in a large percentage of persons who
gave no history of typhoid fever or artificial immunization. — G. H. R.
Erysipelas Migrans and Multiple Abscesses in a Six Months' Old Infant
Successfully Treat^'.d with Vaccines. L. Fischer. (Medical Record,
1916, 89, 734-735.)
Report of a case, which showed no response to treatment with leuco-
descent light, ichthyol, evaporating lotions, or magnesium sulphate, but
in which decided improvement and finally complete recovery followed
the administration of stock streptococcus and autogenous vaccines. —
M. W. C.
Vaccine Treatment. Ludvig Hektoen. (Jour, A, M. A., 1916, 66,
1591-1594.)
A discussion of the theory and practise of vaccine therapy. Follow-
ing accurate diagnostic procedure an autogenous vaccine would seem
the logical method of treatment. Stock vaccines and phylacogens,
which are not standardized and may not be specific, have a limited
application. — G. H. S.
Is the Hyperleucocytosis Following the Injection of Typhoid Bacilli into
Immunized Rabbits Specific^ Helen I. McWilliams. (Journal of
Immunology, 1916, 1, 159.)
The experiments of Gay and Claypole showing hyperleucocytosis in
immune rabbits following the injection of typhoid bacilli, were reviewed,
570 ABSTRACTS
with the special object of determining whether this reaction was spe-
cific. The leucocytosis which McWilhams obtained in immune rab-
bits was no greater than that which normal rabbits showed and the
typhoid immune rabbits reacted as strongly to colon as to typhoid
bacilh.— J. G. H.
Specific Therapy in Certain Acute Infectious Diseases. F. J. Dever.
(New York Med. Jour., 1916, 103, 972-975.)
Treatment of infectious diseases by their specific immune sera holds
more promise of success than an active immunization with either plain
or sensitised vaccines. This is due to the fact that the active immunity
obtained by vaccines is produced relatively slowly and is of questionable
value in an acute infection, while a specific serum, on the other hand,
supphes antibodies without putting any added strain upon the body
cells.— M. W. C.
Intravenous Serohacterin Therapeutics. W. E. Robertson. (New York
Med. Journ., 1916, 103, 777-780.)
In acute infectious diseases, such as typhoid fever and pneumonia
serobacterins, when administered intravenously, shorten the incuba-
tion period, and produce a particularly striking change in the blood
picture, the most noticeable feature of which is a pronounced leucocyto-
sis.
In local and chronic diseases the value of the intravenous use of
serobacterins, as compared with other bacterins, has not as yet been
tested.— M. W. C.
The Value of Autoserum Injections in Skin Diseases. W. S. Gottheil.
(New York Med. Jour., 1916, 103, 1209-1211.)
Autoserum treatment in skin diseases does not itself effect a cure, but
used in conjunction with local treatment it shortens the period of dis-
ease from weeks to days and lessens the probability of relapse.
Autoserum treatment is particularly useful in psoriasis. It is of fre-
quent benefit in chronic urticaria, neurodermatitis, pruritus senilis and
other itchy dermatoses; of some value in pustular acne and chronic
eczema; and of no use in furunculosis, folliculitis, and other pus infec-
tions as well as pemphigus, lepra, lichen planus and syphiHs. — M. W. C.
Autosensitized Vaccines. M. G. Wohl. (Medical Record, 1916, 89,
770-772.)
Serobacterins which are sensitised with the patient's serum are more
efficient than vaccines sensitised with a heterologous serum. This is
due to the fact that the antibodies in the sera of the lower animals are
not identical with those of human serum, and therefore cannot act as
efi'ectively as those found in the patient's serum.
An additional reason why autosensitised vaccines are superior is
that the bacteria do not need to be freed from the immune serum by
washing, but may be injected with the serum used in sensitising. — M.
W. C.
ABSTRACTS 571
Gonococcus-Complement Fixation: A New Lipoid Antigen. C. C. War-
den and L. E. Schmidt. (Jour, of Lab. and Clin. Med., 1916, 1,
333-347.)
In complement fixation tests for gonorrhea, an antigen composed of
an alcoholic solution of the fats of the gonococcus gave a much higher
percent of positive reactions than a commercial watery antigen (Parke,
Davis and Company). With the Warden antigen the positive reac-
tions appeared earlier, persisted longer, and occurred in a larger number
of doubtful cases. Every case which gave a positive reaction with
commercial antigen was positive with the Warden antigen and usually
to a much greater degree. — M. W. C.
The Bio-Chemistry of the Gonococcus in its Relation to Immunity. Carl
C. Warden. (The Urologic and Cutaneous Review, 1916, 20, 181-
182.)
On analysis Gonococcus substance shows 12 per cent nitrogen, 20
per cent fat, together with phosphorus, sulphur, salts and ash. Wash-
ing removes nitrogen and fats. Gonococci possess at least three en-
zymes, one proteoljd^ic, one hydrolytic and one a lipase.
An aqueous or normal salt suspension of gonococci behaves, physi-
cally, as a colloid with an electronegative sign and is flocculated or ag-
glutinated by electrolytes of opposite sign and by other colloids such
as serum, under certain conditions. — C. P. B.
The Complement Deviation Reaction Applied to the Diagnosis of Moni-
liasis of the Digestive Tract. I. G. Martinez. (Am. Jour. Trop.
Dis. and Prevent. Med., 1916, 7, 390-391.)
An extract of 10 cc. normal saline extract of three agar tubes of dif-
ferent strains of monilias was made from seventy-two hours growth on
Sabourand's glucose agar. These were shaken for two hours, 0.5 per
cent carbolic acid added, and the extract heated for one hour at 56°C.
This antigen was titrated according to the usual method, and 0.4 cc.
found to constitute one unit.
In use, following the original Wassermann technic for complement
deviation reaction, two cases of sprue and one suspected case gave a
positive reaction. All other reactions with other sera were negative.
— F. M. J.
Variations in the Pneumococcus Induced hy Growth in Immune Serum.
L. M. Stryker. (Jour. Exp. Med., 1916, 24, 49-68.)
Various strains of highly virulent pneumococci of Types I and II
were grown and subcultui'ed for successive generations in media con-
taining homologous immune serums. The author found that the serum
treated pneumococci became less specifically agglutinable, they could
no longer absorb the agglutinins for normal strains, and, when injected
into the animal body, failed to produce agglutinating sera for normal
strains. Further, the virulence of the various treated strains was de-
creased, which may be due to the absence of capsules on such strains and
572 ABSTRACTS
also to the fact that the organisms so grown are phagocyted in normal
serum. It is interesting to note that the variations in pneumococci
produced by treatment with immune serum do not persist after animal
passage. Reversion to the normal type takes place readily. — B. W.
Further Observations on the Agglutination of Bacteria in Vivo. C. G.
Bull. (Jour. Exp. Med., 1916, 24, 25-34.)
Pneumococci, dysentery bacilU of the Shiga type, and Bacillus muco-
sus-capsulatus are agglutinated immediately when injected into the
circulation of actively immunized rabbits. Staphylococcus aureus and
albus, colon bacilli, meningococci, gonococci, and non-virulent pneumo-
cocci agglutinate in the circulation of normal rabbits. Bouillon cul-
tures of Bacillus avisepticus are highly toxic for both rabbits and dogs.
The fresh sera of these animals have no bactericidal action upon the
bacteria. Dog serum opsonizes the bacilli in vitro, and they are agglu-
tinated and opsonized in the circulation and organs of normal dogs. On
the other hand, this does not occur in connection with normal rabbits.
A very small quantity of culture produces a fatal septicemia in rabbits
but a subtoxic dose is without effect in dogs. The degree of aggluti-
nation and opsonization of bacteria within the animal body is inversely
parallel to the infectiousness of the bacteria for the host. — B. W.
A Simple Method of Quantitative Determination of Complement Fixa-
tion. J. 0. HiRscHFELDER. (Jour. A. M. A., 1916, 66, 1386-1387.)
The author gives his technic for performing complement fixation
tests.— G. H. W.
The "Delayed Negative" Wassermann Reaction. 0. M. Olson. (Jour.
Lab. and CKn. Ned., 1916, 1, 704-705.)
The "delayed negative" reaction differs from the original Wasser-
mann only in the method of reading the test. Instead of one reading
at the end of two hours, readings are taken at twenty minute intervals
while the tubes are in the incubator.
By this method the progress of hemolysis may be observed, and the
relation of the time of reaction between the positive and negative con-
trols and the serum under test may be noted.
Whenever hemolysis begins later and is slower in producing com-
plete lysis than the negative control, the serum under test is called a
"delayed negative" and is considered indicative of syphilis. — M. W. C.
The Antagonistic Action of Negative Sera upon the Wasserman Reaction.
A. W. Sellards and G. R. Minot. (Jour. Med. Res., 1916, 34, 131-
147.)
The authors report observations on the ability of normal negative
sera to antagonize the reaction between a positive syphilitic serum and
antigen. Working on the theory that, with a non-specific antigen such
as is now commonly used for the Wassermann, binding may result from
either an increase of specific complement-fixing antibodies, or a de-
ABSTRACTS 573
crease in the normal antagonistic action, they were able to show that
negative sera possessed, in varying degrees, substances which inhibited
the binding power of positive sera. Of 119 cases tested, only six failed
to show this antagonistic action. All negative sera were inactivated
and none were used which contained two units or more of natural sheep
hemolysin. Although of no practical appKcation in routine Wasser-
rnanns, this factor may be of importance in complement fixation in other
diseases. — H. W. L.
Further Observations on the Schick Test for Diphtheria Immunity. G.
B. Weaver and B. Rappaport. (Jour. A. M. A., 1916, 66, 1448-
1450.)
General discussion of the subject with data upon the presence of the
reaction in healthy adults, in cases of post diphtheritic paralysis, in
scarlet fever patients, and in tonsilUtis patients.
The authors employ toxin neutrahzed to excess with antitoxin as a
control.— G. H. S.
The Schick Test, D. M. Griswold. (Jour. Lab. and CHn. Med.,
1916, 1, 441-443.)
Beside providing a quick and easy method for determining individual
susceptibility to diphtheria, the Schick test affords a means of deter-
mining the duration of the passive immunity conferred by injections
of antitoxin. Of six convalescents tested every second day after they
had recovered from diphtheria, the earliest recurrence of a positive
Schick was 3 weeks after 20,000 units of antitoxin, the longest period
of immunity, six weeks. — M. W. C.
The Pseudoreaction in the Schick Test and its Control. A. Zingher.
(Jour. A. M. A., 1916, 66, 1617-1618.)
The Schick test is due to the action of the soluble diphtheria toxin
while the pseudoreaction is an anaphylactic response to the protein of
the diphtheria bacillus.
To control such pseudoreactions an injection of diphtheria toxin
heated to 75°C. for five minutes may be given. The soluble toxin is
thus destroyed and any reaction following such an injection is due to
the protein of the diphtheria bacillus. — G. H. S.
The Diphtheria Toxin Skin Reaction. H. Koplik and L. J. Unger.
(Jour. A. M. A., 1916, 66, 1195-1196.)
A simphfied method of performing the Schick test is described.
Instead of the diluted toxin, with the necessary sterile accessories,
pipettes, sjTinges, etc. the authors employ undiluted toxin and an ordi-
nary hypodermic needle.
The needle is dipped into the toxin and introduced intradermally.
The advantages of this method, aside from its simphcity, are that un-
diluted toxin will retain its potency, traumatic pseudopositive reac-
574 ABSTRACTS
tions are entirely eliminated, and anaphylactic pseudopositive reac-
tions are largely eliminated.
As controlled by the Schick technic complete uniformity resulted,
showing that the amount of toxin introduced was sufficiently accurate
to secure reliable results. — G. H. S.
Diphtheria Immunity — Natural, Active and Passive. Its Determina-
tion hy the Schick Test. W. H. Park and A. Zingher. (Amer.
Jour, of Pub. Health, 1916, 6, 431^45.)
The test is of great rehability when properly made. In a certain
proportion of individuals a pseudo-reaction is seen but this can usually
be distinguished clinically from the true reaction. The test possesses
great value in determining clinically the immunization of susceptible
individuals, and in clearing up the diagnosis of clinically doubtful cases
of diphtheria. The Schick reaction has added further proof to the
clinical and experimental observations that very toxic cases of diph-
theria do better when given an early intravenous injection of antitoxin
than when it is administered in any other way. The results obtained
with the test in families seem to indicate that besides infection with
virulent diphtheria bacilh, other factors, possibly hereditary in nature
are concerned in the production of natural immunity to diphtheria.
The Schick reaction can be apphed with advantage in testing the pa-
tients, resident staff and nurses of contagious disease hospitals. By its
use a considerable saving can be effected in antitoxin during diphtheria
outbreaks. A more widespread use of an active immunization with
mixtures of diphtheria toxin and antitoxin would help to lessen the
disease. — D. G.
Immunity in Tuberculosis. G. A. Webb. (Jour, of Laboratory and
Clinical Medicine, 1916, 1, 414-^27.)
A comprehensive resume of work upon immunity in tuberculosis.
Attempts of the author to immunize guinea-pigs with living tubercle
bacilli were successful when gradually increasing numbers of individ-
uals of an old human culture were used, but unsuccessful with re-
cently isolated cultures.
Experiments with monkeys inoculated with gradually increasing
doses of a human culture produced in some animals a resistance to
10,000 times the lethal dose. Such a resistance could not be con-
stantly produced, however. Lack of success with monkeys was prob-
ably due to excessive virulence of the culture.
Children inoculated five years ago with a culture of which the mini-
mal lethal dose for a guinea-pig was 125 baciUi are still healthy and
do not respond to the von Pirquet test.
The author is convinced that infection takes place, usually in child-
hood, with very small numbers of bacilli. If an immunity in child-
hood could be raised to even a slight degree, it would probably be pos-
sible to vaccinate successfully against tuberculosis. — M. W. C.
ABSTEACTS 575
Immunity Factors in Pneumococcus Infection in the Dog. C. G. Bull.
(Jour. Exp. Med., 1916, 24, 7-24.)
Intravenous inoculations of from 1 to 3 cc. per kilo of body weight
of a bouillon culture of virulent pneumococci produce septicemia and
meningitis in dogs. The injected penumococci leave the circulation
rapidly, but begin to reinvade the blood from twenty-four to forty-
eight hours later. The septicemia reaches its chmax between the fourth
and fifth days and then abruptly declines, the blood becoming sterile
within from one to three days after the height of the septicemia is
reached. The initial disappearance of the pneumococci from the circu-
lation has been found to be due to agglutination of the diplococci in the
blood stream and accumulation of the clumps in the lungs, liver, spleen,
etc. If the dogs are reinoculated during the ascension of the septi-
cemia, the injected diplococci leave the circulation as rapidly as in nor-
mal dogs. Cultures isolated in this stage of the infection, both before
and from threfe to four hours after the reinoculation, are resistant to
the agglutinins and opsonins of immune sera that agglutinate and opso-
nize the cultures with which the dogs were originally infected. Thus
it follows that the pneumococci are able to reinvade the circulation be-
cause they have acquired a fastness to the existing antibodies and not
because the antibodies have been exhausted. By reinoculating dogs
at the time of the crisis in the septicemia it has been shown that
the agglutination of the pneumococci is more rapid and complete
and that the diplococci leave the circulation much more rapidly than
in normal dogs. Hence acquired antibodies are operative within the
animals at this time although they cannot be demonstrated in vitro
until from twenty-four to forty-eight hours later, Pneumococci iso-
lated as the infection is subsiding are more susceptible to the action of
inmiune sera than the original cultures injected. It is probable that
all the dogs would have survived the infection if a meningitis had not
developed. In the acutely fatal cases of meningitis few pneumococci
are phagocyted, while in the milder and convalescent cases much pha-
goc3rtosis occurs. It is suggested that the incubation period of infec-
tious diseases is due to the fact that the infecting agents must become
adapted to the adverse conditions encountered in the newly infected
host before they can multiply sufficiently to produce the symptoms of
disease. It is further suggested that epidemics may arise because the
infectious agent is passed from person to person in the ascending stage
of the disease and thus enters new hosts in a state of maximum resist-
ance to the natural antibodies of such individuals. When early con-
tacts are avoided, epidemics tend to subside because the infectious
agent is weakened by the action of acquired antibodies during the
period of convalescence. — B. W.
576 ABSTRACTS
LABORATORY TECHNIQUE
Hydrochloric Acid as a Decolorizing Agent for the Tubercle Bacillus. R.
A. Keilty. (Jour. A. M. A., 1916, 68, 1619-1620.)
The following technic is given : Make thin smears, fix with heat, and
stain with cold carbolfuchsin for five minutes. Decolorize for thirty
seconds or more with 30 per cent hydrochloric acid. Counterstain
with Loeffler's methylene blue. — G. H. S.
A Device for Protection against the Tubercle Bacillus. R. A. Keilty.
(New York Med. Jour., 1916, 103, 1074.)
The device described is used to prevent the scattering of tubercle
bacilli from a platinum loop while it is being flamed.
The apparatus consists of a tube of Russian iron, so constructed that
it surrounds the flame of a Bunsen burner, fitting the burner tightly at
the bottom. The platinum loop may be plunged into the flame and
any masses which jump are caught upon the sides or bottom of the
tube.— M. W. C.
Stabilized Gentian Violet. W. D. Stovall and M. S. Nichols. (Jour.
A. M. A., 1916, 66, 1620-1621.)
To prevent deterioration of the gentian violet used in Gram's stain
the authors suggest a stain of the formula:
Anilin 28 cc.
Gentian violet 8 gm.
95 per cent alcohol 100 cc.
N Hydrochloric acid 5 cc.
Distilled water qs. ad 1000 cc.
Dissolve gentian violet in the alcohol. Add hydrochloric acid to the
anihn and dissolve in water to make 900 cc. Filter the aqueous solu-
tion and add to the alcohoHc stain. Filter. — G. H. S.
The Production and Collection of B. coli in Quantity on Synthetic media.
Robert Bengis. (Jour. Infect. Diseases, 1916, 18, 391-39a.)
The best medium was composed of the following: 2.5 per cent agar,
1 per cent ammonium lactate, and 0.2 per cent disodium phosphate,
with or without 1 per cent calcium carbonate. The absence of lac-
tose increased the eflficiency. Inoculation of specially constructed
plates was accomplished by means of a De Vilbiss atomizer. The
growth was scraped off with a safety blade and placed in 75 per cent
cent alcohol.— P. B. H. /
Production of Clear and Sterilized Anti-Hog-Cholera Serum. M. Dor-
set and R. R. Henry. (J. Agr. Res., 1916, 6, 333-338.)
The occasional presence of the foot-and-mouth virus in hog-cholera
serum makes it necessary to devise some means of sterilizing it. Heat-
ing for thirty minutes at 60° kills the foot-and-mouth virus; but unless
ABSTRACTS 577
the corpuscles are removed from the defibrinated hog-cholera-immune
blood, the heat causes coagulation and destroys the commercial value
of the serum. Complete removal of the corpuscles by centrifugaliza-
tion alone has proved impractical ; but if they are first agglutinated by
the addition of extract of the common white navy bean, complete sepa-
ration from the serum is quite easy. The serum thus prepared has
no harmful properties due to the bean extract; and can be heated for
thirty minutes at 60° without undergoing any change. — H. J. C.
PLANT PATHOLOGY
Crown gall Studies; Showing Changes in Plant Structure Due to a Changed
Stimulus. (Preliminary paper.) Erwin F. Smith. (J. Agr. Res.,
1916, 6, 179-182, with six plates.)
This is a continuation of work on crown gall in which its similarity to
human cancer is pointed out. This paper describes some new points
in the pathology of the disease. — H. J, C.
Transmission and Control of Bacterial Wilt of Cucurbits. F. V. Rand
and Ella M. A. Enlows. (J. Agr. Res., 1916, 6, 417-434.)
It was shown several years ago by Erwin Smith that this disease
(due to B. tracheiphilus) was transmitted by the striped cucumber
beetle {Diabrotica vitiata Fab.). The present work confirms this con-
clusion, and shows that the principal summer carriers of the disease
are all species of Diabrotica. There is no evidence of spread through
the soil (unless the roots are injured) or by means of infected seed. The
disease can be controlled by early treatment with Bordeaux and ar-
senate of lead. Experiments are now being carried on to learn how to
control the beetles that spread the disease. — H. J. C.
Further Evidence that Crown Gall of Plants is Cancer. E. F. Smith.
(Science, 1916, 43, 871-889.)
After a brief survey of the main sub-divisions of the forms of cancer
and their outstanding characteristics, the author presents a powerful
argument for the "parasitic origin and essential unity of the various
forms of cancer occurring in men and animals." The evidence is essen-
tially presumptive, being drawn from the striking resemblance between
Crown Gall of plants, wliich the author has proved to be caused by the
Bacterium tumefaciens, and human cancer. The domination of the
morphologists in cancer research is deplored and the attention of the
experimental biologist and the bacteriologist is invited. So firm is the
author's conviction of the parasitic nature of this malady that he ex-
claims: "I am now persuaded that the solution of the whole cancer
problem lies in a study of these plant tumors." The discovery reported
that the Crown Gall organism will produce teratoid tumors with regu-
larity in various plants, not only in dormant buds but in the leaves, is
a most astonishing and important contribution. — C. M. H.
578 ABSTRACTS
Studies on the Crown Gall of Plants: Its Relation to Human Cancer.
Erwin F. Smith. (Journal of Cancer Research, 1916, I, 231-258,
87 pi.)
The author reviews first the geographical and botanical distribution
of crown gall. This disease is differentiated from hypertrophic en-
largements in plants due to a few special parasitized cells as in the root
nodules of legumes and slime mold infections in crucifers, as well as
from granulomatous hyperplasia such as those of the olive tubercle
where the bacteria are imbedded between the cells. Crown gall is a
peculiar hyperplasia caused by Bacterium tumefaciens developing
sparingly and only intracellularly, the parasitized cells being thereby
caused to divide prematurely and repeatedly, which results in a great
mass of non-capsulated small celled tumor tissue in which the bacteria
themselves are invisible.
The tumor resembles cancer in many ways, notably in the exhibition
of growth independently of function, vegetative activity being stimulated,
functional activity depressed. The tumor can be stimulated by feed-
ing or starved into quiescence. It can be grafted upon other plants of
the same species. As in mammalian cancer, the nuclei of the tumor
divide both mitotically and amitotically. Due to the rigid cell walls
of plants, on the other hand, true metastasis seems not to occur.
The writer mentions especially the atypical arrangement of the tis-
sues, their loss of polarity and the anaplasia, or undifferentiation of
the cells. The production of tumors varying in structure according to
the type of tissue experimentally invaded, among which were recog-
nized embryonic organ inclusions analogous to the teratomata of mam-
mals, is recorded for the first time.
The bacteriology of the parasite is discussed briefly, as are also some
experiments with animals. — I. C. H.
PUBLIC HEALTH BACTERIOLOGY
Public Health Laboratories. R. G. Perkins. (New York Med. Jour.,
1916, 103, 721-724.)
A discussion of the activities of public health laboratories. Among
the suggestions made for furthering the development of these labora-
tories it is particularly recommended that there should be close asso-
ciation between the laboratory and a high grade university, in order
that conditions should be made ideal for research work. — Mj W. C.
The Microscopic Examination of Finger Nail Deposits. S. Schneider.
(Jour. A. M. A., 1916. 66, 1615-1617.)
The microscopic examination of finger nail deposits reveals the fact
that the usual methods of cleansing the hands and nails does not result
to any considerable degree in removing deposits.
Bacteriologic examination of deposits shows microorganisms to pre-
dominate in the following order: streptococci; staphylococci; Bacillus
coli; bacilli, cocci, and spirillae derived from diverse sources; yeast
ABSTRACTS 579
cells, spores and filaments of higher fungi ; and larvae of vermes, ame-
bas, diatoms, algae, etc.
Attention is called to the examination of such deposits from the
medico-legal as well as from the hygienic standpoint.— G. H. S.
Controlling the Spread of Sputum. W. A. Manheimer. (Medical
Record, 1916, 89, 997-999.)
Sputum is one of the most dangerous of human discharges because
of its wide dissemination and high content in pathogenic bacteria.
Experiments conducted to determine the viability of a culture of colon
bacilli, when placed upon mailing envelopes as organisms in the spu-
tum would be deposited in ordinary licking showed that of the bacteria
sent through the mail, 5 per cent remained alive. Diphtheria bacilh
placed upon envelopes and dried could not be cultivated in the few
experiments performed for this purpose.
Valuable suggestions are given, which would aid in controlling the
spread of sputum. — M. W. C.
MEDICAL BACTERIOLOGY
Case of Coccidioidal Granuloma. S. T. Lipsitz, G. W. Lawson, and
E. M. Fessenden. (Jour. A. M. A., 1916, 66, 1365-1367.)
Case report with detailed account of the blood picture and bacteri-
ologic findings. — G. H. S.
The Contents of Ovarian Cysts. J. T. Leary, H. J. Hartz, and P. B.
Hawk. (New York Med. Jour., 1916, 104, 16-18.)
Bacteriological examination of the contents of six ovarian cysts re-
sulted in negative findings in every case. — M. W. C.
Neisserian Proctorrhea. Charles C. Mapes. (The Urol, and Cut.
Rev., 1916, 21, 1.)
Infection of the rectal mucosa with the diplococcus of Neisser (Gono-
coccus) occurs more frequently than is generally believed. — C. P. B.
The Treatment of Human Rabies with Quinin and with Phenol. F. S.
Fielder. (Jour. A. M. A., 1916, 66, 1300-1302.)
Several case reports of human rabies treated by injections of quinin
or phenol. No specific action of the drugs was manifested. — G. H. S.
Trichinosis and the Cerebrospinal Fluid. W. Lintz. (Jour. A. M. A.,
1916, 66, 1856.)
An accurate and rapid method of diagnosis in suspected trichinosis is
to be found in an examination of the cerebrospinal fluid for Trichina
spiralis. — G. H. S.
580 ABSTRACTS
The Etiology of Typhus Fever in Mexico. (Tahardillo.) P. K. Olitsky,
B. S. Denver, and C. E. Husk. (Jour. A. M. A., 1916, 66, 1792-
1692.)
An organism was isolated from typhus fever patients in Mexico which
possessed morphological and cultural characteristics identical with
those of the Bacillus typhi-exanthematici. — G. H. S.
The Etiology of Iritis. E. E. Irons and E. V. L. Brown. (Jour. A.
M. A., 1916, 66, 1840-1844.)
An etiologic study of 100 cases of iritis in which the causal relation-
ships of syphilis, gonococcal infection, tuberculosis, dental, tonsillar,
sinus, and genito-urinary infections are discussed. — G. H. S.
The Laboratory Examination of Material in a Case of Suspected Small-
pox. J. N. Force. (Jour. A. M. A., 1916, 66, 1384.)
Pus obtained from a case of suspected smallpox injected intradermally
into immune rabbits, and controlled by injections of vaccine virus, de-
monstrated the absence of small pox. The test required but forty-eight
hours.— G. H. S.
Experimental Studies in the Production of Chronic Gastric Ulcer. A. 0.
Wilensky and S. H. Getst. (Jour. A. M. A., 1916, 66, 1382.)
Cultures of various strains of streptococci and yeasts derived from
human gastric ulcers when injected into lesions artificially produced in
the stomachs of cats failed to cause ulceration or retardation of heal-
ing.—G. H. S.
The Treatment of Genito-Urinary Tuberculosis with Rosenbach's Tuber-
culin. A. Hyman. (Jour. A. M. A., 1916, 66, 1379-1381.)
Rosenbach's tuberculin, a product of the symbiotic growth of the
tubercle bacillus and Trichophyton holosericumalbum, was used thera-
peutically in thirteen cases of urogenital tuberculosis. Two cases
showed improvement, the remainder were not influenced. — G. H. S.
Recurrent Generalized Herpes of Infectious Origin. F. Cohen. (Jour.
A. M. A., 1916, 66, 1598-1599.)
Case report of generalized herpes simplex recurrent over a period of
two years. ,
A streptococcus was isolated from the lesions and admihistered as a
sensitized vaccine. Cure resulted. — G. H. S.
Streptothrix in Bronchopneumonia of Rats Similar to that in Rat-Bite
Fever. R. Tunnicliff. (Jour. A. M. A., 1916, 66, 1606.)
A streptothrix, apparently Streptothrix murisratti was isolated from
several rats affected with bronchopneumonia. Morphological and cul-
tural characteristics are given. — G. H. S.
ABSTRACTS 581
Control of Diphtheria. D. M. Lewis. (Jour. A. M. A., 1916, 66,
1535-1536.)
Emphasis is placed upon the value of epidemiological work in the
control of diphtheria. It is asserted that the examination of the naso-
pharyngeal cavity of patients and contacts is more reliable than cul-
tural work for the detection of carriers. — G. H. S.
Tuberculin in Surgical Tuberculosis. E. Bonime. (New York Med.
Jour., 1916, 103, 726-728.)
Tuberculin, if properly administered, is of great value in surgical
tuberculosis. In cases where other infections occur in connection with
the tuberculous processes, an autogenous vaccine should be used in
addition to the tuberculin. — M. W. C.
Newer Laboratory Methods for the Early Diagnosis of Pulmonary Tuber-
culosis. M. H. Kahn. (Jour, of Lab. and Clin. Med., 1916, 1, 599-
607.)
A description of various laboratory tests used in the diagnosis of tu-
berculosis. The only test of absolute diagnostic importance is the bac-
teriological examination of the sputum. All others are as yet of but
relative value. — M. W. C.
A Case of Infection of Lymph Glands with Bacillus Paratyphosus B.
C. S. Cole. (Jour. Infect. Diseases, 1916, 18, 349-352.)
A condition of multiple lymph-adenitis, first diagnosed as Hodgkins'
disease, and later found to be a paratyphoid infection without the
manifestation of typhoid-like, gastro-enteric symptoms. — P. B. H.
Etiology and Laboratory Diagnosis of Smallpox and Chickenpox. J. N.
Force. (Jour, of Lab. and Clin. Med., 1916, 1, 243-25L)
A review of the methods which have been proposed for the diagnosis
of smallpox and chickenpox.
For absolute diagnosis or differential diagnosis between smallpox and
chickenpox, the intradermal inoculation of the suspected material into
vaccinia immune rabbits appears to be the simplest and most reliable
method.— M. W. C.
The Immune Response in Pulmonary Tuberculosis. E. Bonime.
(New York Med. Jour., 1916, 103, 930.)
In Germany the early use of tuberculin in tuberculosis has caused a
marked diminution in the death rate from the disease. Physicians of
this country should be urged to combine tuberculin treatment with the
usual hygienic measures employed in the early stages of tuberculosis.
M. W. C.
Flagellate Protozoa as an Etiologic Factor of Dysenteric Diarrhea. B.
W. Rhamy and F. A. Metts. (Jour. A. M. A., 1916, 66, 1190-1191.)
The authors assert that Trichomonas intestinalis is a cause of acute
or chronic diarrhea following the drinking of impure water.
582 ABSTRACTS
This conclusion is supported by case histories and by the report of
an epidemic consisting of 78 cases with 17 deaths.
Ipecac and emetin proved valuable in treatment. — G. H. S.
The Causation and Treatment of Pellagra. H. E. Bond. (Medical
Record, 1916, 89, 816-819.)
The theory is advanced that pellagra is caused by bacteria occurring
in the intestinal tract. Toxins produced act primarily upon the sym-
pathetic nervous system and secondarily upon the central nervous sys-
tem. Treatment should consist of the internal use of gastrointestinal
antiseptics and the external use of protective ointments. — M. W. C.
Notes on Grip Epidemic in Chicago. A. M. Moody and J. A. Capps.
(Jour. A. M. A., 1916, 66, 1696.)
An analysis of 53 cases of grip shows that leucocytosis is usually ab-
sent. Bacteriologic examination of 31 cases gave the following results:
Streptococcus hemolyticus, Streptococcus viridans and the pneumococcus
were present 31 times; hemolytic staphylococci, 19 times; Bacillus in-
fluenzae and Friedlander's bacillus twice; Streptococcus mucosas and
Micrococcus catarrhalis once. — G. H. S.
Antimeningococcic Serum in the Joint Manifestations oj Gonorrhea. F.
Malleterre. (New York Med. Jour., 1916, 103, 1024-1026.)
Antimeningococcic serum is of value in generalized polyarticular
forms of arthritis when several joints are involved and where there is
moderate local inflammation without a fluid collection. The serum has
little or no value in cases of gonorrheal monoarthritis with a large
fluid collection.— M. W. C.
Pyelocystitis and Metastatic Abscesses Following Tonsillitis. H. B.
Mills and G. A. Sowell. (New York Med. Jour., 1916, 103, 725-
726.)
Report of a case in which an attack of tonsillitis was followed by
pyelocystitis and metastatic abscesses. Staphylococcus albus was the
predominating organism isolated from the tonsils and was obtained in
pure culture from the urine, blood and abscesses. The authors con-
clude that the tonsillitis was the etiological factor in the pyelocystitis,
while the metastatic abscesses were complications of the latter.
M. W. C.
The Bacillus Epilepticus. C. A. L. Reed. (Jour. A. M. A., 1916, 66,
1607-1611.)
The author asserts that epilepsy is due to infection by Bacillus epi-
lepticus. The organism is a spore bearer, whose primary focus of in-
fection is the cecum but which under suitable conditions may invade
the circulation.
The bacillus has been cultured from the blood and digestive tracts of
epileptics and has proved pathologic for rabbits. — G. H. S.
ABSTRACTS
583
The Diagnosis of Enteric Fever {Tijyhoid and Paratyphoid A and B) by
Agglutination Tests. W. C. Davison. (Jour. A. M. A., 1916, 66, 1297.)
Macroscopic agglutination tests made in accordance with the quan-
titative technic of Dreyer are reliable as diagnostic procedures.
In cases of suspected typhoid in persons who have received prophy-
lactic inoculation a series of tests niade at intervals of 5 to 10 days are
necessary to establish the diagnosis. — G. H. S.
Notes on the Etiology of the Recent Epidemic of Pseudo-Influenza. W.
W. Williams and W. Burdick. (Medical Record, 1916, 89, 876-
877 )
A streptococcus was isolated from cases of pseudo-influenza, which,
immediately after isolation, was virulent for mice and rabbits. Au-
topsy of rabbits, killed forty-eight hours after inoculation with the
strain, revealed a tracheal and bronchial inflammation, suggestive of
a selective action. After four or five generations of subculture, how-
ever, no such selective action was manifest. Upon artificial cultiva-
tion' the organism also reverted to the usual cultural characteristics.
' M. W. C.
Bacteriology in Conjunction with Homeopathic Prescribing. W. W.
Irving. (Jour, of Oph., Otol. and Laryng., 1916, 22, 490.)
The author understands the action of the homoepathic remedy to be
much the same as that of vaccines, in stimulating the body tissues to
greater resistance. " Or does the remedy stimulate the anti body func-
tion or does it act as a germicide?"
By a study based on the above theories he expects to produce a ma-
teria medica which will unify the organism with the remedy.— C. P .B.
Anopheles Punctipennis, a Host of Tertian Malaria W.J. King.
(Am Jour. Trop. Dis. and Prevent. Med., 1916, 8, 426-432.)
In two comparative experiments Anopheles punctipennis was proven
to be an efficient host for the Plasmodium vivax. These mosquitoes
were bred from the larvae and pupae collected in the open. 1 hey were
fed individually on a gametocyte carrier. A high percentage of infec-
tion was obtained. Controls of A. quadrimaculatus were also found
to be infected. The infection in all instances was followed to the sali-
vary glands. — F. M. J.
Simultaneous Injections of Streptococci and Dahlia in the Guinea-pig.
W H. Hoffman, W. B. Ma^clurb, and L. W. Sauer. (Jour. Infect.
Diseases, 1916, 18, 353-357.) ...... ,. ,. r ^^
From a study of the effects of combined injections the authors found
that an injurious reaction resulted, although no change was detected
in the opsonic content of the serum or in the hemolytic reactions in
which the serum was employed. They conclude that ' intravenous in-
jection is not warranted as a therapeutic measure. —P. B. M.
584 ABSTRACTS
The Site and Rate of Destruction of Pneumococci Following Intraperi-
toneal Injection. F. Berry and C. 0. Melick. (Journal of Immu-
nology, 1916, 1, 119.)
This report confirms the observations of Kyes concerning the mode
of destruction of pneumococci injected into an unsusceptible organism
(pigeon). The pneumococci as in Kyes' experiments were found to be
taken up in great numbers by endothelial cells in the liver, and spleen.
They appear in these cells within ten minutes after intravenous injec-
tion and within about two hours after intraperitoneal injection, and
disappear completely in from twenty -four to thirty-six hours. — J. G. H.
A Final Report on the Cultivation of the Tubercle Bacillus from the Spu-
tum hy the Method of Petroff. R. A. Keilty. (Jour. Ex. Med., 1916.
24, 41-48.)
Cultures of the tubercle bacillus were obtained in 12 out of 18 posi-
tives cases of pulmonary tuberculosis and of 7 negative cases 1 showed
growth. From 4 of the 12 positive cases pure cultures were obtained.
In 12 cases sterile cotton swabs were rubbed over the tonsils, fauces,
tongue and gums, the swabs treated with 3 per cent sodium hydroxide
and the neutralized sediment inoculated on the Petroff medium;
and in one case acid fast bacilli were obtained. — B. W.
Post-Operative Tetanus. Kellogg Speed, (Surgery, Gjoi. and Obstet.,
1916, 22, 443.)
A very complete resume of the literature. In addition six cases are
reported all occurring after some abdominal operation. The author
believes that some human beings are tetanus carriers and when opera-
tion is done, any injury to the wall of the intestines may result in
tetanus through invasion from the intestinal contents.
The possibility of haematogenous infection must also be considered;
likewise external contamination from fecal discharges may carry the
infection. — C. P. B.
The Bacteriology and Experimental Production of Ovaritis. E. C. Rose-
now and C. H. Davis. (Jour. A. M. A., 1916, 66, 1175-1180.)
The authors prove experimentally that streptococci derived from
inflammatory conditions of human ovaries show an elective affinity for
the ovaries of animals.
Cultures from ovaries removed at operation yielded Streptococcus
viridans in a large number of cases. The gonococcus, the Welch bacil-
lus. Staphylococcus alhus, the colon bacillus and diphtheroid-like bacilli
were also obtained.
Microscopic preparations of sections of the ovaries showed the pres-
ence of diplococci in several instances.
Cultures of the streptococci isolated were injected into rabbits and
dogs and were recovered in pure culture from the ovaries of the ani-
mals.—G. H. S.
ABSTRACTS 585
Elective Localization in the Bronchial Musculature of Streptococci from
the Sputum of Cases of Bronchial Asthma. S. Oftedal. (Jour. A.
M. A., 1916, 66, 1693-1694.)
Cultures from the sputum of asthmatic patients injected intrave-
nously into rabbits caused marked interference with the respiration.
The respiration was reduced in rate and labored. Microscopic exami-
nation of the lungs showed marked distention of the alveoli with fre-
quent rupture of the alveolar walls. The blood vessels were engorged.
The bronchioles were contracted. Hemorrhage and mud cell infiltra-
tion were present. Streptococci were found in the musculature.
Tissue cultures of the lungs yielded pure cultures of streptococci.
The author considers this an example of the elective locahzation of
streptococci. — G. H. S.
An Epidemic of Appendicitis and Parotitis Probably Due to Streptococci
Contained in Dairy Products. E. C. Rosenow and S. I. Dunlap.
(Jour. Infect. Diseases, 1916, 18, 383-390.)
The authors studied an outbreak occurring in a military academy in
which fifteen cases of appendicitis and thirty-four cases of parotitis
arose between February and the following May. From the appendix
and tonsils of the patients streptococci were isolated that were patho-
genic for rabbits, reproducing lesions in the appendix. Strains iso-
lated from dairy products at the time of the epidemic produced similar
lesions in rabbits. The authors conclude that the epidemic was caused
by infected dairy products. — P. B. H.
The Tuberculocidal Action of Arsenic Compounds and Their Distribu-
tion in the Tuberculous Organism. A. Arkin and H. J. Cooper.
(Jour. Infectious Diseases, 1916, 18, 335-348.)
The authors studied the effect of arsenic upon the tubercle bacillus
and its distribution in the body. They report that (1) Sodium arse-
nite in dilutions from 0.1 to 0.0001 per cent and sodium cacodylate in
dilutions of from 2.0 to 0.002 per cent had no germicidal action in
twenty-four hours at 37°C., although mercury cacodylate showed ger-
micidal power. (2) Atoxyl, arsacetin and neosalvarsan in dilutions of
1.0 to 0.001 per cent showed no germicidal power. (3) These com-
pounds were found in the liver, lungs, kidneys, blood, spleen and in
the tuberculous tissues (lymph glands and eye). No evidence of ac-
cumulation in the tissues was obtained. — P. B. H.
Syphilis in Epilepsy. W. T. Shanahan, J. F. Munson, and A. L.
Shaw. (New York Med. Jour., 1916, 103, 820-824.)
While syphilis may be considered one of the many agencies produc-
ing epilepsy, there is no special type of syphilitic epilepsy.
The percentage of syphilis in epileptic patients is approximately the
same as the percentage in the general population.
Treatment of syphilis in epilepsy does not usually result in improve-
ment, probably because permanent injury has been done to the tissues
long before the beginning of treatment. — M. W. C.
686 ABSTRACTS
Focal Sepsis. J. Daland. (New York Med. Jour., 1916, 103, 1159-
1160.)
Septic foci in the mouth, tonsils, sinuses, or prostate are frequently
the cause of various systemic infections. The organism isolated from
such foci is most frequently a streptococcus, usually Streptococcus hemo-
lyiicus. The severity of the systemic infection resulting from chronic
focal sepsis varies with the virulence and number of the micro-organisms
occurring in the focus, as well as with the resistance of the tissues to
the spread of the infection.
The prompt diagnosis and removal of a septic focus is of the greatest
importance. — M. W. C.
Meningitis. R. C. Rosenberger and D. J. Bentley. (New York
Med. Jour., 1916, 103, 1166.)
A report of seven cases, five of which were of the epidemic variety,
one of pneumococcal, and one of tuberculous origin. The meningococcus
was found in the spinal fluid of the five epidemic cases, and in one of
the five a streptococcus was associated with the meningococcus.
Cultures from a nasal discharge in two of the cases showed beside the
usual bacterial flora, a gram negative diplococcus, which could not be
differentiated from the meningococcus.
Antimeningococcus serum was administered to the five cases of the
epidemic type. Three recovered, two did not.
Tubercle bacilli were demonstrable in the spinal fluid of the case
with tuberculous meningitis two days before death. Pneumococci were
present in the spinal fluid, and at autopsy, in the heart of the patient
with pneumococcal meningitis. — M. W. C.
Tetanus: A Surgical Complication in the Present War. E. K. Tullidge.
(New York Med. Jour., 1916, 103, 1022-1024.)
The cases of tetanus treated during the present war, usually occurred
as secondary infections, where the tetanus bacillus was found in wounded
tissues in association with other organisms — most frequently with
Staphylococcus aureus and Bacillus aerogenes-capsulatus.
The greatest mortality was in those cases of short incubation period,
five days or less.
The most successful treatment was the administration of antitetanic
serums in large doses, varying from 10,000 to 160,000 units.
Cases of long incubation period responded to smaller doses, Ijut in
cases displaying a short incubation period, large doses alone produce
good results.
Chloral hydrate in doses of 5 to 10 grains was of great value in con-
trolling convulsions. Cases where the wounds were treated locally
with iodine did not develop the disease in its severest form. — M. W. C.
The Etiology of Common Colds. G. B. Foster. (Jour. A. M. A., 1916,
66, 1180-1183.)
The conclusion that common colds are due to the action of a filterable
virus is supported by the following observations:
ABSTRACTS 587
The nasal secretions from individuals ill with colds were diluted with
physiological salt solution and filtered through Berkefeld filters. The
filtrates, when cultivated aerobically and anaerobically upon blood
agar were sterile. Of ten men who were inoculated by placing some
of the filtrate in the nostrils, nine developed colds.
The filtrates can be cultivated anaerobically in tissue — ^ascitic fluid.
Stained preparations of the cultures were questionable but dark field
examination showed the presence of active minute bodies possessing
true motility.
Subcultures were prepared, filtered, and used to inoculate 11 men.
After an incubation period of from eight to forty-eight hours all the
men became ill with acute colds.
Filtrates from these experimentally produced colds could be culti-
vated.—G. H. S.
The Production of Amyloid Disease and Chronic Nephritis in Rabbits
by Repeated Intravenous Injections of Living Colon Bacilli. C. H.
Bailey. (Jour. Exp. Med., 1916, 23, 773-790.)
The repeated intravenous injection of rabbits with living Bacillus
com^nunior over long periods has resulted in the formation of amyloid
deposits in the spleen, liver, and kidneys. Suppurative lesions were
not present in most cases and therefore not a factor in their production.
The results have been constant in that amyloid was found in all rab-
bits, eight in number, which were injected over a period of eighty-eight
days or more. Eight rabbits showed amyloid in the spleen, six of
these in the kidneys also, and three in the liver.
The kidneys of these eight rabbits also showed as a result of the in-
jections a subacute and chronic glomerulitis, parenchymatous degen-
eration, some interstitial infiltration with round cells, and a slight
cellular proliferation of connective tissue, thus resembhng the chronic
parenchyinatous nephritis of man which is so commonly associated
with amyloid disease. — B. W.
Anthrax with Report of Cases. S. J. Ullman. (Surgery, Gjm., and
Obstet., 1916, 22, 450.)
Reports two fatal cases of anthrax occurring in negroes who had
helped to skin the carcass of a cow. The animals on this pasture were
vaccinated against anthrax but one cow had escaped while being driven
up. Several months later it was found dead on the range. Showing
no lesion it was ordered to be skinned. Two days after this one of the
negroes was taken sick; he died on the eighth day of the disease, no
physician having been called. The other negro was taken sick a week
later. He was treated with anthrax vaccine and with serum from a
horse inoculated against anthrax, but died one week after symptoms
developed.
Two other negroes who helped do the skinning were taken sick two
weeks afterward. Serum was given immediately; they recovered.
The author is not sure that these two had anthrax but does believe
a standard serum is of considerable value. — C. P. B.
588 ABSTRACTS
An Epidemic of Dysentery at Fort Shafter, Hawaii, with Three Cases of
the Hiss-Russell or " Y" Bacillus Infection. G. M. Van Poole.
(The Military Surgeon, 1916, 38, 525-530.)
An Epidemic of Bacillary Dysentery due to the Hiss-Russell Bacillus.
M. A. Delaney. (Ibid., 531-533.)
Bacillary Dysentery, Recent Epidemic at Fort Shafter, H. T.,from the
Laboratory Aspect. F. H. Foucar. (Ibid., 534-538.)
This epidemic of 34 cases began on October 16. 1915, and the last
case was received November 21, 1915. During this time 3 officers, 2
children, 27 enlisted men and 1 civilian emploj^'ee were stricken. Two
deaths resulted, thus giving a mortality of 6 per cent. The first of
these papers discusses the epidemic from the sanitary standpoint, the
second from the clinical standpoint and the third from the laboratory
standpoint, thus giving a comprehensive study of a sharp outbreak of
bacillary dysentery which was soon brought under control. Perhaps
the most interesting point brought out is found in Captain Foucar's
paper which states that a vaccine was prepared from the " Y" Bacillus
isolated from these cases. The vaccine was made in accordance with
the technique used in preparing the army typhoid vaccine, but was
used in smaller doses. Three doses at ten day intervals were given,
mostly to children, a total of 168 separate doses being given. Only one
severe reaction occurred, and it is stated that the cases among the chil-
dren ceased although adult cases continued for some time. — E. B. V.
•Study of a Strain of B. Welchii Isolated in France Together with Some
Notes on Gastric Ulcers. Mary W. Stewart and Randolph West.
(Journal of Immunology, 1916, 1, 189.)
■Stewart and West studied an organism isolated from gas gangrene
which was a strict anaerobe, was capsulated, non-motile, and formed
spores only in sugar-free broth containing coagulated egg white. It
produced stormy fermentation of milk and in other respects conformed
with the B. Welchii type of the butyric acid forming group of bacteria.
Weinberg and Sacqu(?pee have reported the formation of a solul)le toxin
by a gas gangrene organism which was motile and which sporulated on
sugar media but Stewart and West were unable to detect a soluble
toxin in the cultures of their bacillus. The killed suspensions of washed
bacteria were harmless for guinea pigs. The filtrate of sugar broth cul-
tures which was highly acid was toxic to guinea pigs but this toxicity
could be completely removed by neutralization of the filtrate. The
most noticeable lesions produced by these filtrates were acute gastric
ulcers and such ulcers could be almost as regularly produced by the
injection of acetic acid solutions of similar titre. The necrotic tissue
at the site of an intramuscular inoculation with the bacilli was also
found to be highly acid. Blood cultures from infected pigs were rarely
positive.
The conclusion is drawn that the general toxic effects in gas bacillus
ABSTRACTS 589
infections are due not to generalized infection or to a true toxin, but to
the toxic effect of the acid produced at the site of the localized lesion.
J. G. H.
Bacterial Cultures of Human Spleens Removed hy Surgical Operation.
Andrew W. Sellards. (Journal of Immunology, 1916, 1, 321.)
Eight spleens from cases of pernicious anemia, one showing simple
hypertrophy, and one from infantile primary splenomegaly, all re-
moved at operation, were the subject of this study. Aerobic and an-
aerobic cultures were made on various media including milk and glu-
cose ascitic agar. From four of the spleens micrococci, differing from
the ordinary pyogenic types but not further identified, were recovered.
From three of the spleens, small Gram positive pleomorphic bacilh
were recovered in the anaerobic milk tubes. These organisms cul-
turally and morphologically resembled a culture of the bacillus recov-
ered from typhus by Plotz. Complement fixation reactions were
carried out with extracts of these bacilli against the blood of rabbits
immunized to them. Cross fixation was obtained between the antigens
from the two spleen cultures tested, with the serum of the animal im-
munized to the Plotz bacillus, and an antigen prepared from the Plotz
bacillus gave fixation with the serum prepared by injection of the spleen
cultures. Out of eight human sera tested, one from a case of pernicious
anemia, and one from a case of cholangitis gave complete fixation with
spleen bacillus antigen and with Plotz bacillus antigen. Other sera
were negative. In fermentation tests, two of the spleen cultures dif-
fered from the bacillus of Plotz in fermenting mannite and in failing
to ferment inulin, and one differed only in the absence of inulin
fermentation. Sellards concludes that these organisms represent para-
sitic but non-pathogenic organisms found in the human body. — J. G. H.
Bacteriological and Experimental Studies on Gastric Ulcer. H. L. Cel-
LER and W. Thalhimer. (Jour. Exp. Med., 1916, 23, 791-812.)
Eight chronic gastric ulcers and one ulcer occurring at the ostium of
a gastrojejunostomy were examined bacteriologically and histologi-
cally. From seven of these anhemolytic streptococci were isolated;
streptococci were seen in cultures of the eighth but could not be iso-
lated and from the ninth no streptococci were recovered. Yeasts were
recovered from four. Other organisms including staphylococci. Micro-
coccus tetragenus and B. subtilis were isolated. The streptococci iso-
lated were injected into rabbits and cats intravenously. Of thirty rab-
bits injected in the ear vein, four developed gastric lesions. Of eight
rabbits injected in a branch of the gastric artery six developed gastric
lesions. In two cats a branch of the gastric artery was injected with
streptococci. Both animals developed defects in the gastric mucosa,
which soon began to heal and were observed to have healed completely
in thirty-three days. In addition fourteen of the thirty rabbits devel-
oped cardiac lesions, while two of the eight in which a branch of the
gastric artery was injected developed minute hemorrhages in the endo-
590 ABSTRACTS
cardium. The authors are unable to decide definitely whether or not
the gastric lesions produced by the injection of the rabbits with strep-
tococci are to be considered ulcers. From their experiments they con-
clude "It must be assumed that some cause is operative in certain
cases preventing the healing of defects in the gastric mucosa and is
inoperative in others. Even though anhemolytic streptococci are
present in practically all gastric ulcers, we cannot convince ourselves
that these organisms have been proven as yet to be the factor which
either initiates the ulceration or prevents healing. Nevertheless, the
constant presence of streptococci in this type of lesion is a suggestive
fact and further experiments to determine their significance are being
undertaken." — B. W.
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De Khotinsky Electrically Heated
CREMATOR
No. K1750.
In inoculating culture tubes or making sputum smears the spattering occasioned by
flaming the platinum loop in the Bunsen burner flame has long been recognized as undesir-
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VOLUME I NUMBER 6
JOURNAL
OF
BACTERIOLOGY
OFFICIAL ORGAN OF THE SOCIETY OF AMERICAN
BACTERIOLOGISTS
NOVEMBER, 1916
It is characteristic of Science and Progress that they continually
open new fields to our vision. — Pasteur
PUBLISHED BI-MONTHLY
WILLIAMS & WILKINS COMPANY
BALTIMORE, U. S. A.
THE CAMBRIDGE UNIVERSITY PRESS
FETTER LANE, LONDON; E. C.
Entered as second-claas matter April 17, 1916. at the Post Office at Baltimore, Maryland, under the
Act of March 3, 1879.
Bacteriological Pepton
Fairchild Building
Washington and Laight Sts.
New York
Fairchild Bros. & Foster
Offer to the bacteriologist a Pepton
which is perfectly serviceable for the for-
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employed in the usual proportions and for
whatever purposes pepton of this most
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It may be mentioned that this product
is offered at a price which is intrinsically
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quality of this pepton for all bacteriological
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Pepton, Fairchild
Pepton, Fairchild, is put up in 30 gram
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We shall be pleased to send a 30 gram
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FAIRCHILD BROS. & FOSTER
COPE EXTENSION SCOOP
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This permits the sample to be weighed and introduced into the flask
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SCIENTIFIC MATERIALS CO., Pittsburgh, Pa.
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. PARKER HITCHENS
Glenolden, Pa.
C. C. Bass
R. E. Buchanan
P. F. Clark
H. \V. Conn
F. P. Gay
F, P. Gorham
S. H. Aters
F. Bachmann
D. H. Bergey
O. Bbrghausen
V. Birckner
C. P. Brown
P. E. Brown
H. J. Conn
M. W. Cook
J. T. Emerson
Advisory Editors
F. C. Harrison
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
Chief Abstract Editor
G. H. Smith
Abstract Editors
L. W. Famulenbr
C. P. Fitch
D. Greenberg
P. B. Hadley
I. C. Hall
T. L. Harkey
C. M. Hilliard
J. G. Hopkins
T. G. Hull
A. Itano
F. M. Johns
I. J. Kligler
J. A. Kolmer
H. L. Lang
H. W. Lyall
W. J. MacNeal
E. C. L. Miller
E. H. Nollau
Zae Northrup
L. Pearse
W. T. Sedgwick
F. L. Stevens
A. W. Williams
H. Zinsser
E. B. Phelps
G. H. Robinson
W. Sadler
F. L. Steven.s
F. W. Tanner
R. M. Taylor
A. R. Ward
B. White
CONTENTS
R. E. Buchanan. Studies in the Nomenclature and Classification of Bacteria. The
Problem of Bacterial Nomenclature 591
T. J. Murray. The Oxygen Requirements of Biological Soil Processes 597
Raymond A. Kelser. The Preparation of Culture Media from Whole Blood 615
Max Levine. Preliminary Note on the Classification of Some Lactose Fermenting
Bacteria 019
Myrtle Greenfield. A New Ice Sampler 62,3
George D. Horton. Apparent Recovery of a Hen Infected with Baci'lary White
Diarrhea. ("As Determined by the Macroscopic Agglutination Test) 625
Edgard Zunz and Paul Gyorgy. Observations sur I'lnfluonce Chimique des Milieux de
Culture sur le Developpement et la Production de I'lndol par les Coli-Bacilles et par
les Bacilles Typhiques 627
I. J. Kligler. Some Regulating Factors in Bacterial Metabolism 663
Book Review. Kolmer's Practical Textbook of Infection, Immunity and Specific
Therapy. 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
INFORMATION FOR CONTRIBUTORS AND SUBSCRIBERS
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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STUDIES IN THE NOMENCLATURE AND CLASSIFICA-
TION OF BACTERIA
THE PROBLEM OF BACTERIAL NOMENCLATURE^
R. E. BUCHANAN
From the Bacteriological Laboratories, Iowa State College, Ames, Iowa
Received for publication, July 12, 1916
Erwin F. Smith in the chapter on "Nomenclature and Classifi-
cation" in the first volume of his work on Bacteria in Relation
to Plant Diseases (1905), accepts as valid 33 different names of
bacterial genera. In addition he presents a list of 156 generic
names which he regards as definitely invalid or inappropriate
and to be rejected. In the preparation of material for a course
in systematic bacteriology given at Iowa State College for sev-
eral years, I have had occasion to use this list, and have added
to it. It appears that about 300 generic and pseudogeneric
names have been used by bacteriologists. In addition, about
100 names have been used for orders, classes, famihes, sub-
families, tribes, and subtribes. The problem as to which of
these names are to be regarded as valid, and which invalid, has
been increasingly emphasized as one of considerable importance,
and intimately associated with the development of a satisfactory
classification of the bacteria.
To state that the classification of bacteria is in a chaotic
condition is to express a truism. That this is due to the inherent
difficulties in determining bacterial relationships, and to the
utter disregard of all rules of nomenclature is likewise generally
accepted. Is there any need of action?
Of recent years some bacteriologists have apparently taken
delight in ignoring the well estabhshed customs of biological
nomenclature, and in creating special rules to fit occasions. In
^ Presented at Seventeenth Annual Meeting of the Society of American Bac-
teriologists, Urbana, 111., December 28, 1915.
591
592 R. E. BUCHANAN
this they seem in many cases to have been aided and abetted
by om- technical and scientific periodicals and their editors,
particularly those in the medical or medico-scientific field.
Occasionally an effort is apparently made to depart as far as
practicable from good usage. For example, it is customary in
both botanical and zoological periodicals and generally in the
literature of these sciences to treat the name of a genus as a
proper name, and to capitalize it at least when used with a
specific name. The rule reads, "Genera receive names, sub-
stantives in the singular number and written with a capital
letter." A perusal of technical and medical journals shows the
rule to be commonly ignored. This fact in itself is probably
not of great importance, but is a symptom of a deep seated
trouble. Everywhere we find disregard of law and precedent,
and eveiywhere the loose thinking and writing which are the
consequence.
The whole subject of formal nomenclature, notwithstanding
current lack of interest, is of great importance from the stand-
point of bacteriologists, sanitarians, hygienists, physicians and
pathologists. Our present system, or lack of system, leads to
inaccuracies, misconceptions, and misstatements. We can not
long continue to violate the principle that for every kind of liv-
ing thing there shall be a single valid name, without causing
confusion.
The subject of nomenclature is in part distinct from that of
classification. The latter deals with methods and criteria, of
use in the differentiation of groups from each other, the former
has for its function the determination of the kind of a name that
shall be applied to a particular group, and the validity and
suitability of names that have already been apphed.
The efforts of the Society of American Bacteriologists have
been directed in the main in the past to the problems of differ-
entiation of kinds and groups of organisms, as witnessed by its
descriptive chart. It is time that it should bestir itself concerning
the labels it is to place on the groups which it is learning to sepa-
rate. It is probably safe to state that practically every other
branch of biological science has left us in the rear in this matter.
NOMENCLATURE AND CLASSIFICATION OF BACTERIA 593
Perhaps our fundamental difficulty is to know whether we,
as bacteriologists, are zoologists or botanists. Both of these
groups of scientists have formulated and revised from time to
time in international congresses, elaborate codes of nomenclature,
not perfect perhaps, but helpful and necessary. A study of the
WTitings of protozoologists, helminthologists, and those dealing
with microscopic forms of Ufe definitely on the zoological side
of the fence will show that they have appUed their code with a
considerable degree of satisfaction; information in regard to their
subjects can, in consequence, be quite satisfactorily systema-
tized. The algologists, mycologists, and most of the botanists,
have likewise worked out their schemes of classification in ac-
cordance with definite rules. The bacteriologists on the other
hand have thus far failed to agree on any scheme of classi-
fication, and many are rebellious against any of the restraints
imposed by formal nomenclature. Frequently nomenclature and
classification have been confused in our minds. We have been
unable in many cases to define what is meant by the term bac-
terial species, we hesitate to give a name to that which we can
not accm-ately define. But it is equally true that species have
not been accurately delimited in the older biological sciences,
and this fact has not interfered with at least tentative arrange-
ments of genera and higher groups, nor with the appHcation of
correct names.
A careful search of our hterature fails to show that any group
of bacteriologists has thus far formally agreed upon any code
or system of nomenclature. Inasmuch as the bacteria are to
be regarded as plants, the natural suggestion is that we should
follow the botanists' code. But within recent years bacteri-
ologists have shown a tendency to term themselves microbiolo-
gists in order to make it evident that a portion of the zoological
field is to be covered as well. Furthermore there is no perfect
accord as yet as to where certain microorganisms belong; forms
such as spirochetes, which certainly are within the province of
bacteriology, may be regarded as either animals or plants.
A careful study of the botanical and zoological codes will
show them to be very similar in most essential characteristics.
594 K. E. BUCHANAN
The botanical code is less rigid, and probably has something
more of ambiguity in some of its statements than does the
zoological. Logically it would seem that we should follow the
botanical code with the bacteria and the zoological code with
the protozoa.
The questions at once arise:
Are these codes really applicable to the nomenclature of
microorganisms? Have they not been planned with higher
groups of plants and animals in mind?
A careful study of the provisions of either code will show that
there is no inherent difficulty in application to the lower forms
of life. In the botanical code there are perhaps, one or two
rules which the bacteriologists might be reluctant to accept,
particularly the rule that all new species of plants (in our case,
bacteria) to be recognized as valid, must be pubhshed with a
Latin diagnosis. Furthermore in both botanical and zoological
codes there is a rule that no two genera (or other groups) can
exist with the same name, and that duplicate genera in plant
and animal kingdoms should be avoided as far as possible, but
that such duplicates are not invalid. In other words, two
plants can not have the same name, nor can two animals, but a
plant and an animal may be named alike. It would appear
that for the sake of the microbiologist there might be appended
to each code the rule that in those groups whose position in
plant or animal kingdom is in doubt there should exist no dupli-
cate names.
At the last botanical congress held in 1910, certain points
relative to bacterial nomenclature were definitely referred to a
congress which was to have been held in the summer of 1915,
but which was deferred because of the war. At this congress
two points of interest to bacteriologists were to have been taken
up: 1, the determination of the time or point of departure in
nomenclature of the Schizomycetes, and 2, the adoption of a
list of genera conservanda.
In preparation for this congress Vuillemin (1913), published
a paper in which he discussed bacterial classification and nomen-
clature. He concludes that the best thing to do is to determine
NOMENCLATURE AND CLASSIFICATION OF BACTERIA 595
which generic names are valid, prepare such a list, publish the
names as genera conservanda, and date all bacterial classification
from 1915 when the list should have been adopted by the con-
gress. He comes to the conclusion that all the true bacteria
should be included in the following genera: Planococcus, Strepto-
coccus, Klebsiella, Merista, Planomerista, Neisseria, Sarcina,
Planosarcina, Metabacterium, Clostridium, Serratia, Bacterium
and Spirillum.
A study of Vuillemin's paper, despite his criticism of the
bacteriologists as taxonomists, shows that he himself does not
formulate tenable bases for differentiation of genera, and his
reasons for choosing certain generic names and abandoning others
will scarcely withstand critical analysis.
But after all, is a scientific classification of the bacteria impor-
tant and desirable? Allow me to quote from a paper pubhshed
about two decades ago by H. Marshall Ward. He says:
The only really valid objection to a purely scientific classification is
the old objection of the purely utilitarian "practical" man; and even
there the objection is relative. This leads me to bring out the point
that the bacteriologists in the widest sense of the word, are really
looking at the question of classification from at least two very different
points of view: On the one hand, we have the botanists, who direct
their attention to the organism, the Schizomycete itself, as a biological
phenomenon to be examined and reported upon as thoroughly as
possible, for them no classification is complete which does not record,
or (which amounts to the same thing) imply in its records, all of the
life phenomena of the organism including its pedigree.
On the other hand, we have the pathologists, hygienists, brewers,
chemists, etc., who regard the organism simply as an object to be
named for convenience in reference, because it brings about certain
changes in the tissues, waters, and other media which they are more
specially concerned with. They do not care, and naturally so, what
vagaries the organism exhibits, so long as they can recognize it when
they meet with it. As a matter of experience, however, it is just these
vagaries that bring about the sources of error which beset them on all
hands, and hence they are equally interested with the botanist in
having them cleared up and explained. When we come to the con-
clusion that, whatever may be believed to the contrary, the real
interests of "bacteriologists" of all kinds are identical.
596 R. E. BUCHANAN
Are we yet ready to make a real, systematic, effort to classify
and name the bacteria? As a society we are committed to the
idea that we are ready, as shown by our possessing a committee
on classification. It would seem that just now the time is pro-
pitious for the careful formulation of general rules of bacterio-
logical nomenclature and of a scheme of bacterial classification.
What can we do? Much, it would appear, through the aid
of the committee on classification or a new committee on nomen-
clature or both.
We can ask that the committee make a careful study of the bo-
tanical, and perhaps, too of the zoological codes of nomencla-
ture. They could then report what changes, if any are neces-
sary to make either serve as a working scheme for bacteriology.
They could make recommendations as to the date of departure
for bacterial classification.
They could report on the historical validity of the names
that are used for bacterial groups, particularly genera, and
determine their type species, and adequate diagnoses.
They could prepare a list of recognized generic names, such
as that maintained in the check list of birds by the ornithologists
of this country.
They could seek the active cooperation of committees of other
societies interested in like problems in this country, and as
rapidly as practicable, work with similar organizations in other
countries.
They could prepare a set of resolutions for the next inter-
national botanical congress that would doubtless contribute
greatly to the solution of the problem of nomenclature and
classification among the bacteria.
And lastly, they might with profit give some careful thought
to the preparation of a classification of the chemical changes
brought about by microorganisms. It seems that we are fated
to hear and read discussions of azofication, ferrification, etc.
Could not a system etymologically sound be worked out for
such terms in the interest of uniformity?
REFERENCE
VuiLLEMiN. 1913. Genera Schizomycetum. Annales Mycologici. 11, 512-527.
THE OXYGEN REQUIREMENTS OF BIOLOGICAL SOIL
PROCESSES^
T. J. MURRAY
Deparlment of Plant Pathology and Bacteriology, Virginia Polytechnic Institute,
Blacksburg, Virginia
Received for publication, June 12, 1916
The aim of this work was to find out whether the fundamental
processes carried on by soil bacteria proceed better under aerobic
or anaerobic conditions.
The work was carried out with three soils, first a greenhouse
loam soil, rich in organic matter, second a field soil from the
experiment station plats, — a Hagerstown silt loam, and third, a
clay soil — a Hagerstown clay taken from the side of a hill slop-
ing down to a brook. These were selected to represent three
different types of soil and to secure different flora and different
conditions of microbic development.
The biological processes in soil are influenced according to
Lipman (1911) by moisture, temperature, aeration, reaction, and
food supply. It seems to the writer that the relation of oxygen
to the fundamental soil processes has not been thoroughly in-
vestigated; and that it has not been fully established — taking
soil or synthetic solutions as media — whether nitrogen fixation,
nitrification, ammonification and denitrification will go on under
aerobic or anaerobic conditions only, and whether these processes
will take place better in the presence or in the absence of air.
Preliminary experiments showed that ammonification, deni-
trification, and nitrogen fixation took place readily with or with-
out air. Nitrification on the other hand would not take place
under anaerobic conditions, either in soils or in solution in the
preliminary or subsequent experiments. The results of the work
on nitrification are therefore not included in this paper.
1 Paper No. 45 from the Laboratories of Plant Pathology and Bacteriology,
Va. Agr. Exp. Sta.
597
598 T. J. MURRAY
AMMONIFICATION
Kelley (1915) found that anaerobic conditions greatly retard
the formation of ammonia from all materials except casein and
under anaerobic conditions the formation of ammonia has usu-
ally been found to be considerably less than under aerobic con-
ditions. Aeration (Kelley, McGeorge and Thompson, 1915)
stimulates ammonification but is not essential to the process as
it is to nitrification.
Lohnis and Green (1913) found that aeration is of great
importance in the ammonification of organic matter. "The
most significant cause of variation appears to be that of aera-
tion. Ammonification as a whole proceeds much more rapidly
under aerobic than under anaerobic conditions, but it is believed
that aerobic conditions favor more specifically those latter stages
in the breakdown, which result in the formation of ammonia
itself." Later (Lohnis and Green, 1914) they report that while
aeration is not of preponderating importance ammonification of
such substances as flesh meal, bone meal and blood meal pro-
ceeds better under aerobic than anaerobic conditions.
In my experiments the process of ammonification was tested
both in soils and in solution. One hundred grams of soil of
the different varieties was inoculated with 1 gram of the am-
monifiable substance. Blood meal sterilized with CS2 and sterile
casein (Brown, 1913) solution were used. For solution work,
Dunham's solution (1 per cent peptone plus 0.5 per cent salt)
and urea bouillon (nutrient bouillon and 1 per cent urea) were
used. In each case an easily and a less readily ammonified sub-
stance were used, casein and urea being easily ammonified.
Anaerobic conditions were obtained with the soils by placing
them under a bell jar in a somewhat larger dish with pyrogallic
acid and caustic soda solution. The pyrogallic acid was placed
in the bottom of the dish, the soil in tumblers, and the bell jar
put on, the caustic soda solution added, and then some paraffin
oil. The bell jar was hfted slightly to let the caustic soda come
in contact with the pyrogallic acid. The oil prevented the mix-
ture from absorbing oxygen from the atmosphere. In solution
BIOLOGICAL SOIL PROCESSES
599
anaerobic conditions were obtained by adding one inch of sterile
paraffin oil. The materials were incubated a week at 30° C.
and the ammonia determined by distillation with MgO.
Experiment I. Ammonification of blood meal and casein under aerobic and
anaerobic conditions in soils. Ammonia in milligrams per 100 grams of soil.
Casein
SOIL
AEBOBIC
ANAEROBIC
Greenhouse
90.10
78.05
71.25
97 26
Loam
77 46
Clay
43 18
Blood meal
Greenhouse
Loam
21.42
21.08
10.88
20.70
11 90
Clav
7 60
Experiment II. Ammonification in sterile soil by mass cultures under aerobic
and anaerobic conditions. Ammonia in milligrams per 100 grams of soil.
Casein
Greenhouse.
Loam
Clay
96.39
82.96
91.29
ANAEROBIC
95.61
81.26
81.94
Blood meal
Greenhouse
Loam
98.94
70.72
8.89
105.23
87.72
Clay
14.62
In experiment II the mass cultures were obtained by inocu-
lating Lipman's synthetic media (Lipman and Brown 1911)
with soils of the different types and growing one week at 30° C.
The soil was sterilized in the autoclave. Twenty cubic centi-
meters of this mass culture were added to each 100 grams of
soil.
It is at once apparent that ammonification in soil proceeds
just as readily under anaerobic conditions as it does under
600
T. J. MURRAY
aerobic conditions. The process in general proceeds better in
richer soils. It is best with the greenhouse type and poorest
with the clay. Casein is more easily broken down than blood
meal. Blood meal is ammonified to a greater extent under
aerobic and anaerobic conditions in sterile inoculated soil than
in fresh soil. The soils kept under anaerobic conditions gave a
strong disagreeable odor, showing that other processes were
going on. This was not noticeable with the soils kept under
aerobic conditions.
Experiment III. Ammonification in solution. Ammonia in milligrams per 100
cc. of solution.
Urea bouillon
SOIL
AEROBIC
ANAEROBIC
Greenhouse
Loam
554.71
448.75
553.69
679.66
687 65
Clay
684 22
Dunham's solution
Greenhouse
136.85
134.30
147.90
141 78
Loam
125 12
Clay
148 24
One hundred cubic centimeters of the solutions in 250 cc.
Erlenmeyer flasks were inoculated, respectively with 2 grams of
soil of each type. One inch of sterile paraffin oil was added to
half of them, the other half being kept under aerobic conditions.
Ammonification in solution goes on under anaerobic conditions
as well as under aerobic conditions. The urea is more easily
ammonified than the peptone. The ammonification of urea pro-
ceeds better under anaerobic conditions than under aerobic con-
ditions. The ammonification of peptone proceeds just as readily
in the presence as in the absence of air. Larger amounts of
ammonia were formed in solution than in soil.
It was thought that an excess of air might inhibit or increase
the amount of ammonia formed. A preliminary experiment was
carried out by bubbling washed air through 100 cc. of Dunham's
solution inoculated with 2 grams of garden soil (greenhouse).
BIOLOGICAL SOIL PROCESSES
601
Air was bubbled through sterile water, then through the Dun-
ham solution, and then through 50 cc. of nr H2SO4, colored with
methyl red, by means of a water air pump. Any ammonia
that was drawn across in the process was caught in the acid.
As the acid lost color, more acid was added.
Milligrams of ammonia formed per 100 cc. Dunham's solution
No. I. Excess air 120.53
No. II. Air 155.72
No. III. Without air (oil) 163.20
From these data it would appear that ammonification pro-
ceeded best without air, next with air and least with an excess
of air.
Experiments were carried out with urea bouillon (no pep-
tone) and Dunham solution with the bacteria of the three types
of soil, under anaerobic, aerobic and excess aerobic conditions.
Experiment IV. Ammonification in solution. Milligrams ammonia per 100 cc.
of solution.
Urea bouillon
SOIL
EXCESS AIR
AIR
WITHOUT AIR
Greenhouse
Loam
Clay
357.85
302.94
291.29
313.48
306.51
271.53
570.80
563.04
550.80
Dunham solution
Greenhouse
Loam
Clay
130.17
145.71
55.11
149.36
148.89
93.31
161.23
154.17
137.17
It is again noticeable that urea is more readily ammonified
than peptone. Ammonification proceeds best under anaerobic
conditions. The process seems to proceed equally well with air
or with an excess of air. With urea bouillon the production of
ammonia is slightly higher with an excess of air than under
ordinary air conditions. With Dunham's solution the produc-
tion of ammonia is slightly higher without an excess of air.
602 T. J. MURRAY
Some further experiments with pure cultures were carried out.
A pure culture was isolated from an ammonified urea solution
and grown on urea agar under anaerobic conditions. A very-
simple method was devised for anaerobic plate work. It con-
sisted in adding sterile paraffin oil to agar that had been cooled,
inoculated and poured. Oil was added to the level of the rim
of the plate. This avoided the use of the anaerobic jar and
proved very effective. No spreading colonies were observed,
and colonies were as well isolated as on an aerobic plate. The
plates may be removed from the incubator and examined for
growth at any time. This is a decided advantage over the
anaerobic jar method. The oil may be poured off the plate and
the colonies exposed for further study.
The organism isolated by this method was a diplo-bacillus.
It would not grow on nutrient agar under aerobic or anaerobic
conditions. It grew very well on urea agar under aerobic and
anaerobic conditions, although it had been isolated purely by
anaerobic technique.
Cultures of this organism wete inoculated into 100 cc, respec-
tively of urea bouillon (no peptone), urea solution (glucose, 10
per cent, K2HPO4 5 per cent, MgS04 0.05 per cent, urea 1 per
cent), and Dunham solution. These inoculated flasks were
placed under aerobic and anaerobic conditions.
Experiment V. Ammonificaiion in solution by pure culture. Ammonia in
milligrams per 100 cc. of solution.
MEDIA
AEROBIC
ANAEROBIC
Urea bouillon
308.69
130.63
530.53
Urea solution
Dunham solution
79.31
In this experiment urea bouillon seemed to be the best me-
dium. Ammonification with this material proceeded better under
anaerobic conditions. The reaction did not take place with
Dunham's solution. It did not proceed as well with the urea
solution as with urea bouillon, although the process went on
under aerobic and anaerobic conditions.
BIOLOGICAL SOIL PROCESSES
603
Some further experiments were carried out with pure cultures
of B. mycoides and B. subtilis freshly isolated from the soil.
Experiment VI. Ammonification in solution by B. mycoides and B. subtilis.
Ammonia in milligrams -per 100 grams of solution.
Urea bouillon
OROANISM
AEROBIC
ANAEROBIC
B. mycoides
B. subtilis
42.46
41.28
62.19
62.67
Dunham solution
B. mycoides
B. subtilis
35.71
9.95
14.37
6.07
These two facultative anaerobes not only live under anaerobic
conditions, but carry out their activities as well. Urea bouillon
is more easily ammonified than the Dunham solution by these
organisms. More ammonia is produced under anaerobic con-
ditions with urea and less with peptone.
In general, from a perusal of the preceding experiments, it
appears that ammonification of the substances tested under
laboratory conditions, proceeds readily under aerobic or anaero-
bic conditions in mass cultures using soil as a medium or in media
inoculated with soil or when pure cultures isolated from the soil
are used.
The ammonification of blood meal and casein proceeds as well
under anaerobic as under aerobic conditions in the soil. The
same is true of ammonification in solutions of urea and peptone.
More urea is, however, broken down under anaerobic conditions.
Excess air bubbled through inoculated liquid media does not
inhibit the production of ammonia, although less ammonia was
produced with Dunham's solution under these conditions than
under ordinary air conditions. Pure cultures of B. mycoides
and B. subtilis readily form ammonia under anaerobic condi-
tions. More ammonia is however produced from urea under
anaerobic conditions by these organisms.
604 T. J. MURRAY
NITROGEN FIXATION
The ability of microorganisms to fix atmospheric nitrogen was
first definitely demonstrated to be due to an anaerobic bacillus,
B. Pasteurianus, in 1893 by Winogradski. It remained for
Beyerinck in 1901 to demonstrate an aerobic organism that also
assimilated free nitrogen. Non-symbiotic fixation of nitrogen
in the soil is due to both types of organisms but at times may
be due only to one type. Lipman and Burgess (1915) found
two-thirds of the soils examined by them free from Azotobacter.
Yet these soils were capable of fixing nitrogen when inoculated
into solutions. They ascribed the nitrogen fixation to Clostri-
dium forms. Haselhoff and Bredemann (1906) investigated an-
aerobic nitrogen-collecting bacteria and found results approxi-
mating those of Winogradski. The amount of nitrogen fixed
varies with the amount of carbonaceous matter present, the more
carbon the higher the assimilation. Working with pure and
mixed cultures, they found from 0.42 to 2.74 mgm. of nitrogen
fixed per gram of mannite. Lipman (1908), working with pure
cultures of Azotobacter, found from 0.39 to 10.45 mgm. of nitrogen
per gram of mannite formed in four weeks in mannite solution.
In this work mass cultures were used, either by inoculating solu-
tion with soil or by adding the source of carbon to the soil.
In my work nitrogen fixation was carried out in soils and in
solution. For the solution work, 100 cc. of a nitrogen poor
medium was inoculated with from 2 to 5 grams of soil. The
following solution (N. J. 1908) was used:
H2O 1000 . 0 grams
K2HPO4 0.2 gram
MgS04 0.2 gram
NaCl 0.5 gram
Mannite 20.0 grams
FeCla 1 drop of 10 per cent solution
The solution was neutralized with KOH using phenolphthalein
as an indicator. Anaerobic conditions were obtained by adding
about an inch of sterile paraffin oil or by placing the material
in the anaerobic apparatus described under the ammonification
experiments.
BIOLOGICAL SOIL PROCESSES
605
For nitrogen fixation in soils, the three types of soil used for
ammonification were again studied. One gram of mannite was
added to 100 grams of soil in a beaker. Anaerobic conditions
were again obtained by the absorption of oxygen with pyrogallic
acid and caustic soda solution.
The materials were incubated at about 30° C. for twenty-one
days and then the total nitrogen was determined by the modified
Gunning method, (Hibbard, 1910). Blanks were run at the
beginning and the difference between these blanks and the total
nitrogen at the end of twenty-one days gives the amount of
nitrogen fixed.
Experiment VII. Nitrogen fixation in solution. Nitrogen in milligrams per
100 cc. of solution.
Aerobic
SOIL
NITROGEN AT END
NITROGEN AT
BEGINNING
GAIN
Greenhouse
Loam
Clay
12.00
7.60
5.60
9.38
3.75
1.74
2.62
3.85
3.86
Anaerobic
Greenhouse
Loam
12.60
6.30
5.10
9.38
3.75
1.74
3.22
2 55
Clay
3.36
In this experiment 100 cc. of the solution contained in 250
cc. Erlenmeyer flasks was inoculated with 5 grams of the soils
of the different types. About one inch of sterile paraffin oil
was added in order to insure anaerobic conditions.
All of the soils used are capable of fixing nitrogen under both
aerobic and anaerobic conditions. The greenhouse type of soil
is richer than the loam and the loam richer than the clay in
total nitrogen both at the beginning and at the end of the experi-
ment. The actual increase in nitrogen does not vary much with
any of the three soils. Lipman and Burgess (1915) also noticed
this and remarked in a conclusion that as a rule a high nitrogen
content in the soil seems to mitigate against a vigorous nitrogen
fixation. The nitrogen fixation seems to proceed as readily with
or without the presence of air in this experiment.
606
T. J. MURRAY
In the following two experiments nitrogen fixation was carried
out in solution under anaerobic conditions by placing narrow
bottles containing the mannite solution, inoculated with 5 grams
of soil, in the anaerobic apparatus and absorbing the oxygen
with pyrogallic acid and caustic soda solution. In one case thin
bottles containing a solution in which denitrification was going
on, were added.
Experiment VIII. Anaerobic nitrogen fixation in solution,
grams per 100 cc. of solution.
A
Nitrogen in milli-
Greenhouse
Loam
Clay
Greenhouse
Loam
Clay
NITROGEN AT END
11.34
7.00
8.68
NITROGEN AT
BEGINNING
9.38
3.75
1.74
1.96
3.25
6.94
B
5.62
11.38
4.58
In Series B denitrification was going on in the same apparatus
in three other samples. The nitrogen fixing solution was inocu-
lated with 2 grams of the soils. There was more nitrogen fixed
in B, probably due to the fact that nitrogen was being con-
tinually given off in the denitrification experiments. This might
indicate that the more nitrogen present the more nitrogen is fixed.
It may be that as denitrification takes place in the soil some
part of this nitrogen may be again fixed immediately. Except
in the last experiment anaerobic conditions do not seem to favor
the production of more nitrogen than is produced under aerobic
conditions in solutions. Nitrogen fixation in solution proceeds
as well with or without the presence of air.
Further experiments were carried out in soils by adding 1
gram of mannite per 100 grams of soil. Anaerobic conditions
were obtained with pyrogallic acid and caustic soda solution.
The total nitrogen was determined in 10 gram samples at the
beginning and end of the experiment.
BIOLOGICAL SOIL PROCESSES
607
Experiment IX. Nitrogen fixation in soils. Nitrogen in milligrams per 10
grains of soil.
Aerobic
SOIL
NITROGEN AT END
NITROGEN AT
BEGINNING
GAIN
Greenhouse
27.16
12.46
5.18
26.32
9.38
4.34
0 84
Loam
3 08
Clay
0 84
Anaerobic
Greenhouse.
Loam
Clay
8.50
5.29
4.69
In these experiments greater amounts of nitrogen are fixed
under anaerobic conditions than under aerobic conditions. There
is a gradation shown under anaerobic conditions, most nitrogen
being fixed by the greenhouse soil, less by the loam and least
by the clay.
In the following experiment soil was sterilized in the autoclave
and mass cultures grown under aerobic and anaerobic conditions
in mannite solution were added.
Experiment X. Nitrogen fixation in sterile soil. Nitrogen in milligrams per
10 grams of soil.
Aerobic
Greenhouse.
Loam
Clay
NITROGEN AT END
24.67
12.50
5.60
NITROGEN AT
BEGINNING
26.32
9.32
4.34
LOSS OH GAIN
-1.65
3.10
1.26
Anaerobic
Greenhouse.
Loam
Clay
26.32
1.54
9.38
2.10
4.34
.70
The number of bacteria per gram of soil, capable of growing
on nitrogen poor media, was estunated on three different media
under aerobic and anaerobic conditions. The following media
were used:
608
T. J. MURRAY
Mannite agar
KjHP04 0.2
MgS04 0.2
NaCl 0.5
Mannite 20.0
H2O 1000.0
Agar 15.0
FeCU, 1 drop 10 per cent
Neutralized with KOH.
Ashby agar
K2HPO4 0.2
MgS04 0.2
NaCl 0.2
CaS04 0.2
CaCOs 5.0
H2O 1000.0
Agar 15.0
Winogradski agav
K2HPO4.... 1.00 gram
MgS04 3.00 grams
NaCl 0.01 gram
MnS04 0.01 gram
CaCOa 10.00 grams
FeCl3.2 drops 10 per cent
H2O 1000 grams
ir 15 grams
The soil was plated and the plates were incubated at 30° C.
under aerobic and anaerobic (pyrogallic acid and caustic soda)
conditions for seven days and then counted.
Experiment XI. Number of bacteria per gram on nitrogen-poor media.
Aerobes
son.
MANNITE AGAR
WINOGRADSKI
AGAR
A8HBT AGAR
Greenhouse
Loam
Clay
26,000,000
1,150,000
40,000
29,000,000
1,000,000
21,000
4,000,000
1,190,000
32,000
Anaerobes
Greenhouse
1,050,000
240,000
11,800
1,020,000
240,000
9,600
310,000
Loam
190,000
3,500
Clay
Greater numbers of bacteria develop under aerobic than an-
aerobic conditions. There are more aerobic than anaerobic bac-
teria capable of growing on nitrogen-poor media. The number
of bacteria varies with the type of soil, the greatest number
being present in the greenhouse type and fewest in the clay.
Two cultures of Azotobacter were picked from the aerobic
plates and two cultures were picked from the anaerobic plates.
These cultures were inoculated into 100 cc. of nitrogen poor
media, — Mannite solution, Winogradski solution and Ashby solu-
tion (same composition as the agars without the agar). The
two cultures taken from the anaerobic plates were kept, with
and without, oil. All inoculated material was incubated twenty-
one days at 30°C.
BIOLOGICAL SOIL PROCESSES
609
Experiment XII. Nitrogen fixation in solution by pure cultures. Nitrogen in
milligrams per 100 cc. of solution.
Aerobes
MEDIA
CULTURE NUMBER
NITROGEN FIXED
Mannite
1
2
1
2
1
2
2 02
Ashby
1.86
3 23
Winogradski
1.86
3 23
3.39
Anaerobes (oil)
Mannite
Ashby
Winogradski
3
4
1.75
3
1.90
4
1.75
3
2.02
4
2.21
Experiment XIII. Nitrogen fixation in solution by pure cultures. Nitrogen
in milligrams per 100 cc. of solution.
Anaerobes
Mannite
Ashby
Winogradski
CULTURE NUMBER
NITROGEN FIXED
UNDER OIL
1.75
1.90
1.75
2.02
2.21
NITROGEN FIXED
WITHOUT OIL
2.63
2.63
2.32
2.39
7.21
3.54
More nitrogen is fixed by the anaerobic organisms. More nitro-
gen is fixed by the anaerobic organisms when inoculated into
media with no oil added to insm-e anaerobic conditions than when
oil is added. In the last case more nitrogen is fixed by the anae-
robes without oil than is fixed by the aerobes. The Winogradski
medium seems to be the best for nitrogen fixation by pure cul-
tures. More nitrogen is fixed in Winogradski media than in
the other two materials used.
610 T. J. MURKAY
In general, fixation proceeds better in soils than in solution,
more nitrogen being fixed in soils. The nitrogen fixed per gram
of mannite is higher with the soils than in solution. The greater
amount of nitrogen fixed in soils may be due of course to other
forms of energy in the soil in the shape of decomposed plant
tissue. Nitrogen is fixed readily under aerobic or anaerobic
conditions in solutions. In soils, nitrogen fixation proceeds better
under anaerobic conditions.
DENITRIFIC ATI ON
Broadly speaking, denitrification is the breaking down of
nitrates to nitrites and ammonia and the liberation of free
nitrogen. More narrowly it includes only the latter phase, the
liberation of free nitrogen by microorganisms acting on nitrates
or nitrites. It is with this latter phase that my experiments
were concerned. The importance for agriculture of denitrifica-
tion in the soil has been greatly exaggerated. It is important
if large amounts of fresh manure are added to soil rich in nitrates,
but not otherwise.
Denitrification is carried out by a variety of microorganisms,
chief among which are B. denitrificans, B. pyocyaneus, B. fluo-
rescens-liquefaciens, and B. Hartlebii. Lipman (1902) found
from 1.3 per cent to 25.6 per cent loss in nitrogen with pure
cultures and as high as 35 per cent loss with mixed cultures. He
also states that denitrifying organisms are found in all soils.
These denitrifjdng organisms live in the presence of air but may
live anaerobically.
Koch and Pettit (1910) found that denitrification varies with
the moisture present. With an increase in moisture there is an
increase in denitrification, and as the moisture is increased under
laboratory conditions there is an increase in the nitrogen lost.
Jensen (1909) has pointed out that denitrification is always
accompanied by oxidative processes.
My study of denitrification was carried out both in soils
and in solutions. For the solution work the following medium
was used — Giltay and Aberson's solution:
BIOLOGICAL SOIL PROCESSES
611
H2O 1000.00
KNO3 2.00
MgS04 2.00
Citric acid 5.00
K2HPO4 2.00
CaCl 0.20
NaaCOs 4.25
For the soil work, 100 grams of soil were inoculated with 0.2
gram of KNO3. Anaerobic conditions were again obtained with
sterile paraffin oil for the solution work, and by the absorption
of oxygen with pyrogallic acid and caustic soda for the soil work.
In the following experiment 200 cc. of Giltay and Aberson's
solution were inoculated with 2 grams of the soils of the three
types and then incubated for twenty-one days at 30° C. under
aerobic and anaerobic conditions.
Experiment XIV. Denitrification in solution. Nitrogen in milligrams per WO
cc. of solution.
Aerobic
SOIL
NITROGEN AT END
NITROGEN AT
BEGINNING
GAIN OR LOSS
Greenhouse
20.10
22.47
18.84
56.19
52.19
51.82
-36.09
Loam
Clay
-29.72
-32.98
Anaerobic
Greenhouse
20.10
16.82
16.74
56.19
52.19
51.82
-36.09
Loam
-35.37
Clay
-35.08
Denitrification seems to proceed better under anaerobic con-
ditions than under aerobic conditions. The difference is not
very marked, though noticeable. Again the process seems to
proceed best in the richest soil, probably due to the fact that
there are more bacteria of the denitrifying type present.
In the following experiment washed air was bubbled through
200 cc. of the solution for twenty-one days at 30°C.
612
T. J. MURRAY
Experiment XV. Denitrification in solution. Nitrogen in milligrams per 200
cc. of solution.
Greenhouse
Loam
Clay
NITROGEN AT KND
20.34
24.33
20.48
NITROGEN AT
BEGINNING
56.19
52.19
51.82
GAIN OR LOSS
-35.85
-27.86
-30.34
Bubbling air through the solution does not inhibit the libera-
tion of free nitrogen into the air by bacteria to any marked
extent. In every case there is less nitrogen lost when air is
bubbled through than under ordinary aerobic and anaerobic
conditions, but there is not enough difference to be of any marked
importance.
Loss of nitrogen under different conditions. Nitrogen in millig
of solution.
'•ams per 200 cc.
SOIL
EXCESS AIR
AIR
WITHOUT AIR
Greenhouse
-35.85
-27.86
-30.34
-36.09
-29.72
-32.98
-36.09
Loam
-35.37
Clay
-35.08
From a study of these figures it is evident in each case, with
each type of soil, that there is least nitrogen lost under excess
air conditions and most lost under anaerobic conditions.
In the following experiment denitrification was carried on in
solution (100 cc. of solution and 2 grams of soil) under anaerobic
conditions, in the anaerobic apparatus (pyrogalhc acid and
caustic soda) along with a nitrogen fixation experiment.
Experiment XVI. Denitrification in solution. Nitrogen in milligrams per 100,
cc. solution.
Greenhouse
Loam
Clay
NITROGEN AT END
11.76
12.88
16.24
NITROGEN AT
BEGINNING
28.10
26.09
25.91
GAIN OR LOSS
-16.34
-13.11
- 9.67
Most denitrification goes on in the greenhouse soil and least
in the clay.
BIOLOGICAL SOIL PROCESSES
613
Some further experiments were carried out with soil as a
medium. One hundred grams of soil were inoculated with 10
CO. of a 2 per cent solution of KNO3, and incubated twenty-
one days at 30°C. under aerobic and anaerobic conditions.
Experiment XVII. Denitrification in soils,
grams of soil.
Aerobic
Nitrogen in mi
lligrams per 10
SOIL
NITROGEN AT END
NITROOEN AT
BEGINNING
GAIN OB LOSS
Greenhouse
25.58
12.59
10.57
29.09
12.15
7.11
-3.51
Loam
0.44
Clay
3.46
Anaerobic
Greenhouse
18.05
13.05
11.31
29.09
12.15
7.11
-11.04
0.90
Clay
4.20
With the greenhouse type of soil, denitrification took place,
to a greater extent under anaerobic than under aerobic condi-
tions. With the other two types of soil there was no loss of
nitrogen. It is evident that soil as a medium does not give
as good results as the solutions in regard to denitrification.
In general, denitrification goes on under aerobic and anaerobic
conditions. An excess of air does not seriously inhibit the pro-
duction of nitrogen, although sHghtly less nitrogen is lost. The
process proceeds slightly better under anaerobic conditions, al-
though almost as much nitrogen is lost under aerobic conditions.
Denitrification proceeds better in solution than in soils, nitrogen
being lost only in the greenhouse type of soil and not in the
other two types.
REFERENCES
Brown, P. E. 1913 The effects of barnyard manure. Iowa Agr. Res. Bull.
13, 423^38.
Haselhoff and Bredermann 1906 Investigation on Anaerobic Nitrogen-Col-
lecting Bacteria. Experiment Station Record 18, 429.
HiBBARD, P. L. 1910 Notes on the determination of nitrogen by the Kjeldahl
method. Jour. Ind. and Eng. Chem., 2, 463.
614 T. J. MURRAY
Jensen, O. 1909 Die Hauptlinien des Natiirlichen Bakteriensystems. Centr.
f. Bakt., Abt. II, 22, 314.
Kelley, W. p. 1915 The biochemical decomposition of nitrogenous substances
in soils. Hawaii Agr. Exp. Sta. Bulletin 39, 1-25.
Kelley, W. p., McGeorge, W., and Thompson, A. R. 1915 The soils of the
Hawaiian Islands. Hawaii Agr. Exper. Sta. Bulletin 40, 1-35.
Koch, A. and Pettit, H. 1910 Uber den verschiedenen Verlauf der Denitrifi-
kation im Boden und in Flussigkeiten. Centralbl. fiir Bakt. Abt.
II, 26, 335-345.
LiPMAN, J. G. 1902 Contribution to the morphology and physiology of deni-
trification. N. J. Agr. Exp. Sta. Report for 1902, 183.
LiPMAN, J. G. 1908 Azotobacter studies. N. J. Agr. Exp. Sta. Report for 1908,
138.
LiPMAN, J. G. 1911 Microorganisms as a factor in soil fertility, Marshall's
Microbiology, p. 227. Blakiston.
LiPMAN AND Brown 1910 Centralbl. fur Bakt., Abt. II, 25, 447.
LiPMAN, C. B. AND Burgess 1915 Studies on nitrogen fixation and Azotobacter
forms in soils of foreign countries. Centralbl. fiir Bakt., Abt. II, 44.
481-511.
LoHNis AND Green 1913 Methods in soil bacteriology VI. Ammonification
in soil and in solution. Centralblatt fiir Bakt., Abt. II, 37, 534.
LoHNis AND Green 1914 Methods in soil bacteriology VII. Ammonification
and nitrification in soil and in solution. Centralbl. fiir Bakt., Abt.
II,, 40, 457.
New Jersey 1908 Report, New Jersey Agricultural Experiment Station for
1908, p. 137.
THE PREPARATION OF CULTURE MEDIA FROM
WHOLE BLOOD
RAYMOND A. KELSER
From Pathological Laboratory, Bureau of Animal Industry, United States
Department of Agriculture
Received for publication, June 4, 1916
Several European workers (Szasz, 1915 a, 1915 b; Schmitz,
1916; Lichtenstein, 1916), have called attention to the fact that
it is possible to prepare from whole blood, a very satisfactory
medium for the propagation of bacteria, which while possessing
in some respects, advantages over media prepared from meat
infusions, can be produced at a cost very much less. It was
found that organisms which grew only sparsely or not at all on
the ordinary culture media, would in most instances grow luxuri-
antly on the medium prepared from blood.
Comprehending the economic importance of such a method,
and the advantage of being able to obtain a medium favoring
the growth of obstinate organisms, I have prepared and tested
several lots of this product with very pleasing results. The
method employed is as follows:
Fresh beef blood is obtained in a clean container (a covered
enamel bucket is a good receptacle) and allowed to clot. After
allowing it to remain in the refrigerator several hours to give
the serum a chance to separate, the clot is removed and finely
ground in a meat chopping machine. This ground material is
then replaced in the serum and the whole weighed. Two vol-
umes of distilled water are then added and the mixture placed
in an enamel pot and slowly brought to a boil, stirring con-
tinually to prevent burning. After boiling gently for five
minutes the fluid portion is filtered off through cheesecloth and
the residue put through a fruit press to extract as much more
fluid as possible, using a towel or other heavy material to line
615
616 KAYMOND A. KELSER
the inside of the press in order that the pulp may not be
pressed through. The residue is then discarded and the fluid
placed over the flame and again brought to a boil, the coagu-
lated protein being skinmied off as it collects on the surface.
A sufficient amount of concentrated acetic acid (approximately
0.5 cc. per Hter of fluid) to cause flocculation is added, and
the boiling continued for five minutes. The product is now
ready to be filtered. For this purpose a stand is arranged hold-
ing three funnels, placed one above the other, the first con-
taining absorbent cotton and the other two filter paper, and the
material is filtered. The volume is then ascertained and 1 per
cent peptone and 0.5 per cent sodium chloride are added, heat-
ing sufficiently to effect solution. The medium can now be
titrated and the reaction corrected by neutralizing with sodium
hydroxide, or if a solid medium is to be prepared the usual
amount of agar may be added before titration. Sterilization
is accomplished by autoclaving for one-half hour under 12
pounds pressure.
While the clot and the serum may be handled separately,
i.e., by removing the clot, cutting it up and boiling with dis-
tilled water, filtering, then adding the serum and again boiling,
this is not essential, as equally good results are obtained by
handling the clot and serum together.
In the use of the bouillon alone I have found that the addi-
tion of a shght amount of carbohydrate (0.25 per cent glucose)
favors the growth of some organisms is this medium. This
is undoubtedly due to the fact that bouillon prepared from the
blood is almost sugar-free, containing only a few hundredths
of 1 per cent sugar. When agar is added the addition of carbo-
hydrate is unnecessary.
The nitrogen content is also somewhat less in the blood
medium than in that prepared from beef.
Several tests were undertaken to ascertain the comparative
value of this medium and that prepared from beef. Tubes of
bouillon, plain and glycerin agar were prepared from the blood
bouillon and the same from beef bouillon and inoculated with
various types of organisms, including streptococci, staphylococci,
PREPARATION OF CULTURE MEDIA FROM WHOLE BLOOD 617
B. typhi, B. bipolaris-septicus, B. diphtheriae, B. anthracis, B.
tuberculosis (isolated from a bear), B. abortus, B. pyocyaneus,
B. mallei, fungi, saccharomyces, etc. In all instances where
there was a noticeable difference in growth on the two types of
media, it was in favor of the blood preparation, such organisms
as streptococci and B. diphtheriae growing with special luxuriance.
A test conducted on a lot of the medium prepared without
the addition of peptone demonstrated the fact that it is possible
to grow organisms such as streptococci, etc., on the peptone free
medium but not so luxuriantly as when peptone is added.
In most instances the addition of glycerin is unnecessary;
organisms which are usually grown on glycerin-agar, will, as a
rule, grow very satisfactorily on plain agar prepared from the
blood bouillon.
This medium, as can be seen from the method outlined above,
is very easy to prepare, in fact more simple of preparation than
the ordinary beef infusion, as the time required for trimming
away fat, fascia, etc., is eliminated. This fact, together with
the small cost and the adaptability of the medium for organisms
which do not thrive well on the ordinary culture media should
recommend it as a valuable product in the laboratory. It can
practically take the place of serum-agar which is somewhat
more difficult to prepare.
An effort was made to produce a concentrated extract from
the blood, similar to beef extract. By considerably reducing
the amount of distilled water added to the cut up blood clot
and serum, a much more concentrated extract is obtained and
this can be reduced to the desired consistency through evapora-
tion over a water bath. Media prepared from this concentrated
extract proved satisfactory in every way.
A concentrated extract from blood can also no doubt be pre-
pared by the method used in packing houses for the production
of meat extracts, i.e., through the use of heat and vacuum.
REFERENCES
LiCHTENSTEiN, S. 1916 Centralbl. f. Bakt. Abt. I. Orig. 77, 362-363.
ScHMiTZ, K. E. F. 1916 Centralbl. f. Bakt. Abt. I. Orig. 76, 306-320.
SzASZ, A. 1915 a Centralbl. f. Bakt. Abt. I. Orig. 75, 489-495.
SzAsz, A. 1915 b Centralbl. f. Bakt. Abt. I. Orig. 77, 111-12.
PRELIMINARY NOTE ON THE CLASSIFICATION OF
SOME LACTOSE FERMENTING BACTERIA
MAX LEVINE
From the Bacteriological Laboratories of the Iowa State College, Ames, Iowa
Received for publication, July 22, 1916
The key given below is the result of a study of 333 lactose
fermenting organisms isolated from soil, sewage, and various
animal sources, including man, the horse, the sheep, the cow,
and the pig.
The fermentation reactions were determined in peptone water
containing the test substance.
Motility was observed in a soft agar medium (nutrient broth
with 0.5 per cent agar) after six, and twenty-four, hours incuba-
tion at the body temperature. Six hours seems sufficient for
differentiation.
Gelatin liquefaction was recorded for five weeks.
The methyl red and Voges-Proskauer reactions were deter-
mined in 0.5 per cent glucose-peptone-dipotassium phosphate
solution.
It will be observed that the subdivisions are not based upon
single characters, but upon differences in groups of characters.
Where inspection is impractical, or insufficient to show which
character is best correlated with others, considerable informa-
tion may be obtained from a study of the coefficients of corre-
lation. That character which gives the highest coefficient of
correlation with the greatest number of characters studied is
the best for classification, if subdivision is to be made entirely
upon correlated characters.
It should perhaps be noted that the names assigned to the
species in the key are tentative, and may be changed, if upon
further study of the hterature, they are found to be invaUd.
619
620 MAX LEVINE
A small group of organisms which resemble B. aerogenes with
respect to gas formation from various carbohydrates etc., did
not give the Voges-Proskauer reaction, and were neutral to methyl
red after three days incubation at the body temperature. It
has been previously observed that some organisms do not give
the Voges-Proskauer reaction, and are not alkahne to methyl
red until the fifth or seventh day of incubation. These organ-
isms resemble very closely the B. gasoformans non-liquefaciens
described by MacConkey (1909) who records the Voges-Proskauer
reaction as positive or negative. As this name is a trinomial,
and consequently invalid, the group is included, for the present,
under B. aerogenes.
Key to the more common species of aerobic or facultative non-spore-forming bacteria
which ferment lactose with gas formation
A. Voges-Proskauer reaction negative, usually acid (at least not alkaline) to
methyl red, with no reversion on long standing; indol usually positive;
polysaccharids (starch, dextrin, and inulin) negative.
Coli group.
I. Sucrose positive.
a. Motile; dulcitol, glycerol, and salicin usually positive.
1. B.communior.
b. Non-motile:
1. Salicin positive; dulcitol and glycerol usually positive.
2. B. neapolitanus.
2. Salicin and dulcitol negative, glycerol usually negative.
3. B. coscoroba.
II. Sucrose negative.
a. Salicin positive, dulcitol and glycerol usually positive.
4. B. coli.
1. Motile var. communis.
2. Non-motile. var. immobilis.
b. Salicin negative; dulcitol usually negative.
1. Motile; glycerol usually positive.
5. B. Gruenthal.
2. Non-motile; glycerol usually negative.
6. B. acidi-lactici .
B. Voges-Proskauer reaction positive; (occasionally only after long incubation);
reaction to methyl red alkaline, or if acid at first, it reverts to a distinct
alkaline reaction after long incubation (7 days); indol, usually negative;
polysaccharids, starch inulin and dextrin, negative or positive.
Aerogenes-cloacae group.
SOME LACTOSE FERMENTING BACTERIA 621
I. Non-motile; gelatin rarely liquefied; indol, dulcitol and inulin negative
or positive; sucrose, raffinose, mannitol, glycerol, salicin, dextrin, and
starch positive.
7. B. aerogenes.
II. Motile; gelatin liquefied (often very slowly); indol, dulcitol, glycerol,
inulin and starch usually negative (rarely positive) ; dextrin occasion-
ally positive; sucrose, raflfinose, salicin, and mannitol positive (rarely
negative) .
8. B. cloacae.
A NEW ICE SAMPLER
MYRTLE GREENFIELD
Water and Sewage Laboratory, State Board of Health, Lawrence, Kansas
Received for publication, August 7, 1916
There is much apparent dissatisfaction with the ordinary
methods of sampUng ice. This laboratory is now using an ice
sampler based on the principle of a coal sampler. A brass
cyhnder, with the dimensions given in the diagram, has teeth
cut in the lower end. These teeth are given a set. The appa-
ratus fits into a carpenter's brace, which makes it possible to
bore through a cake of ice with ease. The sample is not a solid
core but consists of small chips of ice which are easily pushed
out by means of a movable plug on the inside of the brass
cylinder. It is necessary to flame the tube a little before the
sample will slip out easily. With this apparatus, it is possible
to get a representative sample of ice without contamination.
623
APPARENT RECOVERY OF A HEN INFECTED WITH
BACILLARY WHITE DIARRHEA
As Determined by the Macroscopic Agglutination Test
GEORGE D. HORTON
Department of Bacteriology, Missouri State Poultry Experiment Station
* Received for publication, September 18, 1916
The following note deals with the case of a Bantam hen which
at one time (1914), gave a positive agglutination test and at a
later date (1916) gave a negative reaction.
The method of testing was essentially that devised by Jones.
A polyvalent test fluid with proper controls was used.
The Bantam was one of a flock of several hundred birds of
which approximately 65 per cent were infected. The bird was
tested first in December, 1914; the reaction was very marked
within 24 hours. In the fall of 1915 the test was applied again;
the reaction was faint after 72 hours. During 1916 two tests
were made, both of which were negative even after 72 hours.
That reacting fowls may lay eggs the yolks of which harbor
the infective organism, B. pullorum, has been demonstrated by
Rettger, Gage and others. The infection in this case was un-
doubtedly of ovarian origin because of the finding and isolation
of pure cultures of B. pullorum (the infective agent in the bacil-
lary form of white diarrhea) from two unhatched eggs of a
sitting of eggs laid by this Bantam in 1915.
In view of the above observation however, we may ask, — ^is
it not possible for an infected fowl to free herself of infection?
or, is she once and for all time a bacillus carrier?
Although this single instance furnishes but limited evidence
it suggests the possibility of recovery, or the throwing off of
ovarian infection.
625
626 GEORGE D. HORTON
Further investigation is necessary to establish this point; and
experiments are now in progress at this Station.
It is not recommended as good practice, to hold over infected
stock in the hope of such a ''recovery," as that indicated by
these observations, because of the great risk from infected eggs.
OBSERVATIONS SUR L'INFLUENCE CHIMIQUE DES
MILIEUX DE CULTURE SUR LE DEVELOPPEMENT
ET LA PRODUCTION DE LTNDOL PAR LES COLI-
BACILLES ET PAR LES BACILLES TYPHIQUES
EDGARD ZUNZ and PAUL GYORGY
From the Institute of Therapeutics, University of Brussels
Received for publication, June 14, 1916
I. INTRODUCTION
Les travaux consacres a la recherche de miheux chimiquement
definis, aptes au developpement de tel ou tel microorganisme,
sont fort nombreux. II serait fastidieux de les passer en revue.
Bornons nous a rappeler les tres interessants memoires d'Armand
Delille, Andre Mayer, G. Schaeffer et E. F. Terroine (1913),
de Frouin (1912), de Galimard et Lacomme (1907), de Franzen
Hartwich (1914), de Proskauer et Beck (1894), de Santon (1912)
de Seliber (1914), de Tiffeneau et Marie (1912), de Trillat et
Fouassier (1912), choisis parmi bien d'autres d'une tout aussi
grande valeur.
Galimard et Lacomme sont partis d'une solution aqueuse
fondamentale renfermant 1.5% de glycerine et 1.5% de con-
stante mine rale de Lepierre (chlorure de sodium 0.5%, sulfate
de magnesie 0.05%, glycerophosphate de chaux 0.2%, bicar-
bonate de potasse en quantite sufhsante pour neutrahser). lis
sont parvenus a cultiver les cohbacilles dans ce miheu additionn<§
de 1% soit de glycocolle, de leucine ou d'arginine, soit de 1%
de tyrosine. Par contre ces micro organismes n'ont pas pousse
dans le miheu fondamental pr^c6dent, auquel on a ajouts 1%
d'acide asfaltique, de phenylalanine, de lysine ou d'uree. Une
seule source d'azote suffit done au developpement des cohbacilles.
Ces microorganismes sont neanmoins plus difhciles dans le choix
de leur nourriture azote e que les bacilles pyocyaniques qui ont
627
628 EDGARD ZUNZ ET PAUL GYORGY
pousse en presence de chacun des acides amines etudies, k Tex-
ception de la phenylalanine. Mais d'autre part les colibacilles
sont bien moins exigeants que les bacilles paratyphiques et les
vibrions du cholera qui n'ont pousse qu'en presence d'arginine
ou de tyrosine et surtout que les staphylocoques pyogenes
oranges qui ne se sont developpes qu'en presence d'arginine.
Quant aux bacilles typhiques, ils n'ont pousse en presence d'aucun
des acides amines pre cites, additionne seul a la solution aqueuse
de glycerine et de constante minerale de Lepierre.
Galimard et Lacomme ont prepare, en partant de leur solu-
tion fondamentale, onze milieux renfermant chacun au moins
deux acides amines. En voici la composition: le premier miileu
contient 0.75% de leucine, 0.2% d'alanine et des traces de tyro-
sine; le second milieu contient 1.5% d'un melange d'acides mono-
amines, de lysine et d'ornithine; le troisieme milieu contient
0.3%) de glycocolle, 0.05%o de leucine, 0.01%o de tyrosine et 0.1%o
d'acide aspartique; le quatrieme miheu contient 0.45% de glyco-
colle, 0.01% de leucine, 0.05% de tyrosine et 0.15% de chlor-
hydrate d'arginine; le cinquieme milieu contient 0.7% de glyco-
colle et 0.1% de chlorhydrate d'arginine; le sixieme miUeu con-
tient 0.45% de leucine, 0.05% de tyrosine et 0.1% de chlor-
hydrate d'arginine; le septieme miUeu contient 0.2% de glyco-
colle, 0.4% de chlorhydrate d'arginine et 0.1% de chlorhydrate
de lysine; le huitieme milieu contient 0.1% de glycocolle et
0.9% de tyrosine; le neuvieme miheu contient 0.5% de glyco-
colle et 0.5% d'uree; le dixieme milieu contient 0.9% d'urce et
0.1% de chlorhydrate d'arginine; le onzieme milieu contient
0.9% de glycocolle et 0.1% de chlorhydrate d'arginine.
Les bacilles pyocyaniques ont pousse dans ces onze miUeux.
Les coUbacilles se sont developpes partout a I'exception du
second miheu. Le developpement des bacilles paratyphiques
n'a pas eu lieu dans les premier et second milieux, mais bien
dans tons les autres. II en a ete de meme des vibrions du cho-
lera, mais ceux-ci n'ont, en outre, pas pousse dans le huitieme
miheu. Les staphylocoques pyogenes orangos ne se sont deve-
loppe s que dans les deux premiers mih eux . G alimard et Lacomme
ne sont point parvenus a cultiver les bacilles d'Eberthdans aucun
des onze milieux.
LES COLIBACILLES ET LES TYPHIBACILLES 629
Dans leurs importantes recherches, Galimard et Lacomme ont
encore etudie la croissance d'autres especes de microorganismes.
lis n'ont pas tente d'evaluer le degre de developpement des
cultures de colibacilles ou des autres bacteries etudiees dans
les divers milieux qu'ils ont employes. lis n'ont pas non plus
procede a des tentatives de reensemencement sur un milieu
solide, tel que I'agar par exemple, apres un sejour plus ou moins
long a I'etuve des divers milieux chimiquement definis ensemences
au moyen de colibacilles ou d'autres microorganismes.
Passons a un autre ordre de faits. On admet depuis longtemps
d4ja que le colibacille donne naissance d'une fagon k peu pres
constante a de Tindol dans les cultures en milieu pepton^, tandis
que le bacille typhique n'en produit pas dans ces conditions
(Kitasato 1889). On s'est demande aux d^pens de quelle sub-
stance contenue dans les melanges commerciaux de produits de
disintegration des proteines, denommes a tort ''peptones," le
colibacille donne la reaction de I'indol. On sait actuellement
que c'est aux depens du tryptophane. L'intensite de la reaction
depend de la teneur du milieu en ce composo. II sufHt de la
presence de 0.03% de tryptophane dans une solution aqueuse
renfermant, en outre, du sucre, de la glycerine, des phosphates
de potasse (0.5%) et de magnesie (0.03%) et du lactate d'am-
moniaque (0.5%) pour obtenir une reaction de I'indol tres nette
apres ensemencement par des colibacilles et sejour de 24 a 48
heures a I'etuve (Zipfel 1913).
Les resultats des experiences de Galimard et Lacomme et de
celles de Zipfel nous ont engage a poursuivre des essais de cul-
ture des cohbacilles et des bacilles typhiques dans des milieux
chimiquement definis, dans I'espoir d'en trouver de particuliere-
ment appropries a la differenciation de ces deux especes de micro-
organismes au moyen de la rctaction de I'indol. Dans ce but,
11 y avait heu de remplacer la "peptone de Witte" ou tout autre
melange de ce genre utilise, en sus du bouillon ou mieux
de I'eau physiologique (Escherich 1903) (Besson 1904) pour
la recherche de la reaction de I'indol, par des proteoses, des pep-
tides, des acides amines, de I'uree, des corps puriques et py-
rimidiques, des substances extractives. On pent ainsi aj outer,
630 EDGARD ZUNZ ET PAUL GYORGY
soit au bouillon, soil a I'eau physiologique, de nombreux pro-
duits azotes en employant un seul ou plusieurs d'entre eux.
Le bouillon renferme, outre ses constituants salins et azotes,
des substances ternaires tels que le glucose et I'inosite. II etait,
par consequent, indique de se preoccuper de I'influence de ces
corps et d'autres hydrates de carbone sur le developpement des
bacilles d'Eberth et des colibacilles et sur la reaction de I'indol.
C'est ainsi que nous avons ete amene a entamer les recherches
expos^es ci-dessous.
Nous av^ons aussi consacre quelques experiences aux bacilles
paratyphiques A et B, aux vibrions du cholera, aux bacilles pyo-
cyaniques, aux staphylocoques pyogenes dores, aux bacilles de
1 'entente de Gartner.
II. TECHNIQUE
Nous soinines partis de quatre miUeux de culture: (1) le bou-
illon de boeuf;^ (2) I'eau physiologique, c'est-a-dire une solu-
tion a 0.5% de chlorure de sodium chimiquement pur; (3) I'eau
physiologique aminee, c'est-a-dire une solution aqueuse renfer-
mant 0.5% de chlorure de sodium et 0.2% de glycyltryptophane;
(4) I'eau peptonee, c'est-a-dire une solution aqueuse contenant
0.5% de chlorure de sodium et 1% de peptone de Witte.
On a re parti aseptiquement les divers milieux, tels quels ou
additionnes d'un ou de plusieurs produits, par portions de 10
centimetres cubes, dans des tubes a essai steriles, puis on a de
nouveau st'rilise ces tubes.
Pour chaque substance ou groupe de substances utilise, ajoute
a I'un ou a I'autre de ces miheux, on a eu soin de preparer deux
tubes lors de chaque essai effectue avec la meme espece de
microbes.
Dans chaque experience, on s'est servi pour I'ensemencement
de la meme culture sur agar de colibacilles, de bacilles d'Eberth
ou d'autres microorganismes. Ces cultures nous ont ete aima-
blement fournies par Messieurs les Professeurs Bordet et Gengou.
On a eu soin de les rajeunir par roensemencement de faQon a
toujours partir de cultures poussant tres vite en bouillon peptone.
' Prepare d'aprfes la formule d^crite dans le Manuel de bact^riologie clinique
de M. Furck, Bruxelles, 1910, p. 12.
LES COLIBACILLES ET LES TYPHIBACILLES 631
Lors de chaque experience, on a examine une premiere serie
de tubes a essai au bout de 15 a 48 heures afin d'e valuer le degr6
de croissance en se basant sur les proprietes optiques (opales-
cence, trouble, voile, etc.) et sur I'examen microscopique. On a
ensuite recherche la reaction de I'indol au moyen de Taldshyde
paradimethylaminobenzoique, de la iagon preconisee par Haenen
(1905). On a parfois proc^de a deux reprises a I'examen des
proprietes optiques de cette serie de tubes, en ayant naturelle-
ment soin, lors du premieur examen, de maintenir les tubes
bien bouches de maniere a eviter toute contamination.
On a examine, en prenant ces precautions, a deux ou trois
reprises, le degre de developpement des diverses cultures dans
la seconde serie de tubes a essai. On a preleve une oese de chaque
milieu liquide, lors de I'un de ces examens, et on a reensemence
sur agar. On a examine a une ou deux reprises le degre de
croissance de ces cultures sur agar. Apres le dernier examen
des proprietes optiques des cultures en bouillon ou en eau physio-
logique (amine e ou non), on a procede a la recherche de la reac-
tion de rindol.
Monsieur le Professeur Kossel (d' Heidelberg) nous a aimable-
ment fourni le sulfate de clupeine. Monsieur le Professeur EmJl
Fischer (de Berlin) a eu la grande obligeance de nous envoyer
de la glycylglycine, de la diglycylglycine et de I'alanylglycyl-
glycine.^ Nous avons prepare les proteoses d'apres la methode
de E. P. Pick (1899), la leucine et la tyrosine de la maniere habi-
tuelle. Les autres produits provenaient des maisons Guibler
(de Dresde et Leipzig), Kalle (de Biebrich) et Kahlbaum (de
Berlin).
III. EXPERIENCES
Nous avons choisi les experiences les plus demonstratives pom-
en reproduire ci-dessous les resultats sous forme de tableaux.
Le plus ou moins grand degre de croissance des cultures dans
les divers milieux experimentes, evalue de la fagon indiquee ci-
dessous, a ete exprime dans ces tableaux, par un nombre plus
* Nous tenons k remercier bien vivement Messieurs les Professeurs Bordet,
Emil Fischer, Gengou, et Kossel de leur extreme obligeance.
632 EDGARD ZUNZ ET PAUL GYORGY
ou moins considerable de signes +. Lorsqu'on n'a pu doceler
aucun developpement microbien dans un tube, on trouvera
en regard le signe — . II est parfois arrive de constater une
legere opacite ou un Icger trouble du milieu, sans qu'on ait
observe de microbes a I'examen microscopique de preparations
colorees provenant du tube en question et sans qu'on soit parvenu
a deceler de developpement des microorganismes apres reense-
mencement sur agar. Ces cas douteux ont ete mentionnes par
le signe +?
Pour ce qui concerne la reaction de Tindol, les signes + et —
veulent dire que la reaction a ete positive ou negative. Dans
certains cas, la reaction a ete douteuse. On n'est parfois pas
parvenu a deceler de coloration rose de la culture de colibacilles
lors de I'addition de I'aldehyde paradimethylaminobenzoique et
de Facide chlorhydrique, mais apres agitation avec le chloro-
forme ou I'alcool amylique, le solvant a pris une legere teinte
rose. Nous avons mentionne, dans les tableaux V et VI, ces
cas par le signe +?. D'autrefois, on a observe I'apparition d'une
coloration rose lors de I'addition a la culture etudiee d'acide
chlorhydrique et d'aldehyde paradimethylaminobenzoique, sans
que cette coloration passe dans le chloroforme ou I'alcool amy-
Uque. On doit considerer ces cas, dont les tableaux V et VI
offrent des exemples, comme tres probablement negatifs; nous
les avons neanmoins, par plus de prudence, qualifies de douteux
en leur attribuant le signe +?.
A. Experiences effectuees seulement avec des colibacilles et des
hacilles typhiques
1. Experiences effectuees en partant du bouillon comme milieu
de culture. A titre d'exemple, nous reproduisons, dans le tableau
I, I'exp^rience la plus demonstrative.
La croissance des colibacilles a eu lieu dans les divers milieux
experiment's, bien qu'a un moindre degre que dans le bouillon
additionne de 1% de peptone de Witte. Le developpement a
6t6 le plus rapide en presence d'heteroalbumose, de glycyltrypto-
phane, de leucine et de gliadine, le plus lent en presence d'alanyl-
glycylglycine, de glycocolJe, d'alanine.
LES COLIBACILLES ET LES TYPHIBACILLES 633
On n'a observe la reaction de I'indol que dans la culture de
colibacilles en bouillon additionne de 0.3% de glycyltryptophane.
Au bout de 48 heures, la reaction de Findol etait, dans ce tube
bien plus intense que dans le tube temoin de bouillon pepton6.
La croissance des bacilles typhiques a ete plus intense en
bouillon additionne de 0.5% de gliadine qu'en bouillon pepton^.
Elle a ete tout aussi abondante en bouillon additionne de 0.5%
d'heteroalbumose qu'en bouillon peptone. Dans les autres mili-
eux, les bacilles d'Eberth n'ont pas pousse ou tres peu.
2. Experiences effectuees en partant de Veau physiologique comme
milieu de culture. On trouvera, dans les tableaux II a IV, les
protocoles des trois experiences effectuees en partant de ce
milieu.
Les colibacilles se sont developpes, tout aussi bien qu'en pre-
sence de peptone de Witte, en presence de leucine et presque
aussi bien en presence de protoalbumose, d'histidine, de diglycyl-
glycine, de glycyltryptophane (seul ou additionne de saccharose),
de caseine, de chlorhydrate de guanidine, de taurine, de dimethyl-
diphenyluree. Le developpement a ete quelque peu retarde en
presence de thioalbumose, de glycocolle, d'alanine, de gliadine,
de xanthine, d'acide glycochohque. II a ete notablement retard^
dans les tubes renfermant de I'acide aspartique, de la tyrosine,
de la phenylalanine, de I'acide urique, de I'acide cyanurique, de
la sarcosine, de la creatine. On n'a observe aucun developpe-
ment dans les tubes contenant de I'heteroalbumose, de I'acide
glutanique, du phenylglycocoUe, du sulfate de clupeine, de I'allan-
toine, de I'hypoxanthine, de la cafeine, de I'alloxane, de la betaine,
de I'acide barbiturique, de I'acide hippurique, de I'acide glyco-
chohque, de I'uree, du chlorhydrate de metaphenylenediamine.
On n'a constate la reaction de I'indol que dans les cultures
de colibacilles en eau physiologique, additionnee soit de peptone
de Witte, soit de glycyltryptophane.
Les bacilles d'Eberth ont tres bien pousse en eau peptonee.
Leur developpement a ete encore plus abondant en eau physio-
logique additionnee de 0.3% de gliadme. lis ont faiblement
pousse dans la solution de chlorure de sodium a 0.5% renfermant
0.3% soit de caseine, soit de sarcosine, soit de taurine, soit de
634
EDGARD ZUNZ ET PAUL GYORGY
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LES COLIBACILLES ET LES TYPHIBACILLE8 635
xanthine. Dans tons les autres milieux, nous n'avons pas con-
stats la moindre croissance des bacilles d'Eberth.
Nous confirmons done les resultats negatifs obtenus par Gali-
mard et Lacomme avec les bacilles typhiques en presence de
glycocolle, de leucine, de tyrosine,, d'acide aspartique, de phenyl-
alanine et d'urSe comme source unique d'azote, les resultats posi-
tifs obtenus par ces auteurs avec les cohbacilles en presence de
glycocolle, de leucine et de tyrosine ainsi que I'absence de d6ve-
loppement de cette derniere espece de microorganismes dans un
milieu renfermant seulement de I'uree comme compose azote.
Par contre, grace a la mSthode de reensemencement sur agar,
nous avons pu mettre en Evidence que Facide aspartique et la
phenylalanine n'etaient pas dSnues de toute valeiu" nutritive
pour les colibacilles.
3. Experiences effectuees en partant de Veau physiologique amin^e
comme milieu de culture. Nous avons remarque, au cours des
experiences effectuees en se servant soit du bouillon, soit de
I'eau physiologique comme base des milieux de culture que I'addi-
tion de glycyltryptophane k ces milieux permet aux colibacilles
un rapide et intense developpement d'indol. II nous a sembie
interessant de rechercher Tinfluence exercSe sur ce phenomene
par Taddition de diverses substances contenues dans le bouillon.
Dans ce but, nous avons prepare une solution aqueuse renfer-
mant 0.5% de chlorure de sodium et 0.2% de glycyltryptophane.
C'est a ce milieu de culture que nous avons donne, comme
nous Tavons, du reste, deja indique plus haut, le nom d'eau
physiologique aminee. On trouvera, dans le tableau V, le pro-
tocole d'une experience de ce genre.
Des divers produits examines, seul la glucose a accru le dSve-
loppement des colibacilles en eau physiologique aminee. La
taurine, I'acide barbiturique, la creatine, la sarcosine, TurSe ont
legerement entravS la croissance de ces microorganismes. L'acide
cyanurique, la guanidine, la betaine et la xanthine Font davan-
tage entravee. L'inosite I'a retardSe de fagon considerable.
Quant k la cafeine et a I'allantoine, elles paraissent I'avoir totale-
ment empechee.
636
EDGARD ZUNZ ET PAUL GYORGY
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LES COLIBACILLES ET LES TYPHIBACILLES
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LES COLIBACILLES ET LES TYPHIBACILLES
641
TABLEAU V
-
COLIBACILLES
BACILLES TTPHIQUE3
1
Reaction
Degr6 de
Reaction
COMPOSITION DES
milieux: eau phtsio-
loqique renfermant
^CHANTILLON
EMPLOY i;
Degr6 de croissance
• apr^s
de ) ' indol
aprSs
croissance
apres
de r indol
aprds
S^
«d
1
S 3
s
s
CO h
M
.a
J3
J3
ja
J3
J3
.4
+?
—
f premier
+ + +
+
Rien d'autre
1^ deuxieme
+ + +
+ + +
+ +
+?
+?
—
f premier
+ +
+ +
+ '!
—
0.2% de taurine
1^ deuxieme
+ +
+ +
+ +
+?
+?
—
0.2% de cafeine ....
r premier
\deuxieme
+ ?
+ ?
+ ?
+ ?
+ ?
+
+
+
-
0.2% d'allantoine. .
fpremier
\deuxieme
+ ?
+ ?
+ ?
_?
_?
_
+?
-
0.2% d'alloxane. . . .
fpremier
\deuxieme
+ ?
+ ?
+
_?
_?
+?
+?
+
-
0.2% d'acide cyanu-
fpremier
+
+
+ !
rique
\deuxieme
+
+
+
+ ?
-
0.2% d'acide barbi-
fpremier
+ +
+
+
turique
\deuxieme
+ +
+ +
+
+
+
fpremier
+ +
+ •!
+v
0.2% de creatine . . .
\ deuxieme
+ +
+ +
+
+ ?
-1-v
—
—
fpremier
+ +
+ +
+
—
0.2% de sarcosine. . .
\ deuxieme
+ +
+ +
+ +
+
+
—
0.2% de guanidine. .
fpremier
\ deuxieme
+
+
+
+
+ +
+
+
+
-
fpremier
+
+
+ ?
—
0.2% de b^taine
\ deuxieme
+
+
+
+v
+v
—
0.2% de xanthine. . .
fpremier
\ deuxieme
+
+
+
+
+
+?
+
-
/premier
\ deuxieme
+ ?
+ 'i
+'.
—
0.2% d'inosite
+ ?
+
+
+'.
+1
1 premier
\ deuxieme
+ +
+
+'
—
0.2% d'ur6e
+ +
+ +
+
+
+
"
fpremier
+ + + -f
+ '
}
—
0.2% de glucose . . .
\deuxieme
+++H-
- + + -|-f
+
+
La reaction de I'indol a ete favorisee, pour les cultures de
coUbacilles en eau physiologique aminee, par la sarcosine et la
taurine. EUe n'a pas ete influencee par la guanidine bon
intensite a diminue dans les tubes renfermant 0.2% d acide
642 EDGARD ZUNZ ET PAUL GYORGY
cyanurique, d'acide barbiturique, de betaine ou d'ur^e et encore
davantage dans les tubes contenant 0.2% de creatine, de glucose
ou d'inosite. Dans ces trois derniers milieux, les tubes examines
au bout de 16 heures n'ont pas montr^ de coloration ros4e lors
de I'addition d'aldehyde paradimethylaminobenzoique et d'acide
chlorhydrique, bien qu'apres agitation avec du chloroforme,
celui-ci soit devenu l^gerement rose. Nous n'oserions pas af-
firmer qu'on se trouve bien en presence de la reaction de I'indol
dans ces cas. II en a aussi et6 ainsi au bout de 48 heures pour
les tubes renfermant 0.2% de cafeine, dans lesquels on n'a, du
reste, pas pu d^celer de colibacilles. Nous n'avons pas non plus
observ6 de reaction de I'lndol dans les tubes renfermant de
I'allantoine ou de I'alloxane, puisque la coloration rose apparue
alors apres I'addition de I'aldehyde paradimethylaminoben-
zoique et de I'acide chlorhydrique n'a pas pass^ dans le chloro-
forme.
Les bacilles d'Eberth n'ont pouss6 que dans I'eau physio-
logique amin^e contenant de I'acide barbiturique, de la cafeine,
de la guanidine, de la sarcosine, de la xanthine ou de I'ur^e.
Rappelons qu'ils ne se sont pas developp^s dans I'eau physio-
logique renfermant de I'acide barbiturique (tableau IV), de la
cafeine, du chlorhydrate de guanidine ou de I'ur^e (tableau III)
sans glycyltryptophane. lis ne se sont developp^s de fagon
appreciable qu'entre la 48^°'*' et la 06^""^ heure dans I'eau physio-
logique additionn^e seulement de xanthine (tableau III) ou de
sarcosine (tableau IV). L'association du glycyltryptophane a
la xanthine a permis aux bacilles d'Eberth de se d^velopper
abondamment entre la 16^°"^ et la 48^°"^ heure. Dans les tubes
renfermant a la fois le glycyltryptophane et la sarcosine, les
bacilles typhiques se sont meme deja developp^s avant la 16 ™^
heure de sejour a I'^tuve.
Ces resultats nous ont engage a etudier les effets de l'associa-
tion de plusieurs produits de glycyltryptophane sur la croissance
des colibacilles et des bacilles d'Eberth et sur la reaction de
I'indol dans les cultures de cohbacilles. Comme Ton a d^ja entre-
pris de multiples recherches relatives k I'addition de la glucose
ou d'autres hydrates de carbone aux miheux destines a cultiver
LES COLIBACILLES ET LES TYPHIBACILLES
643
et surtout a diff^rencier ces deux especes de micro organismes,
nous nous sommes beaucoup pr^occup^s de la presence de la glu-
cose dans les milieux de culture. Nous donnons, dans le tableau
VI, les r^sultats d'une s^rie d'essais ainsi effectu^s avec des
melanges de diverses compositions.
On sait, par les r^sultats relates dans le tableau V, que I'addi-
tion de la glucose a I'eau physiologique amin^e favorise le d^velop-
pement des colibacilles, mais entrave la reaction de I'indol. L'ad-
dition de la sarcosine a I'eau physiologique aminee favorise la re-
action de I'indol, mais entrave le developpement des microorgan-
jsmes. L'addition de la creatine a I'eau physiologique aminee
diminue la rapidite de developpement des colibacilles et I'inten-
site de la reaction de I'indol. Or, d'apres le tableau VI, l'addi-
tion h I'eau physiologique aminee a la fois de creatine, de glucose
et de sarcosine, dans les memes proportions que celles utilis^es
s^par^ment dans les experiences du tableau V, a favoris4 le
developpement des coUbacilles et a accru, du moins dans un
cas, I'intensite de la reaction de I'indol. On ne pent done con-
clure aux effets d'un melange de corps azotes sur la croissance
des coUbacilles et sur I'intensite de la reaction de I'indol dans
leurs cultures en se basant sur les resultats obtenus avec ces
corps examines isol^ment.
La solution a 0.5% de chlorure de sodium renfermant 0.2%
de glycyltryptophane, 0.2%o de glucose, 0.2% de creatine et 0.2%
de sarcosme a amene une abondante croissance des baciUes
d'Eberth entre la IG''"^ et la 88 '"^ heure consecutives k I'ense-
mencement, alors qu'on a obtenu des resultats douteux ou nega-
tifs dans les tubes temoins ne renfermant que du glycyltrypto-
phane et dans ceux contenant du glycyltryptophane et de la glu-
cose ou de la creatine. Les effets favorables paraissent ici etre
dtjs h la presence de la sarcosine, qui amene en eau physiologique
(tableau IV) un developpement faible et tardif des baciUes
d'Eberth et en eau physiologique aminee (tableau V) un deve-
loppement plus pr^coce de ces microorganismes.
Si k la solution aqueuse renfermant 0.5% de chlorure de
sodium, 0.2% de glycyltryptophane, 0.2% de glucose 0.2% de
creatine et 0.2% de sarcosine. Ton ajoute encore de la b^tame
644
EDGARD ZUNZ ET PAUL GYORGY
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LES COLIBACILLES ET LES TYPHIBACILLES
645
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646 EDGARD ZUNZ ET PAUL GYORGY
et de I'inosite, la croissance des colibacilles et la reaction de
I'indol sont entrav^es ou meme completement empechees, et ce
n'est qu'au bout de 88 heures qu'on constate un faible d^veloppe-
ment des bacilles d'Eberth. Ceci s'explique ais^ment, car I'addi-
tion de b^taine et surtout celle d'inosite h Teau physiologique
aminee entrave la croissance des colibacilles. La reaction de
I'indol est plus intense dans les cultures de colibacilles en eau
physiologique aminee telle quelle qu'en ce meme milieu addi-
tionn^ de betaine. Elle est plus intense et plus precoce en eau
physiologique aminee qu'en cette solution additionnee d'inosite.
Substituons la guanidine k I'inosite et partons d'une solution
aqueuse renfermant 0.5% de chlorure de sodium, 0.2% de
glycyltryptophane, 0.2% de glucose, 0.2% de creatine, 0.2% de
sarcosine, 0.2% de betaine et 0.2% de guanidine. II ne se
developpe ni colibacilles ni bacilles d'Eberth, bien qu'on n'ait
decQUvert aucune influence inhibitoire de I'association de la guani-
dine au glycyltryptophane sur la croissance des bacilles typhiques
et que I'addition de guanidine k I'eau physiologique aminee ait,
dans les essais relates dans le tableau V, entrave mais nullement
empech^ le d^veloppement des colibacilles.
Remplagons, dans le miUeu dont nous venons de parler, la
guanidine par la xanthine. Le d^veloppement des colibacilles
s'effectue plus vite et la reaction de I'indol apparait plus tot
que dans les tubes renfermant seulement du glycyltryptophane,
de la glucose, de la creatine, et de la sarcosine. Et pourtant
I'association de la xanthine au glycyltryptophane entrave, d'apres
le tableau V, le, d^veloppement des colibacilles et diminue I'in-
tensite de la reaction de I'indol dans leurs cultures.
Si les r^sultats pr^c^dents se confirment, la solution aqueuse
renfermant 0.5% de chlorure de sodium, 0.2% de glycyltrypto-
phane, 0.2% de glucose, 0.2% de creatine, 0.2% de sarcosine,
0.2% de betaine et 0.2% de xanthine, semble particuUerement
appropri^e h la recherche de la reaction de I'indol dans les cul-
tures de colibacilles. Remarquons toutefois que, dans ce miUeu,
le d^veloppement des bacilles d'Eberth est d^ja tres net au bout
de 16 heures. 11 est vrai, qu'il ne parait, plus s'accroitre par la
suite. L'effet favorable sur la culture des bacilles typhiques
LES COLIBACILLES ET LES Ti'PHIBACILLES 647
est tres probablement dd a Fassociation de la sarcosine et de
la xanthine au glycyltryptophane, si Ton s'en rapporte aux
donn^es du tableau V.
Etudions maintenant un milieu forme de glycyltryptophane,
de glucose, d'alloxane, de sarcosine et de taurine. Le developpe-
ment des colibaciiles est tout aussi intense et precoce que dans
le miheu precedent. Par contre, la reaction de I'indol apparait
plus tard que dans I'eau physiologique aminee et n'est pas plus
intense que dans celle-ci. Les bacilles d'Eberth se developpement
parfois deja au bout de 16 heures dans ce milieu, — d'autresfois
on ne pent les deceouvrir, meme au bout de 88 heures.
Ajoutons de I'inosite au milieu dont nous venons de nous
occuper. Ceci suffit a faire disparaitre les bons effets sur le
developpement des colibaciiles, signales ci-dessus. La croissance
de ces microorganismes est meme legerement entravee par rap-
port a I'eau physiologique aminee. Ceci depend certes de I'ino-
site. En effet, d'apres le tableau III, la taurine ne parait pas, ou
ne parait guere influencer de fagon defavorable le developpement
des colibaciiles. II est vrai que les tableaux IV and V tendent k
d^montrer une action inhibitoire de I'alloxane sur la croissance
des colibaciiles, mais elle n'apparalt point dans le melange de
glycyltryptophane, de glucose, de sarcosine, de tam-ine et d'allo-
xane. Par contre, les resultats obtenus dans ce milieu et les
donn^es des tableaux IV et V tendent a d^montrer que Taction
nocive de I'alloxane s'ajoute a celle de I'inosite pour empecher
la reaction de I'indol dans les cultures de cohbacilles ou tout au
moins retarder I'apparition et I'intensite de cette reaction.
D'apres les donnees du tableau VI, les bacilles d'Eberth n'ont
pas, ou n'ont guere pousse dans le miUeu complexe (glycyltrypto-
phane, glucose, sarcosine, alloxane, taurine, inosite) dont il vient
d'etre question.
L'addition de glucose, de sarcosine, d'acide barbiturique et
d'acide cyanurique k I'eau physiologique aminee ne modifie en
rien la croissance des colibaciiles et la reaction de I'indol dans
leurs cultures. Les bacilles d'Eberth ne semblent pas avoir
davantage pousse dans ce milieu que dans Teau physiologique
aminee.
648 EDGARD ZUNZ ET PAUL GYORGY
Si Ton se base sur les resultats consignes dans le tableau VI,
on constate que I'association de Thistidine au glycyltryptophane
a eu des effets heureux pour la croissance des colibacilles et des
bacilles d'Eberth et pour I'intensite de la reaction de Findol dans
les cultures de colibacilles. Les bacilles typhiques n'ont toute-
fois conimenc6 a se developper de fagon appreciable qu'entre la
16^"°** et la 88^"^ heure consecutives a Fensemencement, alors que
les cultures de colibacilles ont deja pr^sente tres nettement la
reaction de I'indol au bout de 16 heures. On se trouve done
de nouveau en presence d'un milieu specialement approprie a
la diff^renciation des colibacilles d'avec les bacilles d'Eberth par
la recherche de la reaction de I'indol.
Augmentons la complexity du miheu en y ajoutant de la glucose.
Le d^veloppement des cohbacilles et celui des bacilles d'Eberth
sont plus favoris^s qu'en I'absence de cet hydrate de car-
bone. Par contre. la reaction de I'indol est moins forte, et meme
parfois douteuse, dans les cultures de colibacilles. Ce mxiUeu
convient, par consequent, beaucoup moins que le precedent h
la recherche de la reaction de I'indol dans les cultures de coli-
bacilles.
Ajoutons de I'inosite au glycyltryptophane, au glucose et "k
I'histidine. On n'observe plus aucun developpement des bacilles
d'Eberth et tel parait etre aussi le cas pour les colibacilles. L'in-
fluence defavorable de I'inosite ressort k nouveau des faits
precedents.
4. Comparaison de Vaction de Vaddition de divers hydrates de
carhone soil a Veau peptonee, soil a Veau physiologique aminee.
Dans quelques essais, nous avons compare les effets de I'addition
de la glucose ou d'autres hydrates de carbone du groupe des sucres
soit a I'eau peptonee, soit k I'eau physiologique aminee. Le
tableau VII reproduit une telle experience.
II manque malheureusement, dans I'experience rapportee dans
le tableau VII, les tubes temoins formes d'eau peptonee ou d'eau
physiologique aminee. Mais, d'apres les resultats relates dans
les tableaux II, V et VI et d'apres d'autres experiences, nous
Savons que le developpement des cohbacilles est, en general,
plus considerable et plus precoce en eau peptonee qu'en eau
LES COLIBACILLES ET LES TYPHIBACILLES 649
physiologique aminee. Par contre, la reaction de rindol est plus
intense et apparait plus tot dans les cultures de colibacilles en
eau physiologique aminee que dans celles en eau pepton^e. L'eau
physiologique aminee ne constitue pas, en outre, un tres bon
milieu de culture pour les bacilles d'Eberth, qui n'y poussent
pas, ou qui n'y poussent guere. La croissance des bacilles typhi-
ques s'effectue au contraire tres bien en eau peptonee, quoique
beaucoup plus lentement et de fayon bien moins notable que celle
des colibacilles dans ce milieu.
En eau peptonic, la croissance des colibacilles a ^t^ tres
intense en presence de rhamnose, de glucose, de galactose, de
lactose ou de maltose. II en a et^ de meme en presence de
saccharose, mais le developpement des bacilles s'est effectueun
peu moins vite dans ce milieu que dans ceux auxquels nous
venons de faire allusion. Au contraire, le xylose a empech^ les
colibacilles de pousser en eau peptonee.
On n'a observe la reaction de Tindol que dans la culture de
colibacilles en eau peptonee additionnee de saccharose, examinee
au bout de 96 heures. On n'est pas parvenu a d^couvrir cette
reaction dans ce miUeu au bout de 24 heures. Dans les cul-
tures de colibacilles en eau peptonee additionnee de rhamnose,
de glucose, de galactose, de lactose ou de maltose, la reaction
de I'indol a toujours fait defaut. Les hydrates de carbone ont
done exerc4 une action inhibitoire marquee sur cette reaction.
Les bacilles d'Eberth ont pousse tres vite et tres fort en eau
peptonee saccharosee. Leur croissance s'est aussi fort bien
effectu^e, quoique de fagon bien moins intense, en eau peptonic
additionnee de galactose ou de lactose. EUe n'a eu lieu que
tard en eau peptonee additionnee de rhamnose. On a obtenu
des r^sultats douteux en eau peptonee glucosee. Les bacilles
typhiques n'ont pas pousse dans l'eau peptonee additionnee de
xylose ou de maltose.
Passons aux resultats obtenus en eau physiologique aminee.
Sans atteindre le degr^ si notable de developpement observe en
eau peptonee additionnee de glucose, de lactose ou de maltose,
les colibacilles ont pousse de fagon tres intense et tres precoce en
eau physiologique aminee additionnee de I'un ou I'autre de ces
650
EDGARD ZUNZ ET PAUL GYORGY
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LES COLIBACILLES ET LES TYPHIBACILLES
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652 EDGARD ZUNZ ET PAUL GYORGY
hydrates de carbone. La croissance des colibacilles a ete a peu
pres du meme degre en presence d'arabinose. Elle a atteint,
au bout de 96 heures, un degre plus considerable en eau physio-
logique aminee additionnee de mannose qu'en presence des autres
hydrates de carbone dont nous venons de parler. L'addition de
xylose; de galactose ou de levulose a I'eau physiologique aminee
a empeche les colibacilles de pousser.
On a note une reaction de I'indol tres intense en eau physio-
logique aminee additionnee d'arabinose. Elle a ete un peu moins
marquee, mais plus precoce, en presence de lactose. On Ta
encore constatee de fagon nette en presence de glucose. Elle a
ete tardive et tres faible en presence de mannose. Elle a fait
defaut dans les cultures de cohbacilles en eau physiologique
aminee additionnee de maltose.
L'action inhibitoire des sucres vis-^-vis de la reaction de I'indol
est, par consequent, beaucoup moins forte dans les cultures de
colibacilles en eau physiologique aminee que dans celle en eau
peptonee. Ceci tient peut-etre a la presence de glycyltrypto-
phane dans le premier de ces miHeux et de tryptophane dans le
deuxieme, soit a une plus forte teneur en tryptophane de I'eau
physiologique aminee, soit a I'absence dans ce dernier milieu
d'autres composes k effets inhibitoires contenus dans I'eau pep-
tonee.
Les bacilles d'Eberth n'ont pousse que faiblement et tardive-
ment en eau physiologique aminee additionnee de lactose. lis
ont pouss^ de fagon plus precoce en eau physiologique aminee
additionnee de 1% de glucose ou de levulose et le degr6 de
croissance est meme devenu relativement notable entre la 24«°"»
et la 52«'°« heure dans un tube d'eau physiologique aminee glu-
cosee. La croissance de ces microorganismes ne s'est pourtant
pas toujours effectuee en presence de ces sucres, d'apres les
donnees experimentales relatees dans le tableau VII. II y a.
en outre, lieu de rappeler que nous n'avons jamais observe le
moindre developpement de bacilles typhiques dans I'eau physio-
logique aminee renfermant seulement 0.2% de glucose (tableau
V). Les bacilles d'Eberth n'ont pas pouss^ en eau physiologique
aminee additionnee de 1% d'arabinose, de xylose, de galactose,
de maltose ou de mannose.
LES COLIBACILLES ET LES TYPHIBACILLES 653
On devait se demander si les effets inhibitoires de plusieurs
hydrates de carbone sur la reaction de I'indol ne provenaient
pas du fait qu'au fur et a mesure de son developpement aux
d^pens du tryptophane, I'indol se combinerait immediatement
a de I'acide glycuronique provenant d'une action event uelle des
coUbacilles sur les sucres ajoutes a I'eau peptonee ou a I'eau
physiologique aminee. Nous avons en vain recherche au moyen
des di verses reactions des acides glycuroniques (avec I'orcine.
avec la phloroglucine, avec la chlorure de calcium et I'acide
chlorhydrique) a demontrer la presence d'acide indoxylglycu-
ronique. II nous est done impossible d'expliquer pour le moment
pourquoi Ton ne parvient pas a mettre en evidence la reaction
de I'indol dans les cultures de coUbacilles soit en eau peptonee
additionnee de rhamnose, de glucose, de galactose, de lactose ou
de maltose, soit en eau physiologique aminee additionnee de
maltose.
Rappelons que, d'apres Haenen, la presence de glucose ou de
lactose dans les cultures de colibacilles en bouillon pepton^
n'empecherait pas de decouvrir I'indol au moyen de I'aldehyde
paradimethylaminobenzoique, contrairement a ce qui serait le
cas dans le reaction classique de I'indol au moyen d'une nitrite
et d'unacide (Bleisch 1893, Gorini 1893, Smith 1893, Seelig 1897).
B. Experiences effectuees avec d'autres microorganismes que les
coUbacilles et les bacilles d'Eberth
II nous reste a parler de quelques recherches faites avec les
bacilles paratji^hiques A et B, les vibrions du cholera, les bacilles
pyocyaniques, les staphylocoques pyogenes dores et les bacilles
de I'ent^rite de Gartner. Dans I'experience relatee dans le ta-
bleau VIII Ton a compare la croissance de ces microorganismes
et aussi celle des colibacilles et des bacilles typhiques pris comme
t^moin, d'une part en bouillon additionne soit de peptone soit
de glycyltryptophane, d'autre part en eau physiologique pep-
tonee soit telle quelle, soit additionnee de glycyltryptophane.
On a aussi recherche la reaction de I'indol dans les divers tubes
ainsi prepares.
654
EDGARD ZUNZ ET PAUL GYORGY
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LES COLIBACILLES ET LES TYPHIBACILLES 655
Les vibrions du cholera n'ont pousse que dans I'eau physio-
logique peptonee additionnee de glycyltryptophane.
Tous les autres microorganismes ont manifeste le maximum
de croissance en bouilllon peptone et le minimum en eau pep-
tonee. Les bacilles d'Eberth, les bacilles paratyphiques B et
les staphylocoques pyogenes dor^s n'ont pas, ou n'ont guere pousse
dans ce dernier milieu.
Par ordre decroissant d'intensite de developpement, on doit
ranger, pour les colibacilles, les staphylocoques pyogenes dores
et les bacilles de I'enterite de Gartner, de la maniere suivante,
les quatre milieux etudies: bouillon peptone, bouillon additionne
de glycyltryptophane, eau peptonee additionnee de glycyltrypto-
phane, eau peptonee. II en est de meme pour les bacilles para-
typhiques A, sauf que la croissance de ces microorganismes a
ete la meme en bouillon additionn^ de glycyltryptophane et en
eau peptonee additionnee de ce peptide. Les bacilles pyocya-
niques ont manifeste un plus grand degre de developpement au
bout de 96 heures en bouillon additionne de glycyltryptophane
qu'en eau peptonee additionnee ou non de ce peptide, alors qu'au
bout de 36 heures Ton constatait deja une croissance marquee
de cette espece de microorganismes dans les deux derniers mUieux,
mais rien en bouillon additionne de glycyltryptophane. Les
bacilles d'Eberth et les bacilles paratj^^hiques B se sont deve-
loppes da vantage en eau peptonee additionnee de glycyltrypto-
phane qu'en bouillon additionne de ce peptide.
Si nous comparons I'intensite de developpement des diverses
especes microbiennes dans le milieu le plus favorable a leur
croissance, c'est-a-dire le bouillon peptone, nous les rangerons
dans I'ordre decroissant suivant: (1) bacilles pyocyaniques et
bacilles de I'enterite de Gartner; (2) coUbacilles et bacilles para-
tj'^phiques B; (3) bacilles d'Eberth, bacilles paratyphiques A
et staphylocoques pyogenes dores. II s'agit d'une simple con-
statation, car rien ne prouve que cet ordre soit tou jours le
meme dans des conditions experimentales identiques.
On n'a observe la reaction de I'indol que dans les cultures de
cohbacilles et de vibrions du cholera. Pour les coUbacilles, la
reaction a presente le maximum d'intensite et de pr^cocit^ dans
656 EDGARD ZUNZ ET PAUL GYORGY
le bouillon additionn6 de glycyltryptophane, Teau peptonee, le
bouillon peptone. II semble bien que la peptone de Witte et
le bouillon renferment des composes qui entravent soit la forma-
tion de I'indol aux depens du tryptophane, soit la mise en evi-
dence de I'indol au moyen de Taldehyde paradim^thylamino-
benzoique selon la methode preconis^e par Haenen.
IV. Considerations generales
Les resultats experimentaux relates ci-dessus provoquent assure-
ment bien des remarques. Mais comme ces recherches deman-
dent a etre etendues et compl^tees a bien des points de vue, nous
nous bornons a insister sur quelques faits qui paraissent en
ressortir avec une nettet6 suffisante.
Tout d'abord, les colibacilles sont bien moins exigeants pour
leur culture que les bacilles d'Eberth. C'est, du reste, une chose
bien connue.
Les bacilles typhiques ne se sont pas developp^s dans I'eau
physiologique ayant comme seule source d'azote soit un acide
amin^, soit un peptide, soit une proteose. On ne constate de
developpement appreciable de ces microorganismes en eau physi-
ologique qu'en presence d'une proteine ddj^ complexe, la gUadine
(tableau II). Les bacilles d'Eberth parviennent, il est vrai, a
pousser faiblement dans I'eau physiologique contenant de la
xanthine ou de la taurine (tableau III).
Les coHbacilles se developpent fort bien dans I'eau physio-
logique renfermant un seul corps azote approprie. Un acide
monoamine suffit parfaitement dans ce but.
Contrairement k ce qu 'Armand-DeUlle, A. Mayer, G. Schaeffer
et E. P. Terroine ont observe pour les bacilles tuberculeux, les
coHbacilles parviennent a se developper dans un milieu ne ren-
fermant comme source azot^e qu'un acide amine a noyau aro-
matique, la phenylalanine. Ce developpement est toutefois bien
moins accentu^ que lorsqu'on part d'un acide monoamine de la
s4rie grasse.
D'apres les resultats consign^s dans les tableaux I et II, la
leucine parait etre, parmi les acides monoamines, la meilleure
LES COLIBACILLES ET LES TYPHIBACILLES 657
source azotee de developpement pour les colibacilles, alors que
les experiences des auteurs cites ci-dessus tendent a demontrer
que le glycocolle joue ce role pour les bacilles tuberculeux.
En partant du bouillon comme base du milieu de culture, on
observe qu'une proteine (la gliadine), une proteose (I'h^teroalbu-
mose), un peptide (le glycyltryptophane) fournissent une culture
aussi abondante de colibacilles que le bouillon additionn^ de
leucine. Aucun de ces milieux n'amene de developpement com-
parable a celui de ces microorganismes en bouillon pepton^ a
1%. Armand-Delille, A Mayer, G. Schaeffer et E. P. Terroine
ont au contraire constate qu'une solution a 0.2% de glycocolle
dans le bouillon de boeuf donne une culture de bacilles tuber-
culeux aussi abondante que le bouillon peptone a 1%.
Si I'on prend I'eau physiologique comme base du milieu de
culture, la leucine amene un developpement tout aussi abondant
des colibacilles que Feau peptonee et ce developpement s'effectue
meme plus rapidement que dans ce dernier milieu. Si Ton
ajoute a I'eau physiologique un autre acide amin^e, un peptide,
une proteose, une proteine permettant la croissance des coli-
bacilles, on n'observe pas par contre de developpement aussi
intense de ces microorganismes qu'en eau peptonee.
Tandis qu'une seule source d'azote suffit a un developpement
tres net du colibacille, des experiences d'Armand-Delille, A.
Mayer, G. Schaeffer et E. P. Terroine ont etabli que deux sources
d'azote distinctes sont indispensables pour la croissance r^gu-
liere du bacille de Koch, a savoir, un acide monoamine, le glyco-
colle, et un acide diamine, I'arginine.
On obtient un developpement plus abondant des colibacilles
si Ton multiplie les sources d'azote, c'est-a-dire si Ton emploie
par exemple de I'eau physiologique renfermant un peptide (le
glycyltryptophane) et soit un acide diamine (I'histidine), soit
une ou plusieurs substances extractives (creatine et sarcosine).
II se pourrait fort bien que la peptone de Witte intervienne
dans le developpement des colibacilles par I'apport de certains
elements azotes et surtoat par sa teneur en leucine.
Nous avons effectue plusieurs milieux artificiels dans lesquels
les colibacilles poussent rapidement et en grande quantite. Mais
658 EDGARD ZUNZ ET PAUL GYORGY
nous n'avons malheureusement pas compare ces milieux (tableau
VI) a Feau peptonee ou au bouillon peptone. Nous ne pouvons
done affirmer qu'ils soient preferabies a ces deux derniers milieux
au point de vue de la croissance des colibacilles.
Par contre, en comparant les donnees des tableaux I, II, V,
VI, VII et VIII, on constate que de Teau renfermant 0.5% de
chlorure de sodium, 0.2% de glycyltryptophane, 0.2% de glu-
cose, 0.2% de sarcosine, 0.2% de creatine, 0.2% de betaine et
0.2% de xanthine parait tout particulierement appropriee a la
recherche de I'indol dans les cultures de colibacilles.
Peut-etre vaut-il meme mieux pref^rer, pom* la diff^renciation
des colibacilles d'avec les bacilles d'Eberth au moyen de la reac-
tion de I'indol, a ce milieu, qu'on peut deja qualifier de relative-
ment complexe, de Feau contenant en solution 0.5% de chlorure
de sodium, 0.2% de glycyltryptophane et 0.2% d'histidine. En
effect, si les resultats relates dans le tableau VI se verifient, un
tel miUeu donnerait la reaction de Findol de fagon tres precoce.
D'autre part, les colibacilles y poussent certes moins bien que
dans le milieu precedent, mais apparaissent neanmoins bien
avant les bacilles d'Eberth.
II appartiendra a de nouvelles recherches de determiner le
milieu a preferer dans le but dont nous venons de parler. On
ne saurait etre trop prudent a cet egard. II convient d'experi-
menter avec de multiples souches de colibacilles et de bacilles
d'Eberth et d'examiner, toutes les heures apres Fensemencement,
la reaction de I'indol dans ces milieux en les comparant a Feau
peptonee et au bouillon peptone, tels quels ou additionnes de
glycyltryptophane. Rien ne prouve, du reste, qu'il n'y ait pas
interet k changer la proportion de Fun ou I'autre des parties con-
stituantes des deux milieux dont nous venons de nous occuper,
ou meme de remplacer Fun ou Fautre des composes azotes ren-
fermes dans ces miHeux par un compose plus approprie.
En effet, nous n'avons, malheureusement pas ^tudie les effets
exerc^s par Faddition a Feau physiologique aminee de leucine et
d'autres acides monoamines sur la croissance des colibacilles et
sur la reaction de I'indol dans leurs cultures. Or, la leucine a^
en eau physiologique, une action tres favorable sur le developpe-
LES COLIBACILLES ET LES TYPHIBACILLES 659
ment des microorganismes sans amener de croissance des ba-
cilles d'Eberth. Tel est aussi le cas de Thistidine. Des lors, on
doit se demander si Taddition simultan^e de leucine et d'histi-
dine a I'eau physio! ogique amin^e ne realiserait pas un milieu en
quelque sorte ideal pour la diff^renciation des colibacilles d'avec
les bacilles typhiques au moyen de la reaction de I'indol. Nous
ne manquerons pas de nous pr^occuper de cette question des
que les circonstances nous le permettront.
Nos experiences mettent une fois de plus en lumiere que la
formation d'indol dans les cultures de colibacilles depend de la
teneur du milieu en tryptophane. La presence de glycyltrypto-
phane dans le milieu de culture parait particulierement appro-
priee a une formation intensive d'indol tant par les colibacilles
que par les vibrions du cholera.
Un autre point que nous croyons devoir souligner, c'est la
forte action inhibitoire de I'inosite sur le d^veloppement tant
des coUbacilles que des bacilles d'Eberth. II suffit, pour s'en
rendre compte, de comparer les resultats obtenus dans le tableau
VI par I'addition d'inosite soit au miheu forme d'eau physio-
logique, de glycyltryptophane, de glucose et d'histidine, soit au
milieu compose d'eau physiologique, de glycyltryptophane, de
glucose, de sarcosine, d'alloxane et de taurine. II serait certes
fort interessant d'entreprendre des recherches in vivo k propos
de cette action inhibitoire de I'inosite.
Signalons encore que la reaction de I'indol au moyen de I'alde-
hyde paradimethylaminobenzoique est entravee par diverses sub-
stances et surtout, dans certaines conditions, par plusieurs
esp^ces d'hydrates de carbone.
RESUME
1. Les bacilles typhiques ont de plus grandes exigences pour
leur nourriture azotee que les colibacilles.
2. Les cohbacilles parviennent a se developper dans I'eau
physiologique ne renfermant qu'un acide monoamine comme
source d'azote. La leucine permet des cultures extremement
riches de ces microorganismes.
660 EDGARD ZUNZ ET PAUL GYORGY
3. Les bacilles typhiques ne se developpent pas dans I'eau
physiologique renfermant un acide amine proprement dit, un
peptide ou une proteose, mais bien en presence de certaines
proteines ou de certains composes azotes tels que la sarcosine. la
taurine et la xanthine. La gliadine constitute un substrat azot6
particulierement favorable a la culture de ces microorganismes.
4. L'inosite entrave beaucoup le developpement tant des coli-
bacilles que des bacilles typhiques dans divers miHeux de culture.
5. La formation de Tindol dans les cultures de cohbacilles
depend de leur richesse en tryptophane. La presence de gly-
cyltryptophane dans les miHeux de culture est tres utile pour
la recherche de la reaction de I'indol.
6. Certains hydrates de carbone entravent la reaction de
I'indol dans les cultures de colibacilles.
7. On pent substituer a I'eau peptonee des milieux chimique-
ment d^finis tres favorables a la recherche de la reaction de
I'indol dans les cultures de colibacilles. Les deux meilleurs
milieux experimentes ont et3 les solutions aqueuses suivantes:
(1) 0.5% de chlorure de sodium, 0.2% de glycyltryptophane,
0.2% d'histidine; (2) 0.5% de chlorure de sodium, 0.2% de
glycyltryptophane, 0.2% de glucose, 0.2% de sarcosine, 0.2%
de creatine, 0.1% betaine et o.2% de xanthine.
REFERENCES
Armand-Delille, p., Mayer, A., Schaeffer, G., et Terroine, E. P. 1913
Journ. de physiol. et de pathol. gener., 15, 797-811.
Besson, a. 1904 Technique microbiologique et seroth6rapique. 504.
Bleisch, M. 1893 Zeits. f. Hyg. u. Infektionskrankh., 14, 101-113.
EscHERiCH UND Pfaundler 1903 Handb. d. path. Mikroorg. Kolle and
Wassermann, 2, 361.
Frouin, a. 1912 C. R. Soc. Biol., 72, 1034-1037; 73, 640-641.
Frouin, a., and Ledelet, Suzanne 1912 Ibid., 72, 982-983.
Galimard, J., etLacomme, L. 1907 Journ. de physiol. et de pathol. gdn^r.
9, 481-486.
Haenen, G. 1905 Arch, internat. de pharmacodynamie et de therapie, 15,
255-261.
Hartwich, Franjen 1914 Zeits. f. physiol. Chem., 90, 311-354.
Gorini, C. 1893 Zentralbl. f. Bakteriol. 13, I Abt., 790-792.
Kitasato 1889 Zeits. f. Hyg. u. Infektionskrankh. 7, 515, cit6 par F. Neufeld,
Handb. d. path. Mikroorg. Kolle and Wassermann, 2, 211, 1903.
LES COLIBACILLES ET LES TYPHIBACILLES 661
Pick, E. P. 1899 Zeits. f. physiol. Chem. 28, 219-288.
Proskauer, B., xjnd Beck, M. 1894 Zeits. f. Hyg. u. Infektionskrankh. 18,
128-152.
Santon, B. 1912 C. R. Acad. Sciences, 155, 860-861; VIIP™« Congres intern.
de Chimie appliqu^e, 19, 267. 1912.
Seelig, Paul 1897 Virchow's Archiv. 146,, 53-64.
Seliber, G. 1914 C. R. Soc. Biol. 76, 63^641.
Smith, Theobold 1897 Journ. of exper. Med. 2, 54.3-547.
Tiffeneau, M., et Marie, A. 1912 C. R. Soc. Biol. 72, 48-50.
Trillat, a., et Fouassier, M. 1912 C. R. Acad. Sciences, 155, 1184-1186.
ZiPFEL, Hugo 1913 Zentralbl. f. Bakter., 64, 65-80; 67, 572.
SOME REGULATING FACTORS IN BACTERIAL
METABOLISM
I. J. KLIGLER
Department of Public Health, American Museum of Natural History, New York
Received for publication, July 5, 1916
Kendall and his co-workers, in a series of investigations, have
developed the idea that in the metabolism of bacteria sugar has
a sparing effect on the digestion of protein and that, conse-
quently, the continuous feeding of sugar to animals would induce
a change in the intestinal flora from a proteolytic to an acid
forming type. These results on sugar feeding were confirmed
recently by Rettger working on white mice and chickens and
by Torrey on human typhoid patients.
In his investigations Kendall used a constant proportion of
sugar to peptone (1 per cent of each) in standard nutrient broth.
Observations, made in the course of a study on the proteolytic
enzymes of the B. proteus, in media in which the concentration
of sugar was varied, led me to think, however, that there were
other important factors involved in bacterial metabohsm, the
actual effects of which were not known. ^ It became quite evi-
dent from these and other observations that the relative con-
centrations of sugar and peptone were significant ; that different
organisms of closely related groups were capable of utilizing
varying amounts of sugar, and that the amount of sugar that
a particular form could completely ferment varied within limits
with the concentration of the other ingredients, especially pep-
tone and phosphate salts. The following are the results of a
series of preliminary experiments to test more definitely the
validity of these observations:
' Kendall and Walker in an independent investigation of the same problem
obtained identical results.
663
664 I. J. KLIGLER
A. Effect of different concentrations of peptone on the nitrogen
utilization of certain intestinal bacteria. The best measure of the
degree of bacterial metabolism in nitrogenous media is the
amount of ammonia liberated. This criterion was therefore
used. The technique was, briefly, as follows:
Simple solutions of peptone in distilled water containing vary-
ing amounts of peptone and 0.5 per cent glucose were used. Two
series were run; one with Witte's and the other with an American
peptone. The different lots of media from each peptone were
made at the same time, flasked, and autoclaved under the same
conditions. Inoculations were made from young cultures and
the flasks incubated at 30° C. for seven days. One lot of Witte
peptone was incubated for five days, but this period was found
insufficient.
The ammonia determinations were made according to the
Folin micro method. First, 5 cc. of the culture were used but
later 10 cc. were found more satisfactory, due to the small
amounts of ammonia present. The period of aeration was
twenty minutes. The ammonia was caught in 10 cc. -^6 sulphuric
acid and the excess acid titrated with ~ Na(OH).
The effect of different concentrations of peptone is evident
from the results of this experiment. It is, of course, possible
that the greater ammonia production with the higher concen-
tration of peptone is due merely to the favorable action of the
increased amount of buffer in keeping down the hydrogen ion
concentration. That the concentration of the peptone is in
itself an important factor, is however indicated by the different
results obtained with different types of bacteria. B. typhi, for
instance, produced no ammonia in the 0.5 per cent peptone in
one medium and a negative quantity in the other, while in the
higher concentrations small amounts were obtained, though less
than in the sugar-free control. B. coli and B. cloacae, etc., give
increasing amounts of anunonia with the increasing concentra-
tion of peptone; but in all cases the amount still remains lower
than in the sugar-free control.
The results, on the whole, indicate that the concentration of
peptone is an appreciable factor when the concentration of glu-
cose is moderately low. Attention may also be called in passing
REGULATING FACTORS IN BACTERIAL METABOLSIM
665
to the different results with the two peptones. This is, no doubt,
due to an essential difference in the composition of the two prod-
ucts. Since Rettger has shown that certain bacteria do not
TABLE 1
Amount oj ammonia -produced in solutions containing different amounts of Witte's
peptone and 0.5 per cent sugar
ORGANISM
B. coli
B. cloacae
B. paracoli
B. capsulatus
B. typhi
Uninoculated control
AMOUNT OF NHs IN CGM. IN 100 CC. OF MEDIA*
0.5% peptone
0.5% glucose
5 days 7 days
-0.17
0.17
-0.51
0.17
-0.68
0.00
0.17
0.51
0.00
0.17
0.00
0.00
1.0% peptone
0.5% glucose
5 days 7 days
0.0
0.17
0.00
-0.17
0.00
0.34
0.68
0.17
0.51
0.00
0.00
1.5% peptone
0.5% glucose
5 days 7 days
0.34
0.17
0.00
0.00
0.68
0.85
0.17
0.34
0.00
0.00
1.5% peptone
No glucose
5 days 7 days
0.34
1.02
0.85
0.85
0.17
0.00
0.34
0.68
0.51
0.68
0.34
0.00
* The data presented here were obtained from two distinct sets of tests run
on different occasions. In the first a five-day and in the second a seven-day
period of incubation was used. The effects of the different amounts of peptone
on the ammonia yield are brought out in both series.
TABLE n
Amount of ammonia produced in solutions containing different amounts of American
peptone and 0.5 per cent glucose
ORGANISM
B. coli
B. cloacae
B. paracoli
B. typhi
B. capsulatus
Uninoculated control
AMOUNT OF NHj IN CGM. IN 100 CC. OF MEDIA
0.5%
peptone
0.5%
glucose
7 days
0.17
0.34
0.00
0.00
0.17
0.00
1% peptone
0.5%
glucose
7 days
0.17
0.51
0.00
0.17
0.17
0.00
u%
peptone
0.5%
glucose
7 days
0.34
0.68
0.17
0.17
0.68
0.00
Control, 1%
Peptone,
7 days
2.04
2.38
1.19
0.51
1.70
0.00
digest proteoses and peptones, it seems that the American
product is very likely richer in amino-acids and hence gives a
higher yield of ammonia in all instances.
B. Effect of different concentrations of peptone and glucose and
phosphate on the metabolism of intestinal bacteria. Another index
666 I. J. KLIGLER
of the metabolism of bacteria is the change they produce in the
hydrogen ion concentration of the medium as a result of their
activities. The acid titration or the ammonia determination
indicates only the action on either the carbohydrates or the
peptone present in the medium. The hydrogen ion concentra-
tion, on the other hand, gives the resultant of the action on
both the carbohydrate and nitrogenous components. By deter-
mining the hydrogen ion concentration on different days, one
may even trace the progressive increase with the active utiUza-
tion of the sugar and subsequent decrease, if any occurs, during
the active utihzation of the constituents of the peptone. This
test, furthermore, serves as a simple index of (a) the amount of
sugar a particular organism may utilize without producing suffi-
cient acid to inhibit further growth and (b) the rate at which
different organisms can utilize a particular sugar. Experiments
are under way which indicate that distinct and constant differ-
ences exist in both properties among closely related types.
The method with some slight modifications is that used by
Clark and based on Sorensen's colorimetric method of determin-
ing the hydrogen ion concentration. Standard solutions of pri-
mary and secondary phosphate and of sodium acetate-acetic-
acid and the indicators described by Clark and Lubs were used.
Media were made as above containing varying amounts of
peptone, glucose and phosphate. A series of typical cultures
was inoculated into these media, incubated at 30° C. and tests
made at regular intervals. In order to eliminate the color, due
to the breaking down of the glucose during sterilization it was
found necessary to sterilize the peptone-phosphate solution and
the sugar solution separately, and then add the sugar to the
peptone by means of sterile pipettes. This somewhat increased
the difficulty in making the medium but assured a water-clear
solution, which greatly facihtated the color readings. For test-
ing the hydrogen ion concentration 1 cc. of the culture was
mixed with 5 cc. of freshly distilled water^ and three to four
drops of the indicator added.
' Clark has suggested a dilution of 1 : 10 but a few preliminary tests showed
that it was more desirable to use a 1 : 5 dilution as excessive dilution changes the
hydrogen ion concentration.
REGULATING FACTORS IN BACTERIAL METABOLISM 667
(1) Effects of different amounts of peptone with and without the
addition of phosphate. The object of this experiment was to
determine the effects of different concentrations of peptone on
the carbohydrate and nitrogen utihzation by bacteria and the
influence exerted by the addition of an acid regulator such as
primary sodium phosphate (Na2H PO4). The media were made
as outlined above and tests for the hydrogen ion concentration
run on the second and fourth days. With a little care 1 cc.
of the culture can be withdrawn by means of a sterile pipette
without contaminating it. By this simple method the progres-
sive changes in the same culture tube could be followed for a
week or more by daily withdrawals of 1 cc. samples.
The results of these tests are summarized in table III. They
indicate quite clearly that the concentration of peptone plays a
rather significant role in accelerating glucose fermentation, as
well as in favoring its complete utilization. The former effect
is seen in table III, section A, in the differences in the Pj values
in the B. cloacae cultures on the second day and in the B. aero-
genes cultm-es on the fourth day. A similar effect is noted in
table III, section B, in the case of B. coli. The favorable effect
on complete utilization of the carbohydrate is brought out espe-
cially in the case of B. aerogenes in section A and of B. coli in
section B of table III.
The effect of the regulating action of the acid-phosphate is
evident on comparing sections A and B. By keeping down the
hydrogen ion concentration of the medium some of the organ-
isms are enabled to use up all the carbohydrate (0.5 per cent)
without producing sufficient concentration of acid to inhibit
their growth. Once this is accomplished, the active utilization
of the nitrogenous constituents commences and we obtain a
progessively decreasing hydrogen ion concentration (or an in-
creasing Pj value). That the regulating effect of the phosphate
is not the only factor, however, is evident from the results
obtained with B. coli as shown in table III.
It is interesting to note that in the absence of phosphate the
cultures of B. cloacae and B. aerogenes reach a hydrogen ion
concentration practically as high as that of B. coli, which does
668
I. J. KLIGLER
not occur in the same medium when 0.5 per cent phosphate is
present. Another interesting point is that the final hydrogen
ion concentration is higher when no phosphate is present; the
primary phosphate obviously tends to keep down the concen-
tration of the hydrogen ions.
TABLE III
The effects of different amounts of peptone, with and without phosphate, on the
utilization of the carbohydrate and peptone, as indicated by the
hydrogen ion concentration
COMPOSITION OF THE MEDIA
Phosphate .
Glucose
Peptone
Days.
0.0%
0.5%
0 5%
0.0%
0.5%
1.0%
0.0%
0.5%
1.5%
0.5%
0.5%
0.5%
0.5%
0.5%
1.0%
0.5%
0.5%
1.5%
Cultures
B. coli*
B. paracoli. . .
B. cloacae. . .
B. aerogenes.
B. typhi
4.6
5.2
4.6
4.8
4.8
4.8
5.6
4.6
4.6
4.6
5.6
4.8
4.6
4.6
4.8
6.6
5.0
4.7
4.8
6.4
4.8
4.6
4.6
4.6
6.8
5.6
4.8
4
5.0
6.5
6.6
5.0
5.1
5.2
6.8
6.6
5.1
5.4
5.2
6.6
6.4
4.8
6.6
5.2
7.0
6.8
5.0
6.0
5.3
6.8
6.6
5.0
7.2
* This strain of B. coli gave these results constantly. Observations in con-
nection with other experiments indicate that not all strains behave in this man-
ner. It is quite likely that there are two types of B. coli (corresponding to the
B. communior and B. communis respectively), which may be differentiated in
this way.
(2) Effect of different concentrations of phosphate on the metabo-
lism of bacteria. The results of the experiments recorded above
indicated that primary phosphate, acting apparently as an acid
regulator, played an important part in controlling the nutritive
processes of bacteria. In order to get more light on the nature
of this regulatory mechanism and its influence on the carbo-
hydrate and peptone utilization the following tests were per-
formed :
Media were made containing a constant and optimum amount
of peptone (1 per cent), comparatively low concentrations of
REGULATING FACTORS IN BACTERIAL METABOLISM
669
glucose (0.3 per cent and 0.4 per cent, respectively), and varying
amounts of phosphate salt, (0.2 per cent, 0.3 per cent and 0.4
per cent, respectively). As nearly as possible the methods of
preparation of media, inoculation, incubation, etc., were kept
uniform. Tests were made on the first, third, and fourth days,
respectively, with the results given in table IV.
TABLE IV
Effect of different concentrations of acid phosphate on the utilization of glucose by
bacteria
A
B
10%
0.3%
0.2%
1.0%
0.3%
0.37o
1.0%
0..3%
0.4%
1.0%
0.4%
0.2%
1.0%
0.4%
0.3%
1 0%
Glucose
0 4%
0.4%
No. of culture
value
value
P+
value
value
value
value
11
4.9
4.9
5.0
4.9
5.0
5.1
16
4.7
4.8
6.2
4.7
4.8
5.5
17
5.0
5.0
6.5
4.6
4.6
5.0
18
6.0
6.4
7.3
4.8
4.6
7.4
19
5.0
6.2
6.8
4.8
4.7
7.2
22
4.9
5.2
6.5
4.8
4.8
5.0
24
6.6
6.6
7.4
6.2+
6.2+
7.4
40
6.6
6.6
6.8
6.2+
6.2+
6.8
44
4.9
5.0
5.4
4.8
4.9
4.9
135
5.6
5.8
6.6
4-9
4.7
8.5
196
4.8
5.4
5.6
5.4
5.5
5.2
239
5.0
5.0
6.4
4.8
4.8
5.0
To avoid the confusion that might result from unnecessary
detail only the four-day tests are tabulated. The effect of the
phosphate is strikingly shown in both the A and B section
of the Table. The significant results are italicized. The low
figures represent high hydrogen ion concentrations — or the acid
phase — while the high figures represent low hydrogen ion con-
centrations or the alkaline phase. The progressive change pro-
duced in carbohydrate media by members of the colon-typhoid
group, excepting certain types, is from the alkaline to acid and
back. So long as there remains unutilized carbohydrate the
acid phase persists. With the complete consumption of the
670 I. J. KLIGLER
carbohydi'ate the organism actively attacks the nitrogenous com-
ponents of the medium, neutrahzing the acid and gradually
returning to the alkahne phase. The phosphate evidently takes
care of the free acid, thus keeping the ionic concentration below
the lethal point and enabling the organism to proceed in its
activity. As a result of this regulative power the amount of
sugar which a particular species can completely utilize varies
within limits with the relative amount of primary phosphate
salts present in the medium. Thus, in the presence of only 0.2
per cent phosphate, 0.3 per cent sugar is completely digested (as
indicated by the low hydrogen ion concentration) by only four
organisms (18, 24, 40 and 135) while only two (24 and 40) can
use up 0.4 per cent glucose. In the presence of 0.3 per cent
phosphate on the other hand 0.3 per cent glucose is also broken
down by number 19; and when 0.4 per cent phosphate is added
all but one are capable of completely destroying 0.3 per cent
sugar, while five (18, 19, 24, 40 and 135) can even use up 0.4
per cent glucose. On increasing the amount of sugar to 0.5
per cent even in the presence of 0.5 per cent phosphate only
19, 24 and 40 reach the alkaline phase (see table III).
C. Amount of sugar digested by different species. Aside from
the effect of varying concentrations of phosphate, the different
types manifest specific differences in their power to digest defi-
nite quantities of glucose under a given set of conditions. These
differences are not attributable to the toxic action of the high
hydrogen ion concentration on the cell, alone. Why one organ-
ism (11, for instance) (see table IV) should not be able to use 0.3 per
cent glucose while number 18 will do away with 0.4 per cent
with the same amount of phosphate (see table IV), when both
reach approximately the same limiting hydrogen ion concentra-
tion (4.8 and 4.9, respectively, table IV, section B), is not evident
on the surface. The difference is partly explicable on the basis
that number 18 (B. enteritidis) carries the fermentation further
than number 11 {B. typhi), thus removing the acid and its
inhibitive effects. But this would not explain the differences
between numbers 17, 18 and 19, for instance, all gas-producing
forms. We must assume either that there are inhibiting factors
REGULATING FACTORS IN BACTERIAL METABOLISM 671
of a specific character or else that specific differences exist in
the way the glucose molecule is spht by these different forms
giving rise to different amounts of toxic substances. The inter-
esting fact remains that, under the same conditions, these closely
related forms manifest strikingly different powers of utihzation
of sugar. Such a difference exists between B. aerogenes and B.
coli, and Clark, taking advantage of this has devised a simple
method for differentiating the two under a given set of condi-
tions. The difference between B. aerogenes and B. coli is, per-
haps, more fundamental than that between the other members
of the group. Specific differences do exist, however, and experi-
ments are now well under way, which promise to furnish a basis
for separating certain of the other more closely related members
of this group.
SUMMARY AND CONCLUSIONS
A study of some of the factors involved in the regulation of
bacterial metabohsm was made. The effects of different con-
centrations of peptone on the utilization of glucose and amino-
acids, and of different concentrations of phosphate on the fer-
mentation of glucose were studied. The nature of the meta-
bolic changes waa measured by ammonia determinations by the
Folin method and by noting the change in the hydrogen ion
concentration by the Sorensen-Clark method. The results were,
briefly, as follows:
1. The concentration of peptone is an appreciable factor, con-
trolhng the nutrition of bacteria. With a moderate amount of
glucose present the higher the concentration of peptone the
greater the amount of ammonia produced. The amount of
ammonia is in no case as great as in the sugar-free control,
indicating a distinct sparing effect.
2. Primary phosphate acting as an acid-regulator plays a very
important part in the regulating of the carbohydrate utilization
by different bacteria of the colon-typhoid group.
3. The different members of the colon-typhoid group mani-
fest specific differences in their power of completely utihzing
different amounts of sugar under a given set of conditions.
These differences may be utihzed for species differentiation.
BOOK REVIEW
Practical Textbook of Infection, Immunity, and Specific Therapy. John
A. KoLMER, M.D., Dr. P.H. Octavo of 899 pages with 143 illus-
trations, 43 in colors. Philadelphia: W. B. Saunders Company.
1915. $6. Half morocco, $7.50.
Kolmer's work has not been in print a year, but has already won an
important place among the books useful to practitioner and labora-
tory worker.
It is well-planned and, throughout, has the stamp of having been
written by one who is actively working in the subject about which he
is writing. It therefore avoids the fault, so frequently apparent in
textbooks, of consisting largely of the warmed-over teaching gleaned
from other writers. Kolmer is active in his subject, and has the judg-
ment of the trained worker in selecting his material.
The discussions of theory are not too extensive and indeed in the
probably intentional neglect of detailed presentation of evidence on
controversial questions, the book may be somewhat disappointing to
the specializing bacteriologist and serologist. However, one has the
feeling that it is Kolmer's purpose to furnish a very complete manual
for the scholarly practitioner without confusing him by too involved a
discussion of principle, and this purpose is excellently served by the
book as a whole.
The work is divided into five parts treating respectively of general
immunologic technique, principles of infection, principles of immunity,
applied immunity, prophjdaxis and treatment, and a series of experi-
ments excellently compiled for teaching purposes. Especially satis-
factory are those parts of the book which deal with clinical apphca-
tions, for the author seems to possess much judgment in matters in
which his own subject has contact with the clinic, an attribute not
over-common among laboratory teachers. The style is clear, illustra-
tions as a whole are of more than ornamental value, and explanations are
concise.
The book should fulfill an important didactic function in making
easily accessible the principles underlying tests and procedures which
are used in hospital, clinic, and private practice, and which, unfortu-
nately, are too often ordered by the attending physician without
adequate knowledge concerning the principles underlying their perform-
ance and interpretation. Perhaps there has never been an era in medi-
cine in which the physician has relied so much upon the aid of diag-
nostic and therapeutic measures of which he possessed so little direct
understanding, and many a learned and affluent practitioner sends for
typhoid serum when he means vaccine, and thinks that the Wasser-
673
674 BOOK REVIEW
mann reaction is a sort of Widal test done with the Treponema 'palli-
dum. In correcting this state of affairs, the author will be a valuable
missionary, since the entire tone of the work is one which will appeal
to the practicing physician and give him such a wealth of interesting
knowledge, that he will read it — and after all, the fact that it will
be read is not the least important quality of a printed book.
Hans Zinsser.
ABSTRACTS OF AMERICAN BACTERIOLOGICAL
LITERATURE
BACTERIOLOGY OF SOILS
Effect of Grinding Soil on the Number of Microorganisms. E. B.
Fred. Science, 1916, 44, 282-283.
The soil was ground in a ball mill for varying periods of time. Very
marked reductions were obtained after one hour and an "enormous
decrease" followed eight or twenty-four hours grinding. Soil pro-
tozoa were also destroyed by grinding. — C. M. H.
Preliminary Investigations in Comparison of Field with Laboratory
Experiments in Soil Biology. G. P. Koch. Soil Science, 1916, 2,
87-92.
Biological experiments (e.g., in ammonification) can be successfully
carried out in the field. As a rule, a greater amount of organic mat-
ter seems to be ammonified in the laboratory tests than in the field,
and field studies of nitrogen fixation and nitrification are greatly inter-
fered with by rains. The nitrogen content of the soil varies consider-
ably, even over a comparatively small area. — Z. N.
Sources of Error in Soil Bacteriological Analysis. H. C. Lint and D,
A. Coleman. Soil Science, 1916, 2, 157-162.
A comparison is made of the soil shaker with the spatula method of
mixing soil for bacteriological analysis, to the great advantage of the
former. The experimental error is smaller with the soil shaker. The
shaker is easily sterilized; it is also exceptionally well adapted to the
incorporation into soil of very small quantities of materials in a finely
pulverized condition. — Z. N.
The Effect of Soil Reaction on Ammonification by Certain Soil Fungi.
N. KoPELOFF. Soil Science, 1916, 1, 541-573.
Three soil fungi, Rhizopus nigricans, Zygorhynchus vuilleminii and
Penicillium sp. 10, were studied as to their reaction requirements in
soil. They possess a comparatively narrow range of reaction toler-
ance for maximum ammonification which was found to be between the
neutral point and an acidity equivalent to 2000 pounds CaO per acre.
This was true whether sandy or clay soils were used with either dried
blood or cottonseed meal.
Where the soil reaction is unfavorable for the activities of the soil
bacteria concerned in ammonification, the soil fungi might prove to be
an important compensating factor in maintaining fertility.— Z. N.
675
676 ABSTRACTS
Influence of Barnyard Manure and Water upon the Bacterial Activities
of the Soil. J. E. Greaves and E, G. Carter. J. Agr. Res., 1916,
6, 889-926.
This is a report of a fairly extensive investigation of the bacterio-
logical effects of different quantities of manure and water added to
soil. Plate counts are made on Lipman's "synthetic agar." Determi-
nations of ammonifying and nitrifying powers of the soil are made by
the tumbler method, while nitrogen-fixing powers are determined by
adding the soil to Ashby's solution. Part of the work is done on
potted soil, part on field soil. The detailed results are too many to
give in a brief review; but in general the writers find a direct rela-
tionship between the bacterial count, the ammonifying powers, the
nitrifying powers, and the crop produced. As a rule all these factors
are increased with increasing amounts of manure, and with increasing
moisture content up to at least 20 per cent. — H. J. C.
The Effect of Time and Depth of Cultivating a Wheat Seed Bed upon
Bacterial Activity in the Soil. P. L. Gainey. Soil Science, 1916,
2, 193-204.
Call of Kansas presented a paper concerning the effects of different
methods of preparing a seedbed for wheat upon yield, soil moisture,
and nitrates. Gainey's article is concerned with the very marked ef-
fects of the various experimental methods of Call upon the accumu-
lation of nitrates. Gainey determined from his own experimental
evidence that the differences in nitrate content reported by Call can-
not be attributed to a difference in the bacterial content. Some
non-biological condition existing in certain plots under field conditions
prevents the normal activity of the bacterial flora. Among the factors
controlling bacterial activity, the available moisture probably plays a
paramount role. — Z. N.
Azotohacter in Hawaiian Soils. P. S. Burgess. Soil Science, 1916,
2, 183-192.
Thirty soils from different localities on the four large islands of the
Hawaiian group were examined as to their azotobacter content, and
their abilities to fix nitrogen in mannit solutions. Only five of the
soils failed to show azotobacter growth in solutions. Four different
forms of azotobacter were isolated in pure cultures, described and their
abilities to fix nitrogen in solutions and in soils determined. A pos-
sible explanation of the introduction and distribution of azotobacter
in these isolated island soils is given. With the introduction of com-
mercial mixed and single element fertilizers, a fine soil is sometimes
used as a "filler." Azotobacter species may have been carried in the
latter, as it is never sterilized. The imported organic fertilizers, leg-
ume seeds, etc., may also have been carriers of this bacillus. — Z. N.
ABSTRACTS 677
The Effect of Some Manganese Salts on Ammonification and Nitrifi-
cation. P. E. Brown and G. H. Minges. Soil Science, 1916, 2,
67-85.
The effect of the application to soils of manganese chloride, manga-
nese sulfate, manganese nitrate and manganese oxide, upon ammonifi-
cation and nitrification, is given, with the following conclusions:
If manganese salts in small quantities increase crop yields on a
soil, that increase may be due, in part, at least, to a beneficial effect on
ammonification and nitrification, with a consequently greater produc-
tion of available plant food.
On the other hand, if manganese salts when applied to the soil
restrict crop growth, that restriction may be due in part to a depres-
sion of bacterial activit3^ The amounts of manganese salts which
may be applied to any one soil without danger of depressing ammoni-
fication and nitrification are exceedingly variable. — Z. N.
Environmental Factors Infiuencing the Activity of Soil Fungi. D. A.
Coleman. Soil Science, 1916, 2, 1-65.
The type of soil as well as the quality of the organic matter were
found to regulate the activities of the organisms used in the tests.
From the standpoint of pure cultures, every organism will do best
with a definite combination of soil and organic matter. As a general
rule, vegetable matter of high quality was conducive to greatest
activity.
Different species of fungi respond with a very wide divergence to
the mechanical composition of the soil by which the oxygen sup-
ply is determined. Chemicals beneficial to one group or species
may be detrimental to other groups of organisms, suggesting a pos-
sible alteration of group relations among the microbes in the soil.
This is also true of the moisture content of the soil.
The organisms employed were observed to have a very narrow tem-
perature range with an optimum of about 30°. — Z. N.
The Yield and Nitrogen Content of Soy Beans as Affected by Inocula-
tion. J. G. LiPMAN AND A. W. Blair. Soil Science, 1916, 1, 579-
584.
Soy beans were used for this experiment as they are less likely to
become inoculated spontaneously than other legumes which may be
used in tests of the value of commercial cultures for soil inoculation.
Moreover, the plants are rather hardy and may be made to grow with-
out difficulty under a wide range of soil and climatic conditions.
Seven commercial cultures from different firms, and soy bean and
cow pea soil were compared with uninoculated checks. Nitrogen
accumulated was determined by the Kjeldahl method.
The authors conclude that the use of inoculating material may be
very desirable in the growing of soy beans and perhaps other legumes.
It appears that where the soil is lacking in the right type of Ps. radi-
678 ABSTRACTS
cicola, inoculation is eminently desirable and that, even where the
organisms are present in limited numbers, the addition of larger num-
bers may be profitable.
A marked difference in the quality of the different commercial cul-
tures for soil inoculation was noted, as was also apparent with the
inoculated soils employed. The variation was not greater with the
soils than with the commercial cultures. — Z. N.
The Oxidation of Sulfur in Soils as a Means of Increasing the Availa-
bility of Mineral Phosphates. J. G. Lipman, H. C. McLean and
H. C. Lint. Soil Science, 1916, 1, 533-539.
Environmental conditions play an important role in the activities
of sulfur oxidizing microorganisms. In soils containing sulfofying
bacteria under favorable conditions for their development elementary
sulfur is readily oxidized. Besides an abundant supply of oxygen,
moisture and the amount and quality of the organic matter are fac-
tors of direct significance. Moreover, the numbers and physiological
eflticiency of the organisms themselves are always of prime importance.
A strong analogy is shown between nitrification and sulfofication.
The oxidation of sulfur in soils by bacteria may lead to the accumula-
tion of large quantities of sulfuric acid, which in turn readily reacts
with basic substances, e.g., tri-calcic phosphate; this may then fur-
nish available phosphoric acid to crops.
Compost heaps as well as cultivated fields may be so treated as to
provide a congenial environment for sulfofying bacteria and thus may
be utilized for the production of available phosphoric acid out of in-
soluble phosphates. — Z. N.
Bacteriological Studies of a Soil Subjected to Different Systems of Crop-
ping for Twenty-five Years. P. L. Gainey and W. W. Gibbs. J.
Agr. Res., 1916, 6, 953-975.
This is a report of investigations of various soil plats that had been
under the same system of cropping for twenty-five years, some under
continuous cropping, some in rotation. The tests made are: number
of bacteria developing on plates, ammonifying and nitrifying powers.
The writers conclude:
"The soil under continuous corn and wheat contains, in the absence
of any additions of fertilizers or manure, relatively low numbers of
bacteria. In the presence of manure, continuous corn and wheat
soil contain relatively high numbers
"The agricultural practices under study .... produced no
appreciable effect upon the ability of the soil and its organic life to
liberate ammonia from cottonseed meal.
"The ability of the soil complex to oxidize ammonia nitrogen to
nitrate nitrogen has been materially altered by the methods under
study Continuous corn and wheat with no additions of
manure or chemicals have brought about a relative low oxidizing
ABSTRACTS 679
power in the soil complex. The addition of manure materially raises
the oxidizing power, especially under continuous wheat and corn. The
addition of commercial fertilizer brings about a condition similar to
that of manure, though perhaps less marked." — H. J. C.
Studies on the Activity of Soil Protozoa. G. P. Koch. Soil Science,
1916, 2, 163-181.
The approximate time for protozoa to excyst when the cysts come
in contact with free water was determined to be five to seven minutes.
Data are given in regard to the influence of moisture content, physi-
cal character of the soil and the presence of organic matter upon the
activity of protozoa in the soil. About 0.33 gm. of soil was taken for
daily examination for active protozoa. The type of soil determines
whether the addition of organic matter encourages protozoan devel-
opment. It was indicated that the destructive abihty of soil protozoa
(if they possess this power) would be present only for a limited period,
namely, in the early stages of organic decomposition. Increasing the
porosity and aeration of the soils by the addition of sand did not in-
crease the number of motile protozoa.
With the soils used, moisture seemed to be the primary limiting factor
which determined the presence or absence of active protozoa.
With one exception, no correlation between the presence of proto-
zoa in the active condition and numbers of bacteria could be seen.
Increased numbers of bacteria were observed irrespective of the pres-
ence or absence of living protozoa. Inasmuch as the numbers of pro-
tozoa in comparison with the bacterial numbers are so small, even in
the presence of such abnormal quantities of organic matter as were
used in these experiments, it hardly seems that they would be of very
great importance in agricultural practice. — Z. N.
Soil Fungi and their Activities. S. A. Waksman. Soil Science, 1916
2, 103-156.
The author has endeavored in this paper to ascertain: (1) What
fungi are true soil organisms, i.e., occur continually and in most soils;
(2) The part that these organisms play in the fertility of the soil.
The micro-flora of eight soils from various portions of the United
States was studied, from which were isolated nineteen species belong-
ing to the Phycomycetes, several to the Ascomycetes, including uni-
dentified species of yeasts, and eighty species to the Fungi Imperfecti,
all of which were included in thirty-one genera. The most common
genera of fungi as to numbers and species found in the soils investi-
gated are, in the order of their occurrence, as follows: Penicillium,
Mucor, Aspergillus, Irichoderma, Cladosporium, Fusarium, Cephalo-
sporium, Rhizopus, Zygorhynchus, Acrostalagmus, Alternaria and
Verticillium. A hypothetical soil flora is given.
Physiological studies were made of types representative of fungus
groups with regard to their nitrogen-fixing and anmionifying power,
680 ABSTRACTS
their ability to digest starch and to decompose cellulose. The am-
monifying and cellulose decomposing power of the fungi tested proved
to be strong, the starch digesting and the nitrogen-fixing action to be
weak or absent.
Cultivated and uncultivated soils do not differ distinctly in the
species of their fungus flora, though each soil seems to have a more
or less characteristic fungus flora. The numbers of fungi decrease
rapidly with depth, so that at twelve to twenty inches below the sur-
face very few fungi can be found, the largest numbers occurring within
the upper four inches of soil. Zygorhynchus Vuilleminii was often the
only organism present in sub soil.
Many pathogenic fungi have been isolated from the soil, a fact
which leads one to think that they pass certain stages of their life his-
tory in the soil, or are able to live saprophytically in the soil and
perhaps play a part in its fertility. — Z. N.
BACTERIOLOGY OF WATER AND SEWAGE
Some Aspects of Chlorination. Joseph Race. Jour, Am. W. W.
Assn., 1916, 3, 439; Can. Engr., 1916, 30, 603-605.
In the use of liquid chlorin for sterilizing water thorough mechani-
cal mixture is required. Tests at Ottawa show a saving in chlorin
with higher bacterial removals. Data are given indicating that more
chlorin is required for high color and when the temperature is low.
Studies of the surviving types of B. coli did not indicate greater resist-
ance than that possessed by the original culture. — L. P.
A Preliminary Report Upon Purification of Swimming Pools at the
State University of Iowa. J. J. Hinman. Eng. and Contr., 1916,
46, 135-138.
Two types of filters are in use, a pressure filter for the men's pool
and a gravity type for the women's pool. Contract with the filter
company guarantees an effluent which will conform to the government
standard for water on interstate carriers. Out of tests on eighty-three
consecutive days, with 1 cc. samples, only three presumptive tests for
B. coli were secured on the women's pool and seven on the men's pool.
Alum and liquid chlorine are used with the usual apparatus for their
appHcation. The author believes that the 37°C. count gives a bet-
ter indication of the degree of purification than the 20°C. count.
F. W. T.
The Sanitary Control of Swimming Pools. Max Levine. Jour.
Infect. Diseases, 1916, 18, 293-306.
The author reviews the investigations on swimming pool disinfec-
tion and reports his own results involving the use of filtration, calcium
hypochlorite, bleach, and copper sulfate. Continuous filtration
effected a reduction of 60 per cent in the bacterial count, the results
ABSTRACTS 681
on B. coll being better when the temperature of the water was below
23°C. But filtration alone was found insufficient to maintain a sani-
tary condition, and continuous disinfection with copper sulfate is
recommended. The chemical should be added, at the rate of 1 part
per million, three times per week, preferably just before the pool is
opened. This method, together with an occasional sterilization with
calcium hypochlorite keeps the pool in good sanitary condition for
several months. — P. B. H.
CLASSIFICATION OF BACTERIA
Studies on the Paratyphoid-enteritidis Group. C. Krumwiede, Jr.,
J. S. Pratt and L. A. Kohn. Jour. Med. Res., 1916, 34, 355-358,
In a study of a series of cultures of the pathogenic types of the
paratyphoid-enteritidis group the authors found that xylose was fer-
mented by all of the types except paratyphoid A, and suggest this as
a presumptive test for this group. — H. W. T.
A Study of the Grouping of the Meningococcus Strains. Miriam P.
Olmstead, Phoebe L. DuBois, Josephine B. Neal, and Rose
Schweitzer. Collected Studies, Bureau of Laboratories, New
York City, 1914-1915, 8, 180.
By means of complement fixation the meningococcus may be clearly
differentiated from aUied organisms. A differentiation of individual
meningococcus strains is possible by use of refined technique, but the
relationship of strains is so close that it is difficult to obtain abso-
lutely clear-cut and consistent results. Of the 29 strains studied, 14
seem to form one group and 8 another. Three are related to the first
group but have acted so irregularly that they cannot be classed with
it; 2 have shown a relationship with each other only and 2 have shown
a relationship to no other strain. — T. G. H.
A Study of Gas-Production by Different Sti-ains of Bacillus abortivo-
equinus. E. S. Good and S. S. Corbett. Jour. Infect. Diseases,
1916, 18, 586-595.
It was ascertained that this organism produced approximately 2
per cent gas in lactose in 80 per cent of 116 trials; and slightly less
than 2 per cent in sucrose in 50 per cent of 56 trials. Comparisons with
the fermentation power of related organisms cause the authors to
conclude that lactose and sucrose can be employed to advantage in
differentiating B. abortivo-equinus from the colon bacillus, while dul-
cite can be used to differentiate it from the members of Subgroup II
of the colon-typhoid group, although absolute proof must depend upon
other characteristics. — P. B. H.
682 ABSTRACTS
A Non-Gas-Producing Strain of the Hog-Cholera Bacillus Isolated from
an Old Laboratory Culture. C. Tenbroeck. Jour. Exp, Med.,
1916, 24, 213-222.
In a stock culture of the hog-cholera bacillus, which was passed
through a series of rabbits 14 years ago, an organism was found that
differs from the original culture in that it fails to form gas from the
carbohydrates that are usually attacked by this organism, while acid
formation persists. This new strain is agglutinated by an anti-hog-
cholera bacillus serum and produces in rabbits and mice a disease simi-
lar to that caused by the typical cultures. The failure to form gas has
persisted over a period of 18 months and all attempts to cause the
strain to revert to the original condition have failed. It resembles in
many respects Bacillus typhi and it may be that some of the so-
called typhoid cultures that are not agglutinated by antityphoid
serum are non-gas-producing paratyphoids. Attempts to produce a
similar change in a more recently isolated culture of the hog-cholera
bacillus by means of animal passages and changes in the environment
have been negative. — B. W.
IMMUNOLOGY
Report of the Committee on Standard Methods of Preparing Diphtheria
Antitoxin. Amer. Jour. Public Health, 1916, 6, 751-752.
A supplementary report to the one made in 1911. — D. G.
The Wassermann Reaction in Two Hundred and Fifty-one Tuberculous
Dispensary Cases. W. R. Jones. Med. Record, 1916, 90, 418-419.
Of 251 cases examined in the tuberculosis clinic, 73 gave a positive,
and 178 a negative Wassermann. — M. W. C.
Autotherapy in Poliomyelitis. C. H. Duncan. New York Med.
Jour., 1916, 104, 342-343.
Report of a case of poliomyelitis treated by the hypodermic injection
of the spinal fluid removed from the patient. — M, W. C.
A Note on the Serum Treatment of Poliomyelitis (Infantile Paralysis).
S. Flexner. Jour. A. M. A., 1916, 67, 583-584.
A review of the work already reported from the Rockefeller Insti-
tute on the serum treatment of infected monkeys, and the work of
Netter on the treatment of human poliomyelitis with the serum of
persons completely recovered from the disease. — G. H. S.
Vaccine Therapy and Other Treatment in Acne Vulgaris and Furuncu-
losis. H. H. Fox. Jour. A. M. A., 1916, 66, 2064-2067.
From an analysis of 100 cases it would appear that the treatment
of these conditions by vaccines, either autogeneous or stock, does not
effect as high a percentage of cures as do other therapeutic measures.
G. H. S.
ABSTRACTS 683
Inoculation against Tijphoid in Maryland. F. W. Hachtel and H.
W. Stoner. Amer. Jour. Pub. Health, 1916, 6, 703-706.
Of 14,795 residents of Maryland inoculated for from one to five years
with typhoid vaccine but ten have since had the disease, which gives
an attack rate of 6.75 per 10,000 persons. In contrast the attack rate
for the state for a sunilar period (5 years) was 33.30 per 10,000 persons.
D. G.
Autolysis of Anaphylactic and Immune Tissues. W. H. Manwaring
AND Ruth Oppenheimer. Proc. Soc. Exp. Biol, and Med., 1916,
13, 176.
The post-mortem autolysis of livers from normal, anaphylactic and
immune guinea pigs was tested by determination of coagulable and
non-coagulable nitrogen. Autolysis was increased in the immune
livers.— W. J. M.
Universal Immunisation. H. B. Baruch. Medical Record, 1916, 90 >
372-373.
The author proposes that children should be injected at an early
age with serum of adults who have recovered from scarlet fever,
measles, and other infectious diseases. The author maintains that such
serum contains antibodies and should therefore confer immunity upon
the recipient.— M. W. C.
The Bacteriological Aspect of the Abderhalden Test. D. Rivas and A.
C. Buckley. Jour. Med. Res., 1916, 34, 297-304.
The article is essentially a summary of recent opinions regarding
the value of the Abderhalden reaction as a diagnostic test. On the
theory that the presence of products of tryptic digestion may be indi-
cated by the formation of indol by B. coli after a short incubation,
the authors apply this test to a series of cases with negative results.
H. W. L.
The Effects of Exposure to Cold upon Experimental Infections of the Re-
spiratory Tract. J. A. Miller and W. C. Noble. Jour. Exp.
Med., 1916, 24, 223-232.
By subjecting rabbits to sudden changes in temperature, from low
to high, and from high to low, and then inoculating them by spraying
cultures of Bacillus bovisepticus on the nasal mucous membrane, it was
found that any marked change of temperature predisposed the rab-
bits to this infection, the severity of which varies with the amount of
change, and that a change from low to high temperature has an even
more marked effect than that from high to low. — B. W.
684 ABSTRACTS
The Effect of Moderately High Atmospheric Temperatures upon the
Formation of Agglutinins. C.-E. A. Winslow, James Alexander
Miller, and W. C. Noble. Proc. Soc. Exp. Biol, and Med., 1916,
13, 194.
Rabbits kept at a temperature of 29° to 32°C. for five weeks and
injected intraperitoneally twice a week with killed typhoid baciUi,
produced agglutinins somewhat less powerful than those of the con-
trol animals kept at room temperature, 18° to 21°C. — W. J. M.
A Note on Expej'imental Nephropathy from Some Bacterial Poisons.
J. L. Stoddard and A. C. Woods. Jour. Med. Res., 1916, 34,
343-355.
Studying the effect of injections of bacterial poisons of the strepto-
cocci and staphylococci, in comparison with the protein split pro-
ducts of Vaughan, the authors find that these poisons have a special
affinity for the epithelial cells of the kidney. Although somewhat
similar, the bacterial poisons do not correspond completely in their
pathological effect with those of the Vaughan poisons. — H. W. L.
Vaccines in Acute Infection. E. Bonime. Medical Record, 1916, 90,
282-284.
To insure a successful use of bacterial vaccines, care must be taken
that the bacteriological diagnosis of the etiological factor is correct,
that the vaccine is carefully prepared and administered at the proper
tune and at suitable intervals, and that further growth of the causative
organisms is prevented. Without these precautions, failures may
occur in vaccine therapy which cannot be justly ascribed to the vaccine
itself.— M. W. C.
A Bacteriological Study of Pyorrhoea Alveolaris and Apical Abscesses
in Relation to Vaccine Therapy. Charles Krumwiede, Jr., and
Josephine S. Pratt. Collected Studies, Bureau of Laboratories,
New York City, 1914-1915, 8, 166.
In a preliminary study of mouth conditions, the great complexity
of the flora found in pyorrhoea is shown. Vaccines made from a few
types present would not influence the pyorrhoeal condition.
A study of the Streptococcus viridans group should be made, including
as many strains as possible of known invasive capacity, as well as those
from mucous membranes. Much of the vaccine therapy as now
practised, rests on doubtful assumptions. — T. G. H.
The Cause, Treatment, and Prevention of Hay Fever. W. Scheppe-
grell. Medical Record, 1916, 90, 95-98.
Among the effective methods of treatment are immunization with
pollen extracts, the administration of autogenous vaccines, prepared
from the bacteria found in the nasal secretion of the patient, and the
use of calcium chloride and succinimide of mercury.
ABSTRACTS 685
Particular emphasis is laid upon the necessity for preventive meas-
ures, especially the eradication of polhnating weeds. — M. W. C.
The Coexistence of Antibody and Antigen in the Body. B. S. Denzer.
Jour. Infect. Diseases, 1916, 18, 631-645.
Antigen and antibody persisted in both the cells and blood for three
weeks after immunization of a guinea-pig with foreign serum. Anti-
gen was demonstrable in the cells and in the blood for 17 days. Later
it disappeared. Antibody was demonstrable in the cells from the
ninth day and in the blood after the fourteenth day. — P. B. H.
The Action of Antiseptics in Pasteur Antirahic Emulsions. Daniel W.
Poor. Collected Studies, Bureau of Laboratories, New York City,
1914-1915, 8, 191.
In an effort to obtain a substitute for glycerin in antirabic emul-
sions, several antiseptics were tried.
Chinosol in dilutions of 1:10,000; 1:15,000 and 1:20,000 offered
no advantages over other agents, which are non-proprietary and more
easily obtainable. Dilutions of 1 : 10,000 effected the strength of the
rabies virus, and 1 : 30,000 did not hold in check extraneous organisms.
Camphor and menthol were of little value. Carbolic acid (0.2 per
cent) gave the best result, the rabicidal effect being less than that of
20 per cent glycerin, and the antiseptic effect as good. — T. G. H.
An Epidemic of Whooping Cough Treated with Pertussis Stock Vaccine.
Matthias Nicoll, Jr., and Paul Luttinger. Collected Studies,
Bureau of Laboratories, New York City, 1914-1915, 8, 86.
Out of 350 children who had been exposed to whooping cough, half
were treated with pertussis vaccine. None of them took the disease.
If all had been treated, freedom from disease probably would have
been ascribed to the vaccine.
The possibility of immunization against pertussus is still an open
question. — T. G. H.
A Case of Sensitization to Witte's Peptone. K. R. Collins. Proc.
N. Y. Pathol. Soc, 1916, 16, 46.
The patient was a laboratory worker who acquired a very marked
sensitiveness to peptone, such that severe coryza, conjunctivitis and
edema of the glottis would follow inhalation of minute quantities of it.
He appeared not to be sensitized to other substances. — W. J. M.
Preliminary Studies of the Antigenic Properties of Different Strains of
Bacillus Typhosus. Sanford B. Hooker. Proc. Soc. Exp. Biol,
and Med., 1916, 13, 139.
Serum cross-titrations with standardized antigens indicated a divi-
sion of typhoid strains into three groups. Group I strains cross-
fixed with all antigens. Those of Group II cross-fixed with each other
686 ABSTRACTS
but not with antigens of Group I. Strains of Group I-A gave irregu-
lar results. The results suggest that a polyvalent vaccine should be
used in typhoid prophylaxis. — W. J. M.
Characteristics of the Precipitation Reaction. Richard Weil. Proc.
Soc. Exp. Biol, and Med., 1916, 13, 200.
Chemically pure antigen unites with precipitin in proportions which
are definite and constant, so that the Danyz-Dungern phenomenon
cannot be demonstrated in the precipitation reaction when performed
with pure reagents. The reaction is probably a quantitative chemical
reaction and not comparable to the adsorption phenomena of mutually
precipitating colloids. — W. J. M.
Immunity in Syphilis. H. Zinsser. Jour. Lab. and Clin. Med., 1916,
1, 785-802.
From clinical and experimental observations it does not seem prob-
able that an infection of syphilis confers true immunity. Although
the syphilitic subject acquires a definite resistance to reinoculation,
which is most pronounced in the secondary stages of the disease, this
resistance decreases during the tertiary stage and probably disappears
entirely upon cure, so that recovery from syphilis leaves the patient as
susceptible to infection as a normal individual. Recovery from the
disease does not occur spontaneously and any apparent immunity to
reinfection is an evidence of persistence of the disease in a latent
form.— M. W. C.
Endothelial Opsonins. W. H. Manwaring and Harry C. Coe.
Proc. Soc. Exp. Biol, and Med., 1916, 13, 171.
When the blood-free liver of an immunized rabbit is perfused with a
suspension of pneumococci in Ringer's solution, the bacteria are rap-
idly removed from the fluid and adhere to the capillary endothelium.
Immune serum added to the suspension of bacteria gives rise to the
same result when the suspension is perfused through normal livers.
Suitable controls give negative results. The serum component resists
a temperature of 60°C. for 30 minutes.
Extrahepatic capillaries fail to show similar phagocytic properties
but the spleen and bone marrow have not been tested as yet. — W. J. M.
Specific Receptors of Fixed Tissues. W. H. Manwaring and Yoshio
KusAMA. Proc. Soc. Exp. Biol., and Med., 1916, 13, 172.
Ringer's solution containing 1 per cent goat serum, repeatedly per-
fused through blood-free liver of normal, anaphylactic or immune
rabbits shows no diminution in amount of serum, that can be detected
by titration with specific precipitating serum. Analogous results are
obtained if normal anaphylactic or immune rabbit blood is added to the
perfusion fluid. The results furnish no evidence of the existence of
specific receptor apparatus in rabbit livers. — W. J. M.
ABSTRACTS
687
Hepatic Baderiolysins. W. H. Manwaring and Harry C. Coe.
(Preliminary Report). Proc. Soc. Exp. Biol, and Med., 1916, 13,
177.
If pneumococci are deposited by perfusion methods in the liver of a
normal rabbit in the presence of normal rabbit blood and the liver then
incubated at 37°C., the bacteria multiply and overgrow the liver after
six hours. If the analogous experiment is tried with liver and blood
of an immune rabbit a gradual decrease in the deposited pneimiococci
is observed. The bacteria in the larger vessels, not in contact witli
parenchyma, are not destroyed. There is apparently an hepatic
mechanism in the immune animal for the destruction of microoi-gan-
isms.— W. J. M.
Protein Absorption by Blood Corpuscles. W. H. Manwaring and
YosHio KusAMA. Proc. Soc. Exp. Biol, and Med., 1916, 13, 173.
Freshly drawn defibrinated rabbit's blood added to a 1 per cent
solution of goat's serum and incubated for one hour shows only 25
per cent of the goat protein remaining in the fluid portion. If the
serum and corpuscles are now separated and allowed to undergo inde-
pendent autolysis (10 hours at 37°C.) a distinct restoration of the
goat protein occurs in both, but is more pronounced in the corpuscle
fraction. Analogous results may be obtained in vivo, indicating that
parenterally introduced proteins are absorbed in a large measure by
the circulating blood corpuscles. — W. J. M.
Analysis of the Anaphylactic and Immune Reactions by Means of the
Isolated Mammalian Heart. W. H. Manwaring, Arthur R. Mein-
HARD AND Helen L. Denhart. Proc. Soc. Exp. Biol, and Med.,
1916, 13, 175. . . ^ u f ■
The heart of a rabbit sensitized to goat serum, tested by perfusion
with 7 to 10 per cent goat serum is more resistant than a normal heart.
Hearts of rabbits sensitized or immunized by repeated injections are
still more resistant. Normal rabbit serum, corpuscles or defibrinated
blood, added to the perfusion fluid decreases its toxicity. Anaphy-
lactic'rabbit serum similarly added increases the toxicity. The ac-
tive principle responsible for this effect is destroyed by heating to
60°C for 30 minutes and is not restored by the addition of fresh nor-
mal serum, and is therefore not complement and probably not precipi-
tin Such inactivated anaphylactic sera are strongly antitoxic.
— W. J. M.
A Method of Producing Antigen for Complement-Fixation in Tubercu-
losis. H. R. Miller and Hans Zinsser. Proc. N. Y. Pathol.
Soc," 1916, 16, 28. , • , -^u u
A weighed amount of bacillary substance is ground up with salt
and subsequently suspended in distilled water sufficient to give isoto-
nicity. Such antigen is not anticomplementary in quantities of 1.0
688 ABSTRACTS
cc. and has fixed in quantities as low as 0.02 cc. Sera from 190 pa-
tients have been tested and positive reactions were obtained in the 89
actively tuberculous cases while 93 cHnically negative cases gave no
fixation.— W. J. M.
Complement Fixation in Tuberculosis. H. R. Miller and Hans
Zinsser. Proc. Soc. Exp. Biol, and Med., 1916, 13, 134.
The antigen is made by grinding 0.020 gram of moist tubercle ba-
cilli with 0.090 gram salt for one hour, then adding 10 cc. of distilled
water. The results in 602 cases are reported. Out of 226 patients
with clinical diagnosis of tuberculosis, 223 gave positive complement
fixation. In 88 cases of arrested tuberculosis, the reaction was nega-
tive in 54, weak in 21 and positive in 13. Of 140 doubtful cases, 32,
gave positive fixation and in some of these 32 a diagnosis of tuberculosis
was subsequently made. Forty-five positive Wassermann sera were
tested, 2 only giving a positive fixation with the tubercle antigen.
One of these two patients was shown to have tuberculous peritonitis.
The fixation seems to be positive only in active tuberculosis. — W. J. M.
A Review of the Complement Fixation Test in Tuberculosis. H. A.
Miller. Jour. Lab. and Clin. Med., 1916, 1, 816-822.
The complement fixation test in tuberculosis has given fair results
with a variety of antigens. Bacillary emulsions, tuberculins and ex-
tracts of bacilli are all available antigens. Particularly successful
results have been obtained with the antigen of Miller and Zinsser.
This is prepared by triturating living or dead bacilli with dry crystals
of ordinary table salt, then adding distilled water up to isotonicity.
This antigen is ahnost invariably positive with active cases, negative
in arrested cases, and gives no cross fixation with luetic sera. — M. W. C.
A Modification of Romer's Intracutaneous Method for the Determination
of Small Amounts of Diphtheria Antitoxin in Blood Sera. Abraham
ZiNGHER. Proc. N. Y. Pathol. Soc, 1916, 16, 49.
A standard, well-ripened toxin is freshly diluted with salt solution
so that 1 cc. represents y|o L+ dose. The serum to be tested is used
undiluted and in dilutions of 1 : 10, 1 : 100, 1 : 1000 and 1 : 10,000. Of
each serimi dilution, 0.2 cc. is added to 0.2, 0.4, 1.0 and 2.0 cc. of the
diluted toxin in four tubes and salt solution, 0.0, 0.2, 0.8 and 1.8 cc,
is added to the respective mixtures, which are allowed to stand 30
minutes before being injected. The injections are made intracutane-
ously into the abdomen of guinea pigs, four widely separated injections
to each animal, the dose being 0.2 cc in each instance. The local
appearance of the skin is recorded daily for four days. As little as
^ J^ unit of antitoxin in a serum can be estimated with a fair degree of
accuracy. — W. J. M.
ABSTRACTS 689
Gonorrhea and Its Complications. A. Hyman. N. Y. Med. Jour
1916, 104, 308-309.
Twenty-five cases of gonorrhea were treated with the vaccine of
Nicolle and Blaizot. Injections were given intramuscularly or intra-
venously for 6 to 8 treatments. The dose amounted to 3,000,000,000
bacteria and was not increased. Acute cases received injections every
day or every other day, chronic cases every two to four days. No
local therapy was employed.
Although 28 per cent of the cases were definitely cured by the vac-
cine, the results in general were inconstant. It had no effect upon
cases of uncomplicated acute and chronic urethritis. Epididymitis
was but slightly improved. Chronic prostatis responded most favor-
ably to the treatment and gonorrheal rheumatism improved greatly in
a few cases. — M. W. C,
Toluol: Its Value in the Sterilization of Vaccines Made from Nonsporo-
genous Gram Negative Bacteria. L. D. Bristol. New York Med.
Jour., 1916, 104, 360-361.
Toluol is advocated as an agent for killing vaccines. It does not
impair the immunizing power of the vaccine as does heat or strong
germicidal chemicals.
Agar slant growth is covered with toluol and allowed to remain
for a length of time necessary to kill. Most Gram negative nonsporo-
genous bacteria will be killed in 24 hours. The toluol is then poured
off and the cultures returned to the incubator to hasten the complete
evaporation of the toluol. Sterihty tests are made and the growth is
suspended in salt solution, counted, and employed as a vaccine.
The staining characteristics of the organisms are retained if the
contact with toluol has not been too long. — M. W. C.
Immunologic Studies on Hodgkins Disease. J. J. Moore. Jour.
Infect. Diseases, 1916, 18, 569-585.
An attempt to ascertain whether an immune serum could be pro-
duced for this disease. Horses were immunized by intravenous inocu-
lation of bacilli isolated from lymph glands. This serum was found
to fix complement and to cause marked agglutination. Complement
fixation tests made with serum of patients gave in no case inhibition
of hemolysis with mixed antigens and vaccination with these organisms
failed to increase complement-binding antibodies. Agglutination tests
were also negative. Similar negative results were observed in the
case of sera from cases of lymphosarcoma, lymphatic leukemia, chronic
arthritis and tuberculosis. — P. B. H.
Bonime's Modification of Koch's Treatment of Tuberculosis. R. C.
Newton. Med. Rec, 1916, 90, 320-324.
Bonime's method of treating tuberculosis consists in the frequent
administration by injection of minute quantities of tuberculin in
gradually increasing doses. The principle is to provide a stimulation
690 ABSTRACTS
of antibody production without causing an overstimulation, resulting
in the liberation of large amounts of toxic products.
The initial dose consists of 0.1 cc. of a 1: 10,000 dilution of O. T.
Doses are repeated and increased in arithmetical progression, tem-
perature conditions controlling the advance, until the patient can re-
ceive 0.2 cc. of pure 0. T. without a reaction. Tuberculin B. E. is
then employed and the immunization continued, doses being given at
longer intervals.
When a patient can take 0.1 cc. of pure O. T. four times in a year
without a reaction, he is presumably immune from infection with
tuberculosis.— M. W. C.
Sputum Cultures with Subsequent Complement Fixation Control. W.
W. Williams and Ward Burdick. Interstate Med. Jour., 1916,
23, 508-512.
The technique described by the authors deals with infections other
than tubercular. The mouth of the patient is thoroughly cleansed.
The specimen is then raised from the deep pharyngeal region. The
mass of sputum is washed in sterile saline solution and the mucoid
fibers smeared over human blood agar plates. If a vaccine is de-
sired, the growth is washed off the plates with salt solution containing
0.3 per cent tricresol. The suspension is standardized and diluted so
that the tricresol is only sufficient for purposes of preservation.
The patient's serum is tested by the complement fixation test, using
the autogenous antigen and stock antigens of organisms which might
be expected to cause the inflammation. The autogenous antigen
seldom fails to give a positive reaction and the corresponding stock
antigen generally gives positive results, except in the case of strepto-
cocci. This confirmatory test is a distinct advantage in vaccine
therapy. — G. H. R.
Complement-Fixation in Pulmonary Tuberculosis. A. Meyer. Medi-
cal Record, 1916, 90, 232-235.
Report is given of the results of complement fixation tests with
tubercular sera and a new antigen.
The antigen is polyvalent and is made from young cultures of hu-
man strains by grinding 20 mgm. of moist tubercle bacilli with 90
mgm. of salt for an hour, adding distilled water to isotonicity, and
separating heavier clumps by allowing the suspension to stand a few
minutes after shaking. This antigen is not anticomplementary in 1
cc. quantities, and fixes positive sera in 0.02 cc.
The test is carried out with one-half the original Wassermann quan-
tities, using 2 units of amboceptor and 2 of complement. The anti-
sheep rabbit hemolytic system is used.
Of the cases tested 96 per cent of those with positive sputum re-
acted positively; 93 per cent of doubtful cases gave positive results,
and these results were later substantiated hy clinical or skiagraphic
ABSTRACTS 691
findings. In 22 cases of various diseases which gave negative reactions,
86 per cent were proved to be non-tuberculous.
With this antigen complement fixation in tuberculosis is considered
of as much value as the Wassermann test for syphilis. — M. W. C.
Studies on Intradermal Sensitization, I. Intradermal Reactions to
Emulsions of Normal and Pathologic Skin. John H. Stokes. Jour.
Infect. Diseases, 1916, 18, 403-414.
The present work is an effort to explain certain conflicting observa-
tions on cutaneous sensitization. It is reported that normal skin
reacts to intradermal injection of skin emulsions in a definite though
variable manner, comparable with the papular luetin reaction, and a
positive reaction may be judged by the same criteria. No specific
character could be established in normal persons for the response
toward their own as compared with other's skin emulsions, and no
evidence of Sellei's " homoasthesia" was found. A suspension of pro-
teins from blood clots gave rise to more transient reactions. In a
single test positive results were obtained from the use of a boiled
(Kozilek's) emulsion. The emulsion possessed no antigenic properties,
and there was no evidence of active anaphylaxis to the proteins. The
attempt to immunize guinea-pigs passively by means of sermn from the
donors of the emulsion resulted negatively. — P. B. H.
Studies in Intradermal Sensitization, II. An Intradermal Reaction to
Agar and an I nteryr elation of Intradermal Reactions. John H.
Stokes. Jour. Infect. Diseases, 1916, 18, 415-436.
The author presents the results of his study of reaction to agar and
enters upon a general discussion of the mechanism of intradermal
reactions in general, with special reference to anaphylatoxin formations.
He concludes that: Reactions to luetin, pallidin, agar, iodid, placental
tissues and skin emulsions, may be regarded as in part due to the
introduction of antiferment adsorbents, the activity of which un-
covers the ferments normally present. These proteases split up the
proteins of the subject with the formation of anaphylatoxins producing
focal necrosis. The course of reaction is determined by the success or
failure of the body cells in the effort to restore the antienzjTne-pro-
tease balance at the site of the injection. Systemic symptoms may be
due to the escape of toxins into the circulation. Such reactions may
be considered as non-specific, conceivably due to the action of the
patient's own enzymes on his own proteins, not to a specific reaction
between the substance injected and a specific amboceptor in the
blood. The author suggests that reaction or non-reaction may find
its solution in investigations of variability in the ferment balance,
either locally or in the body at large. — P. B. H.
692 ABSTRACTS
Studies on Treponema Pallidum and Syphilis. Hans Zinsser, J, G.
Hopkins, and Malcolm McBurney. Proc. N. Y. Pathol. Soc,
1916, 16, 15.
Five strains have been kept aHve through ten or more generations
in rabbits. Four of these were newly isolated and the fifth was the
strain of Dr. Nichols. Apparent fluctuations in virulence are believed
to depend upon the character of the lesion from which the virus is
taken for inoculation, the size of the testes of the rabbit to be inocu-
lated, the thoroughness of maceration of the material before inocula-
tion, the delay before inoculation and the exact site at which the in-
jected material is placed. The apparent fluctuations are therefore
not considered as real alterations in virulence. Variation in thickness
of the spiral occurred in the different strains and was not characteristic
of any one in particular.
The agglutination tests failed to distinguish Treponema pallidum
clearly from other similar organisms, notably Tr. calligyrum, even when
absorption methods were employed.
Normal rabbit serum was found to be treponemacidal when consid-
erable amounts were used. Immune serum was treponemacidal in
one-tenth the quantity. The germicidal property was destroyed by
heating the serum to 56°C. for half an hour.
Agglutination tests failed to distinguish syphilitic human serum
from that of patients free from syphilis.
Agglutination occurred more readily in treponemata which have
been grown in artificial culture a long time.
The preparation from cultures of a specific antigen for the Wasser-
mann test is being studied. — W. J. M.
Animal Experiments upon the Acquirement of Active Immunity by
Treatment with Von Ruck's Vaccine against Tuberculosis. F. J.
Clemenger and F. C. Martlet. Medical Record, 1916, 90, 135-
142.
Experiments were conducted to determine whether the use of von
Ruck's vaccine was followed by an active immunity to tuberculosis.
Clinical use of the vaccines in cases of lymph gland affections re-
sulted in no permanent changes in the local lesions, but in four out
of six cases there was improvement in the general condition. The sug-
gestion is made that the insusceptibility to treatment of the local
conditions was due to the fact that owing to the minimum of circulation
in the affected tissue, no elaboration of immune bodies, however
marked, would be met by a local response.
Attempts to produce immune sera which would conform to a given
standard as expressed by the complement fixation test were partially
unsuccessful because of the prevalence and interfering action of
pseudotuberculosis.
Bactericidal experiments with tubercular human sera demonstrated
that animals which had received a mixture of serum and tubercle ba-
ABSTRACTS 693
cilli were more resistant to infection than those in which salt solution
or normal serum was substituted for the tubercular serum.
The results obtained after infecting pigs which had been actively
immunized with von Ruck's vaccine were not entu-ely satisfactory.
On the whole, however, it appeared that animals possessing an active
immunity showed a greater resistance to infection than control ani-
mals.—M. W. C.
LABORATORY TECHNIQUE
Culture Media for Paramecia and Euglena. R. M. Strong. Science
1916, 44, 238.
The author describes a simple and convenient medium for the abun-
dant production of Paramecia and Euglena for class work. — C. M. H.
Another Use of the Double Plate Method. W. D. Frost and Freda M:
Bachmann. Science, 1916, 44, 433.
The senior author justly insists upon his priority in the use of the
double plate method, introduced by him for the study of antagonism
towards the Bacillus typhi in 1904. In the present work on anti-
septic values of certain spices and condiments, he describes a modi-
fication of his method, made by substituting semi-discs of muslin for
glass rods in the petri dishes. When the plain agar has solidified in
one-half of the plate, the cloth is removed and the agar containing the
condiment is poured into the other half. — C. M. H.
Studies on Laboratory Media. Jane L. Berry. Collected Studiesi,
Bureau of Laboratories, New York City, 1914-1915, 8, 288.
A medium containing the usual amount of agar and one-twelfth
the amount of meat-extract, peptone and salt gave good results in
milk plates, with much reduced cost. Still better results for milk
work were given by agar made with a one to fifteen dilution of Hot-
tinger's stock broth. This is also good for carrying stock cultures.
Agar which has been used once for cultivation of various organisms
can be melted, poured together, titrated, sterilized, and used over
again, especially where large numbers of bacteria are desired as in
vaccine work.
Tests made on several American-made peptones compared favor-
ably with Witte's peptone. — T. G. H.
A New Culture Medium for the Isolation of Bacillus Typhosus from
Stools. J. E. Holt-Harris and Oscar Teague. Jour. Infect.
Diseases, 1916, 18, 596-600.
The authors point out that the chief difficulty in the use of the
Endo plate is that colonies of B. coli may redden the entire plate and
thus obscure typhoid colonies. They recommend a medium com-
posed of nutrient agar containing peptone, sodium chloride, Liebig's
meat extract, sucrose, lactose, eosin and methylene blue. On such
694 ABSTRACTS
media colonies of B. coli are black, while the colonies of B. typhi
are colorless. Moreover the colon colonies do not affect the media
lying between the colonies. Other advantages are mentioned.
—P. B. H.
The Requirements of the Gonococcus for Its Natural and Artificial
Growth. L. D. Bristol. Medical Record, 1916, 90, 63-65.
From a theoretical standpomt it would appear that the gonococcus
requires for its growth human glycoproteins, especially human mucins
and related compounds. This conclusion is drawn from a study of the
character of the proteins upon which the gonococcus grows in the
hmnan body.
Additional evidence that conjugated proteins are essential to the
growth of the gonococcus is found in the fact that while the gonococcus
will not multiply in the ordinary culture media, containing simple
animal proteins, its best growth is obtained upon media containing
human fluids, such as ascitic, cystic, or hydrocele fluids, blood serum
or urine, in all of which mucoid substances of the conjugated protein
group are found. — M. Vv". C,
Limitations to the Cultivation of Mammalian Tissues in Vitro. R. A.
Lambert. Proc. N. Y. Pathol. Soc, 1916, 16, 63.
Connective tissue is the only mammalian tissue which can be cul-
tivated in vitro for any considerable period. Transplantable tumors
show active growth only for a short time. Human malignant tumors
have not been cultivated successfully although human connective
tissue cells and wandering cells show marked activity in cultures.
The limited growth of tumor cells may be referable to several fac-
tors, mechanical injury, accumulation of metabolic products, presence
of inhibiting antibodies and nutritional disturbances. Probably sev-
eral factors are jointly responsible. — W. J. M.
MEDICAL BACTERIOLOGY
Case of Mastoiditis Complicated by Purulent Cerebrospinal Meningitis;
Operation and Recovery. W. H. Huntington. Jour. A. M. A.,
1916, 67, 201-202.
Report of a case.
Friedlander's bacillus was the only organism found in the spinal
fluid.— G. H. S.
Syphilis and Tuberculosis in the Same Lung. R. A. Keilty. New
York Med. Jour., 1916, 104, 252-253.
Report of a case in which the tubercle bacillus and Treponema
pallidum were demonstrable in the lung tissue. — M. W. C.
ABSTRACTS 695
The Bulgarian Bacillus in the Treatment of Vulvovaginitis. M. B.
Cohen. Jour. Lab. and Clin. Med., 1916, 1, 757-759.
Vulvovaginitis cannot be treated to advantage by means of cul-
tures of Bacillus bulgaricus, since this organism does not thrive in the
human vagina. — M. W. C.
The Types of Pneumococci in Tuberculous Sputum. Harold W.
Lyall. Collected Studies, Bureau of Laboratories, New York City,
1914-1915, 8, 176.
Pneumococci were isolated from the sputum of 9 out of 25 tubercu-
lous patients. The pneumococci were all of the type found in normal
mouths.— T. G, H.
Tuberculosis of the Middle Ear. H. B. Graham. Annals of Otology,
Rhinology and Laryngology, 1916, 25, 105-118.
The author gives histories of nine cases.
Diagnosis was made by finding tubercle bacilli in microscopic prepa-
rations, after animal inoculation or in sections showing the pathological
process. — C. P. B.
Gastro-Intestinal Findings in Acne Vulgaris. L. W. Ketron and J.
H. King. Jour. A. M. A., 1916, 67, 671-675.
While it is conceded that the acne bacillus is the direct cause of
acne vulgaris, an analysis of 30 cases suggests that gastro-intestinal
derangements form an important predisposing factor. — G. H. S.
Focal Infection in Relation to Certain Dermatoses. M. L. Ravitch.
Jour. A. M. A., 1916, 67, 430-^31.
Case reports are presented showing that many types of dermatoses
may be due to focal infection. The removal of the infected part,
teeth, tonsils, appendix, or of a streptococcic throat infection, re-
sulted in cure. — G. H. S.
The Treatment of Chronic Colon Bacillus Pyelitis by Pelvic Lavage. H.
L. Kretschmer and F. W. Gaarde. Jour. A. M. A., 1916, 66,
2052-2053.
Of 14 cases of chronic colon bacillus pyelitis 11 bacteriologic cures
were obtained by means of pelvic lavage employing silver nitrate in
1 per cent solution.
Treatments, at intervals of 5 or 6 days and numbering from 1 to 8,
were required to free the urine of bacteria. — G. H. S.
Laboratory Facts in Poliomyelitis. S. R. Klein. New York Med.
Jour., 1916, 104, 219-220.
An examination of about 400 cases of infantile paralysis showed
that the blood and urine v/ere uniformly sterile. The micrococcus of
Weichselbaum was found in considerable numbers in the spinal fluid.
No other organisms were demonstrable. — M. W. C,
696 ABSTBACTS
Vincenfs Bacillus in the Cervix. G. McConnell. N. Y. Med. Jour
1916, 104, 300-301.
A case report. The fusiform bacillus and the spirillum, so con-
stantly present in cases of Vincent's angina, were found in almost
pure culture in a smear prepared from an edematous cervix. A severe
sore thi'oat was coincident with the vaginal disturbance. — M. W. C.
Is Bad. Abortus {Bang) Pathogenic for Human Beings? L. H. Cool-
edge. Jour. Med. Res., 1916, 34, 459-467.
No proof is offered that Bad. abortus is directly pathogenic for
human beings. Adults drinking large quantities of milk from in-
fected cows show, by the complement fixation test, the presence of
antibodies in their blood. The authors believe this to be a passive
immunity due to absorption of antibodies from the infected milk.
H. W. L.
A Bacteriologic Study of the Causes of Some Stillbirths. J. B. DeLee.
Jour. A. M. A., 1916, 67, 344-345.
Several case reports are presented. Pure cultures of Streptococcus
viridans, the pneumococcus and an anaerobic nonhemolytic strepto-
coccus were isolated from the organs of the fetus. The author sug-
gests that many cases of so-called "habitual abortion" and repeated
"premature labor after viability and before term" may be explained
by bacterial infections. — G. H. S.
The Pathogenesis of Psoriasis. A. H. Cook. New York Med. Jour.
1916, 104, 255-257.
From a study of ten cases it seems probable that psoriasis is due to
infections with staphylococci and streptococci. This conclusion is
based upon the fact that removal of the infections coexistent with
psoriasis frequently resulted in recovery. — M. W. C.
The Etiology and Pathology of Otitic Cerebellar Abscess. Isidore
Friesner. Annals of Otology, Rhinology and Laryngology. 1916,
25, 92-104.
In the eighty-six cases collected the infectious agent is only stated
eighteen times. Streptococcus was found eight times, Staphylococcus
once. Vincent's spirillum and bacillus are mentioned. The author
quotes Michaelsen's series in which the infecting agent was not found in
pure culture even once. — C. P. B.
The Microscope in Dermatology. O. L. Levin. New York Med.
Jour., 1916, 104, 117-120.
A description of the use of the microscope in the diagnosis of the
more common diseases of the skin as well as anthrax, glanders, blasto-
mycosis, leprosy, sporotrichosis, actinomycosis, mycetoma, filarial
elephantiasis, Dhobie itch, tinea imbricata, erythrasma, pinea, and
diseases of the hair. — M. W. C.
ABSTRACTS 697
Connellan-King Diplococcus. Infection of the Tonsil. J. J. King.
New York Med. Jour., 1916, 104, 120-121.
From observation of 100 cases of septic arthritis, it appears that
every case is caused by a focus of infection, which is most frequently
situated in the tonsils. The causative organism has many times been
found to be a Gram negative diplococcus, called the Connellan-King
diplococcus. The administration of an autogenous vaccine, followed by
the removal of the tonsils by enucleation constitutes a successful
method of treatment. — M, W. C.
Studies on the Cultivation of the Rabies Virus. Caroline R. Gurley
AND Charlotte C. Van Winkle. Collected Studies, Bureau of
Laboratories, New York City, 1914-1915, 8, 383.
Using the methods described by Noguchi, work was undertaken to
determine with what regularity the cultivation of rabic virus could be
obtained.
No evidence was found of the multiplication of the virus. The
Negri-like bodies found by Noguchi were very rare and not definite
in the experience of these authors. What may have been the same
were once seen in a control tube of ascitic fluid of the kidney.
T. G. H.
Gas Bacillus Infection with Remarks on Gastric Ulcer. Randolph
West and Mary E. Stewart. Proc. N. Y. Pathol. Soc, 1916,
16, 30.
Filtrates of anerobic broth cultures of B. welchii killed guinea pigs,
but not after neutralization of the acid. Acid solutions of equivalent
strength were found to be toxic also. The guinea pigs frequently
showed gastric ulcers and gastric ulcer without death of the animal
was caused regularly by the intravenous injection of 4 to 4.5 cc. of 1
per cent acetic acid. — W. J. M.
Diphtheria in the First Year of Life. J. D. Rolleston. Amer. Jour.
Diseases of Children, 1916, 12, 47-52.
In an analysis of 2600 cases of diphtheria it was found that only
20 or less than 1 per cent were under one year of age. The mortality
among these 20 cases was 45 per cent as compared with 7.3 per cent
in the total. Three patients showed unmistakable signs of congenital
syphihs and the author believes others were probably syphilitic. It
is therefore concluded that syphilis is probably an important predis-
posing factor.— R. M. T.
Resume on Infectious Diseases. Albert H, Beifeld. Amer. Jour.
Diseases of Children, 1916, 12, 166-200.
A very complete and comprehensive resume of the advances made
in contagious diseases during the last few years is here given, together
with a bibliography consisting of 300 references.
698 ABSTRACTS
The following subjects are considered: hospital care, chicken pox,
epidemic parotitis, measles, German measles, whooping cough, diph-
theria and scarlet fever. — R. M. T.
Chronic General Infection with the Bacillus pyocyaneus. Leonard
Freeman. Annals of Surgery, 1916, 64, 195-202.
The case reported is one of extreme chronicity with typical neural-
gic pains followed by paresis and muscular atrophy, and is of special
interest because of the absence of the B. pyocyaneus in the blood and
its presence in the bile; the absence of any discoverable point of infec-
tion, unless possibly the teeth; recovery through drainage of the gall-
bladder and the use of an autogenous vaccine; the occurrence of cir-
rhosis of the liver (and its apparent disappearance since the recovery
of the patient); the presence of ascites and pleural effusion, both of
which promptly disappeared. — T. L. H.
The Bactericidal and Protozoacidal Activity of Emetin Hydrochloride in
Vitro. John A. Kolmer and Allen J. Smith. Jour. Infect. Dis-
eases, 1916, 18, 247-265.
Emetin possesses bactericidal properties but prolonged contact is
required. A 5 per cent solution failed to kill B. typhi in 15 min-
utes and a 2 per cent solution required 45 minutes to kill various bac-
teria from a case of pyorrhea. On the whole the effect was about
equal to that of phenol in corresponding dilutions. Emetin proved
to have some trypanocidal properties but these were less active than
its amebacidal action. — P. B. H,
The Bactericidal and Protozoacidal Effect of Emetin Hydrochloride in
Vivo. John A. Kolmer and Allen J. Smith. Jour. Infect. Dis-
eases, 1916, 18, 266-276.
In studying the bactericidal action of emetin on Staph, aureus, B.
tetanus and B. anthracis, the authors found that the drug, adminis-
tered intravenously, exerted a slight influence or none on the first named
organism (infection in rabbits), and, when administered intraperitone-
ally to mice exerted no inhibitory action upon the other two. Admin-
istered intravenously to white rats, however, it exerted a slight germi-
cidal influence upon T. equiperdum and T. lewisi. The authors con-
clude that the improvement or cure of pyorrhea alveolaris by emetin
is to be attributed solely to its amebacidal action.— P. B. H.
Experimental Cholera-Carriers. Otto Schobl. Jour. Infect. Dis-
eases, 1916, 18, 307-314.
The investigation was undertaken to ascertain whether animals
could be made carriers of cholera vibrios. Inoculations were made
into the gall-bladder, stomach, small intestine, blood stream and ser-
ous cavity; also by feeding. After direct inoculation into the gall-
bladder, stomach and small intestine the organisms were found in the
ABSTRACTS 699
alimentary canal, but intravesicular inoculation was more successful.
The duration of infection in all cases was limited but appeared sufficient
for therapeutic measures. — P. B. H.
The Localization of Streptococci in the Eye. E. E. Irons, E. V. L.
Brown and W. H. Nadler. Jour. Infect. Diseases, 1916, 18, 315.
A study of the invasive action of haemolytic streptococci from a
case of iridocycHtis. Inoculated into rabbits the cultures produced
iritis for 17 days after isolation, but showed a loss of the quality of
localization. This could not be regained when once lost. The au-
thors conclude that "the invasive power of an organism for special
tissue may change within a short period of time during residence in the
original host, during animal passage, and in culture, without pronounced
or constant changes in cultural characteristics, or in general virulence
for animals." — P. B. H.
Chronic Influenza in Pulmonary Tuberculosis. M. L. Hamblet and
H. L. Barnes. Archives Int. Med., 1916, 18, 313-316.
The purpose of the author's investigation was to determine the
number of tuberculous patients infected with influenza bacilli and to
determine whether the symptoms of patients diagnosed as having pul-
monary tuberculosis could be caused by a chronic influenza infection.
Examination for influenza bacilli was accomplished by cultural and
microscopic methods. Ninety-three to 96 per cent of patients diag-
nosed as having both open and closed tuberculosis appeared not to
harbor influenza bacilli. The sputum of twenty patients in whom
the diagnosis of tuberculosis was doubtful was negative for influenza
bacilH.— G. H. R.
The Examination of the Urine and Feces of Suspect Typhoid Carriers
with a Report on Elaterin Catharsis. F. 0. Tonney, F. C. Cald-
well AND P. J. Griffin. Jour. Infect. Diseases, 1916, 18, 239-246.
The writers report the following results of the examination of 290
specimens of urine and 298 specimens of feces : The use of elaterin ca-
tharsis is of assistance in detection of typhoid bacteria, and at the same
time raises the value of a negative test. Endo plates were found
most satisfactory. Lactose-peptone-bile was found to inhibit typhoid
growth. The infectivity of typhoid carriers, in the absence of diarrhea
is probably negligible. Typhoid bacteria were not found in the urine
after administration of hexamethylenamin. — P. B. H.
A Contrihutioti to the Chemotherapy of Tuberculosis, First Experimental
Report. G. Koga. Jour. Exp. Med., 1916, 24, 107-147.
Inspired by Koch's observation on the remarkable germicidal action
of potassium auricyanide on the tubercle bacillus in vitro, Koga pre-
pared a compound of copper and cyanide (details unfortunately not
given) and studied its action on experimental tuberculosis in the guinea
700 ABSTRACTS
pig. The results following the intravenous injection of this salt
("cyanocuprol") are summarized as follows: The effect of a single
injection upon the lesions is either negative or inconspicuous. But
after repeated injections of the preparation the congestion and
leucocytic infiltration about the lesions are markedly decreased, the
cheesy material resulting from degeneration of the lesions and other
degeneration products are in process of absorption, and young connec-
tive tissue is being actively produced in the periphery. While these
changes are taking place the number of bacilli is also being reduced
until finally they can no longer be detected on microscopic examina-
tion. Whether the preparation brings about these results directly by
kilHng the bacilli or indirectly by favoring the healing process of the
body, it has power to inhibit the growth of or annihilate entirely the
bacilli in vitro. — B. W.
A Contribution to the Chemotherapy of Tuberculosis. First Clinical Re-
port. G. KoGA. Jour. Exp. Med., 1916, 24, 149-186.
The author concludes, from a study of sixty-three cases, that cyano-
cuprol given intravenouoly to tuberculous individuals, greatly improves
or apparently cures pulmonary and surgical tuberculosis in the first
and second stage, and it seems also to produce beneficial effects upon
the disease in the third stage. The duration of these beneficial effects
is still to be established. — B. W.
The Treatment of Tuberculosis with Cyanocuprol. M. Otani. Jour.
Exp. Med., 1916, 2, 187-206.
The paper deals with the clinical treatment of tuberculous patients
with ''cyanocuprol." The author claims marked efficacy for the treat-
ment and gives directions for dosage, intervals for injection, precau-
tions and contraindications. — B. W.
The Treatment of Leprosy with Cyanocuprol. R. Takano. Jour. Exp.
Med., 1916, 24, 207-211.
A brief report of treatment of leprosy with cyanocuprol which
appears to be attended with beneficial results. — B. W.
Elective Localization of Bacteria in Diseases of the Nervous System. E.
C. RosENow. Jour. A. M. A., 1916, 67, 662-665.
A study of the possible etiologic relationship of focal infection to
diseases of the nervous system.
Three strains of organisms, one staphylococcus and two green-
producing streptococci, isolated from tonsils or teeth in cases of mul-
tiple sclerosis produced lesions of the spinal cord in 58 per cent of the
animals inoculated.
A staphylococcus was isolated from a typical case of sporadic ante-
rior poliomyelitis, which caused lesions of the spinal cord in 78 per cent
of the animals injected.
ABSTRACTS 701
Bacteria isolated from pyorrheal pockets and tonsils of a case of
transverse myelitis caused lesions of the meninges in 50 per cent, and
of the spinal cord, in 66 per cent of the inoculated.
Streptococci isolated from cases of brachial, intercostal and post-
herpetic neuralgia produced lesions of the posterior roots in 83 per
cent.
The pneumococcus isolated from multiple neuritis caused lesions
of the peripheral nerves in 79 per cent.
Such elective localization warrants the conclusion that the lesions
in the infected patients were due to an infection from a focal source.
G. H. S.
The Natural Resistance of the Pigeon to the Pneumococcus. Preston
Kyes. Jour. Infect. Diseases, 1916, 18, 277-292, with one plate in
color.
The author analyzes in detail the biologic factors which govern
natural immunity in the pigeon to the pneumococcus. It was found
that the organisms, whether injected intravenously or intraperitone-
ally, were rapidly withdrawn from the general circulation and localized
in the liver and spleen. Here the ultimate localization and destruction
was within a type of fixed phagocyte — the hemophage — having the
normal function of destroying red blood cells. The destruction of the
pneumococci by the hemophages is so rapid and complete as to con-
stitute the determining factor in the production of natural immunity.
The possible effect of temperature, of body fluids, of phagocytosis by
wandering phagocytes (crystalloid acidophiles)^ and of opsonic factors
are considered and ruled out as important factors in the final elimina-
tion of the pneumococci. Especial attention is directed to the func-
tion, in the spleen, of a double or triple layer of cells surrounding the
endothelial intima which borders the lumen of certain smaller blood
vessels. Through the agency of this zone of cells is brought about a
mechanical filtration (interstitial) of the organisms which are even-
tually destroyed by the hemophages of the pulp cords. P. B. H.
Laboratory Aids in the Diagnosis of Poliomyelitis. J. B. Neal. New-
York Med. Jour., 1916, 104, 167-168.
One of the most interesting methods of laboratory diagnosis of polio-
myelitis is the transmission of the disease to monkeys by inoculating
them with the washings from the respiratory and alimentary mucous
membranes. Sections of the brain from one monkey thus inoculated,
showed a few globoid bodies similar to those described by Flexner and
Noguchi.
Another method of some diagnostic value is the so-called neutrali-
zation test. Serum from a suspected case in the stage of recovery is
mixed with a known fatal dose of an active virus. The mixture is incu-
bated and injected intracerebrally into monkeys. In case polio-
myelitis does not develop, there is indication that the virus has been
neutralized. However, such neutralization may also be produced
702 ABSTRACTS
through normal serum. Therefore, this method does not furnish con-
clusive evidence of poliomyelitis. Experiments with monkeys are
too complicated and too costly for ordinary diagnostic use.
Examination of the spinal fluid offers the most trustworthy method
of diagnosis. A spinal fluid increased in amount and showing a slight
to moderate increase in albumin and globulin, a good reduction of Feh-
ling's, and a varying cellular increase, mostly mononuclear, makes the
diagnosis reasonably certain in fairly early cases of suspected polio-
myelitis. A slightly cloudy fluid occurring very early in the disease
must be differentiated from a similar fluid in an early purulent meningi-
tis. Fluids from the cerebral or encephalitic type of poliomyelitis
may sometimes be differentiated from fluids of tuberculous meningitis
only by animal inoculation. — M. W. C.
Cultivation of the Organisms of Vaccinia, Variola, and Varicella. H.
Greeley. Medical Record, 1916, 90, 265-271.
A minute, bipolar bacillus has been found in all specimens of vac-
cinia virus, and varicella and variola pustules. The organism occurs
either in the form of a bacillus or in involution forms from which spore-
like bodies escape. The organisms of vaccinia, variola, and varicella
differ somewhat in size, but the general morphology is the same.
Cultural experiments upon the bacillus of vaccinia virus indicate
growth upon hydrocele fluid, whether diluted in different proportions
with physiological salt, distilled water, or bouillon, or when used alone.
Media consisting of about 70 per cent hydrocele fluid and 30 per cent
bouillon, and containing a lime water equivalent of 10 per cent were
particularly suited to the growth of the organism. Fifty per cent
bouillon in distilled water, saturated with 10 to 100 per cent calcium
oxide was also a good medium. Both glycerin and glucose when
added to the cultures exerted an unfavorable influence. Slants of
Loeffler blood serum gave good growths when washed every day with
one or two drops of limed bouillon mixture.
The virus of variola and also that of varicella grew under the same
conditions as those which were found to be most favorable for vaccinia.
Agglutination tests showed specific results between variola sera and
variola organisms, between vaccinia sera and vaccinia organisms.
There was also agglutination between vaccinia sera and variola anti-
gen. Varicella antigen reacted irregularly with all sera except those
from varicella cases, with which it was uniformly negative.
In complement fixation the results were specific with variola sera
and variola antigen, also with vaccinia sera and vaccinia antigen.
Two vaccinia sera gave slightly positive reactions with variola antigen,
but there was no cross reaction between variola sera and vaccinia
antigen. Sera from cases of varicella gave negative results with
varicella and variola antigens, but normal sera in almost every case
reacted positively with varicella antigen. — M. W. C.
ABSTRACTS 703
A Study of the Etiology/ of Chorea. John Lovett Morse and Cleve-
land Floyd. Amer. Jour. Diseases of Children, 1916, 12, 61-72.
Twenty-six cases of chorea were studied, of which twenty-one gave
no suggestive history of syphihs and only one gave a positive Wasser-
mann and one a doubtful reaction. This percentage is no higher
than that obtained among hospital children in general. It is therefore
concluded that syphilis probably plays no part in the etiology of chorea.
Seven of the patients or 37 per cent gave a history of rheumatism, six
of them had acute endocarditis and six chronic valvular lesions, a
total of 46 per cent. The tonsils were enlarged in eleven or 42 per cent
and had been removed in four; the teeth were carious in nineteen or
73 per cent, pyorrhea being present in two of these and pockets of
pus in three others.
Thirty-one blood cultures were made from the twenty-six cases
and the cerebro-spinal fluid was cultured twenty times in nineteen
cases. Loeffler's blood serum, neutral and acid milk, serum water
glucose, lactose and sucrose, glucose bouillon and at times hydrocele
fluid, alone and with agar were employed, both under aerobic and
anaerobic conditions. In five cases organisms were obtained. One
was a small gram negative "diphtheroid" bacillus, which grew very
poorly and failed to produce any effect upon rabbits. Diplococci were
found in the blood smears once but did not grow. In three cases
streptococci were grown in the original culture but in only one were
transplants successful. The authors think that these organisms were
probably alike. The one subcultured was injected into rabbits with
the production of endocarditis, arthritis and a congestion and round
cell infiltration of the pia over the cortex and extending into the con-
volutions. In one of the three rabbits in which the brain was exam-
ined smears from the cortex revealed streptococci.
The number of cells in the cerebro-spinal fluid taken from ten of the
patients was 2, 5, 7, 8, 10, 10, 10, 18, 24, and 25 respectively showing
a slight increase in 3 or 30 per cent.
The authors conclude that there is a definite relationship between
chorea and rheumatism, endocarditis and infections about the oral
cavity, and that their results suggest that a microorganism or group of
microorganisms may be the cause of chorea. — R. M. T.
PHYSIOLOGY OF BACTERIA
The Destructive Effects of Light and Dnjing and Other Living-Room
Conditions Upon Diphtheria Bacilli, Streptococci, and Staphylococci.
Daniel W. Poor and Charles P. Fitzpatrick. Collected Studies,
Bureau of Laboratories, New York City, 1914-1915, 8, 197.
Membranes from four cases of diphtheria, and mucus from one case,
with mucus from one case of scarlet fever were subjected to various
light and drying tests. When exposed to diffuse Hght, drying and
admixture of dust and other bacteria, diphtheria bacilli, staphylo-
cocci, and streptococci were rapidly reduced in numbers during the
704 ABSTRACTS
first few days, after which there was a more gradual reduction. Strep-
tococci seldom resisted drying and light longer than a week, nor diph-
theria bacilli more than three weeks.
Ascitic broth cultures sometim.es showed the presence of a few sur-
viving diphtheria bacilli and streptococci after plate cultures had
failed.
The reduction in the number of bacteria in mucus and in mem-
branes took place much more slowly if the light was partially ex-
cluded (as would be the case where membranes were deposited under a
bed or in a dark closet). None of the streptococci remained alive as
long as three weeks, nor the diphtheria bacilli as long as ten weeks.
T. G. H.
Life Cycles of the Bacteria {Preliminary Communication). F. Lohnis
AND N. R. Smith. J. Agr. Res., 1916, 6, 675-702.
If the writers' claims are true, they will almost revolutionize our
conceptions of the morphology of bacteria. They claim that a single
species may pass through as many as twelve or fourteen distinct mor-
phological forms, varying from tiny, filterable "gonidia" to large,
unorganized masses of "synplasm" formed by the fusion of smaller
elements. None of these are dead or attentuated forms (although
often described in the past as involution forms), even the "synplasm"
being capable of development into organized cells, passing through an
intermediate stage in which very small granules appear, called by the
writers "regenerative units." The writers distinctly say, however;
"We beg to point out that by discussing the life cycles of the bacteria
we do not intend to revive any of those unclear theories concerning
bacterial polymorphism or pleomorphism. The development of the
bacteria is characterized not by the irregular occurrence of more or
less abnormal forms but by the regular occurrence of many different
forms and stages of growth connected with each other by constant
relations."
The writers' most thorough studies have been made on B. asoto-
bacter. They show that there are four groups of the different forms
of growth of this organism, and that ordinarily any culture shows
only one of these groups of growth forms. It is so seldom that a
culture develops forms of one of the other groups that separate species
have undoubtedly in the past been based upon these morphological
differences. The writers have studied a few other bacteria in lesser
detail, and have evidence that they also pass through similar life
cycles. — H. J. C.
The Inhibiting Action of Certain Spices on the Growth of Microorgan-
isms. Freda M. Bachmann. Jour. Ind. and Eng. Chem., 1916, 8,
620.
The author has studied the antiseptic action of ground spices,
their alcoholic extracts, essential oils and active principles, on pure
cultures of mold spores and filaments, and of bacteria. Molds were
ABSTRACTS 705
represented by species of Rhizo-pus, Penicillium, Aspergillus and Alter-
naria and the bacteria by B. coli, B. prodigiosus and B. subtilis. The
double plate method was used, one half the plate containing plain
agar the other half the medium plus the spice to be tested. Tests
were made by streaking the plate with sterile water suspensions of the
organisms.
Alcoholic extracts of cinnamon, cloves and mustard showed variable
degrees of effectiveness. Cloves was most active; mustard not at all.
Rhizopus was more sensitive than Penicillium to cinnamon but less
so to cloves. In powder form cinnamon was more effective than
cloves, allspice or nutmeg. Rhizopus was not affected by cinnamon,
other molds and B. subtilis were highly sensitive to all spices, except
nutmeg; B. coli and B. prodigiosus were inhibited by allspice but
scarcely at all by the others. Cloves and allspice were about equally
effective. Cinnamic aldehyde in concentration of 1: 2000 inhibits
growth of all molds and bacteria; volatilization inhibits growth even
on spicefree half of plate. Eugenol was antiseptic for Alternaria and
B. subtilis only. Of the oils that of allspice was best; effective
against Rhizopus in 1: 5000, B. subtilis 1: 1000; it does not inhibit B.
coli and B. prodigiosus.
In general mold spores were more sensitive than mycelia and both
more so than bacteria; B. subtilis was most sensitive among latter.
Spices in the amounts used in the kitchen are not good preservatives.
Large amounts of cinnamon, cloves and allspice do act as preservatives.
I. J. K.
PLANT PATHOLOGY
Observations on Fire Blight in the Yakima Valley, Washington. J. W.
HoTSON. Phytopath., 1916, 6, 288-292.
Leaf infection of Bartlett pears is reported, beginning at the mar-
gins. Invasion of the sap wood from twig blight and an abnormal
number of fruit infections are also noted. — F. L. S.
Bacteriological Blights of Barley and Certain Other Cereals. L. R.
Jones, A. G. Johnson and C. S. Reading. Science, 1916, 44,432.
The authors describe the causal organism for blights of barley,
wheat, rye, spelt, and oats. In all but the latter case the organism
seems to be of the same species, a monotrichous rod with a single polar
flagellum. It is seed-borne, which accounts for its general distribution.
The organism survives on infected seeds for at least two years. More
detailed studies will appear at a later date. — C. M. H.
A Bacterial Disease of Western Wheat-Grass, Agropyron smithii.
Occurrence of a New Type of Bacterial Disease in America. P. T.
O'Gara. Phytopath., 1916, 6, 341.
A disease of Agropyron smithii found in several places in Utah, and
similar to the diseases of orchard grass discussed by Rathay is de-
706 ABSTRACTS
scribed. The affected plants are somewhat dwarfed and have upon
their surfaces masses of bacteria which form a yellow ooze which pro-
duces layers between the stem and the upper sheath and between the
glumes of the inflorescence. The organism does not penetrate into the
tissues until some time after it has covered the surface. It is later
found in the substomatic chambers and in the intercellular spaces
but has not been found occupying the cells. Normal seed are rarely
produced. The organism is non-motile and is described by the author
as '' Aplanohader Agropyri sp. nov." It is a short rod with rounded
ends which occurs singly or in pairs and infrequently in chains of four.
They are 0.4 to 0.6 x 0.6 to l.l^t in size. No endospores have been
observed. Capsules have been frequently demonstrated. The organ-
isms stain readily with the common stains.
The group number is 212.2223522.— F. L. S.
Some Properties of the Virus of the Mosaic Disease of Tobacco. H. A.
Allard. J. Agr. Res., 1916, 6, 649-674.
Several theories have been advanced in the past to explain this dis-
ease as of physiological instead of infectious origin. It has been
claimed that the oxidases and peroxidases produced by the plant
cause the disease, and spread the disease to other plants if inoculated
with the sap from diseased plants. The wi'iter gives evidence to dis-
prove this theory. It is true that the virus is not removed from the
sap if filtered through a Berkefeld filter; but by submitting the virus
to various treatments the writer shows that it may be destroyed by
means that do not destroy enzymes, or that the enzymes, on the
other hand, may be destroyed by means that do not destroy the virus.
The writer concludes: "Since this pathogenic agent is highly infectious
and is capable of increasing indefinitely within susceptible plants,
there is every reason to believe that it is an ultramicroscopic parasite
of some kind." — H. J. C.
PUBLIC HEALTH BACTERIOLOGY
Bubbling Fountain Tests. Jane L. Berry. Collected Studies, Bureau
of Laboratories, New York City, 1914-1915, 8, 135.
Two bacteriological tests made on a bubbling fountain showed only
staphylococci present. When the fountain was smeared with a cul-
ture of B. coli, considerable flushing did not entirely remove this or-
ganism. The danger, if pathogens are present, is evident. The best
safeguard for the kind of fountain described is a continuous flow of
water.— T. G. H.
Cigar Cutter Tests. Jane L. Berry. Collected Studies, Bureau of
Laboratories, New York City, 1914-1915, 8, 137.
Of 145 customers purchasing cigars, 74 used the cigar cutter, and
of these, 4 first moistened the cigar in the mouth before cutting.
Streptococci, staphylococci and spore-bearing rods were easily obtain-
ABSTRACTS 707
able from the cigar cutter after such procedure. Cigars moistened
with B. pyocyaneus, when cut, contaminated the cutter, and fresh
cigars were in turn contaminated. Of three cigars held in the mouths
of different diphtheria patients, one contaminated the cutter with the
Klebs-Loeffler bacillus. The author recommends that cigar cutters
be abolished.— T. G. H.
Studies on the Use of Brilliant Green and a Modified Endo's Medium in
the Isolation of Bacillus Typhosus from Feces. H. C. Robinson and
L. F. Rettger. Jour. Med. Res., 1916, 34, 363-376.
Using a preliminary enriching medium consisting of peptone water,
reaction + 1.0 to phenolphthalein, containing brilliant green in dilu-
tions of 1 : 60,000 to 1 : 300,000, and a modified Endo's medium, the
authors report successful results in the isolation of typhoid bacilli from
feces. The modification of Endo's medium consists in the use of sodium
bisulphite instead of the plain sulphite. — H. W. L.
Studies on Diphtheria in Cleveland, I. Diphtheria Carriers. R. G.
Perkins, M. J. Miller and H. O. Rush. Jour. Infect. Diseases,
1916, 18, 607-617.
A paper having special reference to the problem of shortening the
average quarantine period. The points discussed include the incidence
of carriers in Cleveland, morphological studies on the organisms found,
and the results of the application of the Schick test. — P. B. H.
A Study of the Normal Bacterial Flora of Postage Stamps. R. A.
Keilty and p. D. McMaster. Medical Record, 1916, 90, 153-154.
A bacteriological study of 50 postage stamps obtained from various
sources showed the presence of bacteria upon 48 of the 50 stamps.
Methods particularly adapted for the detection of tubercle bacilli
Bacillus tetani, members of the colon group and diphtherial forms
failed to demonstrate any of these organisms. The organisms isolated
were, with possibly two exceptions, all non-pathogenic. Micrococci
predominated. Other non-pathogenic organisms, molds, B. subtilis,
etc., were present in only small numbers. — M. W. C.
INDEX TO VOLUME I
Abstracts of American bacteriological literature 123, 249, 357, 455, 553, 675
Actinomycetes, A possible function of, in soil 197
Advantages of a carbohydrate naedium in the routine bacterial examination
of milk. The 481
Agiar plates, The number of colonies allowable on satisfactory 321
agar culture media, A note on the preparation of 547
Alcohol forming bacterimn, A new species of, isolated from the interior of
stalks of sugar cane infested with the cane-borer Diairaea saccharalis. . . 235
American bacteriological literature. Abstracts of.
Animal pathology.
B. enteritidis as the cause of infectious diarrhea in calves, The. H. F.
Meyer, J. Traum and C. L. Roadhouse 367
Diphtheroid bacillus of Preisz-Nocard from equine, bovine and ovine ab-
scesses. The. Ulcerative lymphangitis and caseous lymphadenitis.
R. C. Hall and R. V. Stone 553
Experiments to determine the relative value of trikresol and carbolic acid
in the preservation of hog cholera serum. John Reichel 359
Filterable organism isolated from the tissues of cholera hogs, A. D. J.
Healy and E. J. Gott 357
Maintenance of virulence of B. abortivus equinus, The. E. S. Good and
W. V. Smith 249
Notes on the histo-pathology of the intestines in young chicks infected
with Bacterium pullorum. C. Edward Gage and James F. Martin.. 553
Report upon an outbreak of fowl typhoid, A. Walter J. Taylor 357
Studies to diagnose a fatal disease of cattle in the mountainous regions
of California. K. F. Meyer 249
Vaccination experiments against anthrax. A. Eichhorn 250
Value of virulent salt solution in the production of anti-hog-cholera
serum by the intravenous method. The. Robert Graham and L. R.
Himmelberger 358
Bacteriology of air and dust.
Recovery of Streptococcus viridans from New York street dust. W. C.
Thro 360
Bacteriology of food.
Bacterial examination of sausages and its sanitary significance. The.
W. E. Gary • 123
Effects of refrigeration upon the larvae of Trichinella spiralis. B. H.
Ransom 123
Feeding experiments with B. pullorum. The toxicity of infected eggs.
Leo F. Rettger, T. G. Hull and W. S. Sturges 455
Food poisoning by the B. paratyphosus B. Harry S. Bernstein and
Ezra S. Fish 361
Indol in cheese. V. E. Nelson 361
709
710 INDEX
American bacteriological literature — continued
Bacteriology of the mouth.
Constitutional conditions caused by oral sepsis. J. Daland 563
Cultivation of Entameba buccalis, On the. Wm. B. Wherry and Wade W.
Oliver 362
Deep seated alveolar infection. M. L. Rhein 362
Dental aspect of the relation of endamoeba to pyorrhea alveolaris, The
W. A. Price 362
path: its importance as an avenue to infection, The. T. B.'Hart-
zell and A. T. Henrici 363
Observations upon the endamoebae of the mouth. I. Endamobea gin-
givalis (buccalis). C. F. Craig 455
Pyorrhea alveolaris and some modern discoveries regarding its cause
and treatment. F. E. Stewart 455
Relation of amoebiasis to pyorrhea alveolaris, The. A. H. Sanford and
Gordon B. New 362
Study of Endameba buccalis in alveolodental pyorrhea, A. F. M. Johns 564
Treatment of pyorrhea alveolaris and its secondary systemic infec-
tions by deep muscular injections of mercury. B. D. Wright 563
Bacteriology of soils.
Actinomyces of the soil. Selman A. Waksman and Roland E. Curtis. . . 558
Azotobacter in Hawaiian soils. P. S. Burgess 676
Bacterial numbers in soils at different depths, and in different seasons
of the year. Sehnan A. Waksman 556
Bacteriological studies of a soil subjected to different systems of crop-
ping for twenty-five years. P. L. Gainey and W. W. Gibbs 678
Can soil be sterilized without radical alteration? David A. Coleman,
H. Clay Lint and Nicholas Kopeloff 560
Comparison of the acid production of the B. coli group isolated from
various sources, A. W. W. Browne 124
Detailed study of effects of climate on important properties of soils, A.
C. B. Lipman and D. D. Waynick 562
Diastase activity and invertase activity of bacteria. George P. Koch. . 556
Effect of grinding soil on the number of microorganisms. E. B. Fred. . . 675
— '■ — of soil reaction on ammonification by certain soil fungi, The. N.
Kopeloff 675
of some manganese salts on ammonification and nitrification, The.
P. E. Brown and G. H. Minges 677
of time and depth of cultivating a wheat seed bed upon bacterial
activity in the soil. The. P. L. Gainey 676
Environmental factors influencing the activity of soil fungi. D. A.
Coleman 677
Incubation studies with soil fungi. S. A. Waksman 555
Influence of barnyard manure and water upon the bacterial activities
of the soil. J. E. Greaves and E. G. Carter 676
of some common humus-forming materials of narrow and of
wide nitrogen-carbon ratio on bacteria,l activities, The. P. E.
Brown and F. E. Allison 559
INDEX 711
American bacteriological literature — continued
Bacteriology oj soils.
Inoculation and incubation of soil fungi, The. Nicholas Kopeloff 557
Nitro cultures and their commercial application. F. C. Harrison 456
Occurrence of bacteria in frozen soil, The. E. C. Harder 555
Oxidation of sulfur in soils as a means of increasing the availability of
mineral phosphates, The. J. G. Lipman, H. C. McLean and H. C.
Lint 678
Preliminary experiments on some effects of leaching on the soil flora.
C. B. Lipman and L. W. Fowler 553
investigations in comparison of field with laboratory experiments
in soil bacteriology. G. P. Koch 675
Quantitative method for the estimation of bacteria in soils. R. C. Cook 554
Relation between certain bacterial activities in soils and their crop-
producing power. Percy Edgar Browne 124
of carbon bisulphid to soil organisms and plant growth. E. B.
Fred 361
of green manures to the failure of certain seedlings. E. B. Fred. . . 361
Soil fungi and their activities. S. A. Waksman 679
Some factors influencing the longevity of soil micro5rganisms subjected
to desiccation with special reference to soil solution. Ward Gilt-
ner and H. Virginia Langworthy 123
factors that influence nitrate formation in acid soils. E. B. Fred
and E. J. Graul 558
Sources of error in soil bacteriological analysis. H. C. Lint and D. A.
Coleman 675
Stimulating influence of arsenic upon the nitrogen-fixing organisms of
the soil. J. E. Greaves 554
Studies in sulfofication. P. E. Brown and H. W. Johnson 553
on soil protozoa. Selman A. Waksman 561
on the activity of soil protozoa. G. P. Koch 679
on the decomposition of cellulose in soils. I. G. McBeth 557
Variation in nodule formation. L. T. Leonard 456
Yield and nitrogen content of soy beans as aff'ected by inoculation, The.
J. G. Lipman and A. W. Blair 677
Bacteriology of water and sewage.
Activated sludge process of sewage treatment. The. G. J. Fowler 251
Air diffusers tested at Milwaukee (Wis.) sewage plant. T. Chalk-
ley Halton 125
Bacteria in commercial bottled waters. Maud Mason Obst 564
Bacterial counts in water examination. J. Race 564
Confirmatory tests for B. coli in routine water examinations. W. H.
Frost 565
Copper sulphate treatment of St. Paul (Minn.) water supplies. N. L.
Huff and G. O. House 458
Electrical treatment of water, The. T. A. Starkey 363
Experiences in the application of the activated sludge process to Chi-
cago stockyard sewage. Arthur Lederer 457
712 INDEX
American bacteriological literature — continued
Bacteriology of water and sewage.
Fundamental principles of the activated sludge process of sewage treat-
ment, The T. Chalkey Halton 250
Grease and fertilizer base from Boston sewage. R. S. Weston 457
Hartford (Conn.) waterworks notes. C. M. Saville 251
Maryland experiences in the disinfection of water supplies. R. B. Morse. 457
Predicts federal control of stream pollution. Earle B. Phelps 125
Preliminary report upon purification of swimming pools at the State Uni-
versity of Iowa, A. J. J. Hinman 680
Sanitary control of swimming pools. The. Max Levine 680
features of Los Angeles aqueduct. B. A. Heinley 250
Some aspects of chlorination. Joseph Race 458, 680
Sterilization of Chicago's water supply, The 457
Study of some organisms which produce black fields in aesculin bile-
salt-media, A. F. C. Harrison and J. Vanderleck 458
Use of ammonia in the chlorination of water, The. Joseph Race 458
Vitality of the cholera vibrio in the water of New York Bay. A. J.
Gelarie 251
Water supplies of interstate common carriers on the Great Lakes, The.
H. P. Letton 458
Winter experience with the activated sludge process at Milwaukee. W.
R. Copeland ' 457
Classification of bacteria.
Correlation of the Voges-Proskauer and methyl red reaction in the coli-
aerogenes group of bacteria. The. Max Levine 566
Grouping of meningococcus strains by means of complement fixation.
Miriam P. Olmstead 364
Non-producing strain of the hog-cholera bacillus isolated from an old
laboratory culture, A. C. Tenbroeck 682
Studies on the classification of the colon-typhoid group. J. B. Thomas
and E. A. Sandman 565
on the grouping of meningococcus strains, A. Miriam P. Olm-
stead, Phoebe L. DuBois, Josephine B. Neal and Rose Schweitzer. . . 566
on the paratyphoidenteritidis group. C. Krumweide, Jr., J. S.
Pratt and L. A. Kohn 681
Study of gas-production by different strains of B. abortivo-equinus, A.
E. S. Good and S. S. Corbett 681
of the grouping of the meningococcus strains. A. Miriam P.
Olmstead, Phoebe L. DuBois, Josephine B. Neal and Rose Schweitzer 681
Dairy Bacteriology.
Agglutination as a means of studying the presence of B. abortus in
milk. L. H. Cooledge 126
Bacterial testing versus dairy inspection. C. E. North 566
Colon bacteria and streptococci and their significance in milk. L. A.
Rogers, W. M. Clark and A. C. Evans 567
Fermented milks. L. A. Rogers 125
Pasteurization as a public health measure. C. M. Hilliard 566
INDEX 713
American bacteriological literature — continued
Dairy Bacteriology.
Present status of the pasteurization of milk, The. S. Henry Ayers 125
Some observations on causes of high bacterial counts in milk. H. D.
Pease 567
Study of condensed and evaporated milks. Ida A. Bengston 126
Disinfection.
Antiseptic action of ether in peritoneal infections, The. J. Saliba 567
Bactericidal and fungicidal action of copper salts. The. L. M. DeWitt
and Hope Sherman 567
Bacterial properties of the quarternar}' salts of hexamethylenetetramine,
The. I. The problem of the chemotherapy of experimental bac-
terial infections. W. A. Jacobs 460
. II. The relation between constitution and bactericidal action
in the substituted benzylhexamethylenetetraminium salts. W. A.
Jacobs, M. Heidelberger and H. L. Amoss 460
. III. Relation between constitution and bactericidal action in
the quaternary salts obtained from halogenacetyl compounds. The.
W. A. Jacobs, M. Heidelberger and C. G. Bull 460
Effect of pasteurization on mold spores. Charles Thorn 459
Further investigation into the precipitation of the typhoid bacillus by
means of definite hydrogen-ion concentration. C. F. Kemper 459
Physical chemistry of disinfection, The. J. F. Norton and Paul H. Hsu 459
Soap. G. K. Dickinson 364
Standardization of disinfectants, The. J. T. Ainslie Walker 364
Immunology.
Action of antiseptics in Pasteur antirabic emulsions. The. Daniel
W. Poor 685
Agglutinability of blood and agar strains of typhoid bacilli. The. C. G.
Bull and I. W. Pritchett 568
Agglutination in pertussis. O. R. Povitzky and E. Worth 127
of bacteria in vivo: its relation to the destruction of bacteria within
the infected host and to septicaemia. C. F. Bull 127
reaction with sera derived from human cases of leprosy and from
the experimental animal upon various members of the acid-fast
group. The. W. H. Harris and J. A. Lanford 569
Allergic skin reaction to diphtheria bacilli, An. J. A. Kolmer 365
Analysis of the anaphylactic and immune reactions by means of the iso-
lated mammalian heart. W. H. Manwaring, Arthur R. Meinhard
and Helen L. Denhart 687
Anaphylatoxin and the mechanism of anaphylaxis. Richard Weil 126
Animal experiments upon the acquirement of active immunity by treat-
ment with Von Ruck's vaccine against tuberculosis. F. J. Clemenger
and F. C. Martley 692
Antagonistic action of negative sera upon the Wassermann reaction.
A. W. Sellards and G. R. Minot 572
Antiblastic immunity. A. R. Dochez and O. T. Avery 254
Antityphoid inoculation 461
714 INDEX
American bacteriological literature — continued
Immunology.
Autolysis of anaphylactic and immune tissue. W. H. Manwaring and
Ruth Oppenheimer 683
Autosensitized vaccines. M. G. Wohl 570
Autotherapy in poliomyelitis. C. H. Duncan 682
Bacterial aspect of the Abderhalden test, The. D. Rivas and A. C.
Buckley 683
Bacteriological study of pyorrhoea alveolaris and apical abscesses in
relation to vaccine therapy, A. Charles Krumwiede, Jr., and Joseph-
ine S. Pratt .684
Biochemistry of the gonococcus in its relation to immunity. The. Carl
C. Warden 571
Bonime's modification of Koch's treatment of tuberculosis. R. C. New-
ton 689
Case of sensitization to Witte's peptone, A. K. R. Collins 685
Cause, treatment and prevention of hay fever. The. W. Scheppegrell . . 684
Characteristics of the precipitation reaction. Richard Weil 686
Coexistence of antibody and antigen in the body, The. B. S. Denzer. . 685
Complement deviation reaction applied to the diagnosis of moniliasis of
the digestive tract, The. I. G. Martinez 571
fixation in acne vulgaris. Albert Strickler, John A. Kolmer and
Jay F. Schamberg 462
fixation in intestinal parasitism of dogs. John A. Kolmer, Mary
E. Trist and George D. Heist 369
fixation in pulmonary tuberculosis. A. Meyer 690
fixation in tuberculosis. H. R. Miller and Hans Zinsser 688
fixation in vaccinia and variola. John A. Kolmer 568
fixation in varicella. John A. Kolmer 568
fixation reactions of the Bordet-Gengou bacillus, The. M. P.
Olmstead and O. R. Povitzky 254
Continuous transfusion; the production of immunity. A. Kahn 366
"Delayed negative" Wassermann reaction, The. O. M. Olson 572
Development of immune reactions in serum disease. W. T. Longcope
and F. M. Rackemann 461
Diphtheria immunity — natural, active and passive. Its determination
by the Schick test. W. H. Park and A. Zingher 574
toxin skin reaction, The. H. Koplik and L. J. Unger 573
Effect of moderately high atmospheric temperatures upon the formation
of hemolysins, The. C.-E. A. Winslow, James Alexander and W. C.
Noble 365
formation of agglutinins. C.-E. A. Winslow, James Alexander
Miller and W. C. Noble 684
Effects of exposure to cold upon experimental infection of the respira-
tory tract, The. J. A. Miller and W. C. Noble 683
Endothelial opsonins. W. H. Manwaring and Harry C. Coe 686
Epidemic of whooping cough treated with pertussis stock vaccine, An.
Matthias Nicoll, Jr., and Paul Luttinger 685
INDEX 715
American bacteriological literature — continued
Immunology.
Equilibrium in the combination and the dissociation of precipitates.
Richard Weil 255
Erysipelas migrans and multiple abscesses in a six months' old infant
successfully treated with vaccines. L. Fischer 569
Experimental study of the effect of emetinized blood on the typhoid
bacillus. Marcus Beekman 252
Frontal and maxillary sinusitis and sequelae. Due to Staphylococcus
-pyogenes albus. Ralph Opdyke 252
Further observations on the agglutination of bacteria in vivo. C. G.
Bull 572
observations on the Schick test for diphtheria immunity. G. B.
Weaver and B. Rappaport 573
Gonococcus-complement fixation: a new lipoid antigen. C. C. Warden
and L. E. Schmidt 571
Gonorrhea and its complications. A. Hyman 689
Hepatic bacteriolysins. W. H. Manwaring and Harry C. Coe 687
Immunity factors in pneumococcus infection in the dog. C. G. Bull. . 575
in syphilis. H. Zinsser 686
in tuberculosis. G. A. Webb 574
, natural and acquired. W. H. Porter 568
Immunization with sensitized bacteria. Homer F. Swift and Ralph
A. Kinsella 462
Immunologic studies on Hodgkin's disease. J. J. Moore 689
Immunological studies in pneumonia. Richard Weil and John C.
Torrey 253
Influence of typhoid bacilli on the antibodies of normal and immune rab-
bits, The. C. G. Bull 365
Inoculation against typhoid in Maryland. F. W. Hachtel and H. W.
Stoner 683
Intravenous serobacterin therapeutics. W. E. Robertson 570
Is the hyperleucocytosis following the injection of tj'phoid bacilH into
immunized rabbits specific? Helen I. McWilliams 569
Late results in active immunization with diphtheria toxin-antitoxin and
with toxin-antitoxin combined with diphtheria bacilli. W. H.
Park and Abraham Zingher 127
Mechanism of anaphylaxis and antianaphylaxis, On the. J. Bronfen-
brenner 127
of the Abderhalden reaction with bacterial substrates. G. H.
Smith and M. W. Cook 128
Method for the rapid preparation of anti-meningitis serum, A. H. L.
Amoss and Martha Wollstein 366
of producing antigen for complement fixation in tuberculosis, A.
H. R. Miller and Hans Zinsser 687
Methods of using diphtheria toxin in the Schick test and of controlling
the reaction. Abraham Zingher 367
716 INDEX
American bacteriological literature — continued
Immunology.
Modification of Romer's intracutaneous method for the determination of
small amounts of diphtheria antitoxin in blood sera, A. Abraham
Zingher 688
Newer practical points in the treatment of typhoid fever. Beverley
Robinson 252
Note on a skin reaction in pneumonia. Richard Weil 252
on experim.ental nephropathy from some bacterial poisons, A. J.
L. Stoddard and A. C. Woods 684
on the serum treatment of poliomyelitis (infantile paralysis), A.
S. Flexner 682
Observations on the typhoid reaction. C. R. Austrian and A. L. Bloom-
field 569
Pollen extracts and vaccines in hay fever. Solomon Strouse and Ira
Frank 255
therapy in pollinosis. S. Oppenheimer and M. J. Gottlieb 368
Possible reasons for lack of protection after anti-typhoid vaccination.
Henry J. Nichols 366
Preliminary notes on skin reactions excited by various bacterial pro-
teins in certain vasomotor disturbances of the upper air passages.
J. L. Goodale 368
studies of the antigenic properties of diiTerent strains of B. typhosus.
Sanford B. Hooker 685
Preparation and preservation of complement. Loyd Thompson 252
Production of a hyperimmune serum for infectious abortion in mares,
The. E. S. Good and Wallace V. Smith 568
Progress in the treatment of skin diseases. G. M. MacKee 253
Protein absorption by blood corpuscles. W. H. Manwaring and Yoshio
Kusama 687
Pseudoreaction in the Schick test and its control, The. A. Zingher 573
Recent developments in the treatment of leprosy. Victor G. Heiser. . . . 252
Report of the committee on standard methods of preparing diphtheria
antitoxin 682
Review of the complement fixation test in tuberculosis, A. H. A. Miller. 688
Schick test. The. D. M. Griswold 573
Simple method of Quantitative determination of complement fixation,
A. J. O. Hirschfelder 572
Simplified method of producing a potent precipitin serum, A. Wal-
lace V. Smith 569
Specific receptors of fixed tissues. W. H. Manwaring and Yoshio Kusama 686
therapy in certain acute infectious diseases. J. F. Dever 570
Sputum cultures with subsequent complement fixation control. W.
W. Williams and Ward Burdick 690
Studies in non-specific complement fixation: I. Non-specific comple-
ment fixation by normal rabbit serum. John A. Kolmer and Mary
E. Trist 369
INDEX 717
Axnerican bacteriological literature — continued
Immunology.
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 370
. 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 370
. IV. The relation of serum lipoids and proteins to non-specific
complement fixation with normal rabbit and dog sera. John A.
Kolmer 371
. V. The effect of heat on normal rabbit and dog sera in relation
to antilytic and non-specific complement fixation reactions. John
A. Kolmer and Mary E. Trist 371
on intradermal sensitization. I. Intradermal reactions to emul-
sions of normal and pathologic skin. John H. Stokes 691
. II. An intradermal reaction to agar and an interpretation of intra-
dermal reactions. John A. Stokes 691
on treponema pallidum and syphilis. Hans Zinsser, J. G. Hopkins
and Malcolm McBurney 692
Therapy as related to the immunology of tuberculosis. E. R. Baldwin. 364
Toluol : its value in the sterilization of vaccines made from non-sporoge-
nous gram negative bacteria. L. D. Bristol 689
Treatment of cases of epidemic meningitis. J. B. Neal 365
of rheumatic fever. Beverley Robinson 254
of typhoid fever by intravenous injections of polyvalent sensitized
typhoid vaccine sediment. Studies in typhoid immunization. VI.
F. P. Gay and H. T. Chickering 128
of typhoid fever with bacterins. Edward Waitzfelder 253
Tuberculin therapy. Henry L. Shively 251
Universal immunization. H. B. Baruch 683
Utilization of "reactor" milk in tuberculo-medicine, The. C. B. Fitz-
patrick 127
Vaccine therapy. G. A. Ehret 253
therapy and other treatment in acne vulgaris and furunculosis. H.
H. Fox 682
treatment. Ludvig Hektoen ^^^
Vaccines in acute infection. E. Bonime "°'*
of favus and ringworm. C. H. Lavinder • 365
Value of autoserum injections in skin diseases, The. W. S. Gottheil 570
Variations in reaction of the blood of different species as indicated by
hemolysis of the red blood cells when treated with acids or alkalis,
The. J. G. Cummings 462
in the pneumococcus induced by growth in immune serum. L. M.
Stryker ; ^^^
in the strength of positive Wassermann reactions in cases of un-
treated syphilis. D. A. Haller • 366
Wassermann reaction in two hundred and fifty-one tuberculous dispen-
sary cases, The. W. R. Jones 682
718 INDEX
American bacteriological literature — continued
Industrial bacteriology.
Importance of B. bulgaricus group in ensilage, The. O. W. Hunter and
L. D. Bushnell 372
Removal of the natural impurities of cotton cloth by the action of bac-
teria, The. B. S. Levine 463
Laboratory technique.
Another use of the double plate method. W. D. Frost and Freda M.
Bachmann 693
Apparatus for filling vaccine ampoules, An. R. G. Davis 373
Colorimetric method of adjusting bacteriological culture media to any
optimum hydrogen-ion concentration. On a. S. H. Hurwitz, H. F.
Meyer and Z. Ostenberg 129
Counting bacteria by means of the microscope. R. S. Breed and J. D.
Brew 375
Culture media for paramecia and euglena. R. M. Strong 693
Device for protection against the tubercle bacillus, A. R. A. Keilty. . . 576
Electrical furnace for sterilizing inoculating loops. An. H. J. Corper. . . 256
Eye-shado for use with the microscope, An. E. Kellert 373
Hydrochloric acid as a decolorizing agent for the tubercle bacillus. R.
A. Keilty 576
Limitations to the cultivation of mammalian tissues in vitro. R. A.
Lambert 694
Method of demonstrating bacteria in urine by means of the centrifuge,
A. With some observations on the relative value of examinations by
culture or stained sediment. E. G. Crabtree 374
of obtaining suspensions of living cells from the fixed tissues, and
for the plating out of individual cells, A. Peyton Rous and F. S.
Jones 256
New culture medium for the isolation of B. typhosus from stools, A. J.
E. Holt-Harris and Oscar Teague 693
method of separating fungi from protozoa and bacteria, A. N.
KopelofT, H. C. Lint and D. A. Coleman 373
Production and collection of B. coli in quantity on synthetic media.
Robert Bengis 576
of clear and sterilized anti-hog-cholera serum. M. Dorset and
R. R. Henry 576
Rapid method of counting living bacteria in milk and other richly
seeded materials, A. W. D. Frost 374
method of cultivating the gonococcus. On a. Wm. B. Wherry and
Wade W. Oliver 373
Requirements of the gonococcus for its natural and artificial growth.
The. L. D. Bristol 694
Simple method for blood cultures, A. Paul G. Weston 256
Stabilized gentian violet. W. D. Stovall and M. S. Nichols 576
Stain for tubercle bacilli, A. Emanuel Klein 256
Studies on laboratory media. Jane L. Berry 693
Study of blood with a new stain. B. Lemchen 374
INDEX 719
American bacteriological literature — continued
Laboratory technique.
Two laboratory suggestions. George B. Lake 256
Use of brilliant green for the isolation of typhoid and paratyphoid bacilli
from feces, The. Charles Krumwiede, Jr., Josephine S. Pratt and
Helen I. McWilliams 129
of the sand tube in isolating the B. typhosus, The. M. D. Levy. . . 373
Medical bacteriology.
Analysis of one hundred and thirty-four cases of bacteriemia. M. War-
ren and W. W. Herrick 467
Anopheles punctipennis , a host of tertian malaria. W. V. King 583
Anthrax, with report of cases. S. J. UUman 587
Antimeningococci serum in the joint manifestations of gonorrhea. F.
Malleterre 582
Bacillary dysentery, recent epidemic at Fort Shafter, H. T., from the
laboratory aspect. H. F. Foucar 588
B. epilepticus, The. C. A. L. Reed 582
Bacteria associated with certain types of abnormal lymph glands. J.
C. Torrey 380
Bacterial cultures of hiunan spleens removed by surgical operation.
Andrew W. Sellards 589
Bactericidal and protoacidal activity of emetin hydrochloride in vitro,
The. John A. Kolmer and Allen J. Smith 698
in vivo. John A. Kolmer and Allen J. Smith 698
Bacteriologic study of the causes of some stillbirths, A. J. B. DeLee. . 696
Bacteriological and experimental studies on gastric ulcer. H. L. Celler
and W. Thalhimer 589
work at the American ambulance. Orville F. Rogers and George
Benet 381
Bacteriology and experimental production of ovaritis. The. E. C.
Rosenow and C. H. Davis 584
in conjunction with homeopathic prescribing. W. W. Irving 583
of the recent grip epidemic, The. Charles Halpin Nammack 261
Bulgarian bacillus in the treatment of vulvovaginitis. The. M. B. Cohen 695
Case of coccidiodal granuloma. S. T. Lipsitz, G. W. Lawson and E. M.
Fessenden 579
of infection of lymph glands with B. Paratyphosus B., A. C. S. Cole 581
of mastoiditis by purulent cerebrospinal meningitis; operation and
recovery "94
Causation and treatment of pellagra, The. H. E. Bond 582
Cause of rat-bite fever, The. Kenzo Futaki, Etsuma Futaki, Tenji
Taniguchi and Shimpachi Osumi 259
Chemical versus serum treatment of epidemic meningitis. Simon Flex-
ner and H. L. Amoss 466
Choroidal tubercle in tuberculous meningitis. The. J. F. Bredeck 376
Chronic general infection with the B. pyocyaneus. Leonard Freeman . . 698
influenza in pulmonary tuberculosis. M. L. Hamblet and H. L.
Barnes ^^^
tonsilitis. Louis Fischer 257
720 INDEX
American bacteriological litei'ature — continued
Medical bacteriology.
Colon bacillus infection of the bladder. R. T. Morris 381
Common affections of the eye. S. D. Risley 257
Connellan-King diplococcus. Infection of the tonsil. J. J. King 697
Contents of ovarian cysts, The. J. T. Leary, H. J. Hartz and P. B.
Hawk 579
Contribution to the chemotherapy of tuberculosis, A. First experi-
mental report. G. Koga 699
. First clinical report. G. Koga 700
Control of diphtheria. D. M. Lewis 581
of diphtheria epidemics. The. W. D. Stovall 257
Cultivation of the organisms of vaccinia, variola and varicella. H.
Greeley 702
Cultural experiments with the Spirochaeta pallida derived from the
paretic brain. Udo J. Wile and Paul Henry de Kriuf 258
Cytology of the exudate in the early stages of experimental pneumonia,
The. Frank A. Evans 464
Diagnosis of enteric fever (typhoid and paratyphoid A and B) by agglu-
tination tests. The. W. C. Davison 583
of genitourinary tuberculosis, The. J. W. Churchman 380
Diphtheria carriers. J. C. Geiger, Frank L. Kelly and Violet M. Bath-
gate 258
in Manila. A. P. Goff 377
in the first year of life. J. D. Rolleston 697
Early tuberculosis of the cervix. T. S. Cullen 376
Effect of continuous electric light in experimental arthritis, The. W.
E. Simmonds and J. L. Moore 130
Elective localization in the bronchial musculature of streptococci from
the sputum of cases of bronchial asthma. S. Oftedal 585
localization of bacteria in diseases of the nervous system. E. C.
Rosenow 700
Epidemic of appendicitis and parotitis probably due to streptococci
contained in dairy products. An. E. C. Rosenow and S. I. Dunlap. 585
of baeillary dysentery due to the Hiss-Russell bacillus. M. A.
Delaney 588
of dysentery at Fort Shafter, Hawaii, with three cases of the Hiss-
Russell or "Y" bacillus infection, An. G. M. Van Poole 588
of typhus exanthemaiicus in the Balkans and in the camps of Europe,
The. Bert. \V. Caldwell 265
Epidemiology of tuberculosis, The. F. C. Smith 264
Etiology and laboratory diagnosis of smallpox and chickenpox. J. N.
Force 581
and pathology of otitic cerebellar abscess. The. Isidore Friesner. . 696
and treatment of rat-bite fever. The. W. Tileston 377
, mode of infection, and specific therapy of Weil's disease (Spirochae-
tosis icterohae7norrhagica) , The. R. Inda, Y. Ido, R. Hoki, R.
Kaneko and H. Ito 379
INDEX 721
American bacteriological literature — continued
Medical bacteriology.
Etiology of common colds, The. G. B. Foster 586
of iritis, The. E. E. Irons and E. V. L. Brown 580
of rat-bite fever, The. Francis G. Blake 259
of Rocky Mountain spotted fever, The. S. B. Wolbach 378
of scarlet fever. The. F. B. Mallory and E. M. Medlar 377
of the current epidemic of respiratory infections in Chicago, The.
George Mathers 259
of typhus fever in Mexico (Tabardillo), The. P. K. Olitsky, B. S.
Denver and C. E. Husk 580
Examination of the urine and feces of suspect typhoid carriers with a
report on elaterin catharsis, The. F. O. Tonney, F. C. Caldwell and
P. J. Griffin 699
Experimental cholera carriers. Otto Schobl 698
studies in the production of chronic gastric ulcer. A. O. Wilensky
and S. H. Geist 580
study of parotitis (mumps). An. Martha Wollatein 379
syphilis in the rabbit produced by the brain substance of the living
paretic. Udo J. Wile 258
Experiments with poliomyelitis in the rabbit. M. J. Rosenau and L. C.
Havens 465
Extent and significance of gonorrhea in a reformatory for women, The.
Elizabeth A. Sullivan and Edith R. Spaulding 263
Final report on the cultivation of the tubercle bacillus from the sputum
by the method of Petroff, A. R. A. Keilty 584
Flagellate protozoa as an etiologic factor of dysenteric diarrhea. B. W.
Rhamy and F. A. Metts 581
Focal infection in relation to certain dermatoses. M. L. Ravitch 695
sepsis. J. Daland 586
Foot and mouth disease in man. R. L. Sutton and A. O'Donnell 376
Further experimentation in animals with a monilia commonly found in
sprue. B. K. Ashford 467
Gall bladder diseases. C. H. Mayo 261
Gas bacillus infection with remarks on gastric ulcer. Randolph West
and Mary E. Stewart 697
Gastro-intestinal findings in acne vulgaris. L. W. Ketron and J. H.
King 695
Immune response in pulmonary tuberculosis. The. E. Bonime 581
Incidence of syphilis among juvenile delinquents. The. Thomas H.
Harris 258
Influenza. A. H. Doty 257
and grippe in infants and children. Carl G. Leo-Wolf 259
Is B. abortus (Bang) pathogenic for human beings. L. H. Cooledge 696
Laboratory aids in the diagnosis of poliomyelitis. J. B. Neal 701
examination of material in a case of suspected smallpox. J. N.
Force 580
facts in poliomyelitis. S. R. Klein 695
722 INDEX
American bacteriological literature — continued
Medical bacteriology.
Leptothrix associated with chronic hemorrhagic nephritis, A. G. R.
Dick, G. F. Dick and B. Rappaport 464
Lesions produced in rabbits by repeated intravenous injections of liv-
ing colon bacilli. C. H. Bailey 130
Localization of streptococci in the eye. The. E. E. Irons, E. V. L. Brown
and W. H. Nadler 699
Meningitis. R. C. Rosenberger and D. J. Bentley 586
Microscope in dermatology. The. O. L. Levin 696
Milk-borne paratyphoid outbreak in Ames, Iowa, The. Max Levine and
Frederick Eberson 464
Mode of infection in pulmonary distomiasis, The. Koan Nakagawa. . . . 465
Natural resistance of the pigeon to the jmeumococcus, The. Preston
Kyes 701
Neisserian proctorrhea. Charles C. Mapes 579
Newer laboratory methods for the early diagnosis of pulmonary tuber-
culosis. M. H. Kahn 581
Notes on grip epidemic in Chicago. A. M. Moody and J. A. Capps. . . . 582
on the etiology of the recent epidemic of pseudo-influenza. W. W.
Williams and W. Burdick 583
Pathogenesis of psoriasis. A. H. Cook 696
Pathogeny of diabetes and fecal disinfection. G. D. Palacios 377
Period of life at which infection from tuberculosis occurs most fre-
quently, The. S. Adolphus Knopf 265
Peritonitis following acute ovaritis of anginal origin. Russell M. Wil-
der 257
Pneumotyphoid, with report of a case. F. Billings 464
Post-operative tetanus. Kellogg Speed 584
Practical points in the prevention of Asiatic cholera. Allan J. Mc-
Laughlin 131, 377
value of guinea-pig tests for the virulence of diphtheria bacilli,
The. John A. Kolmer, Samuel S. Woody and Emily S. Moshage... 382
Preliminary report on pneumonia in children, with special reference to
its epidemiology, A. Godfrey R. Pisek and Marshall C. Pease 383
Present views in respect of modes and periods of infection in tubercu-
losis. Mazyck P. Ravenel 257
Production of amyloid disease and chronic nephritis in rabbits by re-
peated intravenous injections of living colon bacilli. The. C. H.
Bailey 587
Prompt cure of gonorrhea. The. George A. Wyeth 260
Protection of pathogenic microorganisms by living tissue cells, The. P.
Rous and F. S. Jones 466
Pure cultivation of Spirochaeta icterohemorrhagiae (Inada), The. T.
Ito and H. Matsuzaki 464
Pyelocystitis and metastatic abscesses following tonsilitis. H. B. Mills
and G. A. Sowell 582
INDEX 723
American bacteriological literature — continued
Medical bacteriology .
Reactions between bacteria and animal tissues under conditions of
artificial cultivation, The. II. Bactericidal action in tissue cul-
tures. H. F. Smyth 465
. III. The action of bacterial vaccines on tissue cultures in vitro.
H. F. Smyth 465
Recurrent generalized herpes of infectious origin. F. Cohen 580
Relapsing fever in Serbia. J. Rudis-Jicinsky 380
Remarks on B. Welchii in the stools of pellagrins. W. H. Holmes 260
Removal of tonsils and adenoids in diphtheria carriers. S. A. Fried-
berg 257
Resume of infectious diseases. Albert H. Beifeld 697
Rocky Mountain spotted fever. Henry C. Michie, Jr., and Houston H.
Parsons 260
R61e of the Ijonphatics in ascending renal infection. Daniel N. Eisen-
drath and Jacob V. Kahn 258
Routine Wassermann examinations of four thousand hospital patients.
I. C. Walker and D. A. Haller 262
Salvarsan in primary syphilis. Alexander A. Uhle and Wm. H. Mac-
kinney 260
Simultaneous injection of streptococci and dahlia in the guinea-pig.
W. H. Hoffman, W. B. Maclure and L. W. Sauer 583
Site and rate of destruction of pneimiococci following intraperitoneal
injection, The. F. Berry and C. O. Melick 584
So-called grippe. J. B. Rucker, Jr 262
primary tuberculosis of the conjunctivita and the conjunctival
tuberculosis of lupus patients. The. K. K. K. Lunds-Gaard 376
Some fatal ear cases in the writer's practice. O. D. Stickney 376
Streptothrix in broncho-pneumonia of rats similar to that in rat-bite
fever. R. Tunnicliff 580
Studies on diphtheria. II. The treatment of diphtheria carriers by
tonsillectomy. H. O. Ruh, M. J. Miller and R. G. Perkins 381
on Treponema pallidum and syphilis. II. Spirochaeticidal anti-
bodies against Treponema pallidum. H. Zinsser and J. G. Hop-
kins 378
. III. The individual fluctuations in virulence and comparative
virulence of Treponema pallidutn strains passed through rabbits.
Hans Zinsser, J. G. Hopkins and M. McBurney 378
. IV. The difference in behavior in immune serum between cul-
tivated non-virulent Treponema pallidum and viriflent Treponemata
from lesions. Hans Zinsser, J. G. Hopkins and M. McBurney 379
on the cultivation of the rabies virus. Caroline R. Gurley and
Charlotte C. Van Winkle 697
Study of a strain of B. welchii isolated in France together with some
notes on gastric ulcers. Mary W. Stewart and Randolph West 588
of the bacteriology of chronic prostatitis and spermatocystitis, A.
Harry B. Culver 261
724 INDEX
American bacteriological literature — continued
Medical bacteriology .
Study of the etiology of chorea, A. John Levett Morse and Cleveland
Floyd 703
of the etiology of chronic nephritis, A. P. K. Brown and W. T.
Cummins 258
Syphilis and tuberculosis in the same lung. R. A. Keilty 694
in epilepsy. W. T. Shanahan, J. F. Munson and A. L. Shaw 585
Technique of cultivating human tissues in vitro. R. A. Lambert 464
Tetanus: a surgical complication in the present war. E. K. TuUidge. . . 586
Tonsillar endamebiasis and thyroid disturbances. J. S. Evans, W. S.
Middleton and A. J. Smith 468
Treatment of chronic colon bacillus pyelitis by pelvic lavage, The. H.
L. Kretschmer and F. W. Gaarde 695
of diphtheria carriers with iodized phenol, The. W. O. Ott and
K. A. Roy 263
of gastric ulcer. The. A. F. R. Andresen 262
of genitourinary tuberculosis with Rosenbach's tuberculin. A.
Hyman 580
of human rabies with quinin and with phenol, The. F. S. Fielder. . 579
of infections of accessory sinus. The. A. M. McWhinnie 260
of leprosy with cyanocuprol. The. R. Takano 700
of tuberculosis with cyanocuprol, The. M. Otani 700
Trichinosis and the cerebrospinal fluid. W. Lintz 579
Tuberculin in surgical tuberculosis. E. Bonime 581
Tuberculocidal action of arsenic compounds and their distribution in the
tuberculous organism, The. A. Arkin and H. J. Cooper 585
Tuberculosis. Hermann M. Biggs 264
in infancy. C. H. Dunn 130
of the middle ear. H. B. Graham 695
Two unusual strains of diphtheroid bacilli. Ralph R. Mellon 257
Types of pneumococci in tuberculous sputum. The. Harold W. Lyall. ..695
Unique lesion of the heart in systemic blastomycosis. T. B. Hurley. . . 376
Use of kaolin to remove diphtheria bacilli from the nose and throat.
The. B. Rappaport 382
Vincent's bacillus in the cervix. G. McConnell 696
Paleontology.
Mesozoic pathology and bacteriology. Roy L. Moodie 384
Physiology of bacteria.
Action of Schumann rays on living organisms. W. T. Bovie 132
Destructive effects of light and drying and other living-room condi-
tions upon diphtheria bacilli; streptococci and staphylococci, The.
Daniel VV. Poor and Charles P. Fitzpatrick 703
Effect of elemental sulphur and of calcium sulphate on certain of the
higher and lower forms of plant life. Walter Pitz 132
of natural low temperature on certain fungi and bacteria. H.
E. Bartram 131
Inhibiting action of certain spices on the growth of microorganisms,
The. Freda M. Bachmann 704
INDEX
725
American bacteriological literature — continued
Plujsiology of bacteria.
Life cycles of the bacteria. F. Lohnis and N. R. Smith 704
Plant pathology.
Bacterial disease of western wheat-grass, Agropyron smilhii, A. Oc-
currence of a new type of bacterial disease in America. P. T. O'Gara 705
Bacteriological blights of barley and certain other cereals. L. R. Jones,
A. G. Johnson and C. S. Reading 705
Citrus canker. F. A. Wolf 384
Crown gall studies, showing changes in plant structure due to a changed
stimulus. Erwin F. Smith 577
Factors involved in the growth and the pycnidium formation of Pleno-
domus fuscomaculans . George Herbert Coons 133
Further evidence that crown gall of plants is cancer. E. F. Smith 577
studies in the role of insects in the dissemination of fire blight bac-
teria. V. B. Stewart and M. D. Leonard 384
studies on peanut leafspot. Frederick A. Wolf 133
Observations on fire blight in the Yakima Valley, Washington. J. W.
Hotson 705
Serious disease in forest nurseries caused by Peridermium filamento-
sum, A. James R. Weir and Ernest E. Hubert 132
Soil stain, or scurf of the sweet potato. L. J. Tabenhaus 133
Some properties of the virus of the mosaic disease of tobacco. H. A.
Allard 706
Studies on the crown gal! of plants: its relation to human cancer.
Erwin F. Smith 578
Sweet potato scurf. L. L. Harter 133
Transmission ard control of bacterial wilt of cucurbits. F. V. Rand
and Ella M. A. Enlows 577
Protozoa and other animal parasites.
Dermatitis herpetiformis. M. F. Engman and Robert Davis 266
Filaria sanguinis hominis. Codis Phipps 266
Thionin as a diagnostic stain for pyorrhea alveolaris. Martin Dupray.. 266
Trichiniasis. Michael G. Wohl 266
Trichinosis. Arthur R. Elliott 266
Public health bacteriology.
Bubbling fountain test. Jane L. Berry 706
Cigar cutter tests. Jane L. Berry 706
Controlling the spread of sputum. W. A. Manheimer ^ 579
Microscopic examination of finger nail deposits, The. S. Schneider. ... 578
Public health laboratories. R. G. Perkins 578
Studies on diphtheria in Cleveland. I. Diphtheria carriers. R. G.
Perkins, M. J. Miller and H. O. Rush 707
on the use of brilliant green and a modified Endo's medium in the
isolation of B. typhosus from feces. H. C. Robinson and L. F.
Rettger 707
Study of the normal bacteria flora of postage stamps, A. R. A. Kedty
and P. D. McMaster 707
726 INDEX
American Bacteriologists, Society of. Abstracts of papers presented at sev-
enteenth annual meeting 81
Systematic bacteriology.
Characteristics of bacteria of the colon type occurring in human feces,
The. L. A. Rogers, Wm. Mansfield and H. A. Lubs 82
Chromogenic bacillus, The. Frank L. Rector 86
Correlation of the Voges-Proskauer and the methyl red reaction, On the.
Max Levine 87
Hemolytic streptococci found in milk, The. Their significance and
their relation to streptococci of human origin. David John Davis. . 83
Hydrogen-ion concentrations in cultures of streptococci. S. Henry
Ayers 84
Some problems in bacterial nomenclature. R. E. Buchanan 82
Studies on the classification of the colon-typhoid group. C.-E. A.
Winslow and I. J. Kligler 81
Type of colon bacillus occurring in surface waters, The. L.A.Rogers. 82
Value of lactose bile for the B. coli presumptive test, The. John W. M.
Bunker 85
Technique.
Acidity of media. H. A. Noyes 87
Amniotic fluid as a bacterial culture medium. Ward Giltner and L. C.
Ludlum 91
Another use of the double plate method. W. D. Frost and Freda M.
Bachmann 92
Culture medium for maintaining stock cultures of the meningococcus, A.
C. G. A. Roos 88
Modification in staining technic. Zae Northrup 91
Pasteurization applied to mold spores. Charles Thorn and S. H. Ayers. 89
Simple apparatus for isolating anaerobes. Zae Northrup 90
Study of effect of dilution water on bacterial suspensions. H. M.
Weeter 92
Use of 0.01 cubic centimeter pipettes in bacterial milk analj'sis, The.
James D. Brew 89
Variation in plant counts under research conditions. M. J. Prucha 92
Industrial bacteriology.
Are spore-forming bacteria of any significance in soil under normal
conditions. H. Joel Conn 95
Bacteria, actinomyces and fungi in soils. Selman A. Waksman 101
Coli-like organisms of the soil. B. R. Johnson 96
Ferrification in soils. P. E. Brown and G. E. Corson 95
Indirect effect of certain soil treatments upon bacterial activity, The.
P. L. Gainey 98
Influence of soil solution on the longevity of microorganisms subjected
to desiccation. The. Ward Giltner and Virginia Langworthy 96
Media for soil bacteria. H. A. Noyes 93
Possible function of actinomycetes in soil, A. H. Joel Conn 93
Quantitative media for the estimation of bacteria in soil. R. C. Cook. . 100
INDEX
727
American Bacteriologists, Society of. Abstracts of papers— Continued
Industrial bacteriology.
Reaction of the soil solution as an index of biological changes in the
soil. J. F. Morgan and O. M. Gruzit 97
Relation of protozoa to certain groups of soil bacteria, The. T. L.
Hills.
99
Soil solution as an index of the biological changes in the soil. J. Frank-
lin Morgan ^'
Studies on soil protozoa and their relation to the bacterial flora. J. M.
Sherman ^^
Study of the nodule-forming bacteria, A. F. O. Ockerblad 100
Food.
Bacteria in commercial, bottled waters. Maud Mason Obst 103
Bacterial content of market oysters. Fred Berry 107
Comparison of rapid method of counting bacteria in milk with standard
methods. W. D. Frost.
103
of the number of water bacteria growing on agar at 37°C. and on
gelatin at 20°C. Fred W. Tanner 101
Effect of air pressure on potable waters during storage. The. W. D.
Frost and Freda M. Bachmann ^^^
Normal fermentation of sauerkraut. Lester A. Round 108
Notes on brine pickle fermentation. C. W. Brown 104
Pasteurization of dairy by-products. The. Robert S. Breed and W. D.
T-. . , 106
Dotterer
Sampling milk for bacterial analysis. Robert S. Breed 105
Scientific methods of control in the mineral water industry. Frank L.
Rector '
Study of the effect of spices on the growth of certain organisms, A.
Freda M. Bachmann
Sanitary bacteriology.
Diphtheria diagnosis by means of blood serum containing potassium
tellurate. Will Shimer - • ; • • • • • • • "^
Influence of conditions in the barn upon the germ content of milk. M.
J. Prucha and H. M. Weeter ■■■- J"^
Number of bacteria in the air of cow stables, The. G. L. A. Reuh e . . U^
Purification of sewage by aeration in the presence of activated sludge. ^^^
Edward Bartow tt' j- iin
Relation of bacteriology to city milk standards. H. A. Harding liU
Validity of presumptive tests. W. F. Monfort H'*
Infection and immunity. xt -n. , • 1 1 1^
Antigenic properties of autolysed bacteria. George H. Robinson . 115
Bacterial changes in uniced specimens of water. Henry Albert, Jack J.
Hinman, Jr., and Gharrett Jordan ••.■•.■•.••
Bacterium pyogenes associated with a case of multiple arthritis ^^ ^
hog. Archibald R. Ward ■ ; ^^
Behavior of streptococci of human and bovine origin in the cow s
udder, The. George Mathers • • • • • • • • • • • • ' " '
Effect on horses of feed heavily inoculated with B. coh isolated from
oat hay. The. Robert Graham and L. R. Himmelberger 115
728 INDEX
American Bacteriologists, Society of. Abstracts of papers — Continued
Infection and immunity.
Feeding experiments with B. pullorum. The toxicity of infected eggs.
Leo F. Rettger, Thomas G. Hull and William S. Sturges 120
Further studies on the presence of and significance of agglutinins for
B. abortus (Bang) in cows' milk. L. H. Cooledge 117
Spirochaeta hyos — its antigenic value in complement fixation tests on
hog cholera sera. Studies on hog cholera. Walter E. King and R.
H. Drake 114
Studies in bacterial nutrition. The utilization of proteid and non-
proteid nitrogen. Leo F. Rettger, William S. Sturges and Nathan
Berman 121
Study of the bacteria of normal and decayed teeth, A. I. J. Kligler 114
Yeasts, probably pathogenic, recovered from routine throat cultures.
Arthur L. Grover 121
Anaerobic plating, A method of, permitting observation of growth 339
Anthony, Bertha van Houten, and Ekroth, Clarence V. Practical obser-
vations on the titration and adjustment of culture media 209
Apparent recovery of a hen infected with bacillary white diarrhea 625
Are spore-forming bacteria of any significance in soil under normal conditions? 187
Bacillary white diarrhea, Apparent recovery of a hen infected with 625
B. abortus (Bang) as an etiological factor in infectious abortion in swine 415
Bacteria, Are spore-forming, of any significance in soil under normal condi-
tions? 187
, Biological variations of. 1 385
in milk. Spore-bearing 277
, Studies on aerobic spore-bearing non-pathogenic. . . .Part I, 273; Part II, 493
Bacterial nutrition: a brief note on the production of erepsin (peptolytic
enzyme) by bacteria 537
nutrition, Further studies on: the utilization of proteid and non-proteid
nitrogen 15
Bacteriology of the bubble fountain. The 471
. The genesis of a new science 1
, The pedagogics of 5
Bergey, David H. The pedagogics of bacteriology 5
Berman, Nathan, and Rettger, Leo F. Bacterial nutrition: a brief note on
the production of erepsin (peptolytic enzyme) by bacteria 537
, Sturges, William S., and Rettger, Leo F. Further studies on bacterial
nutrition: the utilization of proteid and non-proteid nitrogen 15
Bile compared with lactose bouillon for determining the presence of B. coli
in water 73
Biological variations of bacteria. 1 385
Boas-Oppler bacillus, A study of the 435
Bogart, Franklin B., Clark, Paul F., and Pettibone, Dorothy F. The bac-
teriology of the bubble fountain 471
Book reviews :
A textbook upon the pathogenic bacteria and protozoa, by Joseph Mc-
Farland, M.D. (Rev. by D. Greenberg) 549
INDEX 729
Book reviews — continued
Der Erreger der Maul- und Klauenseuche, by Heinrich Stauffacher.
(Rev. by Gary N. Calkins) 353
Laboratory manual in general micro-biology, by Ward Giltner. (Rev.
by C. M. Hilliard) 453
Practical textbook of infection, immunity and specific therapy, by John
A. Kolmer, M.D., Dr. P.H. (Rev. by Hans Zinsser) 673
The principles of pathologic histology, by Frank B. Mallory, M.D.
(Rev. by F. P. Gay) 550
Breed, Robert S., and Dotterrer, W. D. The number of colonies allowable
on satisfactory agar plates 321
Bubble fountain, The bacteriology of the 471
Buchanan, R. E. Studies in the nomenclature and classification of bac-
teria. 1 591
Burdick, Ward, and Williams, Wm. Whitridge. A new culture medium for
the tubercle bacillus 411
Calkins, Gary N. Rev. of Der Erreger der Maul- und Klauenseuche, by
Heinrich Stauffacher 353
Cannon, Paul R. A rapid and simple indol test 535
Clark, Paul F., Pettibone, Dorothy F., and Bogart, Franklin B. The bac-
teriology of the bubble fountain 471
Classification of some lactose fermenting bacteria. Preliminary note on 619
of spore-bearing organisms 527
Coli-bacilles et par les bacilles typhiques, Observations sur I'influence
chimique des milieux de culture sur le d^veloppement et la production
de I'indol par les 627
Conn, H. Joel. A possible function of actinomycetes in soil 197
. Are spore-forming bacteria of any significance in soil under normal
conditions? 187
Contribution to the bacteriology of silage, A 445
Culture media from whole blood. The preparation of 615
media. Practical observations on the titration and adjustment of 209
medium for maintaining stock cultures of the meningococcus, A 67
Cultures, Miscellaneous 518
Doryland, C. J. T. Preliminary report on synthetic media 135
Dust, Spore-bearing bacteria in 493
Dotterrer, W. D., and Breed, Robert S. The number of colonies allowable
on satisfactory agar plates 321
Ecker, E. E., and Heineman, P. G. A study of the Boas-Oppler bacillus. . 435
Ekroth, Clarence V., and Anthony, Bertha Van Houten. Practical ob-
servations on the titration and adjustment of culture media 209
Erepsin (peptolytic enzyme). Bacterial nutrition: a brief note on the pro-
duction of, by bacteria '537
Ford, W. W. Classification of spore-bearing organisms 527
, and Lawrence, J. S. Spore-bearing bacteria in milk 277
^ Laubach, C. A., and Rice, J. L. Studies on aerobic spore-bearing non-
pathogenic bacteria. Part II 493
. Miscellaneous cultures 518
730 INDEX
Ford, W. W. Studies on aerobic spore-bearing non-pathogenic bacteria.
Part I. (Introduction) 273
Further studies on bacterial nutrition: the utilization of proteid and non-
proteid nitrogen 15
Gay, F. P. Rev. of The principles of pathologic histology, by Frank B.
Mallory M.D 550
Genesis of a new science — bacteriology. The 1
Gonococcus, Testicular infusion agar — a sterilizable culture medium for the 343
Good, Edwin S., and Smith Wallace V. B. abortus (Bang) as an etiological
factor in infectious abortion in swine 415
Greenberg, D. Rev. of A textbook upon the pathogenic bacteria and
protozoa, by Joseph McFarland, M.D 549
Greenfield, Myrtle. A new ice sampler 623
Guinea-pigs, A practical method for the identification of, under treatment. . 541
Gyorgy, Paul, and Zunz, Edgard. Observations sur I'influence chimique des
milieux de culture sur le d^veloppement et la production de I'indol par
les coli-bacilles et par les bacilles typhiques 627
Heinemann, P. G., and Ecker, E. E. A study of the Boas-Oppler bacillus. . 435
Hilliard, C. M. Rev. of Laboratory manual in microbiology, by Ward Gilt-
ner 453
Hills, T. L. The relation of protozoa to certain groups of soil bacteria 423
Hitchens, A. Parker. A practical method for the identification of guinea-
pigs under treatment 541
Hopkins, J. G., and Zinsser, Hans. On a species of treponema found in rabbits 489
Horton, George D. Apparent recovery of a hen infected with bacillary
white diarrhea 625
Indol test, A rapid and simple 535
Infectious abortion in swine, B. abortus (Bang) as an etiological factor in. 415
In memoriara Thomas J. Burrill 269
Jones, Horry M. A method of anaerobic plating permitting observation of
growth , 339
Kelser, Raymond A. The preparation of culture media from whole blood. . 615
Kligler, I. J. Some regulating factors in bacterial metabolism 663
Lactose bouillon. Bile compared with, for determining the presence of B.
coli in water 73
Laubach, C. A. Spore-bearing bacteria in dust 493
, and Rice, J. L. Spore-bearing bacteria in soil 513
. Spore-bearing organisms in water 505
, Rice, J. L., and Ford, W. W. Studies on aerobic spore-forming non-
pathogenic bacteria 493
Lawrence, J. S., and Ford, W. W. Spore-bearing bacteria in milk 277
. Studies on aerobic spore-bearing non-pathogenic bacteria. Part I. 273
Levine, Max. On the significance of the Voges-Proskauer reaction 153
. Preliminary note on the classification of some lactose fermenting bac-
teria 619
Meningococcus, A culture medium for maintaining stock cultures of the 67
Metabolism, Some regulating factors in bacterial 663
Method of anaerobic plating permitting observation of growth, A 339
INDEX 731
Milk, The advantages of a carbohydrate medium in the routine bacterial
examination of 481
Miscellaneous cultures 518
Modification of the hygienic laboratory method for the production of tetanus
toxin, A 333
Murray, T. J. The oxygen requirements of biological soil processes 597
New culture medivun for the tubercle bacillus, A 411
ice sampler, A 623
species of alcohol forming bacterium isolated from the interior of stalks
of sugar cane infested with the cane-borer Diatraea saccharalis, A 235
Nitrogen, Further studies on bacterial nutrition: the utilization of proteid
and non-proteid 15
Nomenclature and classification of bacteria, Studies in the. I. The prob-
lem of bacterial nomenclature 591
Note on the preparation of agar agar culture media, A 547
Number of colonies allowable on satisfactory agar plates, The 321
Observations sur I'influence chimique des milieux de culture sur le deve-
loppement et la production de I'indol par les coli-bacilles et par les
bacilles typhiques 627
Obst, Maud Mason. Bile compared with lactose bouillon for determining
the presence of B. coli in water 73
Owen, Wm. L. A new species of alcohol forming bacterium isolated from
the interior of stalks of sugar cane infested with the cane-borer Diatraea
saccharalis 235
Oxygen requirements of biological soil processes. The 597
Pedagogics of bacteriology. The 5
Pettibone, Dorothy F., Bogart, Franklin B., and Clark, Paul F. The bac-
teriology of the bubble fountain 471
Possible function of actinomycetes in soil, A 197
Practical method for the identification of guinea-pigs under treatment, A. . 541
observations on the titration and adjustment of culture media 209
Preliminary note on the classification of some lactose fermenting bacteria. . 619
report on synthetic media 135
Preparation of culture media from whole blood, The 615
Protozoa, Studies on soil, and their relation to the bacterial flora. . . .1, 35, II, 165
Rapid and simple indol test, A 535
Relation of protozoa to certain groups of soil bacteria. The 423
Rettger, Leo F., and Berman, Nathan. Bacterial nutrition: a brief note on
the production of erepsin (peptolytic enzjrme) by bacteria 537
, Berman, Nathan, and Sturges, William S. Further studies on bacterial
nutrition : the utilization of proteid and non-proteid nitrogen 15
Rice, J. L., and Laubach, C. A. Spore-bearing bacteria in soil 513
, Ford, W. W., and Laubach, C. A. Studies on aerobic spore-forming
non-pathogenic bacteria 493
Roos, C. G. A. A culture medium for maintaining stock cultures of the
meningococcus .
67
Sampler, A new ice 623
Sedgwick, W. T. The genesis of a new science — bacteriology 1
i
i
I
Ihe Specific Treatment of
Lobar Pneumonia
Immune serum treatment in lobar pneumonia has passed
the purely experimental stage.
Lobar pneumonia is caused chiefly by the pneumococcus,
of which there are three different fixed types. Antipneumo-
coccic Serum prepared by the Mulford Laboratories is obtained
from horses which have been injected with the three fixed
types of the pneumococcus.
Forty per cent of all cases of lobar pneumonia are caused
by type 1, and lobar pneumonia caused by this type is the
most amenable to serum treatment, while types 2 and 3 are
less amenable to serum treatment. Antipneumococcic Serum
Polyvalent Mulford is highly potent in its protective power
against lobar pneumonia caused by pneumococcus type 1, and
also contains antibodies to the other types — 2 and 3.
Intravenous injection of 50 to 200 c.c. is advocated by prominent
authorities to insure immediate action.
Antipneumococcic Serum Polyvalent Mulford is furnished in
syringes of 20 c.c. each, and in ampuls of 50 c.c. for intravenous injection.
Further information sent on request.
Pneumo-Serobacterin Mulford is an efficient prophylactic agent
against lobar pneumonia. Wright suggests doses of 1000 million pneumo-
cocci, followed by subsequent doses of 1000 million, for prophylactic
purposes.
Pneumo-Serobacterin Mulford is supplied in packages of four
graduated syringes. A, B, C, D strength, and in syringes of D strength
separately.
Syring-e A 250 million kilied sensitized bacteria
Syringe B 500 million killed sensitized bacteria
Syringe C 1000 million killed sensitized bacteria
Syringe D 2000 million killed sensitized bacteria
P
i
i
I
I
H. K. MULFORD COMPANY, Philadelphia, U. S. A.
I
i
"DIFCO" STANDARDIZED
PRODUCTS FOR CULTURE MEDIA
T\ \ r"TO PTTPTOIVT? ^ ii^iti-ient for bacteria, derived from
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■RATTn PTTT7 HIFT^TTTIV/I^ standard test medium for
DALlU-DlLli MriUlUiVl B.coli,contammgm add-on
to the necessary bile, 1% of Peptone and 1% of Lactose, all reduced to powder
form, ready for use upon dissolving in water and sterilizing.
"D 4 /^XO P1?T AXTTVTT ^ product designed for the peculiar
i3r\\^ 1 vJ"VJl_iljrV 1 111 r!i needs of culture media, with low
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TX \ r^nrO a r^ a T? ^ selected Japanese agar, carefully treated to
-U.^ v>( X Vy"xWJr\A\. reduce pigment, extraneous matter, inorganic
salts, and moisture. Packaged in spongy granular form in tight cartons.
Kcadily dissolved without attention.
PAr^T^O 1^171717 '^ desiccated lean beef meat, especially made for
-iJr\v>4 J. \J'1J±^Lj1. use in place of fresh beef, and eliminating its
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P A r^nrO \7T? at ^ desiccated lean veal, convenient, economical
-L''^V>< J. v7" V l_ix\.J-j and reliable forthe preparation of diphtheria toxin.
RAr'XO T Ar'XO^l? "^ P"-"'^ ^"^ ^^^^^^^ powder, free from other
-Di^ v>( 1 W"l^r\.V>( 1 v/kJ X^ sugars, ashless, no traces of starch, pro-
teid, alcohol, or heavy metals, and completely soluble. A dependable product
for bacteriologists.
"RAr^TTO OYr^AT T ^ ^^^^ ^^^^ powder, manufactured so as to
i3r\V4 1 \J'\J I\.\Ji\Lj±j preserve the essential properties of fluid
bile, and proved against known strains. Widely employed in the presumptive
test for B. coli, on account of its convenience and reliability.
Our Research Laboratories are Constantly Working on the Problems of
Proteid Chemistry, Biochemistry, and the Metabolism
of Micro-organisms
Requests for Samples and Literature Promptly Answered
Digestive Ferments Co., Sicifl^u^s.T:
ONE OUNCL '|i'>
HFIED PYROXn
'III ;SHREDS IMMERSED IN WTtB t
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If^LlillHCKRODT CHEMICAL W*'!'
PURIFIED PYROXYLIN
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WE offer this highly purified preparation of
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M.C.W. PURIFIED PYROXYLIN is supplied in
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MALLINCKRODT CHEMICAL WORKS
ST. LOUIS :: :: :: NEW YORK
Manufacturers of High Grade Medicinal Chemicals
THE JOURNAL OF IMMUNOLOGY
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Official Organ of the American Association for Cancer Research
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Official Organ of the Society of American Bacteriologists
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WILLIAMS & WILKINS COMPANY
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A NEW BOOK
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Explaining their therapeutic action — How, When and Where to
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This book contains over 500 pages, is cloth bound and sells
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HECHT-GRADWOHL TEST
COMPLETE URINE ANALYSIS
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PASTEUR TREATMENT FOR RABIES
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Write us for Free Booklet on
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Catalog Cy. on request
2169/18— Wolfhuegel's Counting Apparatus, coiissisting of
Countint; Plate No. 2169/17 with polished wooden base
containing drawer for accessories $6.25
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by gas or pyrogallate method. In two sizes: 150x80 mm.
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SPECIALTIES:
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HENRY HEIL CHEMICAL CO.
210-214 South 4th Street
St. Louis, Mo.
De Khotinsky
ELECTRICALLY HEATED AND REGULATED
INCUBATOR AND DRYING OVEN
Patent Pending
No. 4867E.
4867E. Drying Oven and Incubator.
The Cabinet, perforated shelves and door are made of asbestos board, which is fire-proof
and resistant to the corrosive action of fumes and chemicals, and minimizes the loss of heat
by conduction. All the walls are double in construction and filled with magnesia-asbestos
packing. No metal frame work which would conduct heat from the inner to the outer walls
is used. All cabinets have a polished white enamel finish and are mounted on nickel plated
steel bases.
Heating of the cabinet is effected by means of de Khotinsky standard heating units,
which are inserted from the outside, and permit of replacement in case of accidental breakage
without stopping service or opening the door. The units are carefully protected by the inner
removable bottom of the cabinet. One unit is connected directly to the relay for tempera-
ture regulation and the remaining units may be placed in service one by one by means of the
switch. The regulating unit will maintain any temperature from 5" to 70° above the sur-
rounding temperature according to the setting of the thermo-regulator. Temperatures from
70 to 120° C. are obtained by the introduction of a second unit; higher temperatures with the
other units. , , x l rr-u
The Thermo-Regulator action is based on the expansion of mercury m a steel tube. 1 he
attached regulating head with glass capillary renders the make and break of the mercury
meniscus visible and permits of cleaning without interruption of the service. The regulator
may be set for any temperature up to 150° C. or even higher. .
The Relay is extremely sensitive and operates with 0.4 watt energy consumption. Ihe
precision of action of the regulating system is easily iV C, and can be held at xk" C. by
careful adjustment. , ^ ., , , x j j j ** u
The cabinet is provided with cartridge fuses, flexible asbestos covered cord and attach-
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Size inside. 7 in. wide by 6i in. deep by 10 in. high. Conrplete with four eO-watt heating unite for 110 volts D. C.
current wei » .
4867F, Dryiag Oven and Incubator. Same as No. 4867E, but 12 in. wide by 11 in. deep by U in. high. Complete
with four 115-watt heating units for 110 volts D. C current «« oa-""
4867G. Drying Oven and Incubator. Same as No. 4867E. but 16 in. wide by 14 in. deep by 19 in. high. Com-
pletewith four 140-watt heating units for 110 volts D. C current Net 135.0U
NOTE:— For 220 volte D.C. or 110 volts A.C., add $2.50 to the above prices; for 220 volte A.C. add J5.00. In ordering
state voltage, and if A.C. the nxunber of cycles and phase
Send for Pamphlet 61 describing the De Khotinsky ElectricaUy Heated and Regulated
Constant Temperature Devices
CENTRAL SCIENTIFIC COMPANY
460 East Ohio Street
(Lake Shore Drive, Ohio and Ontario Streets)
CHICAGO U. S. A.
These moderate
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are giving Excel-
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Thelco Bacteriological Incubators
Electrically Heated and Controlled
Thelco Bacteriological Incubators are built of well seasoned heavy oak,
finished inside and outside with white paint enamel. This finish imparts
to the incubators a handsome and clean appearance. An inner door of
glass is provided to allow inspection of the chamber without opening the
door. No water jacket is used or required.
An exclusive feature is the removable wire resistance heating plate.
This method of heating is distinctly superior to lamps in that it
evenly distributes the heat throughout the chamber; furthermore, it will
not burn out to cause spoilage of cultures, annoyance and inconvenience,
that is so apt to happen when lamps are employed as a heating medium.
The Thelco bi-metallic Thermostat employed will maintain the temper-
ature constant to within a degree without any attention after adjustment
is once made.
The Operation is Very Simple
To place the incubator in use, it is only necessary to screw the attachment plug into an
electric light flocket and ttirn on the current; then adjust the regulator until the thermometer
reaches the temperature desired, which will remain constant to within a degree. The incu-
bator can be operated on direct or alternating current.
When ordering please state voltage of current.
Each Incubator is supplied with perforated metal shelf (Nos. 4 to 7 with two shelves) and
cord with plug ior attaching to regular electric lamp socket.
No.
Dimensions of Chamber
Inches
12 z 12 X 12
18 X 18 X 26
IS X 18 X 26
28 X 36 X 18
28 X 36 X 18
Equipment
Without Stand
Without Stand
With Stand
Without Stand
With Stand
Price Net
$30.00
60 00
6i ,)
110.00
125.00
• Incubators No. 6 and 7 are made with double divided doors.
EIMER & AMEND
Headquarters for all Laboratory Supplies
NEW YORK PITTSBURGH
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