PROCEDURES RECOMMENDED FOR THE STUDY 
OF BACTERIA, 
WITH ESPECIAL REFERENCE TO GREATER 
UNIFORMITY IN THE DESCRIPTION 
AND DIFFERENTIATION OF 
SPECIES. 
BRING THE REPORT OF A COMMITTEE OF AMERICAN BACTERIOL- 
OGISTS TO THE COMMITTEE ON THE POLLUTION 
OF WATER SUPPLIES OF THE AMERICAN 
PUBLIC HEALTH ASSOCIATION. 
Submitted at the meeting of the Association in Philadelphia, Pa., September, 1897 
CONCORD, N. H.; 
THE RUAPORD PRESS. 
1898. PROCEDURES RECOMMENDED FOR THE STUDY 
OF BACTERIA, 
WITH ESPECIAL REFERENCE TO GREATER 
UNIFORMITY IN THE DESCRIPTION 
AND DIFFERENTIATION OF 
SPECIES. 
BEING THE REPORT OF A COMMITTEE OF AMERICAN BACTERIOL- 
OGISTS TO THE COMMITTEE ON THE POLLUTION 
OF WATER SUPPLIES OF THE AMERICAN 
PUBLIC HEALTH ASSOCIATION. 
Submitted at the 7neeting of the Association in Philadelphia, Pa., September, 1897. 
CONCORD, N. H.: 
THE, RUAFORb FRESS. 
1898. PREFACE. 
At the meeting of the American Public Health Association in Mont- 
real, Canada, in 1894, the Committee on the Pollution of Water Supplies 
closed its report with the suggestion of a co-operative investigation into 
the bacteriology of water supplies as a means of bringing order out of the 
chaotic state of the literature of water bacteria, and of throwing light 
from the bacteriological side on questions of practical sanitation. This 
suggestion1 was approved by the Association and the Chairman of the 
Committee was authorized to build up a committee for collective bacteri- 
ological investigation. The bacteriologists promptly acceded to the 
proposition. They recognized that such an investigation would give an 
immense impetus to bacteriological work ; that it would do much to clear 
away the confusion surrounding species, and to increase and systematize 
our knowledge ; and that practical results might also be expected, par- 
ticularly as regards the typhoid and colon bacilli, the unwholesomeness of 
water supplies and the means of lessening the prevalence of typhoid fever 
and diarrheal diseases, A sub-committee consisting of Professor J. 
George Adami, Dr. Wyatt Johnston, Mr. George W. Fuller, and myself, 
appointed to'‘determine the methods of laboratory procedure to be 
adopted by the committee in the practical work of the investigation, 
found it impossible to formulate a satisfactory scheme of work until cer- 
tain questions, mostly relating to technique, had been discussed fully and 
settled in accordance with the most advanced knowledge of the subjects 
concerned. An effort to effect this by correspondence developed so 
much variance in the practice of the different laboratories that it became 
needful to call a convention for a thorough discussion of the points at 
issue. The convention was held in the Academy of Medicine, New York 
city, June 21 and 22, 1895. Most of the prominent bacteriologists of the 
United States and Canada were present, but although the members were 
informed beforehand of the subjects that were to be brought up for settle- 
ment, and although full discussion was given to each under the chair- 
manship of Professor Welch of Johns Hopkins University, many of the 
points presented so much difficulty that the whole series was referred to 
a committee, with the understanding that the convention would accept its 
decision. 
This committee consisted of 
J. George Adami, McGill University, Chairman, 
A. C. Abbott, University of Pennsylvania. 
T. M. Cheesman, College Physicians and Surgeons, New York. 
George W. Fuller, Louisville Water Company. 
IFor which credit is due to Dr. Wyatt Johnston. 4 
REPORT OF COMMITTEE. 
W. T. Sedgwick, State Board of Health, Massachusetts. 
Charles Smart, U. S. Army. 
Theobald Smith, Harvard University. 
W. H. Welch, Johns Hopkins University. 
The committee met in New York city in February, 1896, to digest its 
materials and outline its report which was presented to the American 
Public Health Association at its meeting in Buffalo, New York, in Sep- 
tember of that year. The report was subsequently withdrawn for further 
criticism and amendment, and was finally submitted for publication at the 
meeting of the Association in Philadelphia, Pennsylvania, September, 
1897. 
Charles Smart, 
Deputy Surgeon General, U. S. Army. REPORT OF COMMITTEE. 
5 
INTRODUCTION. 
As explained by Dr. Smart in the preface to this report, a convention of 
bacteriologists from the United States and Canada assembled in the city 
of New York, on June 21 and 22, 1895, in response to the invitation of a 
sub-committee of the Committee on the Pollution of Water Supplies of the 
American Public Health Association. The proceedings of this conven- 
tion, including the papers read and their discussion, were published in 
the Journal of the American Public Health Association, October, 1895. 
These papers and discussions related mainly to technical procedures to 
be followed in the systematic study of bacteria with especial reference to 
their description and identification. There was general agreement of 
opinion as to the importance of securing greater precision and uniformity 
in the methods of studying and describing bacterial species. A com- 
mittee of members of the convention was, therefore, appointed to prepare 
a report, to be presented to the Water Committee of the American Public 
Health Association, this report to contain recommendations concerning 
bacteriological methods based partly upon the deliberations of the con- 
vention and partly upon a wider study of the subject. The members 
selected for this committee were Drs. J. George Adami, William T. Sedg- 
wick, George W. Fuller, Charles Smart, Alexander C. Abbott, T. M. 
Cheesman, Theobald Smith, and William H. Welch, 
A first draft of a report was drawn up by Dr. Adami and submitted to 
the members of the committee, who made various suggestions. The 
final preparation of the report was undertaken by Dr. T. M. Cheesman, 
Instructor in Bacteriology in the College of Physicians and Surgeons, 
Columbia University, New York. The following statement by Dr. 
Adami well expresses the aims and manner of preparation of the report: 
“Naturally with a committee, the members of which are so widely scat- 
tered, it has been found impossible to hold frequent meetings, but at 
these meetings the members have found themselves singularly in accord 
upon everything relating to the main points at issue. Naturally, also, 
correspondence and the circulation of the report in its various stages 
have not been found entirely satisfactory in eliciting the opinions of 
every member upon matters of detail. But all these means accomplished 
much, and it was eventually found possible to place the final drafting of 
the recommendations in the hands of one member. We cannot suffi- 
ciently express our indebtedness to Dr. Cheesman for the amount of 
time, and indeed of independent work which he has devoted to this task. 
“The recommendations thus do not indicate the previous procedure in 
all details of any single member of the committee, but are a concord of 
what has appeared to be the best in the methods and technique of all the 
members and of bacteriologists generally. To have indicated in the 
following pages wherein any single member found himself unable to 
accept in its entirety any one of the many recommendations would have 6 
REPORT OF COMMITTEE. 
counteracted our main object, that, namely, of inducing uniformity and 
precision in procedure in the study and descriptions of species. Each 
member, therefore, to attain this object has voluntarily refrained from 
demanding that one or other method, to which from long employment he 
has become firmly attached, should be inserted in these pages. The com- 
mittee freely admits that there may be other and better methods than 
those here detailed. It has, on the other hand, striven to recommend 
what in the present state of our knowledge would seem to be the best 
and most likely to gain acceptance. It does not demand of bacteriologists 
in general—it does not promise for its own members in particular—that 
these and only these methods shall be employed. It does but ask that 
where new species are being studied for publication the procedure here 
recommended be given a trial, and that, for the direction of other 
workers, where it has been employed a note be given to that effect, 
e.g., ‘ cultures in broth (Method B. C.1) presented the following char- 
acters : ’—or, ‘ save where otherwise indicated, the B. C. methods have 
been used.’ 
“In short, the committee recognizes fully that these recommendations 
must of necessity be provisional. It publishes them in the hope that by 
this act it will direct attention to the urgent need now existing for full 
and accurate descriptions of species of bacteria in which the items have 
been determined by methods common to the main body of workers, and 
as a consequence are capable of verification and control.” 
The report is not intended to be a complete treatise upon bacteriolo- 
gical technique. Its purpose is to make certain recommendations concern- 
ing methods to be pursued in the study of bacteria, with the view of 
securing greater uniformity and exactness in the determination and 
description of the characters of bacterial species. When one considers 
the difficulty, often the impossibility, of the identification of many bacte- 
rial species or varieties described in literature, in consequence of imper- 
fections and carelessness in the determination and description of their 
characters, it is evident that the attainment of the purpose aimed at in 
this report is greatly to be desired. 
The report deals especially with certain ordinary and fundamental pro- 
cedures in bacteriological technique, and it does not attempt to cover fully 
the entire field. In a science so rapidly developing as bacteriology, it 
need scarcely be said that any attempt to present the best technical pro- 
cedures can apply only to the existing state of the science, and that much 
will be added and much corrected in the near future. It is hoped that 
the recommendations in this report may prove useful to workers in bac- 
teriology, and especially may lead to greater accuracy and fullness and 
uniformity in the determination and description of the characters of 
bacteria. William H. Welch. 
1 Bacteriological Committee; the methods recommended by the Committee of American Bacteri- 
ologists. REPORT OF COMMITTEE. 
7 
PROCEDURES RECOMMENDED FOR THE STUDY OF BAC- 
TERIA WITH ESPECIAL REFERENCE TO GREATER UNI- 
FORMITY IN THE DESCRIPTION AND DIFFEREN- 
TIATION OF SPECIES. BEING THE REPORT OF 
A COMMITTEE OF BACTERIOLOGISTS TO 
THE COMMITTEE ON THE POLLUTION 
OF WATER SUPPLIES OF THE 
AMERICAN PUBLIC HEALTH 
ASSOCIATION. 
The various tests applicable for describing species of bacteria may be 
divided into two categories, as follows : 
Necessary tests, and those which in the present condition of bacterial 
science may be regarded as optional. 
The terms “ necessary ” and “ optional ” are here used with consider- 
able hesitation, as many of the tests included among the optional are of 
importance and really necessary for the purposes of species differentia- 
tion in special cases. These tests, as grouped, however, are at the 
present time applicable to a great majority of the species known, those 
classed as necessary being of primary importance and of the greatest 
general utility. 
NECESSARY INFORMATION AND TESTS. 
Information with regard to the following features and properties of 
any species of bacteria that is being studied is held by the committee to 
be necessary and to form the indispensible basis for conclusions as to the 
characters and relationships of that species. 
I. Source and habitat. 
11. Morphological characters. 
1. Form. 
2. Dimensions. 
3. Manner of grouping and arrangement in the growths, 
4. Staining powers, (a) with watery dyes, (b) by Gram’s method. 
5. Presence or absence of capsule. 
6. Presence or absence of flagella (motility). 
7. Spore formation and differentiation of spores from deposits and 
vacuoles within the cell. 
8. Tendency to pleomorphism. 
9. Involution and degeneration forms. 8 
REPORT OF COMMITTEE. 
111. Biological characters. 
A. Cultural characteristics, mode of growth in and upon 
1. Nutrient broth. 
2. Gelatin plates (single colonies, surface and deep). 
3. Gelatin tubes. 
4. Agar plates (single colonies, surface and deep). 
5. Agar tubes. 
6. Potato. 
7. Milk. 
8. Blood serum. 
B. Biochemical features. 
1. Temperature relationship (activity of growth at iB°-22° C. and at 
36°-38° C. and thermal death point). 
2. Relation to free oxygen (aerobic and anaerobic growth). 
3. Relation of growth to acidity and alkalinity of media. 
4. Action upon gelatin (presence or absence of liquefaction). 
5. Action upon proteids (milk and serum), 
6. Action upon carbohydrates (fermentation and gas formation). 
7. Action upon nitrates. 
8. Production of indol. 
9. Production of acid or alkali, 
xo. Pigment formation. 
11. Development of odor. 
C. Pathogenesis. 
OPTIONAL TESTS OF GENERAL USEFULNESS. 
I. Morphological. 
1. Staining reactions with special stains. 
2. Study of flagella by special stains. 
3. Permanency of morphological characters after long-continued 
growth and successive transplantation upon artificial media. 
4. Photographic reproductions of isolated bacteria. 
5. Cover-glass impressions. 
11, Physiological. 
A, Cultural characteristics, mode of growth in or upon 
1. Litmus gelatin. 
2. Loeffler’s blood serum. 
3. Synthesized media. 
Photographic reproduction of characteristic cultures. 
B.« Biochemical. 
1. Minimum, optimum, and maximum temperatures of growth. 
2. Growth in atmospheres of various inert gases (when anaerobic 
power of growth has been determined). REPORT OF COMMITTEE. 
9 
3. Optimum reaction of media and reaction limit, acid and alka- 
line (indicated by phenolphthalein). 
4. Chemical properties and solubility of pigments produced and 
spectroscopic observations upon the pigments. 
C, Pathogenesis. 
1. Inoculation of various species pf animals with minute study of 
the pathological changes produced. 
2. Immunity-producing properties. 
3- Agglutinating properties of specific sera. 
4. Determination and isolation of toxic substances (from non- 
pathogenic as well as from pathogenic bacteria). 
The purpose of this report is to induce uniformity in the employment 
of methods in the study and description of species of bacteria, and to this 
end it is necessary not only to recommend the routine employment of 
various media, and the setting forth in due order of information with 
reference to the morphological and biological features presented by any 
species that has been studied, but also to describe with exactitude the 
usages which the committee have deemed the most acceptable in connec- 
tion with the various tests. 
It is in no sense intended that these recommendations should form a 
complete treatise upon bacteriological technique. Only such matters are 
treated concerning which it is felt that there is need of greater uniformity 
of procedure or a more precise and correct technique. Especially in the 
preparation of media is greater uniformity of procedure necessary. 
The committee feel that these recommendations are in many respects 
imperfect, yet they make them in the belief that, while in no sense final, 
they constitute a step towards a universally accepted method of procedure 
in connection with species differentiation. 
Concerning these important facts, it need only be stated that a possi- 
ble seasonal distribution of species must always be borne in mind. The 
date recorded on the analysis table should bear upon this fact. 
SOURCE AND HABITAT. 
MORPHOLOGICAL CHARACTERS. 
To insure uniformity of description of the three main divisions and 
their subdivisions as determined by their grouping, the following nomen- 
clature is adopted and the terms defined : 
i. Form. 
1. Coccus or micrococcus. 
Forms which are spherical or subspherical. 
2. Bacillus. 
Oblong or cylindrical forms, having one dimension definitely greater 
than any other, more or less straight and never forming spirals. REPORT OF COMMITTEE. 
3. Spirillum. 
Cylindrical and curved forms, constituting complete spirals or por- 
tions of spirals. 
The natural grouping which may be observed in hanging drop cul- 
tures and frequently also in cover-glass preparations, leads natur- 
ally to subdivisions of the main groups, as follows : 
1. Coccus. 
a. Single coccus, grouped irregularly. 
b. Diplococcus, forming pairs. 
c. Streptococcus, forming chains, often showing paired cocci. 
d. Tetracoccus, forming fours by division through two planes of 
space. 
e. Sarcina, forming packets of eight members, by division through 
three planes of space. 
2. Bacillus. 
a. Single bacillus. 
b. Diplo- and Strepto-bacillus, forming twos or longer chains, the 
bacilli attached end to end. 
c. Filaments, or thread-like growths, in which divisions into bacilli of 
the normal length are not apparent, or occur irregularly and 
transversely to the long axis of the growth. 
The determination of morphological characters should always be made 
from fully developed cultures; those which are too young may present 
immature forms, due to rapid multiplication, while in old cultures, altered 
or degenerated forms may be observed. 
When growth is obtained upon different media, variations, especially in 
size, may sometimes be observed. These differences should always be 
described, together with a note of the media upon which they were devel- 
oped, and a statement as to whether such variation is a marked feature of 
the species under consideration. 
The conditions of temperature and of medium which favor growth are 
very various for different species, so that no fixed temperature, medium, 
or age of growth can be determined upon as applicable to all species. 
Morphological descriptions should always be accompanied by a definite 
statement of the age of the growth, the medium from which it was ob- 
tained, and the temperature at which it was developed. 
It is further advisable that the appearances observed in growths 
developed upon a solid and in a liquid medium, should be recorded. 
In accordance with these facts, the following procedure is recom- 
mended as a routine whenever it is applicable, for the determination of 
the form and grouping of bacteria. 
Determine and describe the morphology from growth obtained upon at 
least one solid medium and in at least one liquid medium. Growth at 
36°-38° C. should, in general, be not older than from 24 to 48 hours, REPORT OF COMMITTEE. 
while growth at room temperature (iß° C.-220 C.) should be not older 
than from 48 to 72 hours. Growth on solid media may be studied from 
cover-glass preparations ; while in liquid media growth is best observed 
in hanging drop, preferably in a fresh medium inoculated with a very 
small amount of the culture to be examined. 
It is desirable as a routine procedure in recording form and grouping, 
that growth from nutrient broth, gelatine and agar be microscopically 
examined and described, and that any variation from the morphology 
thus established, found upon examination of growth from other standard 
media, be accurately noted. 
2. Dimensions. 
It seems probable that the remarkable diversity in published state- 
ments with regard to the dimensions of many of the commonest of the 
bacteria, is due largely to the different methods employed of preparing, 
fixing, and staining the cover-glass preparations. 
Spreading the film ; 
The films should be made from a very dilute emulsion of the prescribed 
culture in distilled water, spread thinly upon a perfectly clean cover- 
glass,1 and dried rapidly in the air. 
Fixing the film: 
For ordinary purposes of observation, fixing may be effected by pass- 
ing the preparation three times through a flame, but for specimens to be 
used for accurate measurements this method of fixing is crude. To 
establish a uniform procedure, and to avoid distortions from overheating, 
it is recommended that the film be fixed by heating in an automatically 
regulated air bath for twenty minutes, at a temperature of 1150 C. Care 
must be taken to keep the cover-glass from direct contact with the metal 
shelf of the oven, and in close proximity to the bulb of the thermometer. 
Staining : 
In regard to the dyes that should be used, there is some diversity of 
opinion among the members of the Committee. All agree that, when possible, 
dyes should be used cold, and that a dilute watery solution of fuchsin, made 
by adding 5 c. c. of a saturated alcoholic solution of fuchsin to 95 c. c. of 
distilled water, has a wide application. A dilute watery solution of methy- 
lene blue and one of gentian violet, made in the way recommended for 
preparing the watery solution of fuchsin, also are useful, as is likewise 
Loeffler’s alkaline solution of methylene blue. These dyes, therefore, as 
thus prepared, are recommended by the Committee. It is necessary that 
all dyes be freshly prepared, as the presence of the alcohol, which rapidly 
evaporates, has a distinct effect in their staining properties. 
fermentation tubes, etc., may be freed from all organic matter adhering to them 
by boiling them for an hour in the following solution : 
Potassium bichromate 6 parts, thoroughly dissolved in 100 parts of water, to which is then slowly 
added 5 parts of C. P. sulphuric acid. After boiling, the bichromate solution is allowed to cool, and is 
removed from the glassware by repeated rinsings in water. Cover-glasses thus cleansed are to be 
stored in strong alcohol. 12 
REPORT OF COMMITTEE. 
Mounting in media of different refractive indices makes differences in 
the pictures • obtained. The materials most commonly used for this pur- 
pose are water, and balsam dissolved in xylol, or cedar oil; the former 
having a low index of refraction, and the latter a high index. When exam- 
ined in water the preparation may be ringed with vaseline to prevent 
evaporation and the consequent distortion resulting from partial drying. 
Balsam is much to be preferred, however, for general observation and for 
measurements of the bacteria, although for certain specific purposes other 
media having a higher or lower refractive index are more serviceable. 
Descriptions should always be made from examinations under a magnify- 
ing power not less than that given by a 1-12 homogeneous immersion lens 
and a No. 3 Huygenian eye-piece. 
Mounting: 
Measurement: 
The most accurate method of determining the exact size of bacteria is by 
photography, but as photo-micrography at 1,000 diameters requires much 
special apparatus, the Committee feel that this mode of measurement 
cannot be required of all observers, although it is to be preferred where 
it is practicable. 
Dimensions, especially transverse dimensions, should always be given 
in terms of the micro-millimeter (/x), and if not determined by photography, 
should be by as accurate measurement as can be obtained by an eye-piece 
micrometer. Welch recommends as a ready method for record, comparing 
the size of the bacteria with the diameter of the human red blood cor- 
puscle. This is easily done by obtaining a drop of blood from the finger 
and mixing a portion of the diluted culture with it, a method which insures 
considerable uniformity and gives information as to the size of the organism 
in its living condition. 
3. Methods of grouping and arrangement in the growths. 
Grouping can be accurately determined from growths in liquid media 
and preferably in “ hanging drop ” cultures prepared by placing a drop of 
fresh medium upon a sterilized cover-glass and inoculating the edge with 
a minute portion of the culture, so that the growth and spread of the 
organisms may be watched. 
This method of observation yields in general more accurate information 
than that obtained from “impression preparations” of surface colonies, 
though these latter are of distinct use, and are included among the gener- 
ally useful tests. 
4. Staining powers. 
Little is definitely known about the intimate structure of the bacterial 
cell. Bacteria show, however, marked differences in their staining quali- 
ties, some taking the dye readily and staining uniformly, while others are 
more or less difficult to stain, and some show unstained or slightly stained REPORT OF COMMITTEE. 
13 
portions. These differences may be more or less constant, and they 
appear, to some extent at least, to depend upon the dye or the solution 
used for staining. 
To insure uniformity, it is preferable to use simple aqueous solutions of 
the basic anilin dyes (See p. 64) and when these fail to give satisfactory 
results, Loeffler’s methylene blue1 is to be recommended.2 
The intensity of staining often differs with the dye or solution 
employed, and it is well to study the appearances both in faintly and in 
deeply stained specimens. 
As the action of the decolorizing agents upon stained bacteria has 
been insufficiently studied, much information might be gained by 
research in this direction. The method of staining known as Gram’s 
method3 is the most useful of the differential methods of staining and 
the Committee recommend that it should be applied to all species studied 
5. Capsules. 
All bacteria are believed to have an envelope, slimy or gelatinous in 
character, which causes them to adhere together and to other objects. In 
some cases this gelatinous zone, either from its extent or other characters, 
may be readily demonstrable, and to this the name “ capsule ” was given 
by Friedlaender; but in the majority of species it is very limited in extent 
or difficult of demonstration, so as to be visible only under special condi- 
tions. Many pathogenic bacteria present readily demonstrable capsules 
only in the animal body. 
The term “ capsulated ” as applied to species of bacteria has been 
generally confined to those upon which this zone can, by one or other 
method, be seen. When found in nature, growing in moist places, or fre- 
quently in the animal body, capsules may not infrequently be observed, 
but when bacteria are cultivated artificially the capsule often seems to dis- 
appear entirely, even from the species which develop it in the animal 
body or under natural conditions, and only occasionally is the capsule 
preserved for a few transplantings in cultures in milk and blood serum, and 
in surface growths on moist agar. 
The appearance of clear, unstained zones, more or less regular in con- 
tour, surrounding the bacteria, may be indicative of the presence of 
capsules, but cannot be considered as a demonstration of their presence. 
This demonstration can be considered positive only when the capsules are 
stained differentially from the contained organism and the tissue or other 
matter surrounding them. 
Sometimes a differential stain is obtained by the ordinary staining 
methods, using the watery dyes, anilin-water gentian-violet, or normal or 
1 Mittheilungen aus dem Kaiserlichen Gesundheitsamte, Berlin, 1884, p. 439. 
2 Dyes should be freshly prepared. 
3 Fortschritte der Med. Bd. 11, No. 6, 1884, REPORT OF COMMITTEE. 
dilute carbolic fuchsin, the capsule taking on a paler shade of color than 
the contained protoplasm. In many other instances, however, no stain of 
the capsule is thus obtained, and for purposes of demonstration it then 
becomes necessary to resort to special staining methods to render the 
capsule visible. None of the methods yet devised are universally ap- 
plicable to this end. Welch has found that many of the more delicate 
capsules may be dissolved or rendered invisible by contact with water, 
whether before, during, or after, the application of the dye. Many capsules 
are fixed by glacial acetic acid, and for this group of capsulated bacteria 
Welch’s method of staining the capsules is most satisfactory. This method 
is as follows : The cover-slip specimens, prepared without water, are treated 
first with glacial acetic acid, which is at once allowed to drain off and is 
replaced (without washing in water) with anilin-oil gentian-violet solu- 
tion, which is allowed to run off and is repeatedly added to the surface of 
the cover-glass until the acid has been displaced. The specimen is now 
briskly washed with a one to two per cent, solution of common salt. The 
specimen is to be examined in the salt solution. The proper strength of 
the salt solution to be used varies in different cases, sometimes the ordi- 
nary physiological solution sufficing; at other times over two per cent, may 
be required.1 
In noting the presence or absence of capsules, the methods employed 
for demonstrating them and the conditions under which they have been 
observed should be fully stated. 
6, Flagella. Motility. 
So far as known, the movements of bacteria are produced only through 
the agency of flagella and when true motility of any species, as distin- 
guished from Brownian movement, is observed, the presence of flagella 
may, from this fact alone, be assumed ; although, the converse is by no 
means always true. Different degrees of motility may be observed at 
different ages of growth, young cultures showing much more active move- 
ments than older cultures, while in quite old cultures motion may be 
entirely suspended. Motion may be apparent in specimens from cultures 
of the colon bacillus eight or ten hours old, and absent from cultures 
twenty-four hours old. Other causes not well understood seem to influence 
motility, and an observation has been made by Theobald Smith that a 
specimen of the B. coli communis which was usually non-motile in young 
fluid cultures, was motile in hanging-drop preparations, when made from 
young cultures developed on solid media. Although species maybe found 
which appear motionless under all conditions of growth, but which are 
otherwise undistinguishable from motile forms,2 it may be stated that 
i Welch. The “ Bulletin of The Johns Hopkins Hospital,” December, 1892, p. 128. 
2 Dr. Veranus A. Moore, in 1891, isolatedfrom the organs of a pig a non-motile bacillus which 
proved to be similar in its pathogenic and cultural characters to the (motile) bacillus of hog cholera. 
In spite of the absence of motility, Dr. Theobald Smith determined that he could not do otherwise 
than place this organism in the hog cholera group, this being the solitary mark of distinction. 
V. A. Moore—The nature of the flagella, etc. Journ. Am. Pub. Health Assoc Vol. 20.189=;, p. 436. REPORT OF COMMITTEE. 
15 
generally speaking the presence or absence of motility is a valuable test 
for species differentiation. 
The study of motility is best made in hanging-drop preparations in 
bouillon prepared from young cultures as before described (p. 65), grown 
at or near the optimum temperature for only a few (6 to 18) hours. 
For most bacteria the flagella are so fine and delicate that they are 
invisible in any mounting medium and under any of the magnifying pow- 
ers now obtainable, except when especially stained. 
Considerable practice is usually required for the successful preparation of 
specimens by any of the staining methods yet published ; and while no single 
method is applicable to all species, any or all of these methods are liable 
to prove uncertain, even in the hands of skilled workers. 
From some of the earlier studies on the staining of flagella, it seemed 
probable that the length, number and arrangement of these organs of 
motility might be so constant as to give important morphological data for 
species differentiation,1 but their length has been found to vary so greatly 
and their number and arrangement are so inconstant, that less help can 
be derived from this source than was at first hoped for. It is, however, 
important to determine the arrangement and number of the flagella, to 
describe which the terms monotricha, lophotricha, amphitricha, and peri- 
tricha, introduced by Mesea,2 are serviceable. 
The methods of staining the flagella, most to be recommended, are two, 
one devised by Loeffler and described in Cent. f. Bakt, and Parasit., Bd. 
VII, 1890, p. 625, and the other by Van Ermengem, published in the 
Travaux du Lab. d’ hygiene et de bact. de Gand. T. 1, p. 3, of which 
an abstract is to be found in Cent. f. Bakt. & Parasit., Bd. 15, 1894, p. 
969. 
Other methods, mostly modifications of Loeffler’s, sometimes prove 
useful, among which may be recommended that of Bunge (Fortschritt der 
Med. Bd. XII, 1894, pp. 462-653) and that of Nicolle & Morax (An- 
nales Inst. Pasteur, T. VII, 1893, p. 554). 
In describing motility, the kind of motion observed should always be 
noted, whether active or slow, direct or rotary, vibratory, etc. When 
flagella are stained, the culture from which the preparation is made 
should be fully described and the method of staining noted. 
7. Spores. 
The vegetative form of the bacteria, which we have been considering, 
is the most usual stage of growth observed. While in this stage the bac- 
teria, multiplying by fission, and fulfilling their various life functions, are 
1 Mesea. Revista d’ igiene e sanita publica. No. 14,1889, p. 513. 
Luksch. Cent. f. Bakt. & Parasit. Bd. XII, 1892, s. 427. 
2V. A. Moore. Wilder Quarter Century Book, p. 339. 
V. A. Moore. Journal Am. Pub. H. Assoc., Vol. 20, Oct. 1895, P* 441- 
Stoecklin. Annales Suisses des Sciences Medicals, 1 Serie, Livreson, 6,1894. 16 
REPORT OF COMMITTEE. 
sensitive to external influences, and may be easily destroyed by conditions 
which are inimical to them. Some species have the power to develop 
bodies very much less susceptible to deleterious surroundings, known as 
spores, which, under conditions favorable to their germination, develop 
into the vegetative forms of the organism from which they are derived. 
The spore does not multiply, and from the time of its formation seems to 
be and to remain in an absolutely quiescent or resting stage until it germ- 
inates. The function of the spore seems to be solely the preservation of 
the species; and it is generally believed that spores form only when 
growth conditions are in one or more ways unfavorable. 
It is well known that spores develop within the bodies of many bac- 
teria (endospores) and it is inferred by some that “joint-spores” (arthro- 
spores) may develop in many other species. The former have been accu- 
rately studied, the latter have not, and indeed the whole conception of 
arthrospores for bacteria is rejected by most recent writers. So far as the 
endospores are concerned, they may be seen within the bodies of the bac- 
teria, situated centrally or nearer the ends, and appear as bright, highly 
refractive bodies, which do not stain by exposure for a few minutes to the 
watery dyes. It should be noted whether the spores produce a swelling 
of the bacterial cell at the site of their formation, giving rise to Clostri- 
dium and drumstick shapes. Spores may sometimes be stained with 
comparative ease, by the use of a hot dye, but some one of the special 
methods devised for this purpose must usually be resorted to. 
The methods here recommended are those devised by Hauser (Munch. 
Med. Wochenscrift, 1887, No. 34), by Moeller (Cent, fiir Bakt. & Parasit, 
Bd. X, p. 273), and by Abbott (Principles of Bacteriology, 3d Ed., p. 
146). 
No single method seems applicable to the staining of all spores, how- 
ever, and in many bacteria small, bright, and shiny areas which usually do 
not take up the dye, may often be observed. In some cases it is known 
that these appearances are due to the formation or deposit within the or- 
ganism of vacuoles, fat or crystals, but as the differentiation of these 
bodies from spores is not always possible, either by optical or by staining 
methods, some other means of identifying spores is necessary. 
It is obvious that the only means of positively knowing that a certain 
body is a spore, is to see it perform the only active function it can per- 
form, namely, to observe its development into a vegetative form of bac- 
terium. Such a study is greatly facilitated by a special incubator fitted to 
receive the microscope, and requires patient watching for perhaps many 
hours, so that although it is often possible to prove the identity of spores 
in this way, it is seldom a practical thing to do. The method most in 
vogue for this determination is to test the resistance of the suspected 
bodies to heat (moist heat between Bo° and ioo° C.). This method, as is 
acknowledged, is faulty, for although bodies which will withstand such a 
temperature for any considerable length of time must be spores, yet it is REPORT OF COMMITTEE. 
17 
readily conceivable that spores may also be formed which have not the 
power of resisting such high degrees of heat, or may be capable of with- 
standing only drying or possibly also other conditions which are usually 
inimical to bacterial life. 
This method, however, is the most practical one at present at our com- 
mand for the determination of the presence or absence of spores, and 
while the Committee urge that the germination of spores should be stud- 
ied whenever possible, yet they would recommend that the present test 
for the presence of spores be 
(1). The development of colonies of the species under examination 
from cultures which have been subjected to a temperature of Bo° C. for 
ten minutes ; and also, 
(2). The presence of highly refracting bodies within the bacteria in 
unstained specimens and their demonstration as “spore-like” bodies by 
one or more of the approved special methods for staining spores. 
8. Pleomorphism. 
A separate section is provided for the consideration of the tendency 
of the bacteria to pleomorphism, a subject already referred to, because 
this matter in the past has scarcely received the attention it deserves. 
The well known discrepancies in the descriptions of what is evidently 
one and the same species, given by different and equally competent ob- 
servers, would appear to be largely due to a lack of recognition of this 
tendency; and while careful study will undoubtedly afford a more perfect 
knowledge of the limit of variation in size and shape among the mem- 
bers of a single species of bacteria, .the determination of the extent of 
pleomorphism promises to be of definite value as an additional character 
to be made use of in grouping allied species. 
Attention is called to the variations in size and shape brought about by 
the following conditions of growth : 
(a) at different temperatures; 
(i>) upon or in media of different composition; 
(c) upon or in media of different degrees of acidity and alkalinity; 
(d) in cultures of different ages, 
(e) as well as to the variations in the size and shape of different in- 
dividual bacteria obtained from one culture, and appearing often 
in the same field of view: i. e., subjected to exactly the same 
conditions of growth. 
9. Involutio?i and degeneration fori7is. 
Little need be said in this section beyond recommending that a note be 
made of the changes which occur in the older cultures in the form of the 
bacteria, and of the period at which, under various conditions, such ab- 
normal or irregular forms make their appearance. Up to the present REPORT OF COMMITTEE. 
time there has been little systematic study of the modifications in the 
shape and appearance of bacteria in older cultures. 
BIOLOGICAL CHARACTERS. 
A. Cultural Characteristics. 
The biological characters shown by the bacteria depend so largely upon 
the composition of the media upon or in which these organisms are 
grown, that the Committee urge most strongly the adoption by all work- 
ers in this line of standard media for use in species description and dif- 
ferentiation. 
It is freely conceded that no two batches of media can be made abso- 
lutely identical, but much greater uniformity in the composition of media 
may be obtained than that which now prevails, by adhering closely to a 
uniform method of procedure in their manufacture. 
To this end the Committee have drawn up exact methods of preparing 
culture media, standardized by what have proved, in their experience, the 
best procedures. These methods do not represent as a whole, the pro- 
cedures followed by any one of their number up to the present time, and 
it is not claimed for them that they are in any sense perfect, but it is 
believed that by the adoption of standard methods of preparing media 
much of the divergence that now exists in the descriptions of bacteria, 
both well and little known, will be obviated. 
The preparation of artificial culture media in general. * 
It is recommended that the following ingredients be uniformly em- 
ployed in the preparation of the respective standard culture media: 
1. Distilled water. 
2. Fresh lean meat (beef, or when veal or chicken is substituted, this 
change should be stated). 
3. Witte’s pepton (dry, made from meat), 
4. Sodium chlorid, C. P. 
5. Sodium hydroxid, C. P., in normal solution for alkalinization, 
6. Hydric chlorid, C. P., in normal solution for acidification. 
7. Pure redistilled glycerin. 
8. Carbohydrates, as nearly chemically pure as possible. 
9. Commercial sheet gelatin, wTashed as free as possible from acids 
and other impurities. 
10. Commercial agar in threads (high grade). 
11. Such chemicals as are employed for special purposes, to be as pure 
as practicable. 
Sterilization of media : 
Sterilization may be effected either by the continuous or by the frac- 
tional method. REPORT OF COMMITTEE. 
I. When sterilizing by the continuous method, some form of autoclave 
is to be preferred, and in using this apparatus it is requisite that the con- 
fined air be replaced by superheated steam. To insure this, both the 
manometer and the thermometer should be made use of, and the time 
decided upon for sterilization should begin only when the theoretical tem- 
perature, as indicated by the pressure gauge, corresponds with that re- 
corded by the thermometer. Exposure in the autoclave to a temperature of 
no° C. (6 lbs. pressure) for fifteen minutes is usually sufficient for the 
sterilization of glassware, apparatus, and media in tubes; for the ster- 
ilization of media in bulk, at this temperature about thirty minutes’ heating 
is necessary. 
11. Sterilization by the fractional method may be effected either, a, in 
streaming steam, or b, in an incubator or water bath at some tempera- 
ture not less than 6o° C. 
These methods are so well known as to require no description here. 
The importance of the reaction of media, as a controlling factor in 
the development of biological characters, is well known to be very great. 
Reaction of media: 
The first thing to obtain is a standard “ indicator ” which will give uni- 
form results. These requirements are best fulfilled by phenolphthalein. 
This indicator was first suggested by Schultze in combination with the 
titration method for obtaining the desired reaction for culture media 
(Cent, fur Bakt. & Parasit Bd. X, 1891, p. 53), but its general adoption 
seems to have been retarded largely by Dahmen (Cent, fur Bakt. & Para- 
sit, Bd. XII, 1892, p. 620) who claimed that its use was not feasible, 
owing to complications which might arise from the presence of carbonates 
and ammonium salts in the solution to be tested. These objections to 
the use of phenolphthalein do exist, but may be readily overcome. 
The amount of free and combined ammonia present in culture media 
at the time the reaction is determined, has been found not to exceed 
0.003 Per cer|t-> which is less than one tenth the amount which interferes 
with the accuracy of this indicator,1 while the production of carbon 
dioxid is obviated to a very great degree by neutralizing with sodium 
hydroxid instead of with sodium carbonate, and any of this gas which 
may be absorbed from the atmosphere is practically all driven off by heat 
during the preparation of the media. 
The great advantage in the use of phenolphthalein over other indica- 
tors lies in the fact that it takes into account the reaction of weak organic 
acids and of organic compounds which have an amphoteric reaction, but 
in which the acid character predominates. Turmeric possesses the same 
properties, but the change in color from a yellow to brown is less satisfac- 
tory than the development of purple-red color, and furthermore turmeric 
paper changes color rather slowly, while with phenolphthalein the color 
appears almost instantly. 
1 Ammonia is not produced by the addition of alkali to the nitrogenous compounds, because at no 
time during the preparation of the media is there an appreciable amount of free alkali present. 20 
REPORT OF COMMITTEE. 
Another advantage to be gained from the use of this latter indicator, is 
its behavior towards the phosphates. Petri & Maassen (Arbeiten aus 
dem K. Gesundheitsamte, Bd. VIII, 1893, p. 311) and Timpe (Cent, fur 
Bakt. & Parasit. Bd. XIV, 1893, p. 845 ; Bd. XV, 1894, pp. 394-664; Bd. 
XVII, 1893, p. 416) have shown that the amphoteric reaction of media 
is associated with the presence of phosphates, and that there are present 
in peptone and gelatin proteid bodies which possess both an acid and a 
basic nature, but in which the acid character predominates. These 
observers agree that to determine accurately the reaction of such ampho- 
teric compounds phenolphthalein (or turmeric paper) should be used as 
an indicator. 
It is known that at the neutral point of phenolphthalein any free phos- 
phoric acid present enters into combination, and the monobasic and tri- 
basic salts of this acid are changed to the dibasic form (Na2 H P 04). 
Now disodium hydrogen phosphate reacts alkaline to litmus, lacmoid, 
rosolic acid, and methyl orange, but neutral to phenolphthalein and 
turmeric. 
Studies made at the Lawrence Experiment Station show that this acid 
salt may be added to culture media in amounts greatly exceeding those 
naturally present in the media without producing any apparent influence 
upon bacterial development. 
From these facts it seems clear that the use of any of the above men- 
tioned indicators, other than phenolphthalein and turmeric, in the presence 
of this dibasic phosphate, prevents the addition of a sufficient amount of 
free alkali to effect neutralization, and as the amount of phosphates in 
media varies considerably, the reaction passes beyond accurate control 
when litmus and other substances of its class are used as indicators. 
Datum point to which all degrees of reaction shall be referred: 
From the available evidence it seems advisable to adopt the phenol- 
phthalein neutral point as the fixed point to which all degrees of reaction 
shall be referred. 
The question of the proper reaction of media for the cultivation of bac- 
teria and the method of obtaining this reaction, have been discussed in a 
valuable paper by Mr. George W. Fuller, published in the Journal of the 
American Public Health Association, Vol. 20, October, 1895, p. 321. 
Some of the main results there given have been mentioned above. 
Method of determining the degree of reaction of culture media: 
For this most important part in the preparation of culture media, bur- 
ettes, graduated into one tenth c. c., and three solutions are required. 
1. A 0.5 per cent, solution of commercial phenolphthalein in 50 per 
cent, alcohol. 
2. A— solution of sodium hydroxid. 
3. solution of hydric chlorid. 
20 REPORT OF COMMITTEE. 
21 
Solutions Nos. 2 and 3 must be accurately made up and must corre- 
spond with the normal solutions soon to be referred to. Solutions of 
sodium hydroxid are prone to deterioration from the absorption of car- 
bon dioxid and the consequent formation of sodium carbonate. To pre- 
vent as much as possible this change, it is well to place in the bottle con- 
taining the stock solution a small amount of calcium hydroxid, while the 
air entering the burettes or the supply bottles should be made to pass 
through a “U ” tube containing caustic soda, to extract from it the 
carbon dioxid. 
The medium to be tested, all ingredients being dissolved, is brought to 
the prescribed volume by the addition of distilled water to replace that 
lost by boiling, and after being thoroughly stirred, five c. c. are transferred 
to a 6-inch porcelain evaporating dish; to this forty-five c. c. of distilled 
water are added, and the fifty c. c. of fluid are boiled for three minutes 
over a flame. One c. c. of the solution of phenolphthalein (No. 1) is then 
added and by titration with the required reagent (No. 2 or 3) the reac- 
tion is determined. In the majority of instances the reaction will be 
found to be acid so that the sodium hydroxid is the reagent most fre- 
-20 
quently required. This determination should be made not less than three 
times, and the average of the results obtained taken as the degree of 
reaction. 
One of the most difficult things to determine in this process is exactly 
when the neutral point is reached as shown by the color developed, and 
to be able in every instance to obtain the same shade of color. To aid 
in this regard, it may here be remarked, that in bright daylight the first 
change that can be seen on the addition of alkali is a very faint darken- 
ing of the fluid, which on the addition of more alkali becomes a more 
evident color, and develops into what may be described as an Italian 
pink. A still further addition of alkali suddenly develops a clear and 
bright pink color, and this is the reaction always to be obtained. 
All titrations should be made quickly and in the hot solutions, to avoid 
complications arising from the presence of carbon dioxid. 
When this manipulation is carried out uniformly, as here suggested, 
and the end point having the same intensity of color is always reached, 
very satisfactory and closely-agreeing results may be obtained. 
Neutralization of media: 
The next step in the process is to add to the bulk of the medium the 
calculated amount of reagent, either alkali or acid as may be determined. 
For the purpose of neutralization a normal solution of sodium hydroxid 
or of hydric chlorid is used and after being thoroughly stirred the fluid 
thus neutralized is again tested in the same manner as at first to insure 
the proper reaction of the medium being attained. When neutralization 
is to be effected by the addition of alkali, it not infrequently happens that 
after the calculated amount of normal solution of sodium hydroxid has 22 
REPORT OF COMMITTEE. 
been added the second test by titration will show that the medium is still 
acid to phenolphthalein, to the extent sometimes of from 0.5 to 1 per 
cent. This discrepancy is perhaps due to side reactions which are not 
understood; the reaction of the medium, however, must be brought to the 
desired point by the further addition of sodium hydroxid, and the titra- 
tions and additions of alkali must be repeated until the medium has the 
desired reaction, (i. <?., 0.0 per cent.—0.005 Per cent., see belowr). 
After the prescribed period of heating, it is frequently found that the 
medium is again slightly acid, usually about 0.5 per cent. Without cor- 
recting this the fluid is to be filtered and the calculated amount of acid 
or alkali is to be added to change the reaction to the one desired. 
A still further change in reaction is not infrequently to be observed 
after sterilization, the degree of acidity varying apparently with the com- 
position of the media and the degree and continuance of the heat. 
Manner of expressing the degree of reaction of culture media : 
Since at the time the reaction is first determined culture media are 
more often acid than alkaline, it is proposed that acid media be desig- 
nated by the plus sign and alkaline media by the minus sign, and that the 
degree of acidity or alkalinity be noted in parts per hundred; thus a 
medium marked -f-1.5 would indicate that the medium was acid and that 
1.5 per cent, of” sodium hydroxid is required to make it neutral to 
1 
phenolphthalein, while—l.s would indicate that the medium was alkaline 
and that 1.5 percent, of” acid must be added to make it neutral to the 
1 
indicator. 
Limits of accuracy of the proposed method for the control of the reac- 
tion of media : 
The available data are as yet insufficient to warrant any conclusions 
upon this point. The limits of accuracy seem to vary with the ingredi- 
ents employed in preparing nutrient media, different samples of meat 
infusion, pepton, and gelatin appearing to react differently with the acids 
and alkalis and in a way which is not understood. 
This method, nevertheless, when carefully carried out, and when the 
media before titration are thoroughly mixed and are of the prescribed vol- 
ume, gives fairly uniform results. 
Standard reaction of media (provisional) : 
Experience seems to vary somewhat as to the optimum degree of reac- 
tion which shall be uniformly adopted in the preparation of standard 
culture media. To what extent this is due to variation in natural condi- 
tions as compared with variations of laboratory procedure, it seems 
impossible to state. Somewhat different degrees of reaction for optimum 
growth are required, not only in or upon the media of different composi- 
tion and by bacteria of different species, but also by bacteria of the same 
species when in different stages of vitality. REPORT OF COMMITTEE. 
23 
The bulk of available evidence from both Europe and America points 
to a reaction of -j-i-5 as the optimum degree of reaction for bacterial devel- 
opment in inoculated culture media; and while this experience is at 
variance with that in several of our own laboratories, it has been deemed 
wisest to adopt -{-1.5 as the provisional standard reaction of media, but 
with the recommendation that the optimum growth reaction be always 
recorded in species descriptions. 
The preservation of culture media in stock: 
From what has been said, it is evident that species differentiation can 
be best determined upon media made in a single batch, and it is therefore 
advisable to preserve a special set of media for this purpose. Such stock, 
however, is useful only when properly preserved, and to this end contami- 
nation and drying must be particularly guarded against. 
The following methods of preserving media are suggested: 
For preserving media in bulk the method employed at the Pasteur Insti- 
tute in Paris is most efficient. Bulbs of moderately thick glass, with a 
capacity of from to 2 liters, and provided with a long, narrow neck, 
must be secured. 
The neck of the flask is first drawn out in the flame of a blow-pipe, two 
or three cm. above the bulb; the flask is then filled up to the neck with 
the desired medium; the opening is plugged with cotton and sterilized in 
an autoclave. After sterilization the flask is hermetically sealed in a 
blow-pipe flame at the point of constriction. 
Other very efficient and perhaps more practical methods are, after ster- 
ilization of the media in ordinary flasks, to cut off the cotton plug with a 
hot scissors, singe the free end of the plug in a flame, and cover the top 
of the tube or flask with thin sterilized tin foil1 which may be held in 
place by a small rubber band, or sealed with paraffin ; or, seal with par- 
affin either by dipping the engaged end of the cotton plug in paraffin 
previously sterilized at 150° C., and replacing it while hot in the tube or 
flask, or cover the free end of the cotton plug with paraffin which melts at 
a temperature not lower than 40° C. 
Stored media should always be kept in a dark place, and preferably at 
a low temperature. 
As the exact methods employed by different workers in bacteriology in 
preparing culture media used for species differentiation, are so seldom 
described, and as the majority of published articles dealing with descrip- 
tions of new species make little or no mention of the mode of preparation 
of the commonest, and therefore the most important, culture media, the 
Committee have deemed it necessary to draw particular attention to this 
fact and to devise a standard procedure to be followed in the preparation 
The preparation and use of artificial culture media in particular; 
ITin foil may be sterilized by storing sheets of the desired size in 0.5 per cent, solution of car- 
bolic acid. These should be rinsed in sterilized water, or passed quickly through a flame immedi- 
ately before use. 24 
REPORT OF COMMITTEE. 
of such media as are here described, when used for species description and 
differentiation. 
i. Nutrient broth. 
One part of finely chopped, fresh, lean meat is macerated in two parts 
of distilled water in an ice-chest for from 18 to 24 hours, stirring the 
mixture occasionally. The infusion is strained while cold through a fine 
cloth, and to the clear filtrate 1 per cent, of pepton and 0.5 of sodium 
chlorid are added.1 The mixture is then heated over a water-bath until 
all the added ingredients are dissolved, when by the titration method (see 
p. 75) a sufficient amount of normal solution of sodium hydroxid is added 
to make the whole solution feebly alkaline (practically neutral) to phe- 
nolphthalein. 
The medium is then heated over a water-bath for 30 minutes and boiled 
for five minutes over a free flame. While still hot it is filtered either 
through filter paper, or through a fine cloth and absorbent cotton, and to 
• n 
the filtrate sufficient —. hydric chlorid is added, the amount being de- 
termined by the titration method, to give the medium the desired reaction 
(-J- 1.5). If the medium is perfectly clear it is now distributed in tubes 
and flasks and sterilized (see p. 76). 
Should the medium prove not to be clear after filtration, it must be 
cleared. This is best done by mixing with each 1 liter of the medium, 
cooled to 50° 6o° C, the whites of one or two eggs stirred in a little of 
the medium, and boiling vigorously. The hot fluid is then again filtered 
and when clear is distributed and sterilized.2 
The use of nutrient broth for species differentiation : 
For the purpose of obtaining a more uniform description of the charac- 
ters developed by bacterial growth in nutrient broth in test tubes, attention 
must be given to the liquid itself, and the appearances on its surface and 
at the bottom of the tube.3 
The special appearances to which attention is drawn are given in some 
detail in the chart or analysis table accompanying this report, and the effect 
upon the cultures of the reaction and composition of the medium, and the 
temperature at which growth develops are referred to elsewhere. 
Preparation of fermentation broth : 
Fermentation broth is usually prepared in the same manner as nutri- 
ent broth except that 1 per cent, of sugar is added either at the time 
of adding the pepton or to the completed nutrient broth. It is impor- 
tant that the broth to which the sugar is to be added should be free 
1 Theobald Smith states that if pepton is added before a preliminary boiling most of it is lost for 
diphtheria toxin ; he therefore recommends that the clear filtrate obtained by straining be boiled and 
filtered and that the solid ingredients be added and dissolved thereafter. See Trans. Assoc, of 
American Physicians, 1896. On appearance of toxin in cultures of diphtheria. 
2 Nutrient broth may be made in considerable quantity and kept in stock, as it serves as a basis for 
other nutrient media. (See pp. 79-80.) 
3 See Th. Smith, “ The relative value of cultures in liquid and solid media in the diagnosis of bac- 
teria.” Botanical Gazette, Vol. XI, No. XI, and Medical News, 1886, Nov. 20, p. 57. REPORT OF COMMITTEE. 
25 
from sugar. Ordinary meat broth is likely to contain some muscle sugar.1 
The sugars recommended for the fermentation test are glucose, lactose, 
and saccharose, and these give the reaction most frequently in the order 
named. 
Sterilization of media containing sugars is best effected in streaming 
steam at ioo° C. on each of three consecutive days, for the reason that 
changes in the sugars may occur at the higher temperatures obtained in 
an autoclave. 
For the purposes of this test a tube of special construction is required2 
which is commonly known as the “ Fermentation tube.” 
This is essentially a tube 1.5 c. m. in diameter 
bent at an acute angle, closed at one end and pro- 
vided with a bulb at the other, which latter should 
be large enough to receive all the liquid contained 
in the closed branch, should gas in any considera- 
ble quantity form and collect there.3 
This tube also serves a most important end in 
giving information as to the aerobic and anaerobic 
growth of the species under examination, for the 
connecting tube, being constricted, serves to prevent 
to a great degree, the entrance of the oxygen of the 
air into the closed branch, and the free oxygen4 in 
the medium is driven out by heat during the process 
of sterilization ; from which it may be seen that 
growth in the bulb, to which access of oxygen is al- 
lowed, is aerobic, while growth in the closed branch, 
from which oxygen is excluded, is anaerobic. 
For method of cleansing the fermentation tube 
see p. 64. 
For the study of fermentation alone, ordinary 
tubes of 1.5 cm. calibre sealed at one end and bent 
to an acute angle will serve. They do not answer so well, however, for 
observations upon the anaerobic properties of bacteria, the connecting 
tube being too broad. 
1 The Journal of Experimental Medicine, Vol. 11, No. 5, p. 546, recommends the following simple 
method of preparing dextrose-free bouillon: “ Beef infusion, prepared either by extracting in the 
cold or at 60 degrees C.,is inoculated in the evening with a rich fluid culture of some acid-producing 
bacterium (I use temporarily B. coli) and placed in the thermostat. Early next morning the infu- 
sion, covered with a thin layer of broth, is boiled, filtered, pepton and salt added and the neutraliza- 
tion and. sterilization carried on as usual. Such bouillon when tested with B. coli in the fermentation 
tube will no longer permit any anaerobic growth. The closed branch remains clear, a sign that carbo- 
hydrates are absent.” Bouillon prepared in this way does not contain indol or nitrites. Spronck’s 
method of allowing beef to decompose for several days, is unreliable because bouillon made from it 
frequently contains relatively much sugar (Th. Smith). 
2 See Theobald Smith, Wilder Quarter Century Book, Ithaca, 1893, p. IS7. 
3 Made by Emil Greiner, 146 William street, New York City. 
* The bubbles which collect in the closed branch after each heating during sterilization, should be 
removed by tilting the tube while the medium is still very hot, otherwise they would be again 
absorbed by the fluid. 26 
REPORT OF COMMITTEE. 
2 and 3. Gelatin plates and tubes. 
Nutrient gelatin maybe made from nutrient broth in stock, by the addi- 
tion to the broth of 10 per cent, of sheet gelatin and neutralizing by the 
process already explained. (See p. 74.) 
When prepared in this way it is advisable as a routine procedure to add 
the whites of one or two eggs after the gelatin has been melted and the 
required alkali added, which when brought to a hard boil coagulates 
firmly, clears the medium, and allows of easy and rapid filtration. 
The other procedure recommended is similar to that for the preparation 
of nutrient broth (see p. 77) except that with the pepton and salt, 10 per 
cent, of sheet gelatin is added. 
The solid ingredients are heated in the meat infusion until the gelatin 
is melted and the pepton dissolved, and the medium is then made neutral, 
or faintly alkaline, by the addition of a sufficient amount of normal sodium 
hydroxid solution, the quantity being determined by titrating with 
phenolphthalein. The solution is then heated for 25 minutes in a steam- 
er water-bath, boiled for 5 minutes over a free flame, cleared by filtration 
and rendered of the standard reaction, or other desired reaction, by the 
addition of hydric chlorid in normal solution. The completed medium 
is then distributed in sterilized tubes and flasks and sterilized. 
Sterilization of gelatin can be satisfactorily effected in the autoclave by 
a single heating for 15 minutes at no° C, or even at 1150 C.1 
Cultural characters in and upon nutrient gelatin : 
A great objection to gelatin as a component of media for the differen- 
tiation of species of bacteria lies in its great variability in reaction, melt- 
ing point, etc.,2 nevertheless, nutrient gelatin is indispensible for bacteri- 
ological work. 
From what can be learned of the manufacture of gelatin, the produc- 
tion of an article having a definite melting point and a definite reaction 
presents peculiar difficulties, and therefore the Committee are unable to 
recommend any of the commercial sheet gelatins they have used as being 
always satisfactory. 
All that can be recommended at present is that an acid-free gelatin be 
employed which in a 10 per cent, solution remains solid at 240 C. after 
being subjected to the required sterilization. 
The characters to be recorded which develop either in the deep and 
surface colonies on gelatin plates or from “streaks” and “punctures” in 
tube cultures are referred to in the annexed analysis table. 
It is most important in studying growths upon nutrieht gelatin that the 
medium should be of uniform consistency throughout. To insure this 
and to prevent a greater density of the medium on the surface layers 
iSee Wyatt Johnston, Jour. Am. Pub. Health Assoc., Vol. 20, Oct. 1895, p. 4^2- 
2 For a discussion and record of experiments upon different forms of gelatin, see Sedgwick & 
Prescott, Jour. Am. Pub. Health Assoc., Vol. 20, Oct. 1895, P- 45°- REPORT OF COMMITTEE. 
27 
through drying, the medium in tubes should be preserved as suggested 
(see p. 76) or, where a slight surface drying has occurred, the medium 
may be melted, thoroughly mixed and again solidified immediately before 
using. 
Growths in tubes (punctures) present very different appearances in surface 
dried media from those observed in media freshly prepared ; and in plates, 
either rolled or poured, it may often be observed that colonies approaching 
the surface do not break through and spread upon the surface when the 
medium is in any way dried. To prevent the medium in Petri dishes from 
drying, several dishes may be placed in a pile and weighted, the cotton 
plug of a tube culture or an Esmarch roll tube after being singed in a flame 
may be sealed with paraffin, or covered with sterilized paper, rubber tis- 
sue, or tin foil and bound with a thread or rubber band. 
4 and 5. Agar plates and tubes. 
Nutrient agar, like nutrient gelatin, may be made from nutrient broth 
already prepared and kept in stock, by melting in the broth from 1 per 
cent, to 2 per cent, of thread agar and changing the reaction as may be 
necessary after titration. With regard to the reaction after adding and 
dissolving the agar, it may be found unchanged or may be too alkaline, 
agar being either neutral or faintly alkaline in reaction. Agar may be 
melted in the broth in the autoclave (1150 C. for 20 minutes) without loss 
of volume, but when for any reason the autoclave is not used the mixture 
should be heated over a free flame. As this boiling is necessarily some- 
what prolonged there will be a considerable loss in the volume of the 
medium and consequent concentration, which latter may be avoided by the 
previous addition of 200—300 c. c. of distilled water for each 1 liter 
and the subsequent adjustment of the bulk of the medium before neutral- 
izing by further evaporation if the bulk be too great or by addition of 
distilled water if the bulk be too small. 
Nutrient agar may also be prepared according to the procedure recom- 
mended for the preparation of nutrient broth (see p. 77), with the 
exception that from 1 per cent, to 2 per cent, of thread agar is added with 
the pepton and salt. The suggestions noted above in referring to the 
melting of agar in nutrient broth pertain with equal force here. The steps 
in the procedure are as follows: To the required bulk of clear meat 
infusion add 1 per cent, of pepton, y2 per cent, of sodium chlorid, and 
from 1 per cent, to 2 per cent, of thread agar; melt and dissolve the 
ingredients either in an autoclave or over a free flame, in which latter case 
additional water should be added to the mixture. The medium is then 
brought to the prescribed volume, and rendered neutral or faintly alkaline 
and steamed in a steam sterilizer for 30 minutes. The original bulk is 
then restored by the addition if necessary of hot distilled water, the solu- 
tion filtered through filter paper or through cloth and absorbent cotton, 
and the medium is then brought to the desired degree of reaction 28 
REPOR7' OF COMMITTEE. 
(standard reaction = -J- 1.5 per cent.) by the addition of the calculated 
amount of normal solution of hydric chloride, after titration with phenol- 
phthalein as an indicator. 
The clear medium1 is then distributed in sterilized flasks and tubes, and 
sterilized (see p. 72). 
When “glycerin agar ” is to be prepared, 6 per cent, by volume of pure, 
redistilled glycerin is added and thoroughly mixed with the hot nutrient 
agar just previous to its distribution in flasks and tubes, after which it is 
sterilized. 
Agar tubes should be freshly melted and inclined just previous to use 
and a sufficiency of condensation water should be present, unless for some 
special purpose dried out agar is desired. 
The technical difficulties in the way of successful “ plating ” in agar are 
considerable, and a recital of the experiences of the Committee in this 
particular may not be out of place. Plates may be made either by streak- 
ing the surface of an agar film cooled in a Petri dish, or by making 
dilutions in the usual way and pouring the infected medium into Petri 
dishes or by forming a film within the tube (Esmarch roll tube).2 Streak 
plates require no particular directions for their preparation. Pour plates 
and roll tubes are best made by boiling the agar in the tubes in a water- 
bath for 10 minutes and then transferring them to a bath of 420 C. for 10 
minutes. The inoculations and dilutions should then be made rapidly 
and the medium poured into Petri dishes, or rolled in a groove made in a 
piece of ice, or in a stream of cool water. Some practice is required for 
making serviceable agar roll tubes, but the method is practical. To pre- 
vent the film from slipping down, the tube should be placed in a slanting 
or horizontalp osition for 12-18 hours, to permit the upper thin edge of 
the medium to dry slightly and adhere to the glass ; after this time the 
tubes may be placed vertically. 
The spreading of growth in the condensation of water in agar plates 
may be largely prevented by allowing the excess of moisture to evaporate 
by canting the covers upon the dishes, while protected by a bell-jar, for 
10-15 minutes before putting the plates in the incubator. 
6. Potato as a culture medium. 
In spite of the differences in the reaction and composition of the potato 
as obtained in different varieties, or from various sources or even from the 
same source, and used at different periods of the year, this tuber is very 
valuable as a culture medium. 
Some species of bacteria, it is true, grow with great uniformity on potato 
from whatever source, but others grow so differently on potatoes of dif- 
ferent composition that they would not be recognized as cultures of the 
same organism. 
1 Should the medium prove not to be clear, it must be cleared with the white of egg (see p. 77). 
2Zeit.fur Hyg., Bd. I, 1886, p. 293. REPORT OF COMMITTEE. 
29 
To obtain satisfactory results two courses are open ; the first, and the 
one which is less generally useful, is to employ the old method of boiling 
and sterilizing the whole potato, then cut the tuber in half and inoculate 
one portion with the culture under examination and the other with a con- 
trol culture.1 This method may well be employed in identifying species 
resembling the “ B ” typhosus. 
The second course, which is generally the preferable one, is to correct 
the reaction of the potato, which will often be found to be acid. Much, 
if not all, of the acid may be removed from the prepared potato cubes2 by 
prolonged washing (12 to 18 hours) in cold running water. A quantity 
of potatoes obtained at one time from the same source and prepared 
as suggested may be stored in mass, as proposed by Plaut.3 These will 
generally be found to give quite uniform growth appearances. Should it 
be desired to change the reaction of the potato, this may be readily done 
by steaming the potato cubes in a measured quantity of distilled water for 
half an hour, testing the reaction of the water with phenolphthalein and 
adding the required amount of ~ sodium hydroxid, after which the boil- 
ing should be repeated for thirty minutes, and the potato cubes may be 
then put into tubes. 
Potatoes are preferably sterilized in streaming steam for 30 minutes on 
each of three consecutive days, or the autoclave may be used. 
Milk to be used as a culture medium should be as fresh as possible, and 
should be heated for 15 minutes in a steam sterilizer, in a cotton plugged 
flask, then placed in an ice-box over night to allow the cream to rise and 
the suspended matters to deposit. The milk is then siphoned off into a 
flask, free from the cream and deposit, and its reaction is tested to phenol- 
phthalein, by titration (seep. 74). Should the milk prove to be less than 
2 per cent, acid to this indicator it may be at once filled into sterilized 
cotton plugged test tubes holding about 20 c. c. each and sterilized for 
20 minutes on each of three (preferably four) consecutive days in stream- 
ing steam. Should the milk, however, prove to be more markedly acid, it 
should be rejected, or rendered 1.5 per cent, acid to phenolphthalein by 
7, Milk as a culture medium. 
the addition of sodium hydroxid; it may then be filled into tubes and 
1 
sterilized as above directed. A solution of litmus may be added to milk 
just previous to its distribution in tubes, in sufficient quantity to give the 
milk a pale blue color. The presence of litmus in the medium facilitates 
greatly the determination of its reaction. 
Professor Conn has pointed out that milk is difficult to sterilize, and 
1 Germano u. Giorgio Maurea. Vergleichende Untersuchungen iiber den Typhusbacillus und 
ahuliche Bacillen. Beitrage z. pathol. Anatomic, XII, (1893). S. 494. 
2 Roux. Annales Inst. Pasteur, T. 1888, p. 28. Bolton. Med. News, Vol. 1,1887, p. 318. 
•Cent. f. Bakt. & Parasit. Bd. Ill,pp. 100-127. 30 
REPORT OF COMMITTEE. 
recommends that after heating on each of four consecutive days the tubes 
be kept in an incubator at 37.5° C, for several days before they are used 
for culture purposes. This full procedure is recommended when practi- 
cable, although the Committee do not consider the incubation of the milk 
tubes before inoculation to be indispensable. One or more of the unsowed 
milk tubes made from the same sample should, in any event, be kept under 
the same conditions as the tubes containing cultures and carefully com- 
pared with them from time to time, as without such control errors are 
likely to occur. 
The changes in milk produced by bacterial growth render this medium 
one of the most valuable we have for species differentiation and identifica- 
tion, and the Committee are greatly indebted to Professor Conn for the 
tables he has compiled for recording these various changes, and which are 
embodied in the appended analysis table. 
The formation of peptons and albumoses in milk must be determined 
by chemical tests and of these, that known as the “biuret reaction” 
seems the most available. This reaction is obtained by rendering 
the fluid to be tested distinctly alkaline with an excess of sodium or 
potassium hydroxid in solution and then adding cautiously a very dilute 
solution of cupric sulphate when a fine, more or less deep rose color is pro- 
duced ; by adding an excess of cupric sulphate the rose color is changed 
to violet. This reaction is common to both peptons and albumoses, and 
when, as is probable in many cases, both these substances are present it 
is desirable that a differentiation between them should be made. 
The several albumoses maybe precipitated byreneutralizing the culture 
which has given the biuret reaction, making it faintly acid with a trace of 
acetic acid and adding ammonium sulphate to complete saturation ; the 
precipitated albumoses are then removed by filtration ; the peptons, how- 
ever, are still in solution and their presence in the filtrate may be deter- 
mined by again applying the biuret test, the intensity of the color reac- 
tion giving a rough indication of the proportional amount of albumoses 
removed by precipitation.1 
8. Blood serum 2 as a culture medium 
Blood of the beef or sheep, collected in large, wide mouthed jars 
(preferably straight sided museum jars of from gal. to 1 gal. capacity), 
previously sterilized, should be brought to rest in the laboratory as soon 
as practicable. Just after coagulation has commenced, usually within 
20 minutes, the adhesions betwen the coagulum and the jar are to be 
carefully broken up with a sterilized glass rod and the jars are then placed 
in an ice-chest for 24 hours to allow the serum to separate from the clot. 
At this time one or more inches of a straw colored to reddish serum will 
be found upon the surface which may be readily siphoned off into steri- 
1 See Gamgee’s Physiological Chemistry, Vol. 11, p. 124, et seq. or other treatises on this subject. 
Koch, Berlin Klin. Wochens, ISS2, No. 15. 
Koch, Mittheil, a. d. k. Gesundheitsamte, 1884, Bd. 11, s. 48. REPORT OF COMMITTEE. 
31 
lized cotton-plugged, Erlenmeyer flasks of about 200 c. c. capacity, or 
into “preserve jars” or other convenient vessels which may be tightly 
sealed. These vessels containing serum may be cotton plugged and 
returned to the refrigerator for 24-48 hours to allow of a further depo- 
sition of the coloring matters, or the serum may be preserved1 by adding 
to it a little more than 1 per cent, of chloroform and sealing the flasks' 
tightly; or the serum may be distributed in tubes and small flasks and 
sterilized by heat. 
Blood serum may be sterilized and remain fluid, or may be rendered 
solid by the degree of heat used in sterilizing. 
For the sterilization2 of fluid serum it is requisite that it be exposed to 
a temperature of from 62° C. to 66° C. for one hour on each of six con- 
secutive days. The best apparatus for obtaining and maintaining this 
temperature (about 65° C.) is a capacious and well-regulated incubator, 
into which the tubes and flasks containing serum are to be put each day 
and in which they are to be left for the prescribed time after having been 
warmed to the desired temperature. 
Serum may be solidified3 and still remain translucent at a temperature 
of 76° C., but when heated to a higher degree, a more definite coagulation 
takes place and the medium becomes opaque. Care must be taken in 
coagulating blood serum at the higher temperatures to run the tempera- 
ture up slowly and not to heat above go° C. until the serum has firmly 
coagulated, for, unless these precautions are taken, ebullition is likely to 
occur which will lead to the formation of bubbles and an unevenness of 
the surface upon which growth is to be obtained and studied. Serum 
may be solidified at the temperatures mentioned in an incubator, water 
oven, or hot air sterilizer, and when coagulated firmly (90° C.) the tubes 
and their contents may on the following day be sterilized in streaming 
steam at ioo° C. without danger of the subsequent formation of bub- 
bles. 
Serum preserved with chloroform may be freed from the deposit which 
forms by filtration and then being filled into sterilized culture (test) tubes, 
is to be sterilized by exactly the same methods as are employed in sterilizing 
fresh serum. The chloroform, being volatile, tends to disappear at ordi- 
nary temperatures, but is quickly and surely driven off at the temperatures 
used in sterilizing. 
Serum may be efficiently sterilized by passing it through a Pasteur or 
Berkefeld filter, under pressure. When so treated the fluid is very clear 
and light colored. 
A very important modification of blood serum as a culture medium for 
IM. Kirchner, Zeits f. Hyg. Bd. VIII, p. 465. 
2 Very practical points in the sterilization of blood serum may be found in Hueppe’s Bakterien 
forscbung, 1891, sth Auflage, p. 215, etc. 
8 Tubes containing serum to be solidified should always be placed obliquely, so that the medium 
will present an extended surface for growth. 32 
bacteria has been suggested by Loefffer.1 This consists of the addition 
of one part of a 1 per cent, glucose broth to three parts of the fluid se- 
rum. This mixture, known as “ Loeffler’s blood serum,” forms a very 
important addition to our culture media, and is to be sterilized in the 
same manner as blood serum, being serviceable either as a fluid or solid 
medium. 
REPORT OF COMMITTEE. 
Serum obtained from the serous cavities of the body, whether occurring 
with or without inflammation, may be used in the same manner as serum 
obtained directly from blood. These effusions, being obtained from the 
human body, are believed to be more efficient for the cultivation of 
certain pathogenic species than are sera from blood of the lower animals. 
By suitable precautions they may often be obtained absolutely sterile, thus 
avoiding the necessity of subsequent sterilization, and their manner of use 
corresponds exactly to that of serum. 
Some of the advantages to be obtained from the use of serum and 
exudates are that they offer a better medium for the growth of many bac- 
teria than do the media compounded from meat or vegetable extracts ; a 
number of species do not grow upon or in them at all, and further, certain 
species possess the power of liquefying coagulated serum, all of which 
facts may prove of value in the work of identification and differentiation. 
B. Biochemical features. 
1. Temperature Relations. 
The main points to be determined in studying the relations of the bac- 
teria to temperature are three : 
I. The extreme limits within which development occurs. 
11. The most favorable temperature for development. 
111. The thermal death point for the bacteria without spores and for the 
spores also when they are present. 
The determination of all these points is important and should, when 
possible, be made. For determining the extreme limits within which 
development may occur and the most favorable temperature for develop- 
ment, a number of incubators set at different temperatures are required, 
and as in a small laboratory such an amount of apparatus is not usually 
to be had, the Committee have deemed it wiser to require that only the 
comparative activity of growth at iB° to 220 C. and at 36° to 38° C. must 
be studied, this information to be coupled with such other facts as maybe 
learned during the study of the organism bearing on this point. 
The determination of the thermal death point is of such importance 
that it cannot be dispensed with. 
In determining the thermal death point the facts required to be known 
are 1, the time of exposure to heat; 2, the presence or absence of 
ISee Loeffler, Mittheil Kaiserlichen Gesunderheitsamte, Bd. 11, 1884, p. 461. REPORT OF COMMITTEE. 
33 
moisture ; 3, the presence or absence of spores ; 4, the age of the culture ; 
5, the composition and reaction of the medium in which its resistance is 
tested ; 6, the amount of the culture used for the tests, and 7, the character 
of the containing vessel. 
The temperature required to destroy the species under consideration is 
to be determined within 20 C., thus, if samples are exposed to tempera- 
tures of so°-520-540-560-580-6o° C., and it is found that development 
in a suitable medium occurs after exposure to 56° C., but not after 
exposure to 58° and 6o° C., the thermal death point is to be given as 58° C., 
although further study might show that it was somewhat less than this. 
It is necessary to limit the time of exposure in order that all organisms 
may be subjected to the same conditions, and to this end 10 minutes would 
seem from experience, to be the most suitable period to select. 
In determining the thermal death point cultures should always be moist. 
The experiments of Koch and Wolfhiigel have shown that in a desiccated 
condition and subjected to dry heat, bacteria show a much greater resist- 
ance to high temperatures than when moist. 
The presence or absence of spores may often be determined by the 
observation of stained specimens, but this method of study alone is some- 
times insufficient, as is evidenced by the fact that B. erythrosporus, and 
possibly also other species, contain spores which are obscured rather than 
made evident by any dye having a reddish tinge. For this determination 
specimens of the culture should be observed unstained, and stained with dyes 
of various hues as well as subjected to a temperature of Bo° C. for ten 
minutes. (See p. 69). 
In old cultures the power of resistance of many or all of the cells may 
be somewhat diminished. To avoid errors arising from this possible loss 
of resistance, it is recommended that the cultures to be tested be grown 
for forty-eight hours in standard nutrient broth, and when possible at 
36°-38° C. Three loopfuls1 of this culture, when thoroughly mixed by 
agitating, are to be transferred to tubes containing 10 c. c. of fresh broth 
for the purposes of the experiment. 
Using a measured quantity of standard nutrient broth to heat the 
culture in would seem to best serve for this determination, as its reaction 
has been pretty accurately determined and it is known to be a fluid which 
is favorable to the development of the culture. Placing the measured 
quantity of the culture in a known quantity of the broth defines the degree 
of dilution, and after exposure to a given degree of heat for 10 minutes, 
no transference to other culture media is required, but the tube, just as it 
1 Uniformity in the size of the loop employed is of some importance, and may be obtained by fol- 
lowing the directions here given; Take a piece of No. 27 platinum wire 2yz times the length desired 
for the loop and bend it through the middle over a bit of No. 10 wire. The ends are now twisted 
throughout their full length and the “ loop ” thus formed is fastened into a glass rod to serve as a 
handle. In measuring out a loopful of culture it is important to observe that the loop holds all the 
fluid it can ; that is, that it forms a biconvex body and is not simply a film covering the space within 
the loop. 34 
REPORT OF COMMITTEE. 
has been experimented with, may be placed under conditions favorable 
for the development of any of the organisms which may have survived. 
The culture (test) tubes used in these determinations should be of thin 
glass and uniform in size, approximately 16 mm. in diameter. 
A number of accurate procedures have been used to define the thermal 
death point of bacteria,1 but that which from its simplicity and the em- 
ployment only of ordinary laboratory apparatus particularly recommends 
itself, is as follows : 
Heat a capacious water-bath to the desired temperature, and place in 
the water in immediate contact with the thermometers, the several tubes 
containing xo c. c. of standard nutrient broth. After 15 minutes’ ex- 
posure to this temperature, i. e., when the broth in the tubes has attained 
the temperature of the bath as indicated by the thermometers, the broth 
is to be inoculated with three loopfuls of the culture to be experimented 
with, by simply removing the cotton plugs, not removing the tubes from 
the bath,2 and after exposure to such temperature for 10 minutes the tubes 
are removed and placed at once in a vessel of cold (ice) water to cool them 
rapidly and prevent further action of the heat upon the bacteria after the 
expiration of the specified time of exposure. These tubes when cooled 
are ready for incubation, and should be placed at a temperature favorable 
for the development of the species experimented with ; and to allow for the 
recovery of such cells as have been injured but not killed by exposure to 
these temperatures, all incubated tubes should be kept under observation 
for not less than seven days. 
2. Relation to free oxygen. 
Although the study of obligatory anaerobes is steadily assuming a 
greater importance, the Committee feel that the subject of anaerobic 
culture cannot properly be fully considered here; yet, as the relation of 
growth to free oxygen divides bacterial species into two great classes with 
perhaps more sharpness than any other of the distinguishing tests, it is 
important that this test should always be applied in the determination of 
characters for species differentiation and identification. 
In plate cultures, the study of growth under a “ mica plate ” as sug- 
gested by Koch may be recommended, while for tube cultures the more 
intricate methods of study are to some extent supplanted by carefully 
observing growth in the “fermentation tube” (see p. 78). Growth indi- 
cated by cloudiness in the bulb only, is to be found among obligatory 
aerobes ; in the closed branch only, among obligatory anaerobes, while 
growth occurring in both arms of the tubes indicates the faculty of 
growth either in the presence or absence of oxygen (facultative anae- 
robes). 
1 Sternberg. Proceedings of Bacteriological Congress, Journal ot the American Public Health 
Association, etc. 
2lt is important that the culture should be placed in the broth and not left upon the side of the 
tube where it may dry. The culture may be mixed wiih the broth by slightly agitating the tubes. REPORT OF COMMITTEE. 
35 
3. Relation of growth to acidity and alkalinity of media. 
Numerous investigations have shown that varying relations exist 
between the reaction of media and the development of different species 
of bacteria, but owing to the lack of uniform and accurate methods it is 
difficult to compare many of the results obtained by different observers 
one with the other. 
Nevertheless, while it has been shown that most species grow best in 
nutrient media rendered faintly alkaline to litmus, a sufficient number of 
exceptions to the rule have been found, to prove that a study of the rela- 
tion of growth to the reaction of the medium is an important auxiliary in 
species differentiation. 
In the vast majority of descriptions, no mention whatever is made of 
this matter, and probably no attention has been given to it. For this 
study all that is necessary is to add to tubes containing equal quantities 
of any of the standard nutrient media a calculated amount of standard 
solution of hydric chlorid or of sodium hydroxid, to obtain the desired 
reaction. 
The evidence is still too meager to warrant the recommendation of the 
uniform use of any one kind of medium for this purpose, but in view of 
the valuable characteristics that may be obtained it is recommended that 
a record be made of cultures upon at least one medium reacting -)- 3 per 
cent., -j- 1.5 per cent., neutral, and 1.5 per cent, to phenolphthalein 
(see p. 75). 
4. Action upon gelatin and agar. 
The liquefaction of gelatin, which often produces such characteristic 
appearances in plate and tube cultures, seems to be brought about by the 
action of some enzyme or ferment produced by the bacteria. 
The nature of the ferment is perhaps not as important to the bacteri- 
ologist as a proper interpretation of the effects produced, and it may 
seem not inappropriate here to give a few points indicating the informa- 
tion which may be obtained from an intelligent examination of the growths 
appearing on plates and in tubes. 
Deep colonies in gelatin : 
Colonies developed from a single bacterium entirely surrounded by the 
medium appear at first as minute spots, which increase gradually in size 
and have a high index of refraction. 
At this time they are usually circular (spherical) and sharply defined, 
and refract light differently from the medium and frequently they appear 
to have a definite texture, either coarsely or finely granular. In plates 
crowded with colonies an arrest of growth may be observed and for this 
reason only colonies which are isolated are to be used for the study of 
development. 
The spread of colonies occurs as might be supposed in the direction of 
least resistance, so that if growth be equally vigorous throughout, deep 
colonies in solid media should be spherical. Other forms of colonies are REPORT OF COMMITTEE. 
often observed, however, due to greater activity of certain parts of the 
growth, perhaps to different consistence in parts of the medium, or to the 
development of minute gas bubbles which split the medium, and permit 
the spread of the growth along the surfaces of the fissure. 
If the growth be near the surface and break through upon it, the appear- 
ances presented are often quite characteristic. 
Surface colonies on gelatin : 
Colonies wholly upon the surface present at times certain differences 
from those growing up from the deeper layers of medium, especially in 
the liquefying and chromogenic species. Color is usually produced only 
in the presence of oxygen, but may be developed only in the absence of 
oxygen as in the case of the Spirillum rubrum. 
Liquefaction of the medium often gives rise to appearances which 
viewed under low powers of the microscope, are very characteristic ; and 
the edge and texture of such growths are to be particularly observed. 
Colonies developed from the under surface of the gelatin, between the 
medium and the plate or dish, are usually in the case of aerobic species 
quite pale and thin though they may be spread over a considerable area. 
Facultative anaerobes do not show the same characteristic appearance. 
Deep colonies in a nutrient agar plate seldom show as marked or char- 
acteristic appearances as those in gelatin. 
This medium never undergoes the change in consistence that occurs in 
gelatin, and is much firmer, thus preventing a characteristic development. 
Deep colonies in agar : 
Surface colonies on agar: 
Upon the surface of agar, however, growth is much more apt to spread 
than it is upon gelatin on account of the condensation water which col- 
lects when the medium solidifies. A moderate amount of moisture allows 
of a natural spreading of the growth and occasionally of the development 
of very characteristic colonies. 
Growths obtained in punctures in 
nutrient gelatin are so various and in 
many ways so characteristic that they 
should be accurately recorded. 
As our media hold but a small quan- 
tity of free oxygen, aerobic growth oc- 
curs mostly on the surface, anaerobic 
growth entirely beneath the surface, and 
facultative anaerobes develop propor- 
tionately along the line of puncture, in 
the surface as well as in the deeper lay- 
ers of the medium. 
The growth of species which liquefy 
the gelatin is especially noteworthy, and 
Colonies in gelatin punctures ; REPORT OF COMMITTEE. 
37 
as types of this growth and liquefaction the following may be noted: 
viz., transverse liquefaction may be surface (Fig. i), or deep (Fig. 2), 
or may extend throughout the length of the puncture producing a “ stock- 
ing ”or “ glove finger ” shaped liquefaction (Fig. 3). Liquefaction com- 
mencing on the surface may extend downwards in a “funnel shape” 
(Fig. 4), or when advancing more rapidly just beneath the surface may 
give rise to a “ tulip ” or “ turnip ” shaped fluidification (Fig. 5), and as 
liquefaction occurs there may occur a rapid evaporation of the water of 
the liquefied medium and the formation of an air buble as shown in Fig, 6. 
5. Action upon proteids, milk and serum. 
[See Chart]. 
6. Action upon carbohydrates. 
Careful studies of the chemical action of bacteria upon the media in 
which they grow show that fermentation and gas production, in the presence 
of fermentable compounds, is perhaps the rule rather than the exception. 
The compounds which lend themselves most readily to gas production 
and fermentation are the various forms of carbohydrates, and of these 
dextrose, lactose, and saccharose are the most useful for experimentation. 
For the bacteriologist the simplest test of the occurrence of fermenta- 
tion is the presence of a gas ; and although gas may show itself in solid 
media as imprisoned bubbles or as clefts formed by the escaping gas, yet 
this gives no accurate information as to the amount or kind of gas formed 
by different species, under different conditions of growth and with differ- 
ent sugars. Here it is that the greatest value of the “ fermentation tube ” 
is shown, for the amount of gas formed can be readily recorded at stated 
times by a mark upon the closed branch or accurately measured with a 
millimeter scale ; while the kind of gas may be determined by compara- 
tively simple chemical tests. 
For purposes of comparison it is important that the fermentation tubes 
used for all these determinations should be uniform in size, the amount 
of gas being, under these conditions, expressed in mm.1 
Experience thus far shows that bacteria which are capable of produc- 
ing fermentation, usually act upon two of the three sugars mentioned, and 
rarely upon but one or upon all three ; and there seems to be no method 
except by the direct experiment to determine which two of the sugars will 
be acted upon by them. For this reason it is important that all species 
should be tested for their action upon each of these three carbohydrates.2 
A difficulty met with in the use of meat or meat extracts as the nutrient 
basis of fermentation broth is the presence of muscle sugar, a compound 
lln cases where the closed branches of fermentation tubes are uniform in calibre but vary in 
length, the amount of gas formed should be expressed in terms of the tube length, in percentages. 
2 For preparation of fermentation broths, see p. 77. 38 
REPORT OF COMMITTEE. 
which chemically and in its fermentative reactions to the bacteria, seems 
to be identical with dextrose. When experimenting with dextrose the 
presence of muscle sugar is of no account, but in experiments with lactose 
or saccharose it is most important that this compound should be elimi- 
nated. To this end all broths should be tested when first made by inocu- 
lating a few fermentation tubes containing samples of it with well-known 
active gas-forming species and when gas is absent muscle sugar can not 
be present in the proportion of more than .03 per cent, which, according 
to Dr. Theobald Smith, is an amount which need not be taken into con- 
sideration. 
The dextrose-free broth of Smith has already been described (p. 78). 
To this the sugar should be added. 
To inoculate any species, whether aerobic or anaerobic, in broth con- 
tained in a fermentation tube, it is necessary only to float a little of the 
culture in the fluid in the bulb. It is not necessary, nor is it desirable to 
carry the needle down to the connecting tube or into the closed branch,, 
lest a certain amount of oxygen should be carried into the closed branch 
and thus vitiate part of the results it is desirable to obtain. In case of 
gas formation, at regular intervals, preferably at the end of every twenty- 
four hours for several days, the level of the fluid in the closed branch is 
marked upon the tube or measured with a millimeter scale, and the 
results recorded in percentages of the length of the closed branch ; thus 
if 1 cm, of gas has formed in a closed branch 10 cm. in length 10 per 
cent, of gas is said to have formed. This is sufficiently accurate as only 
comparative values are considered. 
The gases which are most commonly and abundantly formed in the 
fermentation of sugars are carbonic acid and hydrogen, and valuable 
information is afforded by determining their relative proportion in the 
column of gas. 
To test the amount of C02 present a two per cent, solution of sodium 
hydroxid is employed. The bulb is completely filled with this solution, 
the thumb placed over the mouth of the bulb, and the fluid is run back 
and forth through the length of the tube six or eight times and the gas. 
remaining again returned to the upper end of the closed branch. The 
thumb is now removed and the amount of gas remaining is measured in 
millimeters,—the difference between this and the former measurement 
shows in m. m. the amount of C02 absorbed by the alkali. The remain- 
ing gas may now be transferred to the bulb and exploded in a flame. 
A practical method of recording the results of the gas tests will be best 
understood by referring to the chart at the end of this report. It is to 
be remembered that only the relative amount of gases formed is requisite 
for species determination, and that absolute determinations have not 
been sought for.1 
1 For methods of making analyses of gases formed during fermentation, reference must be made to 
text-books on gas analysis, e.g., Hempel’s Gas Analysis translated by Dennis, MacMillan & Co., 
1892. REPORT OF COMMITTEE. 
39 
7. Action upon nitrates. 
Among the principal changes produced in organic compounds by 
bacteria is their action upon nitrogenous substances. Of some diagnostic 
value is the determination of the capacity of certain bacteria to reduce 
nitrates to nitrites and ammonia. For this test a special medium, nitrate 
broth,1 is required. Nitrites and ammonia are commonly found in 
the air and are readily taken up in solution by watery fluids, so that in 
testing for the presence of these substances it is requisite that a control 
tube containing nitrate medium which has not been inoculated, but which 
has been kept under exactly the same conditions as the inoculated tube, 
should also be tested for the presence of these compounds. 
The inoculated tubes together with a sufficient number of uninoculated 
control tubes should be incubated at 20° C. for seven days and then 
submitted to the following tests for nitrites: 
Prepare two test solutions : 
I. Naphthylamin . , . . . . , o. i g.2 
Distilled water ....... 20.0 c. c. 
Boil until the naphthylamin is dissolved, then cool, filter, and add the 
clear filtrate to hydric acetate dilute (1 to 16 of water) 150 c. c. 
11. Sulphanilic acid ....... 0.5 
Hydric acetate dilute . . . . . . 150.0 c, c. 
These clear solutions are best kept separate in glass bottles with 
closely fitting glass stoppers, and mixed in equal parts just before use. 
About 3 c. c. of the culture or of the medium in the control tubes are 
poured into a perfectly clean test-tube, and to this is added gradually 
about 2c.c. of the test solutions previously mixed in equal parts. The 
development of a red color indicates the presence of nitrites, the amount 
of nitrites being in direct proportion to the intensity of the color, there- 
fore a pinkish or reddish color of about the same intensity developed in 
both the culture and uninoculated medium would indicate that a small 
amount of nitrites was present, either having been present in some of the 
ingredients used in making the medium or absorbed from the air; while 
a deeper shade of color in the culture would show the development of 
nitrites from the reduction of the nitrates in the medium. The formation 
of the color showing the presence of nitrites is sometimes delayed, but 
may be hastened by heat. 
When this test shows the presence of nitrites, one half of the remaining 
culture or control fluid is to be tested with Nessler’s solution3 for the 
presence of ammonia, for the purpose of determining the extent to which 
reduction has occurred. The presence of ammonia is shown by the 
immediate development of a yellow color or precipitate on the addition of 
a few drops of the test solution. 
1 Pepton, gm. i, potassium nitrate o-2, water c. c. 1000. 
2 See Centralblatt fiir Bakt & Parasit. Bd. XVI, 1894, p. 943. 
s For the preparation of Nessler’s solution reference must be made to some text-book on chem- 
istry. 40 
REPORT OF COMMITTEE. 
When these tests are positive our inquiry as to whether or not the 
nitrates have been reduced is answered, but when negative, one of two 
conditions may be present, either the nitrates may have remained 
unchanged or may have been reduced to free nitrogen. 
It is therefore necessary to determine whether nitrates are present or 
not, and for this purpose the phenolsulphonic acid and sodium hydroxid 
test is recommended,1 showing the presence of nitrates by the production 
of a yellow color. 
8. Production of indol. 
Among the products formed by bacteria growing in media containing 
proteids is indol,2 a substance which may be easily detected by tests, 
and which has largely been employed for species differentiation. 
The culture fluid usually recommended for this test is the pepton and 
salt broth (Dunham’s solution). It has, however, been shown by Theo- 
bald Smith (Journal of Experimental Medicine, Vol. ii, p. 543), and by 
Peckham (Ibid, vol. ii, p. 554) that the best results in determining the 
capacity of bacteria for the production of indol are not obtained by the 
use of Dunham’s solution. It is important that carbohydrates should 
not be present in the culture fluid, as these inhibit the production of 
indol. Theobald Smith recommends the dextrose-free bouillon whose 
composition has already been given (p. 78). This gives good results, as 
does also the medium recommended by Peckham, for the composition of 
which the reader is referred to the article already cited. The reagents 
used in making the test are 0.01 per cent, solution of sodium (or potas- 
sium) nitrite, and chemically pure sulphuric acid. The date at which 
the largest amount of indol is present in the culture varies in different 
cases. The proportion in which the reagents are to be added in order 
to obtain the most intense color varies also in different cases, so that 
exact rules cannot be laid down as to either of these points. For further 
details the reader may consult the papers of Smith and of Peckham. 
9. Productio7i of acid and alkali. 
The production of acid in culture media is of very wide occurrence 
among the bacteria and may be found under all circumstances, or be 
restricted to growth occurring only under certain conditions. 
As pointed out by Theobald Smith, much information on this point 
may be obtained from an examination of growths in sugar broths3 in fer- 
mentation tubes, it appearing that acid formation is largely wanting in 
those which grow only in the bulb, i. e., obligatory aerobes, while among 
1 Vide Fuller. Differentiation of the Bacillus of Typhoid Fever. Boston Medical and Surgical 
Journal, September 1, 1892. 
2 Indol is not infrequently present in pepton and errors from such a source must be guarded 
against. 
8 In preparing these sugar broths it is important that dextrose-free bouillon be made as already 
described (page 78) and the desired sugar (usually glucose or lactose or saccharose) be added to this 
—without this precaution serious errors may be made in interpreting the results. REPORT OF COMMITTEE. 
41 
those species which grow in both the bulb and the closed branch, i. e. 
facultative anaerobes, the fluid in the closed branch is usually, perhaps 
always acid while that in the bulb, i. e., exposed to the air, may prove to 
be either acid or alkaline. Anaerobes are usually active acid producers, 
especially in the presence of carbohydrates. 
For the differentiation of species, therefore, the production of acid 
quantitatively with reference to any given carbohydrate, and qualitatively 
with reference to different carbohydrates, becomes of considerable import- 
ance. Qualitative tests may be made from day to day by withdrawing a 
drop of the fluid from both the closed branch and bulb of the fermenta- 
tion tubes and testing their reaction to litmus paper, but this method 
is inexact, and to that extent unsatisfactory. A more satisfactory qual- 
itative study may be made by cultivating the species under consider- 
ation in standard solid media to which have been added one per cent, of 
glucose, saccharose, or lactose, and a sufficient quantity of litmus solu- 
tion to produce a blue tinge. If acid is produced as a result of the 
action of the bacteria upon the sugar the colonies assume a pale pink 
color and there may be also a reddening of the surrounding medium. 
Tests should be made with the three sugars mentioned. 
For testing quantitatively the acid or alkali formed a measured quantity 
(say 5 c. c.) of the culture fluid from both the closed branch and bulb 
should be titrated with a 1-20 normal sodium hydroxid solution or a 1-20 
normal hydric chlorid solution as may be required. A sample of the 
uninoculated broth must also be titrated to afford a basis for the compu 
tation of the changes induced. 
The development of color in a colony or in the medium immediately 
surrounding the growth is one of the most obvious characters of many 
species of bacteria and is important as a means of species differentiation. 
Pigment may develop differently according to the composition of the 
medium, its acidity or alkalinity, as the growth is or is not exposed to the 
action of oxygen, the temperature at which growth occurs, and the period 
of its development. 
10. Figment formation. 
To make this character an available one, therefore, in work upon 
species it is necessary : 
1. That the composition and reaction of all media be accurately 
recorded in or upon which color develops. 
2. That it be noted whether pigment is formed in the surface or deeper 
parts of the growth or medium, or in both. 
3. That the temperature be given to which the culture forming pig- 
ment has been exposed and the period at which the pigment forms, and, 
4, That such a description of the color produced be afforded as to 
render it recognizable to workers in other laboratories who may not have 
seen the culture in question. 42 
RE PORI OF COMMITTEE. 
To fulfill these requirements, it is suggested that the media here- 
recommended as standard be employed, and that the composition and 
reaction of other media which may be used be accurately recorded, all 
reactions being referred to the neutral point of phenolphthalein ; that 
both surface growths and deep punctures be employed; that growths at 
iB°-2o° C. and at 36°-38° C. be studied; and that the color produced be 
recorded according to some color scheme which will serve all workers as 
a basis for identification. Such a scheme of colors has been devised by 
Dr. Shuttleworth, and is here appended.1 For the proper use of these 
plates reference must be had to Shuttleworth’s original article “ On the 
Nomenclature of Colors for Bacteriologists,” published in the Journal of 
American Public Health Association, p. 403. 
Characters depending upon the disposition of light, etc.—(Shuttle- 
worth): 
Transparent. 
Vitreous ; transparent and colorless. 
Oleaginous ; transparent and yellow; olive to linseed oil colored. 
Resinous ; transparent and brown ; varnish or rosin colored. 
Translucent. 
Paraffinous ; translucent and white ; porcelaneous. 
Opalescent translucent, grayish-white by reflected light, smoky brown 
by transmitted light. 
Nacreous ; translucent; grayish white with pearly lustre. 
Sebaceous; translucent; yellowish or grayish white ; tallowy. 
Butyrous ; translucent and yellow. 
Ceraceous ; translucent and wax colored. 
Opaque. 
Cretaceous ; opaque and white; chalky. 
Dull; without lustre. 
Glossy; shining. 
Fluorescent. 
Iridescent. 
11. Development of odor. 
As no method of analyzing or recording odors has been devised or 
seems probable, accuracy of record of odors produced is impossible. The 
odors developed by many species of bacteria, however, are peculiarly char- 
acteristic, and although some defy description, quite a number may be 
more or less accurately likened to odors evolved by well-known organized 
material under various conditions, or with specific odors of sundry chemi- 
cal compounds. The fact that certain species develop odor in all media 
or only in media of certain composition, or fail to develop any odor at all, 
is important, and should be accurately ascertained and recorded. 
1 On account of the cost of reproduction this color scheme had to be omitted from the printed 
report. REPORT OF COMMITTEE. 
43 
c. Pathogenesis. 
It may properly be questioned whether the very obscure and variable 
reactions on the part of the living organism towards bacteria and their 
products ought strictly to be included in any scheme of exact tests for 
species differentiation. Again, it is a matter of common knowledge that 
of all features, the pathogenic properties of at least many forms of bacteria 
are those which are capable of the greatest amount of variation, and this 
fact also is against the employment of the test of inoculation for determin- 
ing the affinities of any bacteria under consideration. But, granting all 
this, it has further to be acknowledged that up to the present time a main 
object of bacteriologic study has been in the direction of determining the 
relationships of bacteria and their products towards man and the higher 
animals, or of studying the relationship of bacteria towards organized 
products. 
Hence, a study of the pathogenic properties of bacteria assumes a 
peculiar importance, and the Committee feel it necessary to recommend 
the adoption of the test of pathogenesis as a routine procedure in the 
determination of the characters of all so called species of bacteria. 
The necessary procedure is much simplified by the fact that while many 
forms grow at the temperature of the blood of warm-blooded animals, a 
large number of others will not grow at so high a temperature. The 
products of these latter species, however, may possess toxic properties 
and, as throwing light upon this point, inoculations of older cultures should 
be made in some animal. Therefore, 
i. Where a given form grows only at or below iB° to 20° C. inoculation 
should be made, into the dorsal lymph-sac of a frog, of about 1 per cent, 
of the body weight of the frog of a. liquid culture seven days old. 
2. Where a given species grows at 350 C. or upwards an inoculation 
should be made into the peritoneal cavity of the most susceptible (in gen- 
eral) of warm-blooded animals, i. e., the mouse, either the white or the 
ordinary house mouse; the amount to be inoculated should be about 1 
per cent, of the body weight of the mouse, of a 48-hour standard broth cul- 
ture, or a broth or watery suspensionl of one platinum loop (see p. 86) from 
solid cultures. When such intraperitoneal injection fails it is unlikely that 
other methods of inoculation will be successful in causing the death of 
the mouse. 
If the inoculations of the frog and the mouse both prove negative the 
Committee think it unnecessary to insist upon any further tests upon 
pathogenesis, as being requisite for work in species differentiation, as. 
strongly pronounced pathogenic properties if present would most likely 
have shown themselves from the previous inoculations. 
If, on the other hand, the mouse succumbs to the inoculation, there 
should be appended either to the original description or forming the sub- 
ject of a separate research, the results of inoculation of the species under 
1 One loopful in 5 c. c. of liquid. 44 
REPORT OF COMMITTEE. 
consideration into other animals, more especially into the rabbit and the 
guinea pig. The methods of inoculation are given in text-books on bac- 
teriology. The ordinary hypodermic syringe will answer for inoculations. 
It should be kept in a 2 per cent, solution of carbolic acid and thoroughly 
washed out five or six times with sterilized water, broth, or other indifferent 
fluid, just before use. 
It is not advisable to employ for subsequent experimentation animals 
which have survived inoculation, as but little is known of the influence 
which a previous inoculation may have upon the susceptibility of the ani. 
mal to the same or to other species of bacteria. 
After inoculation the animal should for some days be kept under observ- 
ation, all signs of disorder should be noted, and in the case of larger ani- 
mals the rectal temperature recorded at least twice in each twenty-four 
hours. When the animal has appeared to have survived the inoculation 
it should be kept under observation for not less than a month. 
If the animal dies, a most careful autopsy should be made as soon 
as possible after death; the site of subcutaneous inoculation should 
be opened up and cultures, always in duplicate, should be made from here 
as well as from the peritoneal and pleural cavities, the spleen, liver, kidney, 
lung, and heart’s blood. The autopsy should be made under the strictest 
antiseptic precautions and the surface of each viscus should be seared 
with a red-hot spatula or other metal before obtaining the material from 
which the cultures are to be made. Tissue from the site of the inocula- 
tion and from the viscera mentioned should be subjected to microscopic 
examination to determine histological changes or the presence of bacteria, 
and in every instance a number of so-called “ tissue smears ” on the cover- 
glass should be examined. The procedure for determining the kind and 
number of bacteria present is by the plate method, and although no single 
medium can be said to be sufficient for these determinations, the one 
which generally best answers the purpose is nutrient glycerin agar. It is 
important to distinguish between actual infection and intoxication result- 
ing from the injection of the products of bacterial growth along with the 
bacteria. This distinction is not always easily made but where large doses 
are required to produce symptoms or to cause death, there should be a strong 
suspicion of pure intoxication and especial attention should be given both 
to the distribution of the bacteria in the animal body and to evidences 
of multiplication of the bacteria in the system. 
While fully alive to the importance which attaches to the toxicity of the 
products of species, which are not directly infectious and pathogenic the 
Committee feel that they should not demand as necessary tests, the possi- 
ble toxic products derived from the bacteria or from the media in which 
they are grown, apart from the presence of the living bacteria. 
For a fuller discussion of Pathogenesis see Welch, “ What shall be the 
mode of procedure in determining the pathogenesis of bacteria found in 
water?” Proceedings Bacteriological Convention Loc. Cit. p. 502. REPORT OF COMMITTEE. 
45 
OPTIONAL TESTS OF GENERAL USEFULNESS. 
It is difficult to know how much or how little to say in reference to the 
tests which have been brought together under this heading. It must be 
fully understood that as the morphological characters of these minute 
organisms offer such an insufficiency of characters on which to base a dif- 
ferentiation of species that every biological character, whether positive or 
negative, is of aid in this regard, and it is far from the wish of the Com- 
mittee to limit the amount of careful study which may be applied to bac- 
terial forms; but on the other hand they believe that they can best per- 
form the work which they have been asked to do, by making clear to those 
engaged in the study and differentiation of species of bacteria the abso- 
lute need of recording the results of at least a certain minimum number 
of features in connection with each form. 
Among the tests classed as optional (see p. 61) there are some to which 
fuller attention may be drawn and which perhaps in the near future, when 
more fully understood, may be regarded as tests of primary importance. 
I. MORPHOLOGICAL, 
Experimentally it is not a difficult matter to vary the properties of certain 
species of bacteria to such an extent, that were we given the modified form 
without information as to the previous treatment it had been subjected to, 
it would be an easy matter to believe that we were dealing with a species 
different from the parent organism. How then are we to be assured that 
many of the closely allied forms which are encountered in the bacterial 
study of water, air, soil, etc., and in the bodies of diseased patients, are 
truly distinct species ? 
3. Permanence of morphological characters. 
It must be confessed that at the present time it is impossible to give 
anything like a complete answer to this question; nevertheless, in a 
certain number of cases it would seem that continued growth, under what 
may perhaps be termed more normal conditions, does lead forms which 
appeared to be distinct, to approximate more closely to each other and to 
a common type. 
This is especially noticeable in the so-called experimental varieties; 
forms which by modification of temperature or environment have departed 
widely from the original, tend when grown upon ordinary media and under 
ordinary laboratory conditions, to revert, sooner or later, to the character- 
istics of the original laboratory form. 
This being so, it would seem that important information may be gained 
as to the relationship of any form by successive growths upon the ordi- 
nary standard media for not less than a year, control and parallel growths 
of the form or forms to which it appears to be allied being carried on 
at the same time and on the same media; and it may further be recom- 
mended that all who describe new species be urged to publish a second 
description after a period of 12 months has elapsed, in the same journal 46 
REPORT OF COMMITTEE. 
in which their first communication appeared, this second description to 
state accurately how far the forms have become modified by continued 
growth on the standard media. It is important that a uniform method of 
treating the cultures during this period be observed and to this end the 
following is recommended : 
Obtain approximately the maximum growth on standard nutrient agar 
(see p. 80) at C. (or at iB°-2o° C.) and then place in a refriger- 
ator at about i2°-is° C. Resowings should be made every 4 or 6 weeks.1 
4. Photographic reproductions of isolated bacteria. 
Inasmuch as it is not always possible to procure the publication of pho- 
tographs, the Committee have not classed this as one of the requisites in 
describing species, yet, when possible, photographs of the isolated bac- 
teria, grown under definite conditions, should be published, care being 
taken that the exact magnification is given. In any event such photo- 
graphs should always be taken from which “ blue prints ” could be made. 
What is said here with regard to reproducing photographically the iso- 
lated bacteria is equally true with reference to photographing characteristic 
growths on plates (surface and deep colonies) and in tubes. It goes without 
saying that photographs are far more trustworthy records than drawings. 
11. PHYSIOLOGICAL, 
a. Cultural characteristics. 
3. Synthesised media. 
The variation in chemical composition of many of the ingredients 
employed in preparing nutrient media, more especially in the organic 
compounds, will necessarily lead to a diversity in description which may 
occasion confusion. 
Synthesised media, i. e., media prepared from compounds of exact 
chemical composition, have been largely experimented with, but thus far 
with unsatisfactory results. 
No such medium has been devised on which even the majority of 
bacteria will grow satisfactorily and on most of the synthesised media 
recommended there is a loss of characteristic features such as pigment 
formation, etc.2 
A practically useful synthesised medium is a great desideratum in the 
interest of scientific accuracy in differentiating species. 
b. Biochemical features. 
1. Temperature of growth. 
Among the necessary tests are included the activity of growth at only 
two temperature ranges and the determination of the thermal death point 
1 See Adami—“ How is variability in bacteria to be regarded ? ” Proceedings Bacteriological 
Convention, Loc. Cit. p. 415. 
2 See MacKenzie—“ What new methods can be suggested for the separation of bacteria into 
groups and for the identification of species ? ” Proc. Convent. Loc. Cit., p. 419. REPORT OF COMMITTEE. 
47 
{see p. 85) but for the accurate determination of temperature relationship 
a study should also be made of the minimum and maximum temperatures 
at which growth can occur and the temperature at which growth is most 
prolific, i. e., the optimum growth temperature. It may be stated that the 
apparatus absolutely required for these determinations is comparatively 
moderate, as much information may be gained by the use of a few water- 
baths, fitted with thermostats and a moderately good refrigerator. 
4. Chemistry of bacterial pigments. 
Very little has yet been accomplished in this branch of bacteriology. 
It would seem, however, from what has been learned that fuller studies of 
the reaction of the pigments produced and of their solubility, together 
with spectroscopic observations upon them, might afford valuable informa- 
tion as to the relation existing between various forms. 
c. Pathogenesis. 
The tests of pathogenesis which are included among the necessary tests 
the Committee consider far from complete, and would here briefly urge 
that these tests be conducted upon more than one animal of a given 
species, and upon animals of different species; that whenever inoculation 
leads to the production of definite lesions, a careful study be made of the 
local and general changes produced in the system ; and that where a form 
is found to be pathogenic, further researches be made as to the immunity 
producing properties of the organism and its products. 
Lastly, the researches of the past few years have shown an importance 
in determining and isolating the toxic substances, not only free from 
cultures of pathogenic, but also from those of non-pathogenic, species. 
A study of toxic substances and their properties would seem to promise 
information of so much value not only with regard to the nature of disease, 
but also as to the evolution of pathogenic bacteria, that it is strongly 
recommended that account be taken and studies be made of the toxic 
substances of bacterial origin. 
The studies in recent years of Pfeiffer, Gruber, Widal, and others upon 
the specific properties of the blood serum and other humors of animals 
and human beings during the period of infection or that of acquired im- 
munity, have demonstrated that these properties may be of great value in 
the diagnosis of disease and in the differentiation of bacterial species. 
The Committee would urge that use be made of these properties of 
immune sera for the differentiation of bacterial species, wherever they are 
available, but in the present state of the subjects and within the limits of 
this report it is not deemed necessary to give directions as to the requisite 
procedures or to discuss their precise value. Information upon these 
points is readily accessible in the special journals and works relating to 
bacteriology.1 
1 Consult especially Pfeiffer, Zeitschrift fiir Hygiene u. Infections Krankheiten, Bd. XVII, XVIII, 
XIX, XX, Pfeiffer and Kolle, Ibid, Bd. XXI. Gruber, Archiv fiir Hygiene, Bd. XX. Miinchener 
Med Wochenschrift, 1896, p. 206, and 1897, Apr. 27 and May 4. Widal and Sicard, Annales de P 
Institut Pasteur, 1897, and FliiggeDie Mikroognaismen, 3te Auflage, Leipzig, 1896. STANDARD CHART FOR BACTERIAL DIAGNOSIS. 
Compiled by T. M. CHEESMAN, M. D., 
Instructor in Bacteriology, Columbia University, and Adopted by a Committee of American Bacteriologists, October, 1897 
No Date 
Source Habitat 
Morphological examination of agar culture grown days at iB°-20°C.; ditto grown days at 
“ “ gelatin culture grown days at iB°-20°C. 
“ “ broth “ “ “ ; ditto grown days at 36°-38°C. 
Micrococcus, single, irregularly grouped; diplococcus; streptococcus; tetracoccus ; sarcina. 
Bacillus, single ; in chains ; in filaments. 
Spirillum, comma; spiral. 
Size, in terms of blood cell j ; breadth a. Average length p, Extreme lengths from pto p_ 
Stain, with standard watery dyes easily or with difficulty, uniformly or irregularly. 
by Gram’s method decolorizes or does not decolorize. 
Capsule, none observed or may be easily observed or may be demonstrated and stained by Ziel’s, Gram’s, or Welch’s method. 
conditions under which it is most likely to be present are Broad or narrow. 
demonstrable or not demonstrable in serum, milk, or surface agar cultures. 
Spores, none observed or do form within .... hours at .... °C. on standard ; are located centrally, or on the 
end; do or do not give rise to enlargement of bacterial cell, causing Clostridium or drum-stick forms; germinate within .... hours 
at .... °C. Stain by Moeller’s or Abbott’s method; are killed at ioo°C. within minutes; withstand ioo°C. for minutes. 
Germination inhibited by this latter exposure for days. Germination observed or not observed. 
Vacuoles, none observed or are observed on standard 
Crystals, none observed or are observed on standard ; dissolved by chloroform or ether. 
Motility, not observed (or brownian movement) or is observed; sluggish or active, rotary or direct, most pronounced in cultures obtained 
from grown for .... hours at ... .°C. Flagella stain by Loeffler’s, Van Emengemi’s, or Bunge’s 
method; originate from one or both ends, or irregularly from the periphery; single or in tufts (monotrichal, lophotrichal, amphitri- 
chal, or peritrichal). 
Pleomorphism, not observed or is observed on standard at °C., or on media reacting per cent, acid or per 
cent, alkaline when grown at °C. for days. 
Growth at iB°-20°C. is more abundant or less abundant than that at 36°-38°C. 
Thermal death point °C; time of exposure minutes. Permanence of morphological characters 
Optimum temperature °C. 
Optimum reaction of media from per cent, to per cent Growth limits, maximum °C.; minimum °C. 
MORPHOLOGY) ETC. 
at °C., or on media reacting per cent, acid or per Gelatin plate. 
5 
Deep Colonies. 
Surface Colonies. 
Sketch of Germ and Colony. 
Size. 
Shape. 
£ 
Margin. 
Texture. 
Color. 
C 
Growth under mica 
plate present or 
ai 
absent. 
& 
Agar plate. 
B 
3 
Size. 
<L> 
I Shape. 
£ 
Gelatin and A 
gar. 
Margin. 
M-l 
O 
Texture. 
Color. 
o 
Tube Cultures. 
Growth under mica 
CJ 
plate. 
<U 
cj 
CJ 
Gelatin tube. 
O 
i 
\ 
1 
I 
I 
i 
I 
V/ 
o 
Puncture. 
—- — — 
Depth. 
d 
Form. 
•£ 
Consistence. 
& 
% 
Surface growth. 
u 
bG 
bf) 
Deep growth. 
-b 
cn 
cn 
Streak. 
Size and shape. 
£ 
O 
JZ 
b 
Margin. 
Surface relief. 
\ / 
Light transmission. 
(J 
\ J 
Light reflection. 
<D 
Color. 
& 
Lustre. 
Change in medium. 
Color. 
Consistence. 
. 
CJ 
Odor. 
O 
/' 
o 
/ \ 
' \ 
i 
/ \ 
Agar tube. 
d 
/ \ 
| i 
Streak. 
£ 
Extent and shape. 
% 
& 
\ / 
Margin. 
(D 
O 
!— 
o 
Surface relief. 
£ 
bio 
\ 1 
bO 
Light transmission. 
b 
i- 
\ y' 
CO 
j- 
\ / 
Light reflection. 
b 
o 
Color. 
Lustre. 
Change in medium. 
& 
\ j 
Color. 
Transparency. 
BIOLOGY Nutrient broth in test tube. 
Remains clear. 
Body of Liquid. 
Remains clear after days at °C. 
Growth appears near surface in hours at °C. 
“ forms deposit in hours at °C. 
deposit is granular or flocculent and disseminated through the fluid. 
Surface of Liquid. 
Pellicle, does or does not form. 
thickness in hours at °C. 
color in hours at °C. 
consistence in hours at °C- 
structure when fully formed after days. 
Deposit, forms in hours at °C. 
amount in days at °C. 
color in days at °C. 
character, whether compact or flocculent. 
on agitating appears granular, flaky, or viscid. V. 
Odor does or does not develop. 
Reaction of culture at end of i day days days ... , S 
days days ,21 days S 
w 
Becomes 
clouded. 
Is opalescent or turbid in hours at °C. 
Clears on standing after hours. 
On shaking forms masses which may be fine, granular, or flocculent. 
Becomes colored. 
In hours at °C. 
Different color at °C. 
Color uniform throughout. 
“ differs in surface and deeper layers. 
“ modified by shaking. 
Colors in medium reacting per cent acid. 
“ “ “ per cent, alkaline. 
No visible change in days at °C. Reaction in days. Does or does not 
curdle on boiling. 
Does not curdle in days at i6°-i8°C. 
Dues curdle in days at i6°-[8°C. 
“ “ “ 36°-38°C. 
“ “ 36°-38°C. 
Reaction 
Reaction when curd forms per cent 
Does or does not curdle on boiling. 
in 14 days per cent , in 21 days per cent 
Color is or is not developed.... 
Color is or is not developed. 
Gas is or is not developed. 
Curd, hard or soft, in one mass or in small fragments. 
Digestion, becomes gradually transparent without forming curds. 
fragmented by gas bubbles or smooth. 
2* 
Transoarency complete or incomplete in days at °C. 
changed or unchanged on boiling. 
s 
Reaction after change has occurred. 
Whey, separated from curd or not. 
Effect of boiling after change has occurred. 
amount, transparent or turbid. 
Odor is or is not developed. 
Digestion. Curd remains unchanged or is dissolved. 
Solution is rapid or slow. 
complete or incomplete in days at °C. 
Reacts per cent is clear or cloudy. 
watery or viscid. 
Odor is or is not developed. 
Reaction at end of process, in days at i6°-r8°C. 
“ 36°-38°C. 
BIOLOGY.  
Amount of gas in per cent, formed in 
i day. 2 days. 3 days. 4 days. .... days. 
. 
Reaction to litmus paper of 
fluid in bulb. 
CO2 per cent. 
H per cent. 
r\ 
At i6°-i8°C. 
\ \ 
G. 
\ \ 
<u 
L. 
\ \ s 
s. 
\ \ ( 
c 
.2 
At 36°-38°C. 
\ \ V 
c 
G. 
\ \ 1 
<u 
£ 
L. 
\ \ / 
S. 
\ \ / 
«+H 
.H 
G=Glucose. L=Lactose. S = Saccharose. 
Vh 
bO 
3 
c/j 
In 
In 
bulb, pellicle forms or does not form. 
remains clear, is granular or flocculent. 
becomes clouded, fluid is opalescent or turbid. 
clears on standing in hours. 
color develops or does not develop, 
on surface. 
uniformly throughout, 
in deeper layers only. 
connecting tube, growth forms deposit, is granular or flocculent. 
color does or does not develop. 
In closed branch, growth does or does not develop, 
remains clear, growth is granular or flocculent. 
becomes clouded, in upper or lower layers, or uniformly throughout. 
clears on standing in hours. 
color does or does not develop. 
ch 
/ \ 
c 
:tose litmus, agar o 
gelatin. 
| 
O 
15 
s 
G 
C 
.2 
Color changes to in hours at °C. 
“ “ °C. 
“ “ “ days at °C. 
Decolorized in hours or days in upper or lower layers. 
in da>s throughout at °C. 
Gas does or does not develop. 
Growth more or less abundant than 
Color does or does not develop. 
on nutrient gelatin or agar. 
( \ 
\ , 
\ ) 
media. Blood 
um. Nitrate broth. 
>ton and salt solutic 
rt 
u 
a 
<L) 
& 
V X 
37 
Ph 
0 u 
C (L) 
Potato. 
Reaction to ph 
phthalein .... ] 
cent. acid. 
Growth occurs or does not occur. 
apparent or not apparent, 
restricted or spreading, 
shiny or dull, 
with or without lustre. 
Gas does or does not develop. 
Color does or does not develop in growth. 
in medium. 
Blood serum. Liquid. Character of growth. 
Solidified. “ “ . Is or is not liquefied. 
Nitrate broth. Nitrate is or is not reduced. Nitrites, ammonia, or free nitrogen present. 
Dextrose-free bouillon. Indol is or is not produced. 
It I O li () (» \ . Pigment, is or is not developed ; in presence or absence of oxygen. 
develops in medium, in days at °C., medium reacting per cent 
Color No Changes to No by addition of acid or alkali. 
Soluble in 
Spectrum analysis shows 
Production of acids or alkalis. Carbohydrates absent. 
“ present. 
Relation to free oxygen. Obligatory aerobe. 
Facultative anaerobe. 
Obligatory anaerobe. 
Relation of growth to acidity or alkalinity. 
Per cent, alkali 4. 
3- 
i-S 
0. 
Per cent, acid 0.5 
Pathogenesis 
Cfi 
*(/} 
C 
<D 
W) 
O 
rC 
* 
£ P-( 
fcO 
. 
X c 
0 .2 
Synthesised media. 
Pigment. Relation tc 
Alkali or acid product 
Synthesised media. 
BIOLOGY