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MANUAL
OF METHODS FOR
PURE CULTURE STUDY OF
BACTERIA
J
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^^ ociETY OF American Bacteriologists
65
.S6
1946
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This Manual is published in loose-leaf form so
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1 1 A
MANUAL
OF METHODS FOR
PURE CULTURE STUDY OF
BACTERIA
/'■
EDITED BY
THE
COMMITTEE ON BACTERIOLOGICAL TECHNIC
OF THE
SOCIETY OF AMERICAN BACTERIOLOGISTS
^
GENEVA, N. Y.
PUBLISHED BY THE BIOTECH PUBLICATIONS
The Society of American Bacteriologists disclaims any responsibility for the views expressed in
this Manual. The methods given have not been formally approved by the Society
and are in no sense official or standard.
0^
. .5 (,
^ Copyright, 1923. 1926. 1930, 1936, 1946
^ X Societv of American Bacteriologists
^ Made in the United States of America
\
\
TABLE OF CONTENTS*
(May 1949)
Leaflet I. Introductory
Purpose of the Manual
Historical
I44-2 Use of the Manual
I44-5 Glossary
-9
-13
Leaflet IL Preparation of Media
Sterilization "44-3
Cultivation and storage media 1I44-4
General differential media 1I44-6
Media for special groups of
aerobes 1I44-9
Media for anaerobic bacteria ii.j-14
Leaflet III The Study of Obligately Anaerobic Bacteria
(Introduction)
Biological methods for oxygen
removal
Chemical methods for oxygen
removal
Oxygen removal by combustion
using Laidlaw principle
IIl4,-3
iii,,-10
Plating system using strongly
reducing medium
Preliminary microscopic exami-
nation
Microscopic examination of
pure cultures
Cultivation technics
11I43-I2
1114,-14
11I43-I4
IIl4,-16
Other methods of value
iii4,-20
Leaflet IV. Staining Methods
General principles IV46-3
General bacterial stains
Recommended procedures IV46-5
Alternate procedures IV46-6
Negative staining of bacteria IV46-7
The Gram stain IV46-8
Acid-fast staining IV46-IO
Spore staining
Recommended procedures IV46-II
Alternate procedures IV46-I3
Staining the diphtheria organism
Recommended procedures
Alternate procedure
Flagella staining
Capsule stains
Stains for Spirochaetes
Stain for Rickettsiae
Dye solubilities
IV46-I3
IV46-I4
IV46-I5
IV46-I8
IV46-2O
IV46-2O
IV46-2I
Leaflet V. Routine Tests for the Descriptive Chart
Introduction
V47-3
The Descriptive Chart
V47-3
Determining optimum
conditions
for growth
V47-4
Incubation
V47-5
Variation
V47-6
Study of morphology
V47-6
Relation to free oxygen
V47-9
Action on nitrates
V47-9
Chromogenesis
V47-II
Indole production
V47-II
The production of hydrogen
sulfide
V47-I3
Liquefaction of gelatin
V4rl4
Cleavage of sugars, alcohols, and
glucosides
V4,-15
Hydrolysis of starch
V4,-18
The methyl red and
Voges-Proskauer tests
V47-20
Acid production in milk
V4rS^l
Rennet production
V47-22
*In these page numbers, the Roman numerals refer to the leaflet, the small inferior
numerals to the year of the edition, and the large arable numerals to the page of the leaflet.
Leaflet VI. Further Biochemical Methods
Introduction vik-2
Relation to free oxygen xj^-i
Cleavage of carbohydrates, alco-
hols, and glucosides vi42-5
Cleavage of proteins and their
products VI4..-13
Action on inorganic nitrogenous
compounds vi4i>-14
Action on erythrocytes VI42-I6
Leaflet VII. The Study of Pathogenic Aerobes. — Determination of
Pathogenicity
Introduction vii48-3
General VII48-I
Use of laboratory animals vii48-5
Methods of injection vii48-5
Recovery of organisms
from blood culture VII48-8
Autopsy VII4 8-9
Factors interfering with the de-
termination of pathogenicity VII48-IO
The use of biochemical methods vii48-14
Summary vii4s-14
Le.
\FLET VIII. :
Use of serology in pure
culture study
VTII47-3
Definition of terms
VIII47-4
Bacterial dissociation
VIII47-4
Agglutination
VIII47-4
Serological Methods
Precipitation viii47-10
Complement fixation vni47-ll
Titration of toxins, toxoids and
antitoxins VI1147-I9
Leaflet IX. The Measurement of pH, Titratable Acidity, and
Oxidation-Reduction Potentials
The measurement of pH
Potentiometric methods
The colorimetric method
Titratable acidity, buffer action,
and pH adjustment of cul-
ture media
1X48-2
1X48-2
1X43-6
IX4S-15
The measurement of oxidation-
reduction potentials IX48-17
The potentiometric method IX48-IJ)
The colorimetric method 1X48-21
Leaflet X. Inoculations with Bacterla Causing Plant Disease
Introduction X4J-3 Cognate consideration
Simple representative inoculation Records
methods X45-5
X4rlO
x.s-12
Index
LEAFLET I
INTRODUCTORY
9th EDITION
Pure Culture Study of Bacteria, Vol. 12, No. 1
February, 1944
Revised, October, 1948
LEAFLET I. INTRODUCTORY
PURPOSE OF THE MANUAL
This Manual is intended for use in that type of bacteriological
work known as "pure culture study of bacteria", the meaning of
which is discussed below.
The methods given here are not to be regarded as official. The
committee has always taken the stand that official methods should not
be adopted in the case of research work, because it is continually
necessary to modify research methods in order to keep them up to
date. The standardization of methods tends to hinder the develop-
ment of new technic, while the chief function of this committee is to
stimulate its development. This contention of the committee seems
now to be officially recognized by the Society of American Bacteri-
ologists, and this organization has of recent years left the establish-
ment of official methods to other bodies having closer connection
with regulatory work. At the request of the Society this Manual
now bears upon its title page the statement: ''The methods given have
not been Jonnally approved by the Society, and are in no sense official
or STANDARD."
The methods in this Manual, therefore, are merely claimed to be
the best that have come to the attention of the committee at the time
of publication. Whenever practical, the methods have been tested
by the committee in comparison with other procedures; when this has
not been done, methods are given with a statement to indicate that
they have not been critically tested.
Meaning of Pure Culture Study
There has sometimes been misunderstanding as to the sense in
which the Committee uses the expression "pure culture study of
bacteria". It is occasionally thought that such an expression would
cover nearly the whole field of bacteriological technic. On the other
hand, the definition of pure culture study of bacteria which has been
drawn up by the Committee on Bacteriological Technic is: the study
of bacterial cultures with the object of learning their characteristics
and behavior or determining their identity, or both. Such a study
may be regarded as including: isolation methods; methods for the
cultivation and the storage of various kinds of bacteria; the micro-
scopic study of pure cultures either stained or unstained; determina-
tion of cultural characteristics of an organism; a study of its physio-
logical characteristics; the chemical methods necessary in making the
last-mentioned study; the determination of pathogenicity and study
I44-2
INTRODUCTORY I44-3
of pathological effects; the serological requirements of an organism
when used as a means of characterization.
It is clear from such a statement that Pure Culture Study of Bac-
teria is fairly comprehensive, but that there are many fields of bac-
teriological technic not included within it, e.g. : methods for the enu-
meration of bacteria in their natural habitats; the diagnosis of disease,
and many other phases of pathological bacteriology; methods em-
ployed in the study of food spoilage and controlling the processes of
fermentation, etc. Such a list might be extended almost indefinitely;
for the field of pure culture study, although fairly broad, is actually
merely a small part of bacteriological technic.
Relation to Taxonomy
Clearly, one of the main objects of pure culture study is to deter-
mine the identity of any bacterial culture under investigation. This
brings the subject very close to the field of bacterial taxonomy — i.e.,
the naming and classifying of bacteria. Inasmuch as bacteria cannot
be classified without studying their characteristics in pure culture, it
is an obvious conclusion that pure culture study is a necessary prelude
to bacterial taxonomy.
It must be recognized, nevertheless, that one can consider pure
culture study without regard to taxonomy and that one can study the
taxonomy of bacteria without paying special attention to the methods
of pure culture study. Since this distinction can be made and the
committee editing this series of publications is a Committee on
Technic, care has always been taken to maintain the distinction so as
not to interfere with the functions of other committees that have been
appointed to deal with matters of nomenclature and classification.
It should be remarked, however, that this distinction was not always
observed in the past, as a result of which the original committee,
from which the present Committee on Technic has descended, was
called the Committee on the Identification of Bacterial Species.
Thus considered, it was really a committee on one phase of taxonomy.
Early in its history, however, it began centering its interests on the
technic involved, and about twenty years ago it seemed wise to
change its name to the Committee on Bacteriological Technic.
Publications of the Committee on Technic
Descriptive Charts: The first descriptive chart actually adopted by
the Society of American Bacteriologists was in 1907. The history
of these early developments is given in Leaflet I of the Manual of
Methods for Pure Culture Study of Bacteria and hardly needs
14,-4 PURE CULTURE STUDY OF BACTERIA
to be discussed here. The chart has been revised from time to time
and at present there are two forms — one known as the Standard
Descriptive Chart, and the other as the Descriptive Chart for In-
struction. The latter is very much simpler than the former. The
former is printed on both sides of a 8}/2" x 11" sheet of light cardboard,
the latter on a sheet of heavy paper of the same size.
The object of the Descriptive Chart is to provide a space for record-
ing the most important characteristics of a single culture. The
Standard Chart is the most complete and is intended especially for
advanced work in bacteriology. Unfortunately, however, it does
not meet modern research needs at all perfectly because each group
of bacteria requires its own set of tests and no form can be drawn up
sufficiently detailed to cover all of them. The Chart for Instruction,
on the other hand, is so much simpler and contains so much blank
space that it sometimes is found to be more satisfactory in research
work than the Standard Chart. It is, however, intended primarily
for students to use in characterizing cultures furnished them in con-
nection with their class work.
Manual of Methods for Pure Culture Study: The origin of this
Manual traces back to a Committee report which was printed in the
Journal of Bacteriology in 1918 and was distributed in reprint form
by the Committee. This report was only 14 pages long and was
concerned only with the methods used in carrying out the determina-
tions called for on the Descriptive Chart of those days. The original
report was revised once or twice, and in 1923 was finally issued as an
independent publication under its present name. The first edition
of the Manual was only 48 pages in length. As it was put out in
loose-leaf form, however, it was possible to revise it section by sec-
tion; and each revision has tended to be longer than the preceding.
The result is that the present edition contains about 200 pages.
The present Manual consists of ten leaflets and each leaflet has its
own pagination. The system of page numbering adopted may seem
peculiar and has caused some objections as seeming slightly compli-
cated. It is, however, the simplest form that can be adopted to
avoid confusion in a publication of this kind. Serial paging for the
entire Manual is impossible because the leaflets vary in size from one
edition to the next. As a result serial paging for each separate leaflet
has been adopted, and to avoid confusion in page references made
elsewhere the number of the leaflet together with the year of publica-
tion is given in small figures before the page number itself; thus II40-8
would indicate page 8 of the 1940 edition of Leaflet II, and a reference
to it in that form is very exact.
INTRODUCTORY I44-5
As just stated, the original object of this Manual was to supply the
methods to be used in the study of organisms according to the
Descriptive Chart. As the subject developed, however, it was felt
that there are other fields of pure culture study equally important and
these have been added from time to time. The present Manual
deals with so many lines of tcchnic that it is essentially a laboratory
handbook covering those procedures referred to above as comprising
the field of pure culture study. It is coming to be used more and
more for this purpose, and in a number of institutions is now put in
the hands of all students in certain classes of bacteriology. Thus
used, it has the advantage over conventional texts in that the Com-
mittee behind it is consistently endeavoring to keep it up to date.
The present edition of this Manual contains ten leaflets bearing
the following titles: I. Introductory; II. Preparation of Media; III.
The Study of Obligately Anaerobic Bacteria; IV. Staining Pro-
cedures; V. Routine Tests for the Descriptive Chart; VI. Further
Biochemical Methods; VII. The Study of Pathogenic Aerobes; VIII.
Serological Methods; IX. The Determination of pH and Titrable
Acidity; X. Inoculations with Bacteria Causing Plant Disease.
The system adopted for keeping the Manual up to date is by means
of subscriptions to this quarterly publication, Pure Culture Study
OF Bacteria. Nearly every issue of this quarterly contains a revi-
sion of some one of the ten leaflets. Anyone owning a copy of the
Manual can subscribe to Pure Culture Study of Bacteria by
filling out the card attached to the front of the Manual and sending it
in to the publishing agency with a year's subscription. Thus, any
student who first purchases a copy merely in connection with his class
work, can easily arrange to have it kept up to date if he finds that he
is going into bacteriological work permanently. It is in this way
that the owner is able to profit from the loose-leaf type of publication
which has been adopted.
HISTORICAL
The first efforts toward producing a descriptive chart for character-
izing bacteria were made by two different individual investigators,
H. W. Conn, and S. de M. Gage. The work of these two investi-
gators called the matter to the attention of bacteriologists in general
and it was finally brought before the Society of American Bacteriolo-
gists by F. D. Chester at the Philadelphia meeting in December 1903,
and then again at the 1904 meeting when he explained his idea of a
"group number" which would be descriptive of the salient charac-
ters of an organism. On his recommendation the Society appointed
r44-6 PURE CULTURE STUDY OF BACTERIA
a Committee on Methods for the Identification of Bacterial Species
of which Prof. Chester was made chairman. This committee drew
up the first descriptive chart with which the Society of American
Bacteriologists had any connection.
This chart was put before the Society at its 1905 meeting. It was
presented at this time as a preliminary effort and no endorsement of
it was given by the Society nor apparently was such endorsement re-
quested. The committee was instructed to continue its work and a
second chart was prepared during 1906 and presented at the Society
meeting in December that year. At this meeting it was decided that
the chart should call for more complete data concerning bacteria than
provided for by either of the two charts already submitted; so the
committee was instructed to do further work along this same line.
The committee at this time was composed of F. D. Chester, F. P.
Gorham, and E. F. Smith; but Prof. Chester was largely responsible
for the first two charts presented at Society meetings. Before the
committee undertook a further revision, however, he had left bac-
teriological work and hence was no longer active on the committee.
During 1907, therefore. Dr. Smith acted as chairman of the Commit-
tee and under his supervision the committee drew up another chart
which was presented to the Society at its meeting in December that
year. This chart was officially endorsed by the Society and was put
on sale by the secretary of the Society.
For several years following no changes were made in the chart.
The next step in its development was brought about by H. A. Harding
(1910), who published a paper in which he outlined the complete
history of the chart, with copies of the early charts, and discussed
improvements that might be made. This paper is available for
those desiring more detail concerning this early history than is given
here.
As the Society felt that further modifications were now needed a
new committee was appointed in 1911 consisting of F. P. Gorham,
C. E. A. Winslow, Simon Flexner, H. A. Harding and E. O. Jordan.
This committee gave a report at the 1913 meeting, presenting a
chart which was put on sale by the Society, but was not officially
endorsed. As this committee was unable to continue the work, an
entirely new one was appointed at this time, consisting of H. A.
Harding, H. J. Conn, Otto Rahn, W. D. Frost and I. J. Kligler. This
committee soon lost Dr. Ilahn, who left the country in 1914, and
M. J. Prucha was added in his place. The committee was called
the Committee on Revision of the Chart for the Identification of
Bacterial Species.
INTRODUCTORY I44-7
The new committee was instructed by the Society to make a con-
servative revision of the chart and at the same time to draw up a
manual of methods to be used in connection with it. At the 1914
meeting of the Society, therefore, a chart was presented for approval,
much like the 1907 chart except for its more logical arrangement of
data. This chart was given the Society's endorsement and was issued
during 1915.
The 1914 chart was printed on a sheet with its back entirely blank,
the glossary previously on the back having been omitted. The com-
mittee gave as the reason for this that the glossary should be included
in the manual on methods shortly to be published. The publication
of this manual was delayed, however, pending investigation of the
methods to be included in it. This investigation of methods was to
be undertaken not only for the sake of the manual but also as a pre-
liminary step toward radical revision of the chart, which was felt to
be badly needed. Early in 1917, however, and before this program
could be carried out, the chairman of the committee was forced by
pressure of other duties to drop the work. As he wished to remain
on the committee, however, no change in membership was made,
but H. J. Conn was asked to become chairman.
The committee then undertook the first step toward the preparation
of a manual on methods. A report was presented at the 1917 meeting,
giving the methods recommended at that time for use with the chart.
The report was printed in the Journal of Bacteriology, March 1918,
and was subsequently sold by the Society in the form of reprints.
This report was considered a preliminary manual on methods.
The committee proposed at the same time a much simplified chart
in the form of a four page folder, which it recommended for use in
instruction until the official chart could be given the revision it
needed. This chart was not endorsed by the Society; but was printed
and sold by the Society for two or three years.
This same committee (but now called the Committee on the
Descriptive Chart) issued another report on methods which appeared
in the Journal of Bacteriology, March 1919, dealing with the Gram
stain, production of acid, and the reduction of nitrates. At the 1919
meeting it issued a further report which appeared in the Journal of
Bacteriology, in two parts, March and May, 1920. The first part of
the report was a revision of the one which had been published in
March 1918, and was sold as a revised manual of methods until the
reprints were exhausted in 1922.
At the 1920 meeting the Committee on the Descriptive Chart was
discharged with the understanding that its functions would be taken
i<4-8 PURE CULTURE STUDY OF BACTERIA
over by a committee of broader scope then appointed and called the
Committee on Bacteriological Technic. This committee was appointed
with the understanding that its membership should fluctuate from
year to year in order to keep on it men actively interested in the work.
The new committee made a further revision of the chart, which
was presented at the 1920 meeting and endorsed by the Society.
Later editions of this chart have been drawn up by the committee in
the years of 1924 and 1929, but neither of these have been submitted
to the Society for official endorsement. In order to avoid committing
the Society in favor of any of the methods concerned, recent editions
of the Chart have merely been presented by the committee and per-
mission asked to put them on sale.
The committee issued four further reports in the Journal of Bacte-
riology, (1921, 1922 a, b, & c) before this Manual was prepared.
One of these reports (1922b) proposed certain revisions of methods,
in the case of the Gram stain, fermentation, nitrate reduction, indole
and hydrogen sulfide production. The committee presented this
report at the 1922 meeting of the Society with the recommendation
that the revised material be published as part of a Manual of Methods
for Pure Culture Study of Bacteria. The committee was thereupon
instructed by the Society to publish this Manual, using the loose-
leaf form of binding, with the understanding that new folders be
issued from time to time to keep it up to date.
The Committee on Bacteriological Technic has seen the following
changes in personnel :
1920 H. J. Conn, K. N. Atkins, I. J. Kligler, J. F. Norton, G. E. Harmon.
1921 H, J. Conn, K. N. Atkins, G. E. Harmon, Frederick Eberson, Alice Evans.
1922 H. J. Conn, K. N. Atkins, G. E. Harmon, Frederick Eberson, F. W. Tanner, and
S. A. Waksman.
1923 H. J. Conn, K. N. Atkins, J. H. Brown, G. E. Harmon, G. J. Hucker, F. W.
Tanner, and S. A. Waksman.
1924-5 H. J. Conn, K. N. Atkins, J. H. Brown, Barnett Cohen, G. J. Hucker, F. W.
Tanner.
1926-7 H. J. Conn, Barnett Cohen, Eliz. F. Genung, W. L. Kulp, W. H. Wright; with
G. J. Hucker and S. Bayne-Jones as a sub-committee on serological methods.
1928 H. J. Conn, Victor Burke, Barnett Cohen, Eliz. F. Genung, W. L. Kulp, W. H.
Wright.
1929-30 H. J. Conn, Victor Burke, Barnett Cohen, Eliz. F. Genung, I. C. Hall,
W. L. Kulp, W. H. Wright (deceased, May, 1929).
1931-4 H. J. Conn, Barnett Cohen, Eliz. F. Genung; Victor Burke (pathological
methods); I. C. Hall (anaerobic methods); .J. .\. Kennedy (serological methods).
1935 H. J. Conn, Victor Burke, Barnett Cohen, W. M. Jennison, J. A. Kennedy.
1936-42 H. J. Conn; .1. H. Brown (anaerobic methods) Victor Burke, (pathological
methods); Barnett Cohen, C. H. Werkman, (biochemical methods); M. W.
Jennison, (the Descriptive Chart); J. A.Kennedy (serological methods); A. J.
Riker (plant pathological methods).
INTRODUCTORY i,8-9
1943-5 H. J. Conn, Victor Burke, Barnett Cohen, C. H. Werkman, M. W.
Jennison, J. A. Kennedy, L. S. McClung, A. J. Riker.
1946-7 H. J. Conn, G. H. Chapman, Barnett Cohen, I. C. Gunsalus, M. W.
Jennison, L. S. McClung, A. J. Riker, C. E. ZoBell.
1948- M. W. Jennison, G. H. Chapman, Barnett Cohen, H. J. Conn, I. C. Gunsalus,
J. A. Kennedy, L. S. IVFcClung, A. J. Riker, C. E. ZoBell.
USE OF THE MANUAL
Pitfalls to be Avoided by the Student
In studying bacterial cultures with the object of identifying them
or describing them, the student is apt to run onto certain pitfalls.
Many of these are well known and others less fully appreciated. At
the risk of making comments that are already too well known by stu-
dents of bacteriology, a few words concerning some of these pitfalls
do seem called for here. They arise primarily from three sources:
first, the danger of impure cultures; second, confusing results due
to variation of bacterial species; third, differences in methods of
study.
The danger in impure cultures is, of course, thoroughly understood.
Unfortunately, however, the second consideration just mentioned
makes it more important to emphasize the danger of impure cultures
today than was the case 25-30 years ago. In those days bacteriolo-
gists quite generally accepted the idea of monomorphism; and when-
ever a culture was observed to be noticeably abnormal either in
morphology or physiology, it was promptly discarded as a contami-
nant. When, however, it began to be learned that even the most
strictly guarded pure cultures might show changes in morphology
during their life history, and then later when it was realized that the
same organism might occur in two or more phases showing distinctly
different cultural and physiological characteristics, the old ideas of
monomorphism were decidedly upset. As a result of the changing
point of view, it is very easy for a careless student today to believe
that he is observing two phases of the same pure culture when actually
one of his "phases" is a contaminant. This makes constant checking
as to purity of cultures even more important than it was before dis-
sociation into phase variants was generally accepted by bacteriolo-
gists.
Accepting the idea of dissociation presents other diflSculties to the
student. Without exhaustive study, it is sometimes very easy to
describe two phases of the same species as though they were different
organisms. It is also easy to prepare a description of some culture
which is an illogical jumble of the characteristics of two or more
144-10 PURE CULTURE STUDY OF BACTERIA
phases, due to the fact that it was first studied in an unstable form
and dissociation was taking place during the course of the study. On
the other hand, some of the methods employed in the hopes of induc-
ing phase variation may actually cause contamination and be in-
correctly interpreted. Some of these points are very adequately dis-
cussed by Frobisher (1933).
The third source of error above mentioned (variation in methods)
also needs emphasis. When a species is described in such terms as
one frequently encounters in published descriptions — e.g. "Produces
acid (without gas) from glucose and lactose but not from sucrose;
does not reduce nitrates" — one has to guess at the answers to such
questions as these: What basal medium was used in each instance.''
What indicator of acid production was employed? How thorough a
study was made to show the absence of any acid from sucrose, or of
any reduction of nitrate .f* Or, in the latter instance, is it safe to as-
sume that the author of the species merely failed to find nitrite in
some nitrate medium? Unless such questions are answered cor-
rectly, the description is meaningless, the attempt to identify an un-
known culture with such a description may well give misleading
results.
With all these pitfalls to avoid, it is easy to see how the same set of
data, no matter how carefully prepared, can be differently interpreted
by two different bacteriologists. As a result extreme caution is urged,
both in determining the identity of a culture and in deciding whether
or not to pronounce it a new species.
Practical Hints
Determining the characteristics of a culture: One should always, if
possible, make a complete study of a culture promptly after its first
isolation while it is in vigorous condition. When a culture has be-
come attenuated in the laboratory, it should be restored to vigor by
growth under conditions well suited for its invigoration. When this
is done, however, the possibility should always be recognized that
by such "invigoration" dissociation may be induced so that the
phase subsequently studied may be quite different from the original
isolation. Whenever distinct evidence of dissociation is observed,
each phase should be studied and recorded separately; and efforts
should be made to reverse the change or to obtain the same change
with other strains until the possibility of impure cultures seems to be
out of the question. No importance should ever be attached to a
single determination, unless supported by a duplicate or even by
triplicates giving the same results. In describing morphology, one
INTRODUCTORY I44-II
should not be contented with one or two observations, but should
study several transfers and should follow up each of them day by day
for about a week. When changes are observed, a careful study
should be made to learn whether they indicate morphologic variation,
dissociation, or merely contamination. In making special staining
tests, like the Gram stain, several determinations should be made on
separate transfers of the culture and at different ages, because there
are species that vary in their staining reactions, and such variation
cannot be detected by single determinations. As a check on the
technic, a known positive and a known negative culture should be
included in the study. For example, when making a Gram stain, it
is good practice to place on the slide, beside the culture under study,
a smear containing a mixture of a known Gram-positive and a known
Gram-negative organism (which differ markedly in morphology).
Then it is possible to observe whether the expected results are ob-
tained with the known cultures, and thus to have some degree of con-
trol on the technic.
Identification: After recording the characteristics of an organism,
the next step is identification, if possible, with a previously described
species. This should never be attempted until at least six repre-
sentative strains of the unknown organism isolated from more than
one source, if possible, have been studied. No rules can be given for
identifying the culture. Descriptions of bacteria are scattered so
widely through the literature and vary so greatly in their form that
identification is often extremely different. Bergey's Manual of
Determinative Bacteriology is a great help; but it is usually neces-
sary to go back to original descriptions and often to secure transfers
of authentic strains before certain identification can be made. Diffi-
cult as this procedure is, no one is justified in naming a new species
of bacteria until a comprehensive search through the literature of
species already described has been made. Frequently it is necessary
to refer in some publication to a previously described species on the
basis of such an identification as this. In this case it is important to
state in the publication whether or not an authentic strain of the
species has been obtained for comparison; if so, from where obtained;
if not, what published description of the species was followed in
making the identification. As to a name to use for such a species
one may follow the original author's nomenclature or may give it the
name employed in some modern system (e.g. Bergey). Whatever
name is chosen no confusion will result it if is accompanied by the
name of the original author of the specific name and by that of the
one making the combination of generic and specific names. Thus,
i4,-12 PURE CULTURE STUDY OF BACTERIA
whether one says "Bacillus coll Migula" or '''Escherichia coli (Migula)
Castellani and Chalmers", it is entirely clear what species is intended.
Naming a new species: When it proves impossible to identify a
culture with any species described in the literature, it is often desirable
to publish a description of it as a new species. When publishing such
a description, there are five important points to be kept in mind:
(1) The description should be based on at least six representative
isolations of the organism. (2) If variations are found to occur
among these strains, a critical study must be made to be sure that
they are not the result of contamination. (3) In naming any charac-
teristic of the species, especially if it is a negative character (e.g.
"nitrates not reduced"), the technic by which it is determined must
be stated. (4) Before giving the results of any test as positive or
negative, comparisons must be made with a control culture known
to be positive and one known to be negative. (5) Before actually
assigning a name one should consult a specialist in bacterial taxonomy,
both as to the necessity for a new name and as to the validity of the
name selected. The Board of Editor-Trustees of Bergey's Manual,
for example, are always very glad to offer such advice.
If these hints were followed by all who are trying to identify species
or to publish descriptions of them, much of the confusion in bacterial
nomenclature would be eliminated.
REFERENCES
Harding, H. A. 1910. The constancy of certain physiological characters in the
classification of bacteria. N. Y. Agric. Exp. Sta. Tech. Bui. 13.
Committee on Descriptive Chart. 1918. Methods of Pure Culture Study. Jour.
Bact.. 3, 115-128.
Committee ON Descriptive Chart. 1919. Methods of Pure Culture Study. Progress
report for 1918. J. Bact. 4, 107-132.
C0.MMITTEE ON Descriptive Chart. 1920 a. Methods of Pure Culture Study. Re-
vised. J. Bact. 5, 127-U3.
Committee on Descriptive Chart. 1920 b. Progress report for 1919. J. Bact.
5, 315-319.
Committee on Bacteriological Technic. 1921. Progress report for 1920. J.
Bact. 6, 13.5-141.
Committee on Bacteriological Technic. 1922 a. An investigation of .\merican
Stains. J. Bact. 7, 127-248.
Committee on Bacteriological Technic. 1922 b. Methods of Pure Culture
Study. J. Bact. 7, 519-528.
Committee on Bacteriological Technic. 1922 c. An investigation of American
gentian violets. J. Bact. 7, 529-536.
Frobisher, M. 1933. Some pitfalls in bacteriology. J. Bact. 25, 565-71.
GLOSSARY OF TERMS USED IN THE MANUAL AND ON
THE DESCRIPTIVE CHART
Acid curd, coagulation of milk due to acid production.
Adherent, applied to sporangium wall, indicates that remnants of sporangium remain
attached to endospore for some time.
Aerobic, growing in the presence of free oxygen, strictly aerobic growing only in the
presence of free oxygen.
Agglutinin, an antibody having the power of clumping suspensions of bacteria.
Anaerobic, growing in the absence of free oxygen; strictly anaerobic, growing only in
the absence of free oxygen; facultative anaerobic, growing in both presence and
in absence of oxygen.
Antibody, a speci6c substance produced by an animal in response to the introduction
of an antigen.
Antigen, a substance which when introduced into an animal body, stimulates the
animal to produce speci6c bodies that react or unite with the substance intro-
duced.
Antigenic action, behavior as an antigen.
Antitoxin, an antibody having the power of uniting with or destroying a toxic sub-
stance.
Arborescent, branched, tree-like growth.
Aseptically, without permitting microbial contamination.
Autotrophic, able to grow in absence of organic matter.
Bacteriocidal, capable of killing bacteria.
Bacteriostasis, preventing bacterial growth, but without killing the bacteria.
Beaded, (in stab or stroke culture) separate or semi-confluent colonies along the line of
inoculation.
Bipolar, at both poles or ends of the bacterial cell.
Bleb, vesicle or blister-like swelling.
Brittle, growth dry, friable under the platinum needle.
Butyrous, growth of butter-like consistency.
Capsule, an envelope surrounding the cell membrane of some kinds of bacteria.
Chains, four or more bacterial cells attached end to end.
Cbromogenesis, the production of color.
Clavate, club-shaped.
Compact, refers to sediment in the form of single fairly tenacious mass.
Complement, a non-specific enzyme-like substance, destroyed if subjected to heat
(56°C or over for 30 minutes), which occurs in blood serum, and is necessarj-, in
conjunction with a specific antibody, in order to bring about cytolysis.
Concentrically ringed, marked with rings, one inside the other.
Contoured, an irregular, smoothly undulating surface, like that of a reUef map.
Crateriform, a saucer-shaped liquefaction of the medium.
Cuneate, wedge-shaped.
Curled, composed of parallel chains in wavj- strands, as in anthrax colonies.
Cytolysin, an antibody causing cytolysis.
Cytolysis, a dissolving action on cells.
Diastatic action, conversion of starch into simpler carbohydrates, such as dextrins or
sugars, by means of diastase.
Diphtheritic, diphtheria-like.
1^-13
1,4-14 PURE CULTURE STUDY OF BACTERIA
Dissociation, separation of characters, usually referring to phase variation (q. v.).
Echinulate, a growth along line of inoculation with toothed or pointed margins.
Edema, intercellular accumulation of fluid in a part of an animal body.
Effuse, growth thin, veily, unusually spreading.
Endospores, thick-walled spores formed within the bacteria; i. e., typical bacterial
spores like those of B. anthracis or B. subtilis.
Endotoxin, a toxic substance produced within a microorganism and not excreted.
Enzyme, a chemical ferment produced by living cells.
Erose, irregidarly notched.
Excentric, slightly to one side of the center, between the positions denoted central and
subterminal.
Exogenous, originating outside the organism.
Exotoxin, a toxic substance excreted by a microorganism and hence found outside the
cell body.
Facultative anaerobe, see anaerobic.
Filamentous, growth composed of long, irregularly placed or interwoven threads.
Filaments, applied to morphology of bacteria, refers to thread-like forms, generally un-
segmented; if segmented, the orgahisms are enclosed in a sheath.
Filiform, in stroke or stab cultures, a uniform growth along line of inoculation.
Flagellum (pZ.-la), a motile, whip-like attachment; an organ of locomotion.
Flaky, refers to sediment in the form of numerous separate flakes.
Flocculent, containing small adherent masses of various shapes floating in the fluid.
Fluorescent, having one color by transmitted light and another by reflected light.
Gonidia, asexual spores.
Gonidial, referring specifically to a bacterial phase producing gonidia-like bodies.
Granular, composed of small granules.
Hemolysin, a substance causing hemolysis either alone or in presence of complement.
Hemolysis, a dissolving action on red blood corpuscles.
Hemorrhage, an escape of blood from the vessels.
Histolysis, breaking down of tissues.
Hydrolysis of starch, destruction of starch by the formation of a chemical union with
water; includes diastatic action, but is a more general term.
Immune serum, an animal fluid containing an antibody.
Inactivate, to destroy complement by heat (at 56° for 30 minutes).
Infundibuliform, in form of a funnel or inverted cone.
Intraperotoneal, within the peritoneum.
Intravenous, within a vein.
Iridescent, exhibiting changing rainbow colors in reflected light.
Lesion, a local injury or morbid structural change.
Lobate, having lobes, or rounded projections.
Maximum temperature, temperature above which gro\%'th does not take place.
Membranous, growth thin, coherent, like a membrane.
Metabolite, a substance produced by metabolism.
Microaerophilic, growing best in presence of small quantities of oxygen.
Minimum temperature, temperature below which growth does not take place.
Mucoid, mucus-like, referring specifically to a bacterial phase producing slimy growth.
Mycelioid, colonies having the radiately filamentous appearance of mold colonies.
Napiform, liquefaction in form of a turnip.
Ontogenetic, pertaining to the life history of an individual.
Opalescent, milky white with tints of color as in an opal.
Opaque, not allowing light to pass thru.
GLOSSARY 14,-15
Optimum temperature, temperature at which most growth occurs.
Papillate, growth beset with small nipi)le-like processes.
Parasitic, deriving its nourishment from some living animal or plant upon which it
lives and which acts as host; not necessarily i)athogenic.
Pathogenic, not only parasitic (q. v.) but also causing disease to the host.
Pellicle, bacterial growth forming either a continuous or an interruj)ted sheet over the
culture fluid.
Peptonization, rendering curdled milk soluble by the action of peptonizing enzymes.
Peritrichiate, api)licd to the arrangement of flagella, indicates that they are distributed
over the entire surface of an organism.
Peritrichic, having flagella in peritrichiate arrangement.
Per OS, thru the mouth.
Persistent, lasting many weeks or months.
Phase variation, separation of a species into strains, having somewhat different
characters.
Photogenic, glowing in the dark, phosphorescent.
Polar, at the end or pole of the bacterial cell.
Precipitin, an antibody having the power of precipitating soluble proteins.
Pulvinate, cushion-shaped.
Punctiform, very small, but visible to naked eye; under 1 mm. in diameter.
Raised, growth thick, with abrupt or terraced edges.
Reduction, removing oxygen or its equivalent from a chemical compound; or addition
of hydrogen or its equivalent. Refers to the conversion of nitrate to nitrite,
ammonia, or free nitrogen; also to the decolorization of litmus.
Rennet curd, coagulation of milk due to rennet or rennet-like enzymes, distinguished
from acid curd by the absence of acid.
Rhizoid, growth of an irregular branched or root-like character, as B. mycoides.
Ring, growth at the upper margin of a liquid culture, adhering to the glass.
Rugose, wrinkled.
Saccate, liquefaction in form of an elongated sac, tubular, cylindrical.
Saprophytic, living on dead growth in the absence of organic matter, i. e., neither
autotropic (q. v.) nor parasitic.
Sensitize, to render sensitive, usually to a foreign protein.
Sepsis, a state of infection.
Sheath, an envelope similar to a capsule (q. v.), but surrounding a filamentous or-
ganism.
Spindled, larger at the middle than at the ends. Applied to sporangia, refers to the
forms frequently called Clostridia.
Sporangium (pZ.-ia), cells containing endospores.
Spreading, growth extending much beyond the line of inoculation, i. e., several milli-
meters or more.
Stratiform, liquefying to the walls of the tube at the top and then proceeding down-
wards horizontally.
Strict aerobe, see aerobic.
Strict anaerobe, see anaerobic.
Subcutaneous, under the skin.
Subtermlnal, situated toward the ond of the cell but not at the extreme end, that is
between the positions denoted excentric (q. v.) and terminal.
Synergism, cooperative action of two organisms, resulting in an end-product which
neither could produce alone.
Thermophilic, growing best at high temperatures, i. e. 50°C or over.
1,4-16 PURE CULTURE STUDY OF BACTERIA
Toxic, poisonous.
Transient, lasting a few days.
Translucent, allowing light to pass thru without allowing complete visibility of objects
seen thru the substance in question.
Trituration, thoro grinding in a mortar.
Truncate, ends abrupt, square.
Turbid, cloudy with flocculent particles; i. e., cloudy plus flocculence.
Ulcer, an open sore.
Undulate, wavy.
Villous, having short, thick, hair-like processes on the surface, intermediate in meaning
between papillate and filamentous.
Virulence, degree of pathogenicity (referring to infectiousness).
Virus, a self-propogating cause of disease, often referring to one too small to be seen
with microscope.
Viscid, growth follows the needle when touched and withdrawn; sediment on shaking
rises as a coherent swirl.
LEAFLET II
PREPARATION OF MEDIA
9th EDITION
Pure Culture Study of Bacteria, Vol. 12, No. 2
April, 1944
Including a section prepared by
Committeeman on Anaerobic Methods
LEAFLET II
PREPARATION OF MEDL/^
STERILIZATION
General directions for preparation of media are hardly called for
here as they can be found in all bacteriological laboratory guides. In
the matter of sterilization, however, a few specific instructions seem
advisable.
Ordinary bacteriological media are sterilized for 20 to 30 minutes
in an autoclave under steam pressure at 121°C (15 pounds pressure
after driving out all air) . In determining this temperature dependence
should not be laid upon a pressure gauge; the autoclave should be
equipped wuth a thermometer. In general, the smaller the container,
and the smaller the number of flasks or tubes sterilized at one time,
the shorter the sterilizing time can be. In the case of small batches
of media, 15 minutes at 15 pounds are ordinarily sufficient, a fact
which is worth taking into account when the media contain sub-
stances likely to be decomposed by heat.
Oils are difficult to sterilize, and when they are added to media it is
well to sterilize them separately by dry heat (165-75° for 1 hour)
or by autoclaving in small quantities at 121°C.
Fractional sterilization in flowing steam at 100° for 30-60 minutes
on three successive days was formerly recommended to avoid this
decomposition in the case of carbohydrates. Recent investigation,
however, tends to show that this procedure can be more harmful
than the higher temperature for 15 minutes; fractional sterilization,
therefore, is used much less than formerly. Instead it is recommended
that those sugars especially susceptible to the effects of heat (e. g.,
xylose, arabinose, fructose, maltose, and under some conditions
sucrose and lactose) be dissolved separately and sterilized by filtra-
tion before adding to the rest of the medium after it has been auto-
claved. The Seitz filter or sintered glass filters prove suitable for
this purpose. Where facilities for such filtration are lacking, these
sugars can ordinarily be autoclaved successfully if sterilized separately
from the rest of the medium and in concentrated solution, employing
as brief heating as possible — e. g., 10 minutes at 10 pounds pressure
(115°C) if serological tubes are used.
MEDIA USED IN PURE CULTURE STUDY
It is a matter of some difficulty to decide just what media should
be included here. It would obviously be beyond the scope of this
n„-3
n44-4 MANUAL OF METHODS FOR PURE CULTURE STUDY
Manual to include all the media employed by bacteriologists. In
selecting the ones to include two principles have been kept in mind :
first to include only those known to be in fairly common use among
American bacteriologists; second, reaUzing that this is a Manual for
Pure Culture Study of Bacteria, not to list media that are used purely
for counting bacteria or for the diagnosis of disease. The media
given here are employed either for maintaining pure cultures or for
the identification of species.
For the purposes of this Manual these media may be classified as
follows: A. Cultivation and storage media; B. General differential
media — i.e. media employed in tests for determining the identity of
saprophytic aerobes, in general; C. Media for special groups of
aerobes — i.e. media employed in the identification of bacteria of
certain narrow groups, such as the colon-typhoid group; D. Media
for anaerobic bacteria.
The media included in this leaflet under the heading "C" have
been arranged into three groups the first of which is denoted "Basal
Media". The basal media may be considered as formulae to which
substances under investigation (e. g., sugars) may be added. The
selection of any of these basal media depends upon the group of
bacteria to be studied.
Special reference is made here to Levine and Schoenlein's Com-
pilation of Culture Media, 1930. In the case of the formulae taken
from this source, the number therein assigned to the medium is given
at the head of each formula under the designation "L&S No "
Attention is called to the fact that many of these media are now
on the market in dehydrated form. Use of such dehydrated media is
entirely permissible, and often convenient.
A. Cultivation and Storage Media
Beef -extract broth ordinarily has the following composition :
Beef-extract 3 g.
Peptone 5 g.
Distilled water 1000 ml.
Concerning the peptone called for in the above formula, no definite
specifications can yet be given. Various commercial products are
available, no one of which is superior for all bacteriological purposes.
In the case of reports on standard methods (e.g., those of the Ameri-
can Public Health Assoc.) emphasis is laid on one brand of peptone
PREPARATION OF MEDIA n^o
for the sake of uniformity; for purposes of pure culture study, how-
ever, any brand known to give best results for the purpose at hand
may be employed.
Beef-exiract agar may be of the same composition plus the addition
of 12 grams of oven-dried agar or 15 grams of commercial agar. The
agar is to be dissolved by heat (e.g. autoclaving) and the sediment
removed either by decantation or by filtration through cotton.
Beef-extract gelatin may be of the same composition as the broth
but with the addition of 100 g. of "Bact5-gelatin" (or some other
gelatin of the same jellying strength; i.e., 175-200 by Bloom test).
Carefully adjust reaction (see below) after dissolving gelatin and
heat for 5 or 10 min. at about 100" C. Filter through cotton.
Meat infusion broth. This is usually prepared as follows : Pour 1 litre
of water over 400-600 g. of lean beef or veal, ground through a meat
chopper. Allow to stand in a refrigerator overnight and then skim
off the scam of fat with a piece of absorbent cotton. Squeeze out the
infusion through a strong muslin cloth and make the amount up to
1000 ml. Dissolve 5 g. of peptone in this by adding the infusion
(without heating) little by little to the peptone in a mortar and
rubbing up with the pestle until the solution is complete. (When
making this bouillon as a basis of blood agar or for serological work,
one should also dissolve in it 0.5 g. sodium chloride.) Adjust reaction
(see below). Heat for about 20 minutes at about 100°C without
stirring; filter through wet filter paper and make up to 1000 ml.
Meat infusion agar. In 1000 ml. meat infusion broth prepared
as above, dissolve 12 g. of oven dried agar (or 15 g. commercial agar)
by heating at about 100°C; filter off any sediment formed.
Yeast-extract broth and agar. These may be made the same as beef-
extract broth and agar except for replacing the beef-extract with 2.5 g.
yeast-extract per litre. The latter should be used in powdered form,
as for example the product of the Difco Laboratories.
Semi-solid agar. With some organisms, especially microaerophiles,
more successful cultivation can be obtained by means of semi-solid
media, containing only 0.2 to 0.5% agar. For such purposes any of
the above agar formulae may be followed, merely decreasing the
quantity of agar. The exact quantity of agar recommended varies.
Thus Hitchens' semi-solid medium (see p. 1I44-II) calls for 0.2%, while
Tittsler and Sandholzer (1936) employ a 0.5% agar for the macro-
scopic determination of motility: the latter is almost solid in con-
sistency.
1*4, 6 MANUAL OF METHODS FOR PURE CULTURE STUDY
Adjusting reaction. The reaction of all these media is to be adjusted
to a hydrogen-ion concentration near neutrality (i.e. pH 7.0). The
beef -extract broth and agar normally require no adjustment to
bring them to this reaction; the others need the addition of alkali
ordinarily. In all cases the reaction should be tested, even though no
adjustment is thought to be necessary. For detailed instructions in
testing or adjusting the reaction one may consult Leaflet IX of this
Manual, entitled The Determination of pH and Titrable Acidity
or may follow the directions given by the American Public Health
Association (1936, p. 199). For ordinary purposes, however, good
results will be obtained by adjusting the media to the neutral point
of brom thymol blue;^ the medium is brought to such a reaction as to
turn this indicator a distinct grass-green (neither yellow green nor
blue green). This color corresponds closely to the desired reaction.
Another equally satisfactory method to bring the medium to this
reaction is to add suflBcient alkali to cause the first faint trace of
permanent pink to appear with phenol red.^ Reaction should always
be checked after final sterilization of each batch.
Natural storage media. Recent years have shown quite a tendency
to employ natural media, particularly skim milk or soil, for the
storage of stock cultures. These materials are frequently used in their
natural state, without addition; but more often a small quantity of
calcium carbonate is added to neutralize acids formed. This addition
is absolutely necessary in the case of limestone-free soils or in the
case of milk when the organisms to be stored produce acid from
lactose.
B. General Differential Media
Plai7i gelatin for use in the determination of gelatin liquefaction.
This is made up like beef -extract gelatin but without the beef-
extract and peptone; it consists of 10% "Bacto-gelatin" (or some
other brand of the same jellying strength) dissolved in distilled water
and the reaction adjusted to pH 7.0.
Sugar broths. Just before sterilization 0.5-1% of the required
carbohydrate is ordinarily added to beef-extract broth; the same
proportions are also usually to be employed in studying the
fermentation of any related carbon compound (e.g. alcohol or gluco-
*Use 0.04% brom thymol blue or 0.02% phenol red. Alcoholic solutions may be
employed without neutralizing, or aqueous solutions of the sodium salts prepared as
directed by Clark (1928, p. 91-95) or as explained in Leaflet IX of this Manual (p.
IX4X-11).
PREPARATION OF MEDIA ii„.7
side). The final reaction should be adjusted to pH 7.0. For precau-
tions in sterilization, see above, p. 1141-3.
It is often desirable to put some indicator into such media. In select-
ing the proper indicator read the section below on Indicator Media.
Sugar agar. As with sugar broth, beef-extract agar media of the
formula given on p. 5 may be made up with 1% of the required
carbohj^drate or related carbon-compound. The latter may be mixed
with the other ingredients only if it is known not to be appreciably
changed by the heat employed; otherwise it should be dissolved and
sterilized separately as above suggested. The reaction should be
adjusted to pH 7.0. An indicator may be added if desired.
Indicator media. Carbohydrate media with some indicator to show
acid production are frequently of value. Litmus and Andrade's in-
dicator (acid fuchsin decolorized with alkali) are much used, but they
do not give accurate results in terms of hydrogen-ion concentration;
so, except for certain special purposes^, it is recommended that
sulphonphthalein indicators be employed. The indicators of most
value are: phenol red, brom thymol blue, brom cresol purple, brom
cresol green, and occasionally brom-chlor phenol blue. Their use is
governed by the following considerations:^
Phenol red indicates changes to the alkaline side of neutrality, as its
range is pH = 6.8-8.4. For use in indicator media it is best kept in a
1.6% alcoholic solution and 1 ml. of the solution added to 1 litre of
medium.
Brgm thymol blue has a sensitive range extending slightly in either
direction from neutrality. It is useful in media carefully adjusted to
pH 7.0, but indicates such small changes in reaction as to be often
impractical. It is best added to media at the rate of 1 ml. of a 1.6%
alcoholic solution to the litre.
Brom cresol purple indicates slightly greater changes to the acid
side of neutrality, as its range is pH = 5.2-6.8. For indicator media
1 ml. of a 1.6% alcoholic solution should be added to the litre. It
seems to be the most generally useful indicator for indicator media of
any at present available. It has, however, the defect of dichromatism.
If this is troublesome, it may be replaced by brom phenol red, which
covers the same pH-range.
Combinations of brom cresol purple and cresol red are often
satisfactory when looking for changes in either direction from neu-
trality. When this combination is employed, the media should be
*See next page; also Lea6et V, p. v^j-iO
'See also Leaflet IX.
11^4-8 MANUAL OF METHODS FOR PURE CULTURE STUDY
carefully adjusted to pH 7.0 with brom thymol blue before adding
any indicator; then 1 ml. of a saturated aqueous solution of each
indicator should be added. This mixture of indicators changes very
slowly from purple to yellow through a long range (from about
pH = 8.0 to about pH = 5,2) extending for a considerable distance on
each side of neutrality. By comparing with a blank tube of the
neutral medium it is easy to detect an increase either in acidity or in
alkalinity.
Brom cresol green (introduced by Cohen, 1922) indicates moder-
ately great changes to the acid side of neutrality as its range is pH =
3.8-5.4. It is best kept for this purpose in a 2% alcoholic solution,
adding 2.0 ml. to each litre of medium. Used in agar media it shows
appreciable change from green to yellow if the reaction is as high as
pH = 5.2; and from that point to the acid end of its range it is very
satisfactory.
Brom phenol blue is now suggested by Cohen (1927) to replace
brom-chlor-phenol blue which he described earlier. As its range is
from pH = 3.0 to pH = 4.6 it is of value in indicator media only with
organisms showing a very high final hydrogen-ion concentration.
For this reason it is very seldom called for; but it is valuable in dis-
tinguishing the most vigorous acid formers.
In spite of all the arguments in favor of the sulphonphthaleins as
H-ion indicators, litmus still remains popular among bacteriologists,
aad no perfect substitute for it has been obtained. Its advantages
are that it is a long-range (even if not highly accurate) indicator,
showing changes on both sides of the neutral point, and at the same
time indicates changes in oxidation-reduction potential. This makes
it useful for diagnostic purposes when employed in certain media,
notably in milk; and no combination of indicators showing all the
characteristics of litmus has yet been proposed. Unfortunately, at
the time when this (9th) edition of this leaflet goes to press, the source
of the lichens from which litmus is manufactured has been cut off
from the United States, and this indicator is becoming harder and
harder to obtain.
Nitrate broth. For routine work 0.1% ICNO3 is added to the regular
formula for beef-extract broth and reaction adjusted as usual.
Similarly routine nitrate agar should contain 0.1% KNO3 added to
the ordinary formula for beef-extract agar, with the reaction properly
adjusted. Modification of these formulae is often necessary as ex-
plained on p. V42-IO Leaflet V of this Manual. A synthetic nitrate
medium often found useful is given below (p. 1144-I4).
PREPARATION OF MEDIA ii«^9
Media for H2S production. In previous editions, four media have
been listed containing lead or iron salts, designed to show blackening
when hj^drogen sulfide is produced. As the present procedure given
in Leaflet V calls for lead acetate test-strips in the mouths of the
tubes, these media are no longer recommended for routine use.
Those who wish to use such media are referred to the papers of
Bailey and Lacy (1927) and of Wilson (1923), who describe lead and
iron salt media, respectively; or they may consult the manual of the
Difco Laboratories, who manufacture dehydrated media for the
purpose in question.
Churchman's gentian violet agar for selective bacteriostasis. To
ordinary beef- extract -peptone agar add a definitely determined
amount of crystal violet of about 85% dye content. If the medium
is to be used to inhibit Gram-positive organisms and permit the
growth of Gram-negatives the dye concentration should be about
1 :100,000. If it is to be used for differentiation between the Gram-
positives its concentration should be between 1 :400,000 and 1 :800,000;
if for differentiation between Grajn-negatives it should be between
1:1,000 and 1:40,000. In either of the two latter cases the exact
concentration depends upon which particular bacteria it is desired
to inhibit and which to permit to grow.
C. Media for Special Groups of Aerobes
1. BASAL MEDIA
Douglas trypsin broth {Hartley) (L&S No. 1123). Mix 150 g. of
lean minced horse meat with 250 ml. tap water and heat at 80°C in
a steamer. Add 250 ml. of an 0.8% Na2C03 (anhydrous) and cool to
45°C. Add 5 ml. of chloroform and 5 ml. of pancreatic extract pre-
pared as directed by Cole and Onslow (1916) and Douglas (1922).
Preparation of pancreatic extract: To 1000 g. minced fresh pig pancreas (free from
fat) add 3000 ml. distilled water and 1000 ml. 95% ethyl alcohol. Place in a large
clean bottle; shake repeatedly; and allow to stand 3 days at room temperature. Strain
through gauze and filter through paper. (Filtration is slow.) Add 1 ml. cone. HCl.
to each 1000 ml. of filtrate. This causes a cloudy precipitate which settles in a few
days and can be filtered off. The liquid keeps indefinitely if placed in a stoppered
bottle; no additional antiseptic is needed.
Estimation of activity: Centrifuge fresh milk and discard the cream; add 1% CaCl,.
Make a series of dilutions (1:100, 1:200. 1:500, 1:1000, 1:2000, 1:-1000, etc.) of the
pancreatic extract, and place in tubes, 1 ml. to the tube. To each tube add 1 ml. of
the milk. Place in a water bath at 50°C for 30 min. The highest dilution of trypsin
which causes clotting is a measure of its potency. Alcoholic pancreatic extract usually
causes clotting at 1:1000; Bacto-trypsin at 1:5000.
II44-10 MANUAL OF METHODS FOR PURE CULTURE STUDY
Incubate at ST^C for 6 hours, shaking frequently. Add 40 ml.
normal HCl and heat in the steamer for 30 minutes. Cool and filter.
Adjust to pH 8.0. Distribute as desired.
Pass steam through the autoclave for one hour then raise the pres-
sure slowly to 10 pounds and turn off the steam. For sterilization of
larger quantities (one litre in a flask) maintain the pressure at 10
pounds for 30 minutes.
Use: Preparation of diphtheria toxin, for growth of numerous
pathogens, and as medium for blood culture.
KracJce and Teasley medium. Dissolve 500 g. finely ground fat-
free heart muscle in 1000 ml. water. Place in ice-box overnight.
Filter through four layers of gauze, heat to boiling, and filter through
fine wire mesh or copper gauze.
Mix separately 500 g. finely ground brain in 1000 ml. water. Place
in ice-box over night. Filter and heat slowly to boiling; keep stirring.
Do not filter after heating.
Prepare medium as follows :
75 ml. heart muscle extract 1 g. glucose
25 ml. brain suspension 1 g. peptone
0.1 g. sodium citrate (NajCeHsO^+gH^O) 0.5 g. Na^HPO^ (anhydrous)
Heat until ingredients are in solution, adjust to pH 7.4, autoclave at
15 pounds for 15 minutes.
Use: Blood culture of pathogens.
Ascitic fluid agar. Various formulae have been proposed. A simple
one is as follows:
Melt 100 cc. of sterile Douglas' agar, pH 7.4-7.8, in a flask. Cool to about 48° to
50°C. With sterile pipette add 20 ml. of sterile, bile-free, ascitic fluid. Pour into
tubes or plates and allow to harden.
Use: Cultivation of pathogenic cocci.
Loeffler's blood serum. A common formula for this calls for glucose
beef-extract broth as its basis. This is prepared as follows:
Beef extract 3 g. Peptone (Difco or Witte) 10 g.
Glucose 10 g. NaCl 5 g.
Distilled water 1000 ml.
Mix the ingredients and dissolve by warming over a flame. Do not adjust the re-
action. Filter through paper. When the broth is cool add one volume to three vol-
umes of clear serum of horse, beef, or pig. Tube, 3 to 4 ml. per tube, and place tubes in
a slanting position in a pan or rack. Take care to prevent the occurrence of bubbles
and frothing. Cover tubes with newspaper. Sterilize in autoclave at 15 pounds for 15
minutes without letting the air out, and repeat on two successive days. Or if it is pre-
PREPARATION OF MEDIA 1I44-II
ferred to complete the sterilization in one day, heat for 15 minutes at 15 pounds without
letting the air escape; then let the air escape slowly while maintaining pressure, after
which the air vent should be closed and sterilization continued at 15 pounds for 16
minutes longer. After completion of the sterilization the pressure should be allowed to
fall very slowly.
Use: Cultivation of diphtheria organism.
Hunloons Hormone Heart Infusion Broth {L&S No. S'37). Mix 10 g. Bacto peptone,
10 g. gelatin, 5 g. NaCl, one whole egg and 500 g. of finely chopped beef heart in a
litre of water. Place in an enamel-ware vessel, e.g. a large coffee pot. Heat over a free
flame with constant stirring until the red color of the meat infusion changes to brown at
a temperature of about 68°C. Do not go beyond this temperature. Adjust to slightly
alkaline to litmus and then add 1.0 ml. additional A^/1 NaOH per litre of medium.
Cover the vessel and place In an Arnold sterilizer or in a water bath at 100° for one
hour. Remove the vessel from the sterilizer and separate with a glass rod the firm clot
which has formed from the side of the vessel. Return to the Arnold sterilizer at 100°
for 11^4 hours. Remove the vessel and allow to stand at room temperature for about
10 minutes in a slightly' inclined position. Pipette ofif the fluid portion or decant. If it
is poured through a fine wire sieve, many of the fine pieces of meat clot may be caught.
(Avoid filtering through cheese cloth, cotton or other absorbent materials.) Allow it to
stand in tall cylinders for 15 to 20 minutes until the fat present has risen to the surface
and been removed. The medium may be further cleared by filtering through glass
wool, asbestos wool, sedimentation or centrifugation. Add 0.15% dextrose and enough
laked blood to give a slight pink tint. Tube in 10 ml. lots. Sterilize by the inter-
mittent method.
Use: To cultivate highly pathogenic organisms.
Hitchens' Semi-solid Glucose Agar {Mulsow) (L &S No. 879) . Add 500 ml. of water to 1
pound of ground lean beef, and allow to stand at 37°C. for 48 hours. Express the juice
and add 20 g. peptone, 2 g. KNO3 and an equal amount (500 ml.) of a 0.2% agar so-
lution heated and cooled to 60°C. Adjust the reaction to + 0.9 to phenolphthalein.
Heat in the autoclave at 15 pounds pressure for 25 minutes. Filter and readjust the re-
action if necessary. Add 2 g. glucose. Final method of sterilization not specified.
Use: Cultivation of gonococcus and microaerophilic bacteria in general.
Egg Medium vnth Glycerol. Break several eggs into a graduated cylinder. Add y^
as much meat infusion or Douglas' broth as the amount of eggs. Add 1% glycerin.
Stir to mix, taking care not to cause the formation of air bubbles or frothing. Filter
through gauze. Tube and slant in a pan or rack. Sterilize in the autoclave at 15
pounds for 15 minutes.
Use: Cultivation of tubercle organism.
2. DIFFERENTIAL MEDIA
Endo medium. Dissolve 5 g. beef extract and 10 g. peptone in
1000 ml. water. Add 30 g. agar and cook in autoclave 45 minutes at
15 lbs. pressure. Filter; then add 10 g. lactose and sterilize 15
minutes at 10 lbs. pressure in small containers 100ml. in each. Just
before use prepare a 3% solution of basic fuchsin (85-90% actual dye
II44-12 MANUAL OF METHODS FOR PURE CULTURE STUDY
content) in 95% ethyl alcohol. Add 1 ml. of this fuchsin solution to
100 ml. of the agar (melted) also 0.125 g. anhydrous sodium sulfite
dissolved in about 5 ml. distilled water and pour plates immediately.
The medium should be light pink while hot and almost colorless after
cooling; as varying results may be obtained with different batches of
fuchsin, it is sometimes necessary to use a weaker stock solution of
that dye (e. g., 2% or occasionally only 1%).
Brilliant-green-bile medium. Dissolve 20 g. dried oxgall and 10 g.
peptone in 1000 ml. boiling water; cook in a double boiler or steam
for an hour. Add 10 g. lactose, and filter through cotton or cotton
flannel. Adjust reaction to between pH 7.1 and 7.3. Add 0.013 g. of
brilliant green (85-90% dye content). This concentration of bile and
dye is adjusted to permit the growth of bacteria of the colon-aero-
genes group, but to restrain or prevent the growth of Gram-positive
organisms, which often confuse diagnostic routine. When used in
water analysis, and more than 1 ml. of water is added to each tube, the
medium should be made of suflSciently greater concentration so that
the final dilution will be the same as that above indicated.
Levine's eosin-methylene-hlue agar. Dissolve by boiling:
Distaied water 1000 ml. K^HPO^ 2 g.
Peptone 10 g. Agar 15 g.
Before sterilizing add to 100 ml. of the above: 2 ml. sterile 2.0%
aqueous solution eosin Y (dye content about 85%), and 2 ml. sterile
0.325% aqueous methylene blue (dye content about 85%). Just
before use add aseptically 5 ml. sterile 20% lactose solution. Re-
action not adjusted. Do not filter.
Buffered peptone solution for methyl red and Voges-Proskauer
tests. Dissolve 7 g. peptone (Witte or Difco Proteose Peptone),
5 g. glucose and 5 g. K2HPO4 in 1000 ml. distilled water. Adjust
reaction to pH 6.9-7.0, and sterilize in the autoclave.
Blood broth. Add 5% of rabbit, sheep, or horse blood, drawn
aseptically and defibrinated, to beef extract broth or meat infusion
broth.
Blood agar. Prepare beef extract or meat infusion agar containing
2% (instead of 1.2%) agar. Melt 100 ml. of this, cool to 45°C,and
add 5 ml. of rabbit, sheep, or horse blood, drawn aseptically and
defibrinated. The medium should be poured into plates or slanted in
tubes very soon after adding the blood.
Bismuth-sulphite agar (Wilson and Blair, 1926; formula from Diagnostic Procedures
and Reagents, A. P. H. A., 1941, p. 25). To 1 litre nutrient agar (2% agar, 0.5% beef
PREPARATION OF MEDIA ii„-13
extract, and 1% peptone) add 45 ml. of 1% aqueous ferric citrate containing 11% of
1% aqueous brilliant green, also 200 ml. of bismuth sulfite mi.xture prepared as follows:
dissolve 6 g. bismuth ammonium citrate scales in 50. ml. boiling water, and 20 g.
anhydrous Na^SOj in 100 ml. boiling water, mi.x, bring to a boil, and dissolve 10 g.
anhydrous Na2HP04 in the mixture while boiling, cool and add 10 g. glucose dissolved
in 50 ml. boiling water, restore lost water. After mixing these two solutions with the
melted agar pour immediately into petri dishes; after 1-2 hr. at room temperature
these plates may be stored in a refrigerator, but must be used within 4 days.
Use: Enrichment of typhoid and paratyphoid groups.
Tellurite agar. (Anderson, et al. 1931). Add l}^ to 2 lbs. minced meat to 1000
ml. tap water at 48° C; after an hour squeeze out juice through cloth, leave in refrigera-
tor overnight and filter through filter paper. To 1000 ml. filtrate add 20 g. peptone
and 5 g. NaCl and dissolve at 45° C. Adjust reaction to pH 7.6. Filter first through a
Seitz K clarifying film; then sterilize by filtration through a sterile Chamberland candle,
collecting in sterile flasks and tubes. Incubate a few tubes for a check on sterility
but store the rest in a refrigerator. For use, mix with equal parts of 5% sterile agar
in water. Add 7-10% freshly drawn defibrinated rabbits' blood and 0.04% potassium
tellurite. Heat at 75° C for 10-15 minutes before pouring into plates.
Use: DifiFerentiation of diphtheria organism.
Desoxycholaie agar. (Lief son, 1935).
Water 1000 ml.
Peptone 10 g. Ferric ammonium citrate 2 g.
Agar 12-17 g. K^HPO 2 g.
NaCl 5 g. Sodium desoxycholate 1 g.
Lactose 10 g. Neutral red (1% aqu. sol.) 3 ml.
Dissolve the peptone in the water, adjust to pH 7.3-7.5, boil briefly and filter through
paper. Add the agar and dissolve by autoclaving; add 6 ml. of N NaOH, then the other
ingredients in the order named, omitting the neutral red until after a final adjustment
of the reaction to 7.3 or 7.5 as desired. Sterilize by heating in flowing steam only long
enough (i.e. about 15 minutes) to kill vegetative cells.
Use: Isolation of colon organisms from milk.
Desoxycholate-citrate agar. (Liefson, 1935). Mix 333 g. fresh, lean, ground pork
with 1000 ml. distilled water and allow to infuse for about an hour; add 3.3 ml. N HCl
and boil for about one minute; filter through paper and add 3.3 ml. N NaOH; boil for
one minute and filter through paper; bring volume up to 1000 ml. by adding distilled
water. Add 10 g. peptone and adjust reaction to about pH 7.5. Boil 2-3 minutes and
filter through paper; then add 20 g. agar and 5 ml. N NaOH; after at least 15 minutes
standing, melt agar by boiling or autoclaving. Add as rapidly as possible in the follow-
ing order: 10 g. lactose, 25 g. sodium citrate (NajCaHjOj-l-o^HjO), 3.5 mg. PbClj
(optional). Just before using, and while melted and held at a temperature of 80-
100° C, add 0.2% ferric ammonium citrate (green scales); adjust reaction to pH 74.
and add to each 100 ml. 0.2 ml. of 1% aqueous neutral red. (It is important that the
temperature of the medium at the time should be high enough to kill vegetative cells.)
Pour into plates without further sterilization.
Use: Isolation of typhoid organism from milk.
II44-14 MANUAL OF METHODS FOR PURE CULTURE STUDY
3. SYNTHETIC MEDIA
Ashby^s mannitol solution. In one litre of distilled water dissolve
the following:
Mannitol 20.0 g. NaCl 0.2 g.
K.HPO4 0.2 g. CaS04+2H,0 0.1 g.
MgS04+7H,0 0.2 g. CaCOi 5.0 g.
Method of sterilization not specified by author; autoclaving presumably satisfactory.
Use: Cultivation of Azotobacter.
Synthetic carbohydrate media. Peptone-free media are often valu-
able in measuring increases in hydrogen-ion concentration when only
small quantities of acid are produced. A formula slightly modified
from one proposed by Ayers, Rupp and Johnson (1919) is as follows:
NH4HaP04 1.0 g.
KCl 0.2 g.
MgS04+7H.O 0.2 g.
Water 1000 ml
Sugar (or other carbon source) .... 10 g.
This may be employed as a liquid medium without or with the addi-
tion of indicator; or as a solid medium with the addition of 15 g. of air-
dry agar. Used with agar for the detection of acidity, it is necessary
to have an indicator present.
Synthetic nitrate medium. A modification of the above is valuable
in detecting nitrate reduction in the case of some organisms that do
not produce nitrite from nitrate in a peptone medium.
Adjust to pH 7 by the addition of
> NaOH. About 6 ml. normal NaOH
required.
K2HPO4 0.5 g.
CaCla (anhyd.) 0.5 g.
MgS04+7H.O 0.2 g.
Glucose 10 g.
KNO3 1 g.
Distilled water 1000 ml.
To prevent precipitation of calcium phosphate, one or
the other of the first two salts listed should be dis-
solved separately in a portion of the water and added
after the other ingredients have been brought into
solution. No adjustment of reaction required.
D. Media for Anaerobic Bacteria^
Before listing the various media which are to be used for anaerobic
bacteria, it is necessary to introduce briefly the related topic — oxida-
tion reduction (0/R) potential.
The 0/R potential required for obligate anaerobes is in general low (Hewitt (1937),
Knight (1931), and Reed and Orr (1943).) The usual fluid medium is a complex of
active oxidation-reduction systems, but if the medium is prepared from peptone or
more simple constituents, usually it is necessary to include special substances to bring
the potential to the desired low level. The addition of a small amount (0.1%) of agar
will aid in the prevention of diffusion of atmospheric oxygen into the medium, but this
*This section has been prepared for the Committee by L. S. McClung.
PREPARATION OF MEDIA ii4,-15
is not sufficient aid for many species. If other actively reducing sul)stances are neces-
sary, the following are the most suitable: glucose, sodium thioglycollate (and thiogly-
collic acid), sodium formaldehyde sulfoxylate, ascorbic acid, sodium formate, gluta-
thione, and cysteine. Glucose seems to be generally satisfactory, but some of the
other compounds are toxic for certain types. Methylene blue (1-500,000) may be
added to culture media to serve as an O/ll indicator. Obligate anaerobes will grow
only in the portion in which the dye remains decolorized following cooling after steriliza-
tion.
The spore-forming anaerobes frequently have been divided into proteolytic and
saccharolytic groups. An organism of the former group possesses the ability to de-
compose complex proteins, usually with the production of offensive odors, sometimes
attacking a small variety of the simpler carbohydrates. The saccharolytic group, on
the other hand, usually show little action on complex proteins (except such com-
pounds as gelatin), but ferment a wide variety of the carbohydrates, usually with
copious production of gas.
Reference in this Leaflet is also made to the "pathogenic group" and the "butyric-
butyl group". The former term is used to designate such organisms as Clostridium
tetani, C. septicum, C. histolyticum, C. chauvoei, C. perfringens, (C. welchii), C. sporogenes,
and C. parabotulinum, etc., which grow best in the richer animal tissue infusions and
require a high degree of anaerobiosis. Representatives of the butyric-butyl group
include C. butyricum, C. beijerinckii, C. butylicum, C. pasteurianum, C. acetobutylicum,
C. felsineum, C. roseum, and C thermosaccharolyticum; they are less exacting with re-
gard to oxygen exclusion and grow best when supplied a fermentable carbohydrate.
Due to the diversity of physiological types within the anaerobic group it will be neces-
sary frequently to recommend two or more media for the same purpose.
All liquid media (except the thioglycollate medium and the semi-solid corn liver
medium) should be boiled 10 minutes, or heated in flowing steam for a similar period,
immediately prior to inoculation unless the medium is used on the same day it is initially
sterilized. The use of vaseline, mineral oil, or other seals at the surface of liquid media
is not recommended. If a liquid medium is used which will not remain reduced during
the desired incubation period, incubate the tubes in an anaerobic jar (see Leaflet III,
^tk Ed.).
1. ENRICHMENT AND GENERAL CULTIVATION MEDIA
Dehydrated Thioglycollate Medium^. This medium (Brewer, 1940a,
b) is obtained in dehydrated form from the manufacturers. After
dissolving, it is essentially a liquid (the percentage of agar being too
small to affect the fluidity) in which sodium thioglycollate acts as a
reducing agent. It also contains meat infusion, peptone, NaCl and
a phosphate, with or without glucose and methylene blue; for most
purposes the presence of these last two ingredients is recommended.
The medium compares favorably with other infusion media in ability
to initiate growth from small inocula (McClung, 1940, 1943).
The appropriate amount (indicated on bottle) of the dry powder is
dissolved in distilled water by brief heating, tubed or dispensed in
^Dehydrated thioglycollate medium. Baltimore Biological Laboratory, Baltimore,
Maryland, or Difco Laboratories, Detroit, Michigan. If the commercially prepared
medium is not available, a satisfactory substitute can be prepared by adding 0.1%
agar and 0.1% sodium thioglycollate to a meat infusion base medium.
ii,,-16 MANUAL OF METHODS FOR PURE CULTURE STUDY
deep columns in flasks or bottles, and sterilized 20 minutes at 15
lbs. pressure. Upon cooling, if methylene blue is present, a greenish
blue color should develop at the surface and sometimes to some distance
below the surface if the medium is disturbed; upon standing a short
time, however, the usual amber color indicative of anaerobiosis will
return. The medium may be stored {at room temperature, not in a
refrigerator) for several days, or even a few weeks, and used without
the heating, required by most media, to expel absorbed oxygen.
Use: Recommended as the medium of choice in the enrichment of
the pathogenic anaerobes. Particularly useful in hospital labora-
tories where small amounts may be made as needed from the dry
powder. Not recommended for isolation of the butyric-butyl group.
Since it is claimed that the thioglycollate not only maintains a low
0/R potential, but also combines with and inactivates most of the
mercurials, (Daily and Blubaugh, 1941; Blubaugh and Reed, 1943;
Nungester et at., 1943), this medium is suggested for use in the routine
sterility testing of biological materials including vaccines, serums,
catgut, etc. (Marshal et al., 1940; Federal Register, 1942).
Beef Heart {or beef tissue) Infusion Medium. Several different
formulae are available for this medium; although there seems to be
little reason to choose any particular one, in preference to another,
the following is satisfactory: Allow 500 g. of beef heart (or lean beef
meat) to stand overnight in refrigerator in 1,000 ml. of tap water.
Trim fat from the meat, and mince or grind before adding to the
water. Remove from icebox and boil over free flame for 15 minutes
or steam in Arnold sterilizer for 30 minutes. Separate tissue from
liquid by passing through two layers of cheese cloth in a fluted glass
funnel, and save both portions. Add 10 g. peptone and 5 g. NaCl to
the liquid after restoring to volume. If necessary, heat briefly to dis-
solve peptone. Adjust to pH 7.6 with 1 N NaOH and boil for 15-20
minutes or heat in Arnold sterilizer for 30 minutes. Filter through
paper. If needed immediately, tube broth over a 2 cm. column of
tissue, and sterilize 45 minutes at 15 pounds pressure. If not needed
immediately, sterilize broth in screw-capped bottles, and rapidly dry
tissue in incubator with forced circulation. These may be used at
any later time. Check the sterility of the medium before use by in-
cubation for at least 24 hours at 37° C.
Use: For enrichment or general cultivation of pathogenic anaerobes;
not suitable for the butyric-butyl group of the thermophilic anaerobes.
Has some diagnostic value as certain species produce a reddening of
the tissue. (Strongly proteolytic organisms cause a disintegration of
the meat tissue with the release of offensive odors.) Suitable for
stock cultures of most of the pathogenic types, as in most instances
(exception C. perfringens) spore production may be detected after
48 hours. Certain proteolytic species deposit crystals of tyrosine in
this medium upon extended incubation.
PREPARATION OF MEDIA ii,,-17
Beef liver infusion medium. Remove fat from 500 g. of fresh beef
liver, grind, and heat, with occasional stirring, in 1,000 ml. of tap
water for one hour in the Arnold sterilizer. Cool and strain through
cheese cloth. Restore filtrate to original volume and add 1% peptone
and 0.1% K2HPO4. Dry tissue (at 55° C. if available) as rapidly
as possible. Tube broth over several chunks of tissue. Use the
broth (before addition of peptone and phosphate) in the original
strength, or diluted five times. Sterilize 30 minutes at 15 lbs. pres-
sure. Avoid longer heating of medium as this diminishes its value
with respect to initiation of growth from small inocula.
Use: Recommended especially for enrichment, from spore stocks or
other sources, of the butyric-butyl group and C. perfringens. May
replace beef heart medium for pathogenic types. Useful for enrich-
ment medium in detection of thermophilic contamination of sugar,
starch, canned foods, etc. (Sometimes difiiculty is encountered
with this medium and the following one due to a Gram-positive rod
which develops as a contaminant during the drying of the liver
tissue.)
Corn Liver Medium. Add 50 g. of ordinary (white or yellow) corn meal and 10 g.
of dried liver powder^ to 1,000 ml. of tap water (McClung and McCoy, 1934). Heat in
flowing steam for 1 hour with occasional stirring. Remove from steam and cool al-
most to room temperature. Dispense in tubes, flasks, or bottles as may be needed.
Sterilize for 45 minutes at 15 pounds pressure. The resulting medium, on cooling,
should be semisolid with the coarser particles of corn settling to the bottom leaving a
2-3 cm. layer of starchy material at the top.
Use: A useful enrichment medium in studies of anaerobic population of natural
samples. (It remains anaerobic throughout prolonged incubation periods) . Especially
suited for the butyric-butyl group, and recommended for the detection of thermophilic
contamination. A very inexpensive and convenient medium suitable for sampling
surveys and other studies involving a large number of tubes. Has some diagnostic
value, as certain of the butyl groups give a characteristic "head" (a slimy mass of un-
fermented cellulosic material raised and collected at the top of the liquid) in this
medium in contrast to the butyrics which usually do not give this reaction.
2. MEDIA FOR PLATING FOR PURIFICATION
For the pathogenic types a good medium can be made from the
liquid obtained by the infusion of beef heart or lean beef tissue, as
discussed above, either with or without 0.5% glucose or defibrinated
blood or both. Similarly, the butyric-butyl group grow well on a
solidified medium prepared from liver broth, with the addition of
0.5% glucose.
T hi ogly collate agars. For the pathogenic types Reed and Orr
(1941) suggested two other media which may be prepared from de-
hydrated ingredients which are available commercially. One of
these is made by adding 2% agar (for surface colonies) or 0.75%
agar (for subsurface colonies) and 0.1% glucose to Brewer's thiogyl-
collate broth, adjusted to pH 7.6 before sterilization. (The medium
^Dried liver powder. Difco Laboratories, Detroit, Michigan.
n„-18 MANUAL OF METHODS FOR PURE CULTURE STUDY
with the smaller percentage of agar is preferred by some for seeded
plates as an aid in securing discrete colonies.) ' An alternate formula
is as follows:
Proteose peptone 20 g. Na2HP04 2 g.
Glucose 1 g. Sodium thioglycollate 1 g.
Agar 20 g. (or 7.5 g. for subsurface colonies)
Distilled water 1000 ml.
Adjust 7.6 pH. If used for subsurface colonies, clarify medium
by filtration through paper using reduced pressure.
Use: Satisfactory for routine purification and colony study of
pathogenic types. Convenient to prepare, since the ingredients are
usually available and for fresh meat extracts are not needed.
Yeast infusion glucose agar. Prepare yeast infusion as follows (although other
methods, sometimes preferred, are equally satisfactory): Obtain fresh yeast (starch-
free if possible) from a fermentation company and add 10% by weight to several liters
of tap water. Autoclave for 3 hours or more. Allow cells to settle by standing for
several days at room temperature. Remove liquid infusion by syphon or with the
Sharpies centrifuge. Sterilize the liquid, after removal from the cells, in screw-capped
bottles and store indefinitely. For plating medium add 0.5% glucose and 2.0% agar.
Adjust to 7.0 pH; sterilize for 20 minutes at 15 pounds pressure. (Note: An equally
satisfactory, but considerably more expensive, basal medium may be prepared from
dehydrated yeast extract, adding 0.5% yeast extract to distilled water).
Use: Recommended as plating medium for butyric-butyl group.
Peptone-try ptone-glucose agar. If a source of yeast for the preparation of yeast in-
fusion is not readily available, the following plating medium may be substituted which
is only slightly less satisfactory than the one above.
Peptone 0.5% Glucose 0.5%
Tryptone 0.5% Agar 2.0%
Adjust 7.0 pH before sterilization. (The medium is improved by the addition of
100 ml. of liver infusion, if available).
Use: A satisfactory plating medium for the butyric-butyl group, calling for ingre-
dients which are usually available.
3. MEDIA FOB DETERMINATION OF PHYSIOLOGICAL REACTIONS
Sugar-free Base for Qualitative Fermentative Reactions'. Two
basal media for use in anaerobic fermentation reactions are given
here. Certain general directions are necessary: Indicators should
be used to test reaction after incubation or on small samples with-
drawn during incubation; they should not be incorporated in the
medium, as many anaerobes reduce them to their leuco form. The
^Some workers have used a meat infusion broth or other medium which has been
rendered sugar-free by fermentation with Escherichia coli or Clostridium perfringens
This seems unnecessary at the present time as most species will grow quite well in one
or the other of the media suggested here. If a particular strain should not grow well
in the basal medium plus glucose, it is probable that some needed nutrient is not
present. For these, as with fastidious aerobes, ascitic fluid may be added, though this
will rarely be necessary.
For quantitative studies on fermentation of the sugars the usual problem requires a
base medium suitable for the butyric-butyl group. Perhaps the most generally useful
basal medium is yeast water infusion prepared according to the method discussed for
yeast infusion glucose agar.
PREPARATION OF MEDIA ii„.19
following fermentable carbon sources are usually suflBcient for
differentiation of the common species: lactose, glucose, salicin, sucrose
and maltose. The next most useful list includes: mannitol, glycerol,
starch, pectin, and cellulose. If there is question concerning the
effect of heat on the carbon compound, a concentrated solution may
be sterilized by filtration and added aseptically to the basal medium
after heat sterilization. In the establishment of the characteristics
of new species list the reaction on all the commonly available carbo-
hydrates.^
Fermentation Basal Medium of Reed and Orr (19/^1). Dissolve the
following in 1,000 ml. of distilled water;
Peptone or proteose peptone 20 g. Sodium thioglycollate 1-0 g.
NaCl 5 g. Agar 1.0 g.
Carbohydrate 10 g.
Use: Recommended for pathogenic group but not for butyric-
butyl group.
Fermentation Basal Medium of Spray (19S6). Dissolve the follow-
ing in 1,000 ml. of distilled water:
Neopeptone 10 g. Agar 2.5 g.
Tryptone 10 g. Carbohydrate 10 g.
Adjust to pH 7.3 or 7.4.
Use: Recommended for all types.
Medium for Testing Action on Litmus Milk. This medium is as
important with the anaerobes as it is with the aerobes and in fact
Spray (1936) used the reactions in this medium as one of the primary
characters in his system of classification.
Use either fresh skimmed milk or spray-dried milk powder. In
the latter case, mix 90-100 g. of powder with 1000 ml. of distilled
water. Prepare a paste with a small amount of water and then dilute
this with the remainder of the water. Use the Waring Blendor^ or
other mixing machine if available. Strain through cheesecloth and
adjust to pH 6.8. Dispense in tube to which 0.05-0.1 g. of reduced
iron^'' is added before the tubing process. If reduced iron is not
available, replace the iron powder with a strip of No. 26 gauge black
stove-pipe iron. Sterilize by intermittent process or by autoclaving
for 15 minutes at 15 pounds. Immediately on removal from auto-
clave cool the tubes by standing them in cold water. Anaerobic
seal is unnecessary as the reduced iron keeps the oxidation-reduction
potential at a low level.
*When interpreting results, make note of the following:
If an organism fails to grow in the basal medium, unless a fermentable carbon source
is present, presence of growth indicates ability to ferment the compound in question.
Gas production, per se, is not proof of carbohydrate fermentation, as many anaerobic
species are highly proteolytic and may produce gas in the cleavage of protein.
'Waring Corporation, 1697 Broadway, New York City.
^"Iron reduced by hydrogen, from Merck Company, Rahway, New Jersey.
1144-20 MANUAL OF METHODS FOR PURE CULTURE STUDY
Use: Satisfactory for the determination of those characters usually
revealed by litmus milk. Of diagnostic aid in the search for C.
perfringens, due to the fact that this organism gives a stormy fer-
mentation.
Note: This test is not strictly specific for C. perfringens as certain non-pathogenic
motile species of the butyric-butyl group also give this reaction. They may be separated
from C. perfringens by virtue of the non-motility of the latter. Robinson and Stovall
(1939) recommend the addition of 1.0 ml. of 20% Na^SOj solution and 0.1 ml. of 8%
FeClj solution to 10 ml. of milk as an additional aid in the diagnosis of C. perfringens.
This organism produces a blackening reaction.
Medium for Liquefaction of Gelatin. For some species standard
nutrient gelatin plus 0.25% glucose may serve as a base medium for
testing for liquefaction of gelatin. If the organism in question will
grow in such a medium, it is recommended for use. For other species
choice may be made between the two formulae which follow:
Gelatin Medium of Reed and Orr (19^1). Dissolve the following ingredients in
1,000 ml. of distilled water:
Gelatin 50 g. Na2HP04 2 g.
Peptone 10 g. Glucose 1 g.
Sodium thioglycollate 1 g.
Gelatin Medium of Spray (1936). Dissolve the following ingredients in 1,000 ml. of
distilled water:
Difco Nutrient Gelatin 128 g.
Glucose 1 g.
Dissolve gelatin in water taking care not to scorch the gelatin. Include a strip of No.
26 gauge black stove-pipe iron in each tube.
Use: Either of the above media may be used for the pathogenic group. The medium
of Spray has the additional advantage of being a presumptive medium for C. his-
tolyiicujti as this organism gives an orange to wine-red color within the first 48 hours of
incubation.
Other Media for Testing Proteolytic Action. The action on gelatin
represents action on a simple and incomplete protein and positive
action is not necessarily an indication that the organism can hydrolyze
the complex proteins. The beef heart infusion represents one of the
media in which putrefactive action on complex proteins may be re-
corded. Coagulated serum slants, prepared in the usual manner,
inoculated and incubated in an anaerobic jar, represent another type
of protein to be tested. Evidence of proteolytic action in this
medium is shown by partial or complete liquefaction of the medium.
For action on coagulated egg albumin include a small cube of the
white of a hard boiled egg in a tube of 1% peptone and 0.2% glucose
broth or other liquid medium. Disintegration of this cube during the
incubation is evidence of proteolytic action. Peptonization of
litmus milk reveals caseinolytic ability. In addition to the above
three other media are recommended. It may not be necessary to
use all of these but more than one should be included in taxonomic
studies because of possible differential reactions.
Alkaline Egg Medium. Mix the yolk of two and the whites of four
eggs (preferably in Waring Blendor). Add 1,000 ml. of distilled
water and 12 ml. of 1 A^ NaOH. Stir well or mix in Waring Blendor.
Add one part of the above to 5 parts of nutrient broth (beef extract
PREPARATION OF MEDIA ii,,-21
and peptone). Tube in deep columns and autoclave for 20 minutes at
15 pounds. The final medium should be an opaque whitish liquid.
Proteolysis is indicated by progressive clearing of the medium.
Brain Medium. Secure fresh sheep (or calf) brains which are as
free as possible from injury. Using forceps clean blood and mem-
branous material from brain tissue. Add distilled water, in the
ratio of 100 ml. of water to 100 g. of brain, and boil slowly for one
half hour. Put brains through potato ricer. Add 1.0% peptone and
0.1% glucose to the resulting mixture and heat slightly to put peptone
in solution. Tube in deep columns while the mixture is stirred in
order to effect an even distribution of the brain tissue. Reduced
iron or a strip of black stove-pipe iron or iron wire may be added to
the tube before tubing the liquid mixture. Sterilize in autoclave for
30 minutes at 15 pounds and check sterility by incubation at 37° C.
for a minimum of 24 hours. The finished medium has approximately
an equal amount of liquid broth above the brain particles. Proteoly-
sis is indicated by putrefactive odors, a disintegration of the particles
and a blackening reaction.
Use: The blackening reaction of this medium has some diagnostic
significance (Hall and Peterson, 1924). This medium is also valu-
able for many species for the production of spores and hence as a
stock culture medium.
Milk Agar for Testing Proteolytic Action. Reed and Orr (1941) suggest the follow-
ing medium: Mix equal parts of skim milk (reconstituted from powder) and a plating
agar (see their media in section on plating media for purification). Autoclave the
two media separately and mix just before pouring. Proteolysis is indicated by a wide
clear zone surrounding the growth.
Medium for Production of H2S. Probably most, if not all, species
of anaerobes produce H2S, at least in trace amounts. From the dis-
cussion of McCoy, et al. (1926), Spray (1936), Pacheco e Costa (1940)
and Reed and Orr (1941), we conclude that there is, as yet, no stand-
ard medium for this reaction. The media listed below were found to
be satisfactory by Reed and Orr (1941); and it is recommended that
the exact method of preparation be listed in published reports for
any additional medium which may be devised.
Medium 1
Proteose peptone 20 g. Glucose 1 g.
Na.HP04 2 g. Agar 2 g.
Water 1000 ml.
Dissolve ingredients, adjust to pH 7.6, and add 10 ml. of 2% lead
acetate. This results in a cloudy precipitate which, however, re-
mains after autoclaving in a reasonably stable suspension.
Medium 2
Proteose peptone 20 g. Glucose 1 g.
NajHP04 2 g. Water 1000 ml.
Dissolve ingredients, adjust to pH 7.6, and add 10 ml. of a 1.5% bis-
muth and ammonium citrate solution. This ordinarily produces a
solution which remains clear after autoclaving.
Medium for the Formation of Indole and Skatole. The foUov/ing
medium will usually be found satisfactory:
11,4-22 MANUAL OF METHODS FOR PURE CULTURE STUDY
Tryptone (Bacto) 20 g. Sodium thioglycollate (for
NejHPO, 2 g. pathogenic group only) Ifg.
Glucose 1 g. Agar 1 g.
Water 1000 ml.
Application of test (see Roessler and McClung, 1943): Place 2 drops offthe culture
(withdrawn by pipette) in a spot plate; add 2 drops of vanillin (5% in|95% ethyl
alcohol) and then 3 drops of concentrated HCl. The addition of one drop of 0.1%
NaN02 causes the violet-pink of skatole to become dark purple but the orange"color
characteristic of indole is not changed.
Medium for Nitrate Reduction. (See Reed, 1942). As certain
species reduce nitrites as well as nitrates, there should be included a
test for the presence (or disappearance) of nitrates as well as the ap-
pearance of nitrites. A negative nitrite test is of no significance.
The medium of Reed and Orr (1941) is satisfactory:
Tryptone (Bacto) 20 g. Agar 1 g.
Na.HP04 2g. KNOj 1 g.
Glucose 1 g. Water 1000 ml.
Adjust pH to 7.6 before autoclaving.
4. OTHER MEDIA OF VALUE
Medium for demonstration of capsules and spores. It is sometimes
inconvenient to use animal autopsy material for demonstration of
capsules. Svec and McCoy (in press) recommend the following
medium for demonstration of capsules and spores of C. perfringens.
Presumably it will be suitable for other species.
Casein hydrolysate (acid) 35 ml. K2HPO4 5 g.
Ovalbumin hydrolysate (acid) . . 15 ml. Sodium thioglycollate 1 g.
Yeast water (prepared by auto- (NH4)2S04 2 g.
claving 20% wet weight of Tryptophane 12 mg.
yeast in water) 100 ml. Glucose 2.5 g.
Sodium lactate 5 ml. Distilled water to make 1000 ml.
Adjust pH to 7.4 and sterilize 25 minutes at 15 pounds.
To prepare acid hydrolysates : Autoclave 200 g. casein (or egg
albumin), 110 ml. concentrated HCl and 170 ml. distilled water for
45 minutes at 12 pounds. If desired, decolorize with norite.
Medium for spore production by butyric-butyl group. If cultures of
this group do not sporulate readily on plain corn mash (prepared ac-
cording to directions for corn-liver medium except that the liver
powder is omitted), use potato infusion prepared as follows:
Irish potatoes 200 g. (NH4)2S04 1 g.
Glucose 5 g. CaCOj 3 g.
Tap water to make 1000 ml.
Peel potatoes and add water. Steam for one half hour or boil
slowly until soft and put through potato ricer. Add other ingredients
and bring up to original volume. Cool and tube, with stirring, so as
to obtain an even distribution of the potato particles.
Medium for toxin production. In Leaflet III there is mention of
the fact that beef heart infusion or glucose meat infusion is satis-
factory for toxin production by most toxigenic species. Another
medium, proposed by Reed, Orr, and Baker (1939), may be recom-
mended for the gangrene group. This is prepared from commercially
available ingredients as follows:
PREPARATION OF MEDIA ii,,-23
NaCl 2.0 g. Gelatin, Difco 50.0 g.
MgS04 0.02 g. Peptone, Bacto 10.0 g.
Na,HP04 5.76 g. Glucose 2.0 g.
KH.PO4 0.24 g. Water 1000 ml.
Adjust to pH 7.7 and autoclave at 15 pounds.
REFERENCES
American Public Health Association. 1936. Standard methods of Water .Analy-
sis. Eighth edition. Published by the Association, New York, 1936.
Anderson, J. S., Happloid, F. C, McLeod, J. W. and Thomson, J. G. 1931. On
the existence of two forms of diphtheria bacillus — B. diptheriae gravis and
B. diphtheriae mitis — and a new medium for their differentiation and for the
bacteriological diagnosis of diphtheria. J. Path. & Bact., 34, 667-81.
Aters, S. H., Rupp, p., and Johnson, W. T. 1919. A study of the alkali-forming
bacteria in milk. U. S. Dept. Agric, Bui. 782.
Bailey, S. F., and Lacy, G. R. 1927. A modiGcation of the Kligler lead acetate
medium. J. Bact.. 13, 183-9.
Blubaugh, L. v., and Reed, W. 1943 Sodium thioglycollate as an antibacteno-
static agent. Its use in sterility testing. J. Bact., 45, 44.
Brewer, J. H. 1940a. A clear liquid medium for the "aerobic" cultivation of
anaerobes. J. Bact., 39, 10.
Brewer, J. H. 1940b. Clear liquid mediums for the "aerobic" cultivation of
anaerobes. J. Amer. Med. Assoc, 115, 598-600.
Clark, W. M. 1928. The Determination of Hydrogen-ions. Third edition. Williams
and Wilkins, Baltimore.
Cohen, Barnett. 1922. Brom cresol green, a sulfonphthalein substitute for methyl
red. Proc. Soc. Exp. Biol. Med., 20, 124.
Cohen, Barnett. 1927. Synthesis and indicator properties of some new sulfon-
phthaleins. Public Health Repts., 41, 3051.
Cole, S. W. and Onslow, H. 1916. A substitute for peptone and a standard
nutrient medium for bacteriological purposes. Lancet, 1916, II, 9-11.
Daily, F. K., and Blubaugh, L. V. 1941. The elimination of bacteriostatic action
by the use of sodium thioglycollate medium. J. Bact., 42, 147-148.
Douglas, S. R. 1922. A new medium for the isolation of B. diphtheriae. Brit. J.
Ex]3. Path., 3, 263-7.
Federal Register. 1942. 7, No. 26, 781-2.
Hall, I. C. 1921. Criteria in anaerobic fermentation tests. J. Inf. Dis., 29, 321-43.
Hall, I. C. and Peterson, E. 1924. The discoloration of brain medium by an-
aerobic bacteria. J. Bact., 9, 211-24.
Hewitt, L. F. 1937. Oxidation-reduction potentials in bacteriology and bio-
chemistry. 4th edition. London County Council.
Hitchens, a. p. 1921. Advantages of culture mediums containing small percent-
ages of agar. J. Inf. Dis., 29, 390-407.
HuNTOON, F. M. 1918. "Hormone" medium; a simple medium employable as a
substitute for serum medium. J. Inf. Dis., 23, 169-72.
Knight, B. C. J. G. 1931. Oxidation-reduction potential measurement in cultures
and culture media. Chapter XIII (pp. 165-73) in Vol. IX of System of
Bacteriology. (Gt. Brit.) Med. Research Council.
Kracke, R. and Teasley, H. E. 1930. The eflBciency of blood cultures. J. Lab.
& Clin. Med.. 16, 169.
II44-24 MANUAL OF METHODS FOR PURE CULTURE STUDY
LiEFSON, EiNAR. 1935. New culture media based on sodium desoxycholate for the
isolation of intestinal pathogens and for the enumeration of colon bacilli in
milk and water. J. Path. & Bact., 40, 581-99.
Levine, Max, and Schoenlein, H. W. 1930. A Compilation of Culture Media for
the Cultivation of Microorganisms. Williams and Wilkins, Baltimore.
McClung, L. S. 1940. The use of dehydrated thioglycollate medium in the enrich-
ment of spore-forming anaerobic bacteria. J. Bact., 40, 645-8.
McClung, L. S. 1943. Thioglycollate media for the cidtivation of pathogenic
Clostridia. J. Bact., 45, 58.
McClung, L. S., and McCoy, E. 1934. Studies on anaerobic bacteria. L A corn-
liver medium for the detection and dilution counts of various anaerobes.
J. Bact., 28, 267-77.
McCoy, E., Fred, E. B., Peterson, W. H., and Hastings, E. G. 1926. A cultural
study of the acetone butyl alcohol organism. J. Inf. Dis., 39, 457-83.
Marshall, M. S., Gunnison, J, B., and Luxen, M. P. 1940. Test for the sterility
of biologic products. Soc. Expt. Biol, and Med., Proc, 44, 672-3.
Nungester, W. J., Hood, M. N., and Warren, M. K. 1943. The use of thiogly-
collate media for testing disinfectants. J. Bact., 45, 44.
Pacheco, G., E Costa, G. A. 1940. Produgao de H2S pelos clostridios anaerobios.
Mem. Inst. Oswaldo Cruz, 35, 311-6.
Reed, G. B., and Orr, J. H. 1941. Rapid identification of gas gangrene anaerobes.
War Med., 1,493-510.
Reed, G. B., and Orr, J. H. 1943. Cultivation of anaerobes and oxidation-reduction
potentials. J. Bact., 45, 309-20.
Reed, G. B., Orr, J. H., and Baker, M. C. 1939. Gas-gangrene- toxin production.
Soc. Expt. Biol, and Med., Proc, 42, 620-1.
Reed, R. W. 1942. Nitrate, nitrite and indole reactions of gas gangrene anaerobes.
J. Bact., 44, 425-31.
Robinson, C. L., and Stovall, W. D. 1939. A clinical bacteriological test for the
recognition of C. welchii in wounds. Amer. Jour. Clin. Path., Technical
supplement, 9, 27-32.
Roessler, W. G., and McClung, L. S. 1943. Suggested method for use of vanillin
as a test reagent for indole and skatole production by bacteria. J. Bact.,
45, 413.
Spray, R. S. 1936. Semisolid media for cultivation and identification of the sporu-
lating anaerobes. J. Bact., 32, 135-55.
Svec, M. H., and McCoy, E. In Press. A chemical and immunological study of the
capsular polysaccharide of Clostridium perfringens. J. Bact., in press.
TiTTSLER, R. P. and Sandholzer, L. A. 1936. The use of semi-solid agar for the
detection of bacterial motility. J. Bact., 31, 575-80.
Wilson, W. J. 1923. Reduction of sulphites by certain bacteria in media containing a
fermentable carbohydrate and metallic salts. J. Hyg., 21, 392-8.
Wilson, W. J. and Blair, E. M. M'V. 1926. A combination of bismuth and sodium
sulphite affording an enrichment and selective medium for the typhoid-
paratyphoid groups of bacteria. J. Path, and Bact., 29, 310-1.
Winogradsky, S. and Omelianski, V. 1899. Ueber den Einfluss der organische
Substanzen auf die Arbeit der nitrifzierenden Mikroben. Centbl. f. Bakt.
II Abt., 5, 329-43, 377-87, 429-40.
LEAFLET III
THE STUDY OF OBLTGATELY ANAEROBIC
BACTERIA
Prepared by
COMMITTEEMAN ON ANAEROBIC METHODS
4th EDITION
NOVEMBER. 1943
Note — The first edition of this leaflet was written, and the second edition revised,
by Ivan C. Hall. The third edition, prepared by J. Howard Brown, represented
revision of certain sections of the second edition. This edition has been prepared for
the Committee by L. S. McClung.
LEAFLET III
THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA'
It is impossible to list here all of the methods which have been
proposed for the study of anaerobic bacteria; an attempt is made,
however, to outline a number of technics which have been used
widely and which should ordinarily be suitable for routine studies
of anaerobic species. Those interested in other technics are advised
to consult Section B of the subject index bibliography relating to
the anaerobic bacteria (McCoy and McClung, 1939; McClung and
McCoy, 1941). The worker who has had no experience with anaero-
bic bacteria should study some of the articles which deal with prin-
ciples of anaerobic culture or which record the results of a study
of a considerable number of strains (Committee upon anaerobic
bacteria and infections, 1919; Fildes, 1931; Hall, 1922, 1928, 1929;
Heller, 1921; Knorr, 1923, 1924; McCoy, et al, 1926, 1930; Mcintosh,
1917; Meyer, 1928; Reed and Orr, 1941; Robertson and O'Brien,
1929; Soule, 1932; Spray, 1936; Zeissler, 1930; Zeissler and Rassfeld,
1928). These are suggested rather than the monographs (Hibler,
1908; Weinberg et Seguin, 1918; Weinberg et Ginsbourg, 1927;
Weinberg, Nativelle, et Prevot, 1937) which are not distributed
widely.
The organisms which we call obligate anaerobes, are those that
require strict exclusion of atmospheric oxygen from the immediate
environment in which they are to grow. It is not easy to answer
the question of the best method of determining whether or not a
given organism is an obligate anaerobe. The catalase reaction,
when applied to pure culture, gives presumptive evidence, for obligate
anaerobes usually are catalase-negative. For this reaction a plate
culture of the organism in question is flooded with a 10% solution
of H2O2. The evolution of gas bubbles from the colonies denotes
the presence of catalase.
If the proper material for the catalase reaction is not available.
'The methods and technics suggested herein are those recommended for use with
the more common spore-forming anaerobic species. Many of these methods are suit-
able, also, for the study of the non-spore-forming types, and for the present no attempt
will be made in this Leaflet to outline particular methods of study for these. If the
technics herein outlined do not prove satisfactory, the worker interested in the patho-
genic non-spore-formers should consult the re%'iew of Dack (1940) and the publications
of Prevot (1924, 1925, 1938, 1940a, 1940b). Non-pathogenic types exist, as for
example, the methane organisms discussed by Barker (1936). For the complete
literature on all types refer to Section Id (non-spore-formers) in the bibliography of
McCoy and McClung (1939) and McClung and McCoy (1941).
III. ,-3
III43-4 MANUAL OF METHODS FOR PURE CULTURE STUDY
or in case of doubt, the following technic will usually suffice to
characterize an anaerobic strain and to differentiate if from the
aerobes: Inoculate, while the agar is molten, several deep tubes
(8-9 cm. columns of medium) of a suitable nutrient agar medium
(see Leaflet II) containing 1.0% glucose; allow these to solidify
in an upright position, and incubate the tubes at several tempera-
tures or at the optimum temperature for the organism in question;
adjust the seeding so that relatively few (e.g., 25-50) colonies per
tube will result. With an obligate anaerobe, all of the colonies
should be localized in the bottom of the tube and none should appear
on the surface or in the upper 1 cm. layer. Likewise, with pathogenic
organisms cultured in fluid thiogly collate medium, the growth should
be confined to the lower section of the medium and no growth
should result in the upper layer wherein the methylene blue is
recolorized. If growth does occur in the upper layer of either
medium, the culture is either not an obligate anaerobe or is con-
taminated with an aerobic or a facultative species.
ANAEROBIC CULTURE METHODS AND EQUIPMENT
All of the procedures which have been devised for the cultivation
of anaerobic bacteria have the single purpose of excluding atmospheric
oxygen from the environment in which the growth is to take place.
With certain tubed media the oxygen potential may be reduced
sufficiently by constituents of the medium to permit anaerobic
growth (Hewitt, 1937; Knight, 1931; and Reed and Orr, 1943).
Since, however, this is rarely possible for surface cultures on a solid
medium, usually plate and slant cultures are incubated within a
closed container from which the oxygen is removed by one or another
means. A study of the various methods shows that no single
procedure may be proposed as the best technic but that the method
of choice will depend upon the prevailing circumstances. A pro-
cedure which is ideal for one situation may be impractical or im-
possible to apply with 'other conditions. Each of the technics out-
lined below is recommended within the limits proposed in the dis-
cussions.
Use of Methylene Blue as Indicator of Anaerobiosis. For all types of anaerobic jars
and containers, except individual plating or tube culture systems, it is convenient
to include an indicator tube which will serve as a check on the development of anaero-
biosis. The most commonly used system utilizes the change of methylene blue from
the colored (oxidized state) to the leuco form (reduced state) Using the solution
prepared as given below, any system which gives sufficient degree of removal of
oxygen from the atmosphere for anaerobic growth to develop will cause the blue color
of the solution to disappear or will maintain the colorless condition if the solution is
THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA in„-5
boiled (heat reduction) immediately prior to its being placed in the container. A some-
what less sensitive system can, in an emergency, be prepared by adding a tinge of color
from Loeffler's alkaline methylene blue to a tube of glucose broth.
Tiie procedure recommended (Fildes, 1931) is: Prepare three stock solutions: (1)
CO ml. N/10 NaOH diluted to 100 ml. with distilled water; (2) 3.0 ml. 0.5% aqueous
methylene blue diluted to 100 ml. with distilled water; (3) 6.0 g. of glucose in 100 ml.
distilled water to which has been added a small crystal of thymol.
Each time the indicator solution is needed, mi.x equal parts of the three solutions
in a test tube and boil in a cup of water until the color disappears. Place tube in
anaerobic container immediately and begin process of securing anaerobic conditions.
If the container is satisfactorily deoxygenated, the color in the solution should not
reappear. If the blue color does return it is a sign that the container leaks or has not
been satisfactorily exhausted of oxygen. (In the vegetable tissue jar, to be described,
the color may appear but will disappear with the development of anaerobiosis during
the incubation period).
Biological Methods for Oxygen Removal
vegetable tissue jar
Materials for method of McClung, McCoy and Fred (1935):
(1) Jar, or other container which may be sealed air tight {Recom-
mended: 6" X 18" or 6" X 12" Pyrex cyhnder^); (2) square (7" X 7")
of plate glass or a glazed plate; (3) plasticene^, 3^ pound; (4) glass
tumbler; (5) supply of oats or other grain (other tissues, particularly
chopped Irish potatoes, may be used, but are less conveniently
stored for occasional use, and in some cases produce objectionable
odors which are evident when the jar is opened) ; (6) tap water.
Method: Place inverted tumbler (if plates are to be used), or
other support, in bottom of cylinder. Add oats to fill at least one
tenth of the capacity of the cylinder. Add sufficient tap water to
moisten the oats. Stack plates (or other cultures) on support.
Add tube of methylene blue solution (see above). Place layer
of plasticene (previously softened by placing in incubator) on rim
of cylinder. Push plate glass square firmly against plasticene;
using fingers, press the clay against both the square and the cylinder
until a satisfactory seal is obtained. Place jar in incubator immedi-
ately. (A 40-48-hour incubation period is recommended).
If plate cultures are employed, use unglazed porcelain ("clay")
tops'* to replace the ordinary petri dish cover to absorb the moisture
which collects within the cylinder. If porcelain tops are unavailable,
add a petri dish lid containing CaCU to absorb the moisture.
^Pyrex cylinder. Corning Glass Works, Corning, New York or supply house.
Pyrex Catalogue No. 850.
^Plasticene The most satisfactory product of this type seems to be the English
clay called "Plasticene" (gray or green colored). This is obtainable in this country
from J. L. Hammet Company, Cambridge, Massachusetts, and perhaps other supply
houses. Other types may be found which are satisfactory but these must be tested
individually for suitability as some have been encountered which dry to a hard cake
upon incubation.
^Unglazed porcelain ("clay") tops for Petri dishes. The Coors porcelain dish, sold
by Arthur H. Thomas Company, has been found to be more uniform in size and quality
than others tested.
iii43-« MANUAL OF METHODS FOR PURE CULTURE STUDY
Advantages: The method is inexpensive and employs easily available materials.
No special apparatus is required — an advantage in laboratories where anaerobic
cultures are not usually prepared. It may be used at any incubation temperature
without danger of explosion. It is particularly suitable in problems requiring large
numbers of plate cultures. It is recommended especially for cultural and physiologi-
cal studies of strains which have been purified by other methods. Disadvantages:
Several hours may be needed for anaerobic conditions to become established and there-
fore the method is not suitable when the results are required quickly. It is not recom-
mended for routine clinical use where speed of isolation of pure culture is an important
factor. With certain enrichments it is not suitable for purification of species con-
taminated with aerobic spore-forming bacteria due to the quick growth of these forms.
In plate culture experiments, as in the isolation of new strains, no one plate may be
removed from the cylinder for observation until the end of the incubation period, for
to do so would destroy the anaerobic conditions within the cylinder.
USE OF AEROBE TO ABSORB OXYGEN
Another biological method for oxygen removal utilizes the growth
of an aerobic organism (usually Staphylococcus aureus, Serratia
marcescens, or Saccharomyces cerevisiae). A wide variety of applica-
tions of this system have appeared in the literature. The technics
suggested^ below involve the growth of the aerobic organism in
pure culture on a medium separate from that on which the anaerobe
is to be cultured.
Method A
Materials for method of Snieszko, 1930: (1) Two petri dishes of
ordinary size; (2) paper tape, scotch tape, adhesive plaster, or
plasticene; (3) culture of Serratia marcescens or other fast growing
aerobic organism; (4) tube of nutrient agar.
Method: Select two petri dishes which have bottoms of exactly
the same size and sterilize these in position in their usual top sections.
Pour nutrient agar into the bottom half of plate A, and after solidifica-
tion, streak the medium heavily (or flood across surface with 0.5 ml.
of broth culture) with the aerobic organism. (As an alternate
method, seed the agar before pouring.) Pour into plate B, a medium
suitable for the anaerobe (see Leaflet 11, 9th Ed.); when hard streak
with the sample or culture of the anaerobe (or seed with the latter
prior to pouring).
Remove the two bottoms from their respective tops and fit to-
gether at their rims. Use tape or other sealing device around the
juncture to provide an air-tight seal. Place plate in the incubator
immediately. If thermophilic anaerobic cultures are to be made,
replace the »S. marcescens by a thermophilic aerobe, or before placing
plates in thermophilic incubator, incubate for 18 hours at 32° C. to
allow S. marcescens to grow and to use the oxygen.
Advantages: No elaborate equipment is needed, since the method uses ordinary
peLri plates and other common materials. Thus it is available as an emergency
'These are similar to the Fortner method and are recommended in place of it. In
the Fortner method the aerobe is streaked on one half of the plate and the anaerobe
on the olluT lialf of the same dish.
THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA iii„-7
method in almost any laboratory at any time. The technic is so simple that no
previous experience with the method is necessary for success. Since each set of plates
is an individual unit, observation of the growth of the anaerobe may be made at any
time without destroying the anaerobic conditions. Disadvantages: It is somewhat
time-consuming when large numbers of platings are to be made, and, therefore, not
suitable in laboratories where routine plating of a number of cultures is not an unusual
event. Anaerobic conditions may not be attained sufficiently quickly to prevent
death of the inoculum of non-spore-forming species or vegetative cells of anaerobic
spore formers.
Method B
Materials for method similar to that of Marshall and Nordby
(1942) : (1) One petri plate of usual size (bottom should be 15 mm.
deep); (2) one small petri plate" (75 mm. X 10 mm.); (3) culture
of »S. marcescens; (4) tube of nutrient agar.
Method: Pour nutrient agar in bottom half of the regular size
plate, and streak or flood surface with aerobe. Pour agar for anaerobe
in bottom half of small plate. Remove this bottom from its top and
press down in agar of the regular size dish.
Advantages: A simple method suitable for small numbers of plates. The pur-
chase of the small-sized plates is less expensive than some of the more elaborate ap-
paratus required by certain other methods. Disadvantages: Necessity of purchase
of the small-sized jilates.
Chemical Methods for Oxygen Removal
Many of the methods proposed for removal of oxygen from the
environment for anaerobic culture involve the initiation of a chemical
reaction in which oxygen is consumed. Of the various systems
which have been suggested, those which are recommended have been
tested and used sufficiently to show their utility and do not require
elaborate apparatus.
PHOSPHORUS JAR
Materials: (1) Sticks of yellow (or white) phosphorus (which
must be kept under water in tightly stoppered wide mouth bottle; the
small sticks, y^ inch diameter, are the most useful); (2) Pyrex
cylinder or any convenient jar or container which may be sealed
air tight; (3) pair of long forceps or chemical tongs; (4) plasticene;
(5) small amount of tap water.
Method: Place small amount of tap water in bottom of cylinder
to remove the P2O6 which forms. Stack inoculated plates or tubes
on support. Add tube of methylene blue solution (see p. 11143-5).
Place small (50 ml.) beaker on top of cultures. Remove two or
three short {\}/2 to 2 inch) pieces of phosphorus from water with
forceps or tongs and place in beaker. Immediately put lid on jar
and seal with plasticene. (Upon drying for a few minutes, the
phosphorus should ignite spontaneously and remain burning as
long as there is oxygen present). If experience shows that the
^Small petri plates. Central Scientific Company, Chicago. Illinois.
iii„-8 MANUAL OF METHODS FOR PURE CULTURE STUDY
phosphorus used does not ignite spontaneously but merely gives off
a grey smoke, ignite it before the jar is sealed by a match held with
the forceps. Since considerable heat is developed, place beaker,
unless resistant glass is used, three inches from the top of the con-
tainer and put a "blank" plate under the beaker rather than an inocu-
lated plate. After the phosphorus ignites, and the jar is tightly
sealed, place it directly in the incubator. At the time the container
is opened, have available a crock or pan filled with water. As soon
as the lid is taken from the jar, remove the beaker containing the
phosphorus with the tongs and submerge under the water in the pan
and save for later use. After this, remove the cultures from the jar.
Advantages: Quick method of obtaining anaerobiosis. It is relatively inexpensive
since the only materials are phosphorus and a container which may be sealed. Dis-
advantages: Care must be exercised to prevent accidental burns which are very pain-
ful. Inexperienced technicians should be cautioned concerning the dangers.
ALKALINE PYROGALLOL METHODS
Another chemical method for removing oxygen in order to promote
anaerobic growth is to utilize the oxygen absorptive capacity of the
reaction between alkali and pyrogallic acid. Of the technics and
devices reported which make use of this reaction, two may be recom-
mended as being especially useful. One of these concerns a technic
applied to individual plate culture and the other relates to a system
for individual tube cultures.
Spray (or Bray) Plate Cultures
Materials: (1) Spray (1930) anaerobic dish''; (2) plasticene
(see footnote 3) or tape for sealing; (3) 20% aqueous NaOH. (4)
40% aqueous pyrogallic acid.
Note: The Spray dish consists of an ordinary glass petri dish top and a special
bottom which is deep and which has a raised ridge across the center. The top of the
bottom dish has a lip into which the top section of the dish fits. Although constructed
of heat resistant glass, in practice considerable breakage during sterilization and hand-
ling of the Spray dish may be encountered. This is eliminated in the Bray^ dish,
which is Pyrex, and which is essentially the same in design as the Spray dish. In the
Bray dish, however, the need for the lip is eliminated since the top of the bottom
section is slightly smaller in diameter than the remainder of the bottom section.
This allows the top to fit down over the rim of the bottom section.
Method: Pour anaerobic medium in the top half of the dish,
and after solidification, streak from sample or culture, or pour
seeded plate. After inverting dish, place 10 ml. of 20% aqueous
NaOH solution in one section of the bottom dish and 4 ml. of 40%
aqueous pyrogallic acid in the other. Seal dish with plasticene or
tape. Tilt dish to mix solutions and place in incubator.
''Spray anaerobic dish. Fisher Scientific Company, Pittsburgh, Pennsylvania, or
E. H. Sargent Company, Chicago, Illinois.
^Bray anaerobic dish. Corning Glass Works, Corning, New York, Pyrex No. 3155,
or dealer.
THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 11143-9
Advantages: Anaerobiosis is attained quickly. It is a useful method for single
p'ate culture. Since each plate is a single unit, observations may be made at any
time and any particular plate of a series may be opened when visual inspection reveals
growth to be at the desired stage. Recommended for clinical laboratory technicians
seeking a quick method of purification of possible pathogenic types. Disadvantages:
Some time is required to prepare the individual dishes; therefore laboratories doing
a great deal of routine work may desire to use instead some of the anaerobic jars.
Special plates must be purchased.
Tube Culture
Method A
Materials: (1) Agar slant of suitable anaerobic medium; (2)
pyrogallic acid crystals; (3) 10% aqueous NaOH; (4) rubber stopper.
Method: Inoculate agar slant with anaerobic organism or from
sample to be cultured. Flame mouth of tube before replacing plug.
Cut off the end of the cotton plug which extends beyond the mouth
of the tube and push the remaining portion into the tube for a dis-
tance of about 2 cm. Fill this space with pyrogallic acid crystals
and pour 2 ml. of 10% NaOH upon the crystals. Immediately
insert rubber stopper and invert tube in such a fashion that the water
of condensation does not run across the slant. Incubate tube in
inverted position.
Method B
Materials for method of Griffin (1932) : (1) Two test tubes with
approximately ^i inch diameter (one empty and the other containing
a liquid or slant culture of the anaerobe); (2) two one-holed rubber
stoppers to fit tubes; (3) short piece of small diameter rubber tubing;
(4) two short pieces of glass tubing of diameter to fit tightly in holes
of rubber stoppers; (5) small glass vial; (6) dry pyrogallic acid;
(7) strong aqueous NaOH.
Method: Put a column of pyrogallic acid, approximately 1}^
inches high, in the bottom of the empty tube. Stand empty vial
in this acid. With pipette, fill vial two thirds full of NaOH solution.
Fashion a connecting unit from the rubber stoppers, and rubber and
glass tubing. Insert one of the stoppers in the tube with the chemi-
cals. Push down cotton plug in culture tube to a level one inch
above the medium. Insert second stopper in this tube. Tilt
tube containing chemicals sufficiently to allow NaOH solution to
spill over the acid.
Advantages: Good method for single tube culture. If a supply of chemicals is
at hand, it is useful as an emergency system, when the special equipment required
by other systems is not available. Disadvantages: Not suitable for large numbers
of cultures, or, at least, such use would be more time consuming than other methods.
CHROMIUM AND SULFURIC ACID METHOD
Rosenthal (1937) introduced a new system for creating an anaero-
bic environment using the reaction of H2SO4 on powdered chromium
to release hydrogen. This flushes out the oxygen by replacing the
iii^rlO MANUAL OF METHODS FOR PURE CULTURE STUDY
air normally present within the container. The method has been
modified by Mueller and Miller (1941) and their report forms the
basis of the description below.
Materials: (1) A suitable container (see below); (2) fruit jar
rubber ring moistened with glycerol or plasticene; (3) chromium
powder^; (4) H2SO4, 15% by volume (3 vol. cone, acid to 17 vol.
distilled water); (5) Na2C03.
Note: In this method a desiccator equipped with a stopcock may be used if
available; or, for tube cultures a 2-quart Ball fruit jar, prepared as follows, can be
recommended: Have a metal casting of the glass cap made. Solder a short length
of brass tubing into a hole drilled through the cap. Attach a short LT-tube of 5 or
7 mm. glass tubing by a rubber connection. Dip the other end of the U-tube below
the surface of mercury (about 2 ml.) in the bottom of a small tube about 2 inches
in length. Plug the open end of this tube with cotton to prevent spattering of
the mercury. Tie this latter tube to the brass tubing or hold in place by a rubber
band.
Method: Place inoculated tubes in jar. Add tube of methylene
blue solution (see p. 11143-5). Add 3 g. of chromium powder and 1 g. of
Na2C03. Using a funnel, introduce 30 ml. of 15% H2SO4. Clamp
lid on jar immediately; if plasticene is used, prepare the seal around
the lid, and allow the hydrogen and CO2 to escape through the mer-
cury trap tube. As soon as the bubbling subsides, place the jar in
the incubator.
Advantages: Quick method of obtaining anaerobiosis for tube culture. With
other containers the system may be used for plate cultures. Relatively inexpensive
chemicals are employed, though the powdered chromium may not always be avail-
able. Disadvantages: Necessity of securing metal castings of jar top. Outsides
of tubes become covered with chemicals necessitating rinsing when they are removed
from container for examination.
Oxygen Removal by Combustion Using Laidlaw Principle
For laboratories which are engaged in problems where anaerobic
plating is to be done frequently, it is advisable to plan for this and
to purchase equipment accordingly. Although the systems discussed
above may be adequate for this purpose, it is well to consider one
of the jars which utilize, on the Laidlaw (1915) principle, combustion
as a means of securing the anaerobic environment. These methods
were designed especially for incubation of plates, but other culture
vessels (flasks, tubes, bottles, etc.) may be used. Jars using this
principle are those of Brewer (Brown and Brewer, 1938) and Mc-
intosh and Fildes (Fildes and Mcintosh, 1921).
BREWER ANAEROBIC JAR^°
Materials for method of Brown and Brewer (1938): (1) Brewer
jar complete with electric cord; (2) source of illuminating gas or
^Chromium powder — 98% pure; e.g., from Fisher Scientific Company, Pittsburgh,
Pennsylvania or Eimer and Amend, New York, New York.
^"Brewer jar. Baltimore Biological Laboratory, Baltimore, Maryland and Fisher
Scientific Company, Pittsburgh, Pennsylvania.
THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA in „-l 1
hydrogen; (3) tube of soda lime; (-1) plasticene (see footnote 3);
(5) water vacuum pump for evacuation.
Method: Place plates in Brewer jar. Add tube of methylene
blue solution (see p. iii.i3-5). Include a tube of soda lime in the jar
to absorb excess CO2. Place roll of (warmed) plasticene around
rim of jar. Put on lid and press down on plasticene to form seal.
Add the lid clamp but tighten only slightly. If used with illuminat-
ing gas, attach the jar by the rubber tubing to the water vacuum
pump. Evacuate until the manometer or gauge reads approximately
20 cm. or 8 inches. After this degree of evacuation is reached, con-
nect the rubber tube to the gas supply (a three way stop-cock facili-
tates this change without loss of vacuum). Attach the electric
plug (110 volt AC or DC) and allow the gas and electric current to
remain attached for 30 to 45 minutes. At the end of this time clamp
the rubber tube tightly, remove the electric cord, and place the jar in
the incubator. (Formation of water droplets on the inside walls of
the jar indicates the proper functioning of the apparatus.) To open
the jar, remove the clamp and insert a knife blade between the lid
and rim of the jar. // used with hydrogen, attach the jar, without
evacuation, to the hydrogen tank and admit the gas at a pressure of
1-2 lb. per square inch. Attach the electric connection and allow
the current and gas both to remain on for 30 minutes. Then treat
the jar as above.
Advantages: Convenient system for incubation of a number of plates in experi-
ments where speed of obtaining anaerobiosis is essential. Recommended for clinical
laboratories. Inexpensive system after the initial outlay for apparatus. Danger
of explosions is less in the Brewer jar than in the Mclntosh-Fildes jar. Disadvantages:
Some possibility of explosion or cracking of jar. Initial expense of equipment is
more than for other methods discussed above — but this may be a good investment
if routine work is to be done over a period of time. Requires source of hydrogen or
illuminating gas and electricity; while these are available in most laboratories, they
are not available in others such as some mobile laboratory units, temporary labora-
tories in field surveys, etc.
Mcintosh and fildes jar"
Materials: (1) Mcintosh and Fildes jar; (2) protective box or
cage of galvanized wire; (3) cylinder of hydrogen {■preferable) or
hydrogen generator; (4) reducing valve for hydrogen cylinder;
(5) resistance coil (approximately 175 ohms for 110 volts or 350 ohms
for 220 volts); (6) electrical wire for connections; (7) three-foot
length of rubber tubing.
Method: (Adapted from various sources, including directions
issued with jar purchased from Arthur H. Thomas Company) :
Clean surfaces of jar and lid with xylol. Apply suitable sealing
medium or hard tallow to these. Grease tips and threads of needle
valves. Place cultures in jar and add tube of methylene blue
indicator solution (see p. 11143-5). Place lid on jar and tighten the
^^Mclntosh and Fildes jar. Arthur H. Thomas Company, Philadelphia, Pennsyl-
vania. Model No. 1085 (glass) or 1085-B (aluminum). A convenient cage is Model
No. 1085-F.
11143-12 MANUAL OF METHODS FOR PURE CULTURE STUDY
large milled head sufficiently to make the lid gas-tight but not to the
point at which the action of the coiled spring is ineffective. Tighten
the lock nut (the smaller and concentric milled head). Introduce
hydrogen from cylinder, through reducing valve set for two pounds,
and keep flowing for two minutes or more. Test whether or not
all the air has been removed by attaching a rubber hose to the exit
valve and allowing the gas to excape in a cup of soapy water. If
the gas bubbles fail to "explode" when a lighted match is applied
but ignite to burn with a non-luminous flame, the concentration of
hydrogen is sufficient to proceed. Close both valves and connect
the wiring terminals to an electric source of correct voltage and
through a 0.6-0.7 ampere resistance. Formation of droplets of water
on the inside walls of the jar indicates correct functioning of the
apparatus. After a negative pressure develops (a few minutes)
add more hydrogen slowly. Continue the current for 30 minutes.
Then tighten the valves of the jar and remove the electric connection.
Advantages and disadvantages: See above for Brewer jar. Apparently there is
greater danger of explosions with the Mcintosh and Fildes jar than with the Brewer
jar. Inexperienced technicians are warned to proceed with caution when using this
apparatus.
Plating System Using Strongly Reducing Medium
Recently there has been introduced by Brewer (1942) another
single plating device which has much to recommend it. Because
of its promise it is introduced here even though it has not as yet been
used sufficiently widely to establish a reputation. The dish must
be used with an agar containing highly reducing agents. The design
of the dish is such that the top of the dish rests, at its periphery,
on the medium to form a seal, and the remainder of the dish is slightly
raised. Thus only a small amount of air is trapped over the surface
of the agar and this is removed by means of the reducing action of
the medium.
brewer culture dish'^
Materials: (1) Brewer anaerobic culture dish; (2) regular petri
dish with bottom either 15 mm. or 10 mm. deep; (3) infusion agar
suitable for anaerobes which contains suitable reducing agents, such
as the following: 0.2% sodium thioglycoUate, 0.1% sodium form-
aldehyde sufoxylate, and 0.0002% methylene blue.
Method: Pour sterilized medium in bottom of regular petri dish
(25 ml. minimum in 10 mm. dish, and 40 ml. minimum in 15 mm.
dish). Streak center area from sample or culture. Replace the
lid of the regular dish with the Brewer anaerobic lid. (The lid
at its periphery, should touch the agar at all points in order that
a perfect seal be obtained. In the successfully prepared dish, the
agar in the center of the dish remains colorless while the blue color
returns to the agar at the edge of the dish due to oxygenation of the
^Brewer anaerobic dish. Baltimore Biological Laboratory, Baltimore, Md., and
Kimble Glass Company, Vineland, New Jersey.
THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA iii«-13
dye which serves as an oxidation reduction potential indicator.)
Place plates in the incubator immediately after they are prepared
and examine as needed during the incubation period. When trans-
fers are to be made from the plate, break the seal by a slight turn
of the lid.
Advantages: A useful, quick method of single plate culture. An extremely simple
method which is easy to learn and use. The only trick in the technic is to have
sufBcieut agar in the original dish that a perfect seal is formed when the special lid
is added. Recommended for routine use in hospital laboratories, and particularly
for mobile laboratories, where anaerobic cultures for pathogens may be encountered.
Disadvantages: Surface moisture may result in film formation in some instances;
this may be reduced by using a porcelain top ( see footnote 4) on the regular dish prior
to the Brewer anaerobic lid or drying the plates in incubator before streaking. Some
organisms apparently are inhibited by the reducing agents. This is not serious since
the reports indicate that all pathogenic types are easily cultured by this method.
The Brewer anaerobic lids are, at the present time, relatively expensive.
There are other anaerobic systems which are satisfactory as, for
example, the Novy jar which depends upon evacuation and gas
replacement in a specially designed desiccator. These will not be
discussed, however, as they are less commonly used at the present
time, and it is believed that the methods discussed above will be
satisfactory in most instances.
TECHNICS FOR STUDY OF ANAEROBIC BACTERIA^^
In the above section the various pieces of apparatus and methods
for their use with anaerobic bacteria have been considered. Formu-
lae for the particular media which are recommended may be found in
the 9th edition of Leaflet 11^^. The remainder of this Leaflet will
be devoted to a discussion of the details of certain technics which
should aid the worker who has not had previous experience w^ith
anaerobes.
It may not be amiss to insert here a precautionary note concerning
the necessity of very careful inspection of the purity of cultures.
There are instances on record, in the older literature, where two
species grew symbiotically on plate culture with such constancy
that recorded observations were made of the colony type of mixture,
the investigator being unaware of the existence of more than one
type. In all studies concerning obligate anaerobes, a check on the
purity of the culture should be made with regard to aerobic contami-
•'In this Leaflet reference will be made to the "pathogenic group" and the "butyric-
butyl group". The former term is used to designate such organisms as Clostridium
tetani, C. septicum, C. histolyticum, C. chauvoei, C. pcrfriiigens, C. parabotulinum, C.
botulinum and C. sporogenes. In the butyric-butyl group are included C. bntyricum, C.
beijerinckii, C. butylicum, C. pasteurianum, C. acetobutylicum, C. felsineum, C. roseum,
and C. thermosaccharolyticum.
"To be published about February, 1944.
iii„-U MANUAL OF METHODS FOR PURE CULTURE STUDY
nants. The following test is suggested: For most cultures, streak
a glucose nutrient agar slope and incubate it at 37° C; but for
anaerobic species having a lower or higher optimum temperature,
incubate a second agar slope at the temperature which is optimum
for the anaerobe. If the culture appears free of aerobic types, in-
vestigate the purity with respect to anaerobic contaminants. Make
repeated platings and scrutinize intensely the colonies which develop.
Preliminary Microscopic Examination
If the sample is suitable, one should make preliminary examina-
tion using the Gram stain. The conventional method of staining
a smear, heat fixed on a glass slide, should be used, except that the
decolorizer should be either 95% ethyl alcohol {'preferred) or 25 parts
acetone and 75 parts ethyl alcohol. The use of greater amounts
of acetone must be avoided because of the ease with which anaerobes
are decolorized. The usefulness of the Gram method is limited in
smears prepared from blood, fibrin or albumin. In samples of patho-
logic material, large, Gram-positive rods are likely to prove to be
anaerobic bacilli, but a final diagnosis must not be based on micro-
scopic observations unsupported by cultural tests. Of the strictly
aerobic Gram-positive species, Bacillus anthracis Koch is the only
usual pathogen. The characteristic morphology of Clostridium
perfringens (syn. C. welchii) and the regularity of its appearance in
certain clinical conditions frequently combine to give presumptive
evidence of value; similarly, the typical microscopic picture presented
by a spore-bearing C. ietani culture should be remembered when such
forms are encountered in pathologic material. All anaerobic species
are non-acid fast; therefore, this stain has no diagnostic importance.
Microscopic Examination of Pure Cultures
GRAM stain
If the organism in question will grow within this period, apply
the Gram stain to a 16-18 hour culture and observe the same caution
with reference to the decolorizer as noted above. Ordinarily the
stain is satisfactory when prepared from any enrichment medium
in which the organism will grow. In recording the Gram reaction
of a new species, state the medium from which the smear was made
and the age of the culture.
examination for motility
The majority of the spore-forming anaerobic bacilli are motile;
the most important exception is C. perfringens (C. u-elchii). The
technic by which the motility examination is made is often of utmost
importance in securing the correct results. Unless the culture is
known to he nonpathogenic, discard all cover slips and slides into a
disinfectant solution or sterilize by steam before washing. Use young
cultures (12-18 hours) except as noted. Accept the results of hang-
ing drop or wet-mount preparations under coverslips only if observa-
THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA iii,,-l,5
tioii reveals positive motility. If motility is doubtful or appears
to be negative, initiate other procedures. For example, use a flat-
tened capillary tube sealed at each end. Heat glass tubing, of
small diameter, and flatten a small area. Prepare a capillary
tube from the flattened section. Draw a small amount of culture
into this tube and seal the tube in the flame on both sides of the drop
of culture. Examine this preparation with the high power objective.
If the motility is still recorded as negative, make further observations
on younger (4-6 hour) cultures. For these, examine the 3rd or
4th tube of a serial passage series, using the medium which appears
to give the best growth of the culture. Because of the relatively
small number of species which are non-motile, considerable caution
should be exercised in reporting cultures which appear to be non-
motile. Naturally occurring non-motile variants of motile species,
however, have been encountered.
FLAGELLA STAIN
For material for preparation of flagella stains use young cultures
growing in the medium which is most favorable to the organism being
studied. If difficulty is encountered in securing positive slides
from cultures known or thought to be motile, consult the directions
given by O'Toole (1942) for suggestions in technic which refer
particularly to anaerobic bacteria.
CAPSULE STAIN
For the capsule stain one may use any of the conventional methods.
The most important capsulated species is Clostridium perfringens
(C loelchU). Material taken from artificially infected laboratory
animals generally serves as the origin of smear preparations. If
stains from in vitro cultures are desired, the medium of Svec and
McCoy (See Leaflet II) is useful if other media prove unsuccessful.
DEMONSTRATION OF SPORES
Cultures surviving 20 minutes heating at 80° C. may be presumed
to be spore-formers. It is, however, useful to demonstrate the spores
microscopically. The exact method of making the spore-stain is
of little importance, in comparison with other factors, as each of
the common methods (Dorner, Moeller, and malachite green)
appears satisfactory. One must, however, pay some attention to
the medium in which one expects to induce sporulation. Media
containing fermentable carbohydrates are not satisfactory, in
general, for the pathogenic group. The media naturally containing
carbohydrate {e.g., corn mash or potato infusion), on the other hand,
appear ideal for most of the butyric-butyl group. For the patho-
gens one should use the deep brain, or beef heart, or alkaline egg
medium. In some instances spores may be demonstrated within
24-28 hours after inoculation, but, if the culture is negative at this
time, older cultures should be examined. Protection from evapora-
tion must be given cultures which are to be incubated longer than
iii«-16 MANUAL OF METHODS FOR PURE CULTURE STUDY
one week. C. perfringens (C. welchii) appears to be one of the most
difficult species in which to demonstrate spores microscopically
with regularity. If success is not attained using the above-men-
tioned media in cultures having the characteristics of this organism,
one may use the medium recommended by Svec and McCoy (See
Leaflet II).
Since some taxonomic systems give considerable attention to the
size and position of the spore, these characteristics should be recorded
when the original laboratory examination is made. The characteris-
tic appearance of Clostridium tetani spores has been noted above;
these are round in shape and borne at the end of a slender vegetative
rod. This is almost the only instance in which the picture of the
spore and sporangium assumes importance in species diagnosis,
and this observation must be supported by cultural or pathologic
information as nontoxic organisms of similar microscopic characters
occur.
GRANULOSE REACTION
The cells of certain species, particularly during the early stages
of spore formation, store granulose. To test for this, add a drop
of Lugol's iodine to a wet mount preparation. Cells containing
granulose will stain blue or violet while others will appear yellow.
Cultivation Technics^^
preliminary enrichment methods
Ordinarily the best method to be followed in initiating growth of
an anaerobe from a sample is to inoculate one of the tubed media
rather than to proceed directly to plate culture. Certainly this
should be done if there is question concerning the possible success
of the preliminary culture, and it is advised that parallel tube cul-
tures be inoculated to serve as reserve cultures at the same time the
plating is done, if the plating technic is favored. The medium
to be used will be a matter of choice, as discussed in Leaflet II (9th
Edition), depending upon the nature of the sample. If aerobic
contamination is suspected and the anaerobe is thought to be in the
spore state, a duplicate primary culture should be heated briefly
(boil for one or two minutes, or hold at 80° C. for 20 minutes). This
should be a duplicate culture, however, in case the anaerobic form
is a non-spore-former or is a spore-former in the vegetative state.
Almost all types of tubed media should have the dissolved oxygen
driven off by boiling or heating in flowing steam.
For the gas gangrene and tetanus group in infected wounds, Reed and Orr (IQll)
recommend a technic to those who work in clinical laboratories and examine such
material. The technic would appear to involve more cultures than is necessary but the
importance of the success of the preliminary culture, and the speed with which it is
attained, necessitate the routine suggested. Colonies which appear in the plates are
transferred to tubes of thioglycollate medium and species identification begun im-
mediately. It should be remembered that gas gangrene frequently is a polymicrobic
i^The use of vaseline, mineral oil or other materials as a seal at the surface of liquid
media is not recommended.
THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA iii4,-17
infection and therefore more than one colony type from a single sample is not to be un-
expected. With slight modifications their suggestions are as follows:
(1) Inoculate heavily tubes of beef heart medium. Use these subsequently only
if the primary plating fails.
(2) Introduce swabs or fragments of tissue into 8 ml. amounts of thioglycollate
broth, mix well, and make 1:10, 1:100, and 1:1000 dilutions in the same medium (not
saline) .
(3) From each dilution prepare surface plates on clear peptone-thioglycollate
agar and pour plates in semisolid agar. As an additional or alternate medium, use blood
agar; in which case hemolysis, if present, is an additional helpful characteristic. Incu-
bate the plates at 37° C. in a Brewer or Mcintosh and Fildes jar. Place a petri dish
lid containing granular CaCL at the bottom of the stack of plates, and another at the
top, to absorb the moisture which forms in the jars. Use the Brewer or Spray plate
if an anaerobic jar is not available.
PRELIMINARY PURIFICATION PROCEDURES
It is often difficult to isolate anaerobic bacteria from enrichments
which also contain aerobic bacteria. It would be presumed that
aerobic bacteria could ordinarily be eliminated merely by the
anaerobic environment when this is introduced. Often in practice
this is not the case, and other procedures must be instituted. It is
of value frequently to attempt partial or complete elimination of
the contaminants in tube culture using a liquid medium before
plating is done. Materials derived from human or animal sources,
other than feces, are usually contaminated with non-sporulating
aerobic rods and cocci. Cultures derived from milk, soil, water,
grains, feces, etc., contain, in addition, spore-forming aerobes. In
fecal and perhaps other samples the contamination may include
non-spore-forming anaerobes. If the non-spore-forming anaerobe
is wanted, then anaerobic plating, and picking of isolated colonies,
should be combined with optimum temperature and selective medium
to secure the culture. In all cases the original enrichment tube
should be preserved in the refrigerator, after growth is evident,
until the purification routine is successfully completed. This will
insure a supply of starting material should something go wrong with
the purification.
Generally one of the easiest practices to be followed to get rid of
non-spore-forming types is as follows: Heat subcultures from the
contaminated enrichment, retaining the original tube, of course,
unheated. Heat the newly inoculated tubes 20 minutes at 80° C.
or a shorter time at higher temperatures. Take care to insure the
presence of the spores of the anaerobe. Use old cultures in a sugar-
free medium as the best source of material to be heated, although
other cultures may be satisfactory in special situations.
For enrichments contaminated with spore-forming aerobes the
above procedure may not be satisfactory, due to the heat resistance
of the aerobic spores. In this case, one may employ dyes as bacterio-
static agents. Nearly all, if not all, aerobic spore-formers are
inhibited by crystal violet, and most of the anaerobic types are
relatively resistant. Two or three serial transfers may, therefore.
ni„-18 MANUAL OF METHODS FOR PURE CULTURE STUDY
be made in a medium containing this dye (approximately 1-100,000
final concentration) to eliminate the aerobe. The exact concen-
tration of the dye to be used may vary with the medium and the
conditions at hand. If used in some of the complex media the effec-
tiveness of the dye may be reduced during sterilization; therefore,
the dye should be added to such media after sterilization. Either
liquid or solid media may be used.
Another method for elimination of aerobic spore-formers utilizes
the fact that while growth of the aerobe may take place in an anaero-
bic environment the conditions for sporulation are unfavorable.
Under such conditions the anaerobe will be expected to sporulate
freely. Thus liquid cultures in tubes or plate cultures taken from
an anaerobic jar are chosen for material for heating as in the case
of the non-spore-forming contaminants.
ISOLATION PROCEDURES
From a purely theoretical viewpoint, microscopic single cell
methods of isolation are ideal, but the low percentage of successes
with these procedures excludes them from any uses except research.
Several reports are in the literature indicating success with anaerobes
using the Chambers micromanipulator, or similar instruments,
and wherever there is great need for strains of single cell origin,
the technic should be attempted. Due to the sensitivity of the vege-
tative cells toward oxygen, it is recommended that spores be picked
rather than vegetative cells. One should use freshly exhausted
media showing highly reducing activity for the subcultures and
naturally the medium should be suited to the organism being purified.
If growth is not evident within the first 48 hours, the tubes may be
protected from evaporation and incubated indefinitely. Reputable
workers have reported dormancy of spores for six months or longer
duration.
In routine problems either plating or deep agar tube methods are
available for purification of cultures from the original enrichment
tubes. As stated above, the usual procedure in the isolation of
anaerobes from samples in which contamination is excessive is best
done by attempting partial purification in tube culture. This,
however, need not be the case if the population of the sample is
dominated by one species. In these the plating routine may be
started without the preliminary enrichment procedure. Perhaps
a few words should be included concerning details of technic. Since
some of the anaerobes tend to spread rapidly over the surface of the
agar, in many instances it will be found that "poured" agar plates
are to be preferred to plates inoculated by streaking the surface.
Two common methods are available for preparing these: (1) melt
tubes of the plating medium, cool, and inoculate before pouring;
(2) place a small amount of sterile tap water in the culture dish,
inoculate, and pour the agar into the dish immediately. If condi-
tions warrant, use crystal violet in the agar. Place the plates in
the anaerobic environment as soon as possible. (The size of inoculum
to be used will vary so that some practice may be necessary to give
THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA ni^.-is
a dilution sufficient that well isolated colonies will appear.) If
difficulty is encountered in obtaining discrete colonies, reduce the
agar concentration in the plating medium to 0.75 to 1.0%.
Another method is available for colony isolation which may be
preferred, particularly if the special apparatus needed for some of
the plating methods is not at hand. This method involves the inocu-
lation of a column of medium as mentioned in the opening pages
of this Leaflet in the discussion of methods useful to determine
whether or not a particular strain is an obligate anaerobe. For
isolation purposes the fewer the number of colonies appearing in
the medium the better. The percentage of fermentable sugar
should be reduced to the lowest amount which gives good growth
of the organism in order to prevent the production of gas which may
crack the medium. Assuming that we have available a deep tube
of agar in which there appear several isolated colonies, two methods
of isolation are available: (1) If soft glass tubes are used, cut the
glass and break the tube at a short distance below the desired colony.
Deposit the agar quickly in a sterile petri dish. Using a hot needle
or small blade cut across the plug of agar near the colony and trans-
fer it to a suitable liquid medium. (This method is preferred if
the tube shows aerobic contamination in the upper layers.) (2) If
Pyrex tubes are used, eject the plug of agar into the sterile dish by
applying a Bunsen flame to the bottom end. Before this heat the
sides of the tube and sterilize the mouth of the tube in the flame.
During the ejection step of the technic, hold the mouth of the tube
so that it points directly into the sterile dish. After the column of
agar is deposited in the dish, proceed as discussed above.
INOCULATION TECHNICS
The following points of culture transfer and other routine technics
are sufficiently different from the procedures used with aerobes
so that some note is needed:
Steam or boil most liquid media for a few minutes immediately
prior to inoculation in order to drive off oxygen which may have been
absorbed following sterilization. Attempt to deliver the inoculum
to the bottom of the new tube of medium, for it is this portion of the
medium which will stay reduced the longest. Although it is possible
to initiate growth from a small number of cells, in routine studies
use a more adequate inoculum. To facilitate the placing of the
inoculum in the bottom of the tube with liquid and semisolid media
substitute a Wright or Pasteur pipette (used with small rubber
bulbs) for the inoculation needle. By this means transfer a small
drop (0.1 or 0.2 ml.) of the culture to the new tube. Use pipette
also in the isolation of subsurface colonies particularly from media
in which the concentration of agar is reduced. Prepare these pipettes
from 6 to 8 inch lengths of sterile 8-9 mm. soft glass tubing (with
cotton plug in each end) by applying heat to the center of the glass
and pulling to form two capillary pipettes.
In general use a culture from 16-20 hours old. With the jjatho-
genic types this time may be extended a few hours with no harm.
iii«-20 MANUAL OF METHODS FOR PURE CULTURE STUDY
With the butyl-butyric types, however, which sporulate readily
in many media, there is a critical period in which the culture is not
very satisfactory for transfer purposes. As the culture goes into
the spore stage it is less and less suitable until sufficient time elapses
for the spores to mature. When spores are present in the inoculum,
with these cultures and perhaps others as well, the new tube should
be given a heat treatment (80° C. for 20 minutes) after inoculation.
Generally, if an anaerobic spore-forming culture is desired in an
experiment, inoculate a tube of a favorable medium from a stock
culture which contains spores, heat-shock it, and use the resulting
culture for the experiment rather than the inoculation of the latter
tube or flask directly from the spore containing culture. Maintain
the stock culture in the spore state and follow the above transfer
routine, rather than carry the anaerobe in a serial passage, and
use such cultures for sources of inoculum for experimental flasks or
tubes. This is particularly true with the actively fermentative types,
where serial passage may yield a culture of undesirable characters —
even though it is descended in pure state from a culture that was
satisfactory.
Other Methods of Value
stock culture methods
The anaerobes are susceptible to freezing-drying technic as a
means of preservation of cultures over a long period of time as shown
by Roe (1940). This technic is unnecessary, however, as species of
Clustridium are usually viable in spore state over a long period of
time. For the pathogenic group, one should use beef heart infusion,
alkaline egg medium, and brain mash, with the latter perhaps being
the best. With the butyric-butyl group, use plain corn mash or
potato infusion. Prepare the plain corn mash in a manner similar
to the method given for corn-liver medium with the exception that
the liver powder is omitted. Brain medium may be suitable also.
(See also Leaflet II, 9th Edition.)
In any medium after all gassing has subsided and spores have been
demonstrated microscopically, the tube should be sealed in the flame
or the stopper covered to protect the medium from evaporation,
and the tube placed in a cool room or refrigerator. Viable sub-
cultures may be obtained from such tubes for months or even years
in some instances. Another method which has been used with suc-
cess is worthy of mention. This involves the storage of cultures
on sterile soil: Dry fresh garden soil and sift through a fine mesh
screen; add 5% of CaCOs to neutralize any acidity of the culture.
Place soil in tubes in 2 inch columns and autoclave overnight. Test
each tube for sterility using both aerobic and anaerobic media. If
sterile, add 2 or 3 ml. of a well sporulated culture with a sterile
pipette and dry the tube (preferably in a vacuum desiccator). To
obtain an active culture from this stock (which may be stored at
room temperature) transfer a small amount of the soil to an enrich-
ment medium and heat shock. By the soil stock method a relatively
permanent source is available from which cultures may be revived
as needed without destroying the stock culture.
THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA iii„-21
SEROLOGICAL REACTIONS
The serological relationships of the spore-forming anaerobes
have been reviewed (McCoy and McClung, 1938) and it is sug-
gested that this paper should be consulted as a background and for
further references by those who are interested in this topic. The
toxin-antitoxin reaction is of value as a taxonomic aid with certain
species. In such an instance one takes advantage of the fact that
relationships may be established by the success or failure of the re-
action of antitoxin, prepared against the toxin of a known organism,
with the toxin from the unidentified strain. In some instances the
anaerobic species are monotypic with respect to toxin formation. In
other species this is not true and subgroups have been established
within these species or species groups on the basis of non-cross
neutralization tests.
The problem of toxin production may be briefly mentioned. Although studies
have been initiated on the possibilities of synthetic media for this purpose, such studies
are designed to provide toxin for chemical purification investigations and for produc-
tion of toxoid. If it is desired to test for the possibility of production of toxin by a
particular culture, it is unnecessary to use a synthetic medium since one of the complex
media will serve as well and because less diflSculty with regard to growth is encountered.
For organisms producing the tetanus or botulinus toxin use the beef heart infusion.
For the gangrene group use the same medium or glucose meat infusion or the medium
of Reed, Orr and Baker (1939). For formulae consult Leaflet II. Use the Berkefeld
or Mandler filter to remove cells from the liquid of a 24-72 hour culture. Discard
the first 25 ml. of filtrate before collecting the test sample.
For the agglutination reaction, cells for antigen suspensions may be prepared by
centrifuging from broth cultures in which maximum growth is attained quickly. For
the pathogenic group ^ucose meat infusion broth or perhaps thioglycollate broth
should be used. For the butyric-butyl group, one should employ 1% tryptone broth
or yeast infusion broth with 0.5 to 1.0% glucose, with a heavy inoculation from a liver
broth culture into deep tubes or bottles of the medium chosen. Care should be taken
to collect the cells before excessive slime formation is evident in order to produce a
stable antigen.
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7, 420-438.
Bergey, D. H., Breed, R. S., Murray, E. G. D., and Kitchens, A. P. 1939. Bergey's
Manual of Determinative Bacteriology, 5th Ed. Williams and Wilkins, Balti-
more. 1032 pp.
Brewer, J. H. 1942. A new petri dish cover and technique for use in the cultiva-
tion of anaerobes and microaerophiles. Science, 95, 587.
Brown, J. H., and Brewer, J. H. 1938. A method for utilizing illuminating gas
in the Brown, Fildes, and Mcintosh or other anaerobe jars of the Laidlaw princi-
ple. J. Lab. and Clin. Med.. 23, 870-874.
Committee Upon Anaerobic Bacteria and Infections. 1919. Report on the anaerobic
infection of wounds and the bacteriological and serological problems arising there-
from. (Gt. Brit.) Med. Research Council, Spec. Rpt. Ser., 39, 1-182.
Dack, G. M. 1940. Non-spore-forming anaerobic bacteria of medical importance.
Bact. Rev., 4, 227-259.
Fildes, P. 1931. Anaerobic cultivation. Chap. VI in System of Bacteriology, Vol.9,
(Gt. Brit.) Med. Research Council.
III43-22 MANUAL OF METHODS FOR PURE CULTURE STUDY
Fildes, P., and Mcintosh, J. 1921. An improved form of Mcintosh and Fildes
anaerobic jar. Brit. J. Exp. Path., 2, 153-154.
Griffin, A. M. 1932. A modi6cation of the Buchner method of cultivating anaerobic
bacteria. Science, 75, 416-417.
Hall, I. C. 1922. Differentiation and identi6cation of the sporulating anaerobes.
J. Inf. Dis., 30, 445-504.
Hall, I. C. 1928. Anaerobiosis. Chapter XIII in The Newer Knowledge of
Bacteriology and Immunology. Edited by Jordon, E. O., and Falk, I. S. Univ.
of Chicago Press, Chicago.
Hall, I. C. 1929. A review of the development and application of physical and
chemical principles in the cultivation of obligately anaerobic bacteria. J. Bact.,
17, 255-301.
Heller, H. H. 1921. Principles concerning the isolation of anaerobes. Studies in
pathogenic anaerobes. II. J. Bact., 6, 445-470.
Hewitt, L. F. 1937. Oxidation-reduction potentials in bacteriology and biochemis-
try, Jfth Ed. London County Council.
Hibler, E. von. 1908. Untersuchungen iiber die pathogenen Anaeroben, iiber die
anatomischer und histologischen Veranderung bei den durch sie bedingten
Infektionskrankungen des Menschen sowie der Tiere und iiber einige nicht-
pathogene Anaerobenarten. Gustav Fischer, Jena. 438 pp.
Knight, B. C. J. G. 1931. Oxidation-reduction potential measurement in cultures
and culture media. Chapter XIII in System of Bacteriology, Vol. 9, (Gt. Brit.)
Med. Research Council.
Knorr, M. 1923. Ergebnisse neurer Arbeiten iiber krankheitserregende Anaerobien.
I. Teil. Krankshcitserregende anaerobe Sporenbildner, ausschliesslich Tetanus
und Botulinus. Zentbl. Gesam. Hyg.. 4, 81-100, 161-180.
Knorr, M. 1924. Ergebnisse neuerer Arbeiten iiber krankheitserregende Anaerobien.
II. Teil, 1: Botulismus. Zentbl. Gesam. Hyg.. 7, 161-171, 241-253.
Laidlaw, P. P. 1915. Some simple anaerobic methods. Brit. Med. J., 1, 497-498.
McClung, L. S., and McCoy, E. 1941. The anaerobic bacteria and their activities
in nature and disease: a subject bibliography. Suppl. 1: Literature for 1938
and 1939. Univ. of California Press, xxii and 244 pp.
McClung, L. S., McCoy, E., and Fred, E. B. 1935. Studies on anaerobic bacteria.
II. Further extensive uses of the vegetable tissue anaerobic system. Zentbl.
Bakt., II Abt., 91, 225-227.
McCoy, E., Fred, E. B., Peterson, W. H., and Hastings, E. G. 1926. A cultural
study of the acetone butyl alcohol organism. J. Inf. Dis., 39, 457-483.
McCoy, E., Fred, E. B., Peterson, W. H., and Hastings, E. G. 1930. A cultural
study of certain anaerobic butyric acid-forming bacteria. J. Inf. Dis., 46, 118-
137.
McCoy, E., and McClung, L. S. 1938. Serological relations among the spore-form-
ing anaerobic bacteria. Bact. Rev., 2, 47-97.
McCoy, E., and McClung, L. S. 1939. The anaerobic bacteria and their activities
in nature and disease: a subject bibliography (i?i two volumes). Univ. of California
Press, xxiii and 295 pp.; xi and 602 pp.
Mcintosh, J. 1917. The classification and study of the anaerobic bacteria of war
wounds. (Gt. Brit.) Med. Research Council, Spec. Rpt. Ser., 12, 1-58.
Marshall, M. S., and Nordby, H. 1942. Anaerobic plates. J. Bact., 44, 619.
Meyer, K. F. 1928. Botulismus. In Kolle, W., Krau.se, R., und Uhlenhuth, P.
Handbuch der pathogenen Mikroorganismen, 3 Aiifl., 4, 1269-2364.
Mueller, J. H., and Miller, P. A. 1941. A modification of Rosenthal's chromium-
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O'Toole, E. 1942. Flagella staining of anaerobic bacilli. Stain Techn., 17, 33-40.
Prevot, A.-R. 1924. Les streptocoques ana^robies. Thesis, Paris. 144 pp.
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447.
THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA iii„-23
I'revot, A.-R. 1938. fitudes de syst^matiquc bactdrienne. III. Invalidile dii
genre Bacleroides Castellani et Chalmers. Demcmbremcnt et reclassification.
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Pr^vot, A.-R. 1940a. Etudes de syst(5matique hactdrienne. V. Essai de classifi-
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Prdvot, A.-R. 1940b. Manual de Classification et de Ddtermination des Bacleries
Anadrobies. Masson et Cie., Paris. 223 pp.
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War Med., 1,493-510.
Reed, G. B., and Orr, J. H. 1943. Cultivation of anaerobes and oxidation-reduction
potentials. J. Bact., 45, 309-320.
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Soc. Expt. Biol, and Med., Proc, 42, 620-621.
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Roe, A. F. 1940. Report on viability of 200 cultures of anaerobes desiccated for
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Rosenthal, L. 1937. "Chromium-sulfuric acid" method for anaerobic cultures
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Snieszko, S. 1930. The growth of anaerobic bacteria in petri dish cultures. Centbl.
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Weinberg, M., Nativelle, R., and Prdvot, A.-R. 1937. Les Microbes Anaerobies
Masson et Cie., Paris. 1186 pp.
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P., Handbuch der pathogenen Mikroorganismen, 3 Aufl., 10, 35-144.
Zeissler, J., und Rassfeld, L. 1928. Die anaerobe Sporenflora der europaischen
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5, Heft 2. 99 pp.
LEAFLET IV
STAINING METHODS
9th EDITION
Pure Culture Study of Bacteria. Vol. 14, No. 2-3
AUGUST 1946
Committee members assisting in the revision: Barnett Cohen, M. W. Jennison,
L. S. McClung, and A. J. Riker
LEAFLET IV
STAINING METHODS
9th Edition
General Principles
The staining of bacteria depends in general upon the same prop-
erties of dyes as docs the staining of animal or plant tissue for histo-
logical purposes. Short discussions of the nature of dyes, with special
reference to staining are given elsewhere (Conn, 1940; Churchman,
1928) and only the briefest summary of the subject need be given
here.
All bacterial dyes are synthetic products — anilin dyes, or coal-tar
dyes, as they are generally called. Although the synthetic dyes vary
greatly in their chemical nature and staining properties, they are
for practical purposes often divided into two general groups, the acid
dyes and the basic dyes. These terms do not mean that the dyes in
question are free acids or free bases. The free color acids and bases,
when obtainable, are colored, to be sure, but they are often insoluble
in water, and rarely have appreciable staining action— i. e., the colors
do not "stick." The salts of these compounds, on the other hand,
are more soluble, penetrate better, and stain more permanently;
they are the true dyes.
An acid dye is the salt of a color acid, a basic dye the salt of a
color base. In other words, acid dyes ow^e their colored properties
to the anion, basic dyes to the cation. The actual reaction of an
aqueous solution of a dye, however, depends on several factors; and
an acid dye may well be basic in reaction, while a basic dye may be
acid. This is because the reaction of such a solution depends on the
relative strengths of the dye ion and of the anion or cation with
which it is combined in the dye salt.
Basic dyes have greatest affinity for the nuclei of cells, probably
because of the acid nature of the nuclear material. Acid dyes have a
stronger tendency to combine with the cytoplasm. As bacteria do
not show typical cell structure and the nuclear material seems to be
distributed throughout their bodies, they tend to stain fairly uniform-
ly with nuclear, i. e., the basic, dyes. Hence, the stains in common
use by the bacteriologists are rarely acid dyes.
PREPARATION OF SMEARS
Pure cultures of bacteria can ordinarily be prepared for staining
by the simple process of making an aqueous suspension and drying
a drop of it on a slide or cover glass, without any fixation other than
gentle heat. The use of this simple procedure depends upon the fact
that most bacteria, because of their small size or their stiff walls,
can be dried without great distortion. For this reason it is not
IV46-4 MANUAL OF METHODS FOR PURE CULTURE STUDY
usually necessary, as with higher organisms, to coagulate the tissues
before microscopic preparations can be made; although it has been
well demonstrated that for accurate determinations of size and shape
of the cells, some form of fixation other than heat is needed.
The best bacterial smears are usually made by removing a small
amount of surface growth from some solid medium and mixing it
with distilled water. It is often possible to use a drop of a culture
growing in a liquid medium, but such a smear is not always so satis-
factory, since certain constituents of the medium may prevent the
bacteria from adhering to the slide or may interfere with the staining.
The suspension used should always be sufficiently dilute. Ordi-
narily, only a faint turbidity should be visible to the naked eye;
for it is always best to avoid the occurrence on the slides of solid
masses of bacteria, piled one on top of the other. If a smear after
staining does not show any portions where the bacteria are well
separated one from another, a new, more dilute smear should be
made. This is particularly important in the case of the Gram stain,
or flagella staining.
The usual method of fixing the suspension to the slide or cover
glass is to pass it rapidly after drying through a Bunsen flame two or
three times. Another very satisfactory method is to allow the drop
of material to dry on a slide lying on a flat, moderately hot surface,
such as a plate of some non-rusting metal resting on a boiling water
bath. With many bacteria an aqueous suspension of the surface
growth from agar can be dried in the air at room temperature and
stained without any fixing; this method is not universally successful,
however.
For special staining procedures special methods of making bac-
terial preparations are necessary, sometimes calling for fixing solu-
tions rather than heat. It is beyond the scope of this leaflet, however,
to discuss them here, but it must be recognized that the technic
described above for staining dried smears is too crude for accurate
measurements of cells or for studying their cytological details.
It is also beyond the scope of this publication to give staining
methods for other than pure culture work, although a few (e.g.,
blood stains) have been given in previous editions.*
In using any of the methods it must be remembered that blind
adherence to a staining technic is no guarantee that the result will
be satisfactory. Even experienced workers sometimes discover to
their dismay that they took too much for granted as to the purity of
their reagents, cleanliness of slides and covers, or proper compound-
ing of the staining solutions. A technic should, therefore, be checked
upon known organisms as controls. It is, furthermore, important to
know that the solutions and water used for dilution are reasonably
free from bacteria and their spores.
*Those interested in other stains for microorganisms and for blood are referred to
the following leaflets of Staining Procedures (Conn and Darrow, 19-13-5):
I D. Miscellaneous methods (blood, bone, marrow, fat).
Ill A. Stains for microorganisms in smears.
Ill B. Stains for microorganisms in sections.
These leaflets can be purchased separately and are punched so as to 6t the cover to
this Manual.
STAINING METHODS iV46-5
STAINING FORMULAE
There has always been a surprising amount of inaccuracy in the
hteraturc concerning staining sohitions. This is due to a variety
of causes: indefiniteness in the original j)ublication; mistakes of
copying by later authors; modifications of the original which are
not described as modifications and come later to be ascribed to the
original author; failure of authors to cite references when giving
their methods. For such reasons it has proved necessary in this
publication to give in many instances both the original (rather
indefinite) formula and an emended formula as interpreted by the
Committee. The Committee, however, assumes no responsibility
for the identity of the tivo, and offers the emendation merely to prevent
the perpetuation of formulae which are clearly ambiguous or indefinite
as to their ingredients. Recent cooperation between this Commit-
tee, the Biological Stain Commission, and the National Formulary
Committee of the American Pharmaceutical Association, has re-
sulted in the virtual adoption of these emended formulae.
In the present edition of this leaflet the practice is still continued
of giving both the original and the emended formulae in such in-
stances. It is anticipated, however, that the latter will be regarded
as sufficiently standard, in a few years, so that the original formulae
can be dropped in future editions.
In early editions of this leaflet staining formulae and methods
were merely taken from the literature without any endorsement
by the Committee. At present, greater experience in such matters
permits the Committee to recommend certain of the procedures,
and they are now grouped according to whether or not they are thus
endorsed. Several of the less frequently used methods formerly
given are now omitted. One or two new methods are included
among those recommended by the Committee.
Staining Schedule. Tap vs. distilled water. When washing
slides after applying any stain, tap water is ordinarily more con-
venient to use than distilled water; and in the staining schedules
that follow, tap water is specified in those instances where its use
is considered to be ordinarily unobjectionable. It must be remem-
bered, however, that the use of distilled water is never contraindicated
for such purposes; and many bacteriologists prefer it for all steps
where washing is called for, because it is not subject to variation
in composition, buffer content, etc.
General Bacterial Stains — Recommended Procedures
ziehl's carbol-fuchsin
OLD STATEMENT OF FORMULA EMENDED STATEMENT OF FORMULA
Solution A
Sat. ale. sol. basic fuchsin 10 ml. Basic fuchsin (90% dye content)^ 0.3 g.
5% sol. carbolic acid 100 ml. Ethyl alcohol (95%) 10 ml
Solution B
Phenol 5 g.
Distilled water 95 ml.
Mix Solutions A and B.
^It is not necessary that dry stains of the exact dye content specified be used in this
or in the following formulae. Samples of higher or lower dye content may be employed
by making the proper adjustment in the quantity used.
IV46-6 MANUAL OF METHODS FOR PURE CULTURE STUDY
AMMONIUM OXALATE CRYSTAL VIOLET (hUCKER's)
Solution A Solution B
Crystal vioiet (90% dye content) 2 g. Ammonium oxalate 0-8 g.
Etiiyl alcohol (95%) 20 ml. Distilled water 80 ml.
Mix solutions A and B.
CRYSTAL VIOLET IN DILUTE ALCOHOL
Crystal violet (90% dye content) 2 g.
Ethyl alcohol (95%) 20 ml.
Distilled water 80 ml.
LOEFFLEr's alkaline METHYLENE BLUE
OBIGINAL STATEMENT OF FORMULA EMENDED STATEMENT
Solution A
Cone. sol. methylene blue in al- Methylene blue (90% dye con-
cohol 30 ml. tent) 0.3 g.
Sol. KOH in distilled water Ethyl alcohol (95%) 30 ml.
(1 :10,000) 100 ml. Solution B
Dilute KOH (0.01 % by weight) 100 ml.
Mix Solutions A and B.
METHYLENE BLUE IN DILUTE ALCOHOL
Methylene blue (90% dye content) 0.3 g.
Ethyl alcohol (95%,) 30 ml.
Distilled water 100 ml.
CARBOL ROSE BENGAL
Rose Bengal (80% dye content) 1 g.
Phenol (5% aqueous solution) 100 ml.
CaCla . 0.01-0.03 g.
(The amount of CaCl2 added determines the intensity of staining.)
Staining schedule: Follow the general procedure given under "Pre-
paration of Smears", p. 3-4 above, allowing 5-60 seconds for ap-
plication of the stain. Overstaining rarely occurs except with
carbol fuchsin; understaining does not have to be feared except
with rose Bengal.
Results: The results depend on which of the above staining fluids is
selected. They are listed in the order of intensity of action;
i.e. carbol fuchsin gives the most intense stain, and is not indicated
when selective staining is desired or when much debris is present
on the slide. The crystal violet solutions are very good for
routine purposes. The methylene blue solutions are much more
selective, with special affinity for metachromatic granules. The
rose Bengal solution is much less commonly used; it is specially
valuable when mucus or colloidal organic material is present, as
such material is not ordinarily stained by it.
General Bacterial Stains — ^Alternate Procedures
Kinyoun's Carbol Fuchsin
Basic fuchsin (dye content not specified; probably 90%) 4 g.
Phenol crystals 8 g.
Ethyl alcohol (95%o) 20 ml.
Distilled water • 100 ml.
This formula is preferred in some quarters to the Ziehl carbol fuchsin. It is attri-
buted to Kinyoun, but the reference to its original publication has not been located.
STAINING METHODS iv«-7
Carbol Crystal Violet (Nicolle)
original statement of formula emended statement
Solution A
Sat. ale. gentian violet 10 ml. Crystal violet (90% dye content) 0.4 g.
1% aqu. sol. phenol 100 ml. Ethyl alcohol (95%) 10 ml.
Solution B
Phenol 1 g.
Distilled water 100 ml.
Mix solutions A and B.
This formula is sometimes preferred either as a general stain or in the Gram technic
If properly prepared it is permanent; but it has a tendency to gelatinize if the amount
of dye is too great. To prevent this sort of deterioration the quantity of dye in the
above amended formula has been reduced to 0.4 g. from the 1.0 g. recommended in
previous editions of this leaflet. Even when the solution is so prepared as to be
permanent, however, it seems to have no advantage over the ammonium oxalate
crystal violet given above.
Anilin "Gentian Violet" (Ehrlich)
original statement of formula emended statement
Solution A
Sat. ale. sol. gentian violet 5-20 ml. Crystal violet (90% dye content) 1.2 g.
Anilin water (2 ml. anilin shaken Ethyl alcohol (95%) 12 ml.
with 98 ml. water and filtered) 100 ml. Solution B
Anilin 2 ml.
Distilled water 98 ml.
Shake and allow to stand for a few min-
utes, then filter.
Mix Solutions A and B.
This formula is given largely for its historic interest. It is a quite unstable solution,
and has no special value today. It was, however, one of the first important bacterial
staining fluids and was formerly regarded as the standard formula for the Gram stain.
It is not, however, certain what was the "anilin gentian violet" originally employed in
the Gram stain, even though ascribed to Ehrlich. As a matter of fact Ehrlich seems to
be properly credited only with the idea of using anilin water in the formula, as he ap-
parently did not recommend any one definite formula.
Negative Staining of Bacteria — Recommended Procedures
dorner's nigrosin solution
Nigrosin, water soluble (nigrosin B Gnibler recommended by Dorner;
American nigrosins certified by Commission on Standardization of Biologi-
cal Stains ordinarily satisfactory) 10 g.
Distilled water 100 ml.
Immerse in boiling water bath for 30 minutes; then add as preservative:
Formalin 0.5 ml .
Filter twice through double filter paper and store in serological test tubes, about 5 ml.
to the tube.
This staining solution is used for the negative demonstration of
bacteria, in place of the Burri India ink. For its use in Dorner's spore
stain, see p. IV46-II.
Staining schedule:
1. Mix a loopful of the bacterial suspension on the slide with an
equal amount of the staining solution. (If prepared from
growth on solid media, the suspension must not be too heavy.)
2. Allow the mixture to dry in the air, and examine under micro-
scope.
Results : Unstained cells in a background which is an even dark gray
if the preparation is well made.
IV46-8 MANUAL OF METHODS FOR PURE CULTURE STUDY
BENIANS' CONGO RED
Congo red (80% dye content) 2 g.
Distilled water 100 ml.
Staining schedule:
1. Place a drop of the above staining fluid on a slide.
2. Mix culture with the drop and spread out into a rather thick film.
3. After film has dried, wash with 1% HCl.
4. Dry, either in the air or by blotting.
Results: Cells unstained in a blue background. Good results are
not to be expected from broth cultures or from cultures in salt
solutions unless the cells are first removed by centrifuging.
The Gram Stain — Recommended Procedures
There are numerous modifications of the Gram stain, many of
which have been listed by Hucker and Conn (1923, 1927). The
two modifications given below have proved especially useful to the
Committee. The Hucker modification is valuable for staining smears
of pure cultures, that of Kopelofl^ and Beerman for preparations of
body discharges such as gonorrhoeal pus, also for pure cultures of
strongly acid-forming organisms. The latter is itself a variation
of the modification by Burke (1921).
hucker modification
AMMONIUM OXALATE CRYSTAL, VIOLET
(See p. IV46-6)
gram's MODIFICATION OF LUGOl's SOLUTION
Iodine 1 g.
KI 2g.
Distilled water 300 ml.
COUNTERSTAIN
Safranin O (2.5% solution in 95% ethyl alcohol) 10 ml.
Distilled water 100 ml.
Staining schedule:
1. Stain smears 1 min. with ammonium oxalate crystal violet.
This formula has sometimes been found to give too intense
staining, so that certain Gram-negative organisms (e.g. the
gonococcus) do not properly decolorize. If this trouble is
encountered, it may be avoided by using less crystal violet.
2. Wash in tap water.
3. Immerse 1 min. in iodine solution.
4. Wash in tap water and blot dry.
5. Decolorize 30 sec. with gentle agitation, in 95% ethyl alcohol.
Blot dry.
6. Counterstain 10 sec. in the above safranin solution.
7. Wash in tap water,
8. Dry and examine.
Results: Gram-positive organisms, blue; Gram-negative organisms,
red.
STAINING METHODS 1V46-9
BURKE AND KOPELOFF-BEERMAN MODIFICATIONS
ALKALINE GENTIAN VIOLET
Solution A Solution B
Gentian or crystal violet- 1 g. NaHCOs 1 g.
Distilled water 100 ml. Distilled water 20 ml.
BURKe's IODINE SOLUTION
Iodine, 1 g.; KI, 2 g.; distilled water, 100 ml.
KOPELOFF AND BEERMAN's IODINE SOLUTION
Iodine 2 g.
Normal NaOH (40.01 g. per liter) 10 ml
After the iodine is dissolved, make up to 100 ml. with distilled water.
burke's counterstain
Safranin O (85% dye content), 2 g.; distilled water, 100 ml.
KOPELOFF AND BEERMAN's COUNTERSTAIN
Basic fuchsin (90% dye content), 0.1 g.; distilled water, 100 ml.
Staining schedule:
1. Dry thinly spread films in the air without heat.
2. Flood with Solution A; mix on the slide with 2-3 drops (or
more, depending on size of flooded area) of Solution B, and
allow to stand 2-3 min.
Kopeloff and Beerman mix the two solutions in advance,
1.5 ml. Sol. A to 0.4 ml. Sol. B, and allow to stay on slide
5 min. or more.
3. Kinse with either of the above iodine solutions. (The Com-
mittee indicates no preference between the two; some work-
ers prefer one, some the other.)
4. Cover with fresh iodine solution and let stand 2 min. or longer.
5. Rinse with tap water; then blot water from surface of smear,
ivithout drying. (Kopeloff and Beerman omit the washing.)
The amomit of drying is important in this step. One must
get rid of all free water, but not allow the cells to dry.
6. Follow the blotting very quickly with decolorization in ether
and acetone (1 vol. ether to 1-3 vol. acetone), adding to
the slide drop by drop until practically no color comes off
in the drippings (usually less than 10 sec.) In this step
the speed of decolorization can be varied by varying the
ratio of ether to acetone; the more acetone the more rapid
the process. It is sometimes desirable to slow down the
process by using a ratio of 1:1.
7. Dry in the air.
8. Counterstain 5-10 sec. in one of the above given counter-
stains. Burke's (i.e. safranin) is preferred. The Kopeloff
and Beerman counterstain is too powerful to be used when
the shorter staining time recommended by Burke is followed.
'The authors specify either crystal violet or methyl violet 6B. Probably any of the
gentian violets now sold under the Commission certification are satisfactory; i. e. either
crystal violet or one of the bluer grades of methyl violet (e. g., methyl violet 2B).
IV46-10 MANUAL OF METHODS FOR PURE CULTURE STUDY
9. Wash in tap water.
10. Dry and examine.
Results: Gram-positive organisms, blue; Gram-negative organisms,
red. This technic is claimed to have the advantage of not giving
false positives due to vacuolar bodies that resist decolorization by
other Gram-staining procedures.
INTERPRETATION OF THE GRAM STAIN
A word of caution is necessary as to the interpretation of the Gram
stain. The test is often regarded with unjustified finality because
organisms are generally described as being either Gram-positive or
Gram-negative. Many organisms, however, actually are Gram-
variable. Hence, one should never give the Gram reaction of an un-
known organism on the basis of a single test. He should repeat the
procedure on cultures having different ages and should use more than
one staining technique in order to determine the constancy of the
organism toward the stain. Two phenomena deserve consideration.
(1) Henry & Stacey (1943) and Bartholomew and Umbreit (1944)
have shown that Gram-positive organisms can be made Gram-nega-
tive by treatment with ribonuclease, and that their Gram-positive
reaction can be restored subsequently by treatment with magnesium
ribonucleate. (2) Some organisms have granules which resist
decolorization and which may cause misinterpretation. Such
observations show that the Gram stain does not always give a clear
cut reaction and that the results must be interpreted with care.
Acid-fast Staining — Recommended Procedure
ziehl-neelsen method
Ziehl (1882); Neelsen (1883)
Staining schedule:
1. Stain dried smears 3-5 min. with Ziehl's carbol fuchsin (p. 5),
applying enough heat for gentle steaming.
2. Rinse in tap water.
3. Decolorize in 95% ethyl alcohol, containing 3% by volume of
cone. HCl, until only a suggestion of pink remains.
4. Wash in tap water.
5. Counterstain with one of the methylene blue solutions given on
p. 6.
6. Wash in tap water.
7. Dry and examine.
Results: Acid-fast organisms, red; others, blue.
AciD-FAST Staining — Alternate Procedures
Fluorescence Method
Richards and Miller (1941)
Although this method is not of special importance in pure culture work, special
mention should be made of it because of the amount of attention now given to it in
diagnostic work. Its real advantage is that it can be used with relatively low magnifi-
cation, and the large fields that can be examined assure positive diagnoses in cases
where the numbers of tubercle organisms are few.
STAINING METHODS iv«-ll
Solution A Solution B
Auramine O (90% dye content) . . 0.1 g. Ethyl alcohol (70%) 100 ml.
Liquefied phenol 3 ml. Cone. HCl 0.5 ml.
Distilled water 97 ml. NaCl 0.5 g.
Staining schedule:
1. Stain dried smears 2-3 min. in Solution A.
2. Wash in tap water.
3. Destain 3-5 min. in Solution B, freshly prepared.
4. Dry, and examine under a monocular microscope, using 8 mm. dry objective and
a 20X ocular; illumination should he a low voltage, high amperage microscope
lamp, supplied with a l)lue (ultraviolet transmitting) filter, a complementary
yellow filter having been provided for the ocular.
Results: Acid-fast bacteria, bright yellow, fluorescent; other organisms, not visible;
background, nearly black.
Much's Method
Much (1907)
Much's method No. 2, which is now quite widely used, employs carbol gentian violet
of essentially the formula given on page iv^t-S for carbol fuchsin except that in the
place of basic fuchsin the author calls for methyl violet BN. Preparations are
stained cold for 24 hours or by gentle application of heat until steaming. They are
then washed in water and treated with Lugol's iodine (see p. iv^6-8) from 1 to 5
minutes. After a second washing they are treated with 5% nitric acid for 1 minute
followed by 3% hydrochloric acid for 10 seconds. They are then decolorized 1 minute
in equal parts of acetone and 95% ethyl alcohol. Weiss (1909) has modified this
procedure by staining with a mixture of 3 parts of carbol fuchsin to 1 part of carbol
gentian violet and counterstaining with 1% aqueous safranin (5 to 10 seconds) or with
Bismarck brown (1 minute). The counterstain is applied immediately after the
decolorization, the acetone-alcohol being removed merely by blotting. In some
laboratories this method of counterstaining is employed following the Much technic
with carbol gentian violet alone for the primary stain.
Cooper's Method
Cooper (1926)
The Cooper method calls for staining in Ziehl's carbol fuchsin to which 3% of a 10%
aqueous sodium chloride solution is added just before use. Smears are stained either
by steaming 3 to 4 minutes, then allowing them to cool until a precipitate forms, or
else by standing overnight in a 37° incubator and cooling in an ice box for 20 minutes
to allow precipitation to occur. After the precipitation, the smears are washed with
tap water and decolorized 1 to 10 minutes in acid alcohol (5 ml. of nitric acid, sp. gr.
1.42, to 95 ml. of 95% ethyl alcohol); washed again with water, and finally for 1 minute
with 95% ethyl alcohol. They are counterstained with 1% brilliant green, or if the
smear is heavy, with a greater dilution of this same stain; washed with water, dried,
and examined.
Spore Staining — Recommended Procedures
dorner's method
Dorner (1922, 1926)
Staining schedule:
1. Make a heavy suspension of the organism in 2-3 drops of dis-
tilled water in a small test tube.
2. Add an equal quantity of freshly filtered Ziehl's carbol fuchsin
3. Allow the mixture to stand in a boiling water bath 10 min. or
more.
4. On a cover slip or slide mix one loopful of the stained prepara-
tion with one loopful of Dorner's nigrosin solution (p. 7).
5. Smear as thinly as possible and do not dry too slowly.
IV46-12 MANUAL OF METHODS FOR PURE CULTURE STUDY
Note: If even backgrounds for exhibiting or photographing are re-
quired, especially in the case of slime-producing bacteria, the
following procedure is recommended:
1. Make the suspension in 0.5 ml. nutrient broth or water.
2. Add 1 ml. of 10% gelatin solution.
3. Add 1 ml. of carbol fuchsin and stain as in (1) and (2) above.
4. Wash out the colloids with warm tap water, with the help of
centrifuge or sedimentation.
5. Mix with nigrosin and proceed as above.
Results: Spores, red; vegetative cells, unstained; background, gray.
DORNER METHOD — SNYDER MODIFICATION
Snyder (1934)
Staining schedule:
1. Prepare a dried smear on a slide and cover with a small piece of
blotting paper.
2. Saturate blotting paper with freshly filtered Ziehl's carbol
fuchsin (p 5).
3. Allow to steam 5-10 min., keeping paper moist by adding more
staining fluid.
4. For neat preparations, decolorize instantaneously with 95%
ethyl alcohol (but omit this step if the organisms do not hold
color well.)
5. Wash with tap water.
6. Apply a drop of saturated acjueous nigrosin (or Dorner's fluid)
and spread evenly.
7. Allow slide to dry quickly with gentle heat, without prior
washing.
Results: Same as with original method; but this modification proves
applicable to some bacteria (e.g. Bacillus subtilis) that are difficult
to stain by Dorner's technic.
conklin's modification of wirtz method
Wirtz (1908); Conklin (1934)
Staining schedule:
1. Make smears as usual and fix by heat.
2. Flood slide with 5% aqueous malachite green, and steam for 10
minutes, keeping slide flooded by addition of fresh staining
fluid.
3. Wash 30 sec. in running water.
4. Counterstain 1 min. with 5% aqueous mercurochrome.
5. Wash in running water.
6. Blot dry and examine.
Results: Spores, green; rest of cell, red. Trouble is sometimes
experienced with the green fading after the slides have stood a few
days. Apparently this is due to an alkaline reaction and can be
prevented by treating the slides in acid before making the smears.
(The alkalinity may be due to an invisible film of soap or washing
powder.)
STAINING METHODS iV46-13
Spore Staining — Alternate Procedure
SCHAEFFER-FULTON MouiKICATION OF WiRTZ MeTHOD
Schacffer & Fulton (1933)
Bacterial smears are made as usual and fixed in a flame. They are flooded with 5%
aqueous malachite green for 30 to GO seconds, and heated to steaming three or four
times. The excess stain is washed off in running water for about lialf a minute, and
0.5% aqueous safranin is added for about 30 seconds. The smears arc tiien washed
and blotted. The spores sliould be stained green, the rest of the cells red.
Staining the Diphtheria Organism — Recommended Procedures
Various special procedures have been devised for staining the
diphtheria organism in such a manner as to render it distinctive in
appearance by differentiation of its characteristic metachromatic
granules.
staining with methylene blue
Staining schedule:
1. Prepare smear as usual, and fix with gentle heat.
2. Stain for a few seconds with either of the methylene blue solu-
tions (i.e. Loeffler's, or dilute alcoholic) given on p. 6.
3. Wash in tap water.
4. Dry and examine.
Results: Metachromatic granules, dark blue to violet; bacteria with-
out such granules, evenly stained. The picture varies a little ac-
cording to which of the two methylene blue solutions is employed.
The Loeffler formula gives purplish shades of staining because of
the oxidation of methylene blue caused by the alkali. Some users
consider the polychrome effect thus obtained to give better differ-
entiation; others think the metachromatic granules show more
sharply with the clear blue of the unpolychromed dye.
Albert's diphtheria stain
Albert (1920)
Toluidine blue 0.15 g.
Methyl green 0.20 g.
Acetic acid (glacial) 1 ml.
Ethyl alcohol (95%) 2 ml.
Distilled water 100 ml.
laybourn's modification
Laybourn (1924) has modified the Albert stain by replacing the
methyl green with an equal amount of malachite green.
Staining schedule:
1. Make smears as usual and fix with gentle heat.
2. Stain 5 min. in either Albert's staining fluid or Laybourn's
modification of it. The latter is claimed to give deeper
staining of both granules an<l body of the cells, without lessen-
ing the contrast between them.
3. Drain without washing.
4. Treat 1 min. in a modified Lugol's solution (iodine, 2 g. ; KI, 3 g.;
distilled water, 300 ml.).
IV46-14 MANUAL OF METHODS FOR PURE CULTURE STUDY
5. Wash briefly in tap water.
6. Blot with filter paper, and examine.
Results: Metachromatic granules, black; bars of diphtheria cells,
dark green to black; body of cells, light green.
LJUBINSKY STAIN
(from Blumenthal and Lipskerow, 1905)
ORIGINAL FORMULA EMENDED FORMULA
Solution A Solution A
Pyoktanin (Merck) 0.25 g. Methyl violet 2B or crystal vio-
5% acetic acid 100 ml. let (85% dye content) 0.25 g.
Glacial acetic acid 5 ml.
Distilled water 95 ml.
Solution B Solution B
0.1% vesuvin Bismarck brown Y 0.1 g.
Distilled water 100 ml.
Staining schedule:
1. Make smears as usual and fix with gentle heat.
2. Stain 30 sec. to 2 min. in Solution A.
3. Wash in tap water.
4. Stain 30 sec. with solution B.
5. Wash in tap water.
6. Dry and examine.
Results: Metachromatic granules, dark blue or black; rest of cell,
reddish or yellowish.
Staining the Diphtheria Organism — Alternate Procedures
Neisser's Diphtheria Stain
Neisser (1903)
Solution No. 1 Solution No. 2
Methylene blue (dye content Crystal violet (dye content not
not specified; probably 90%) . . 1 g. specified; probably 85%) 1 g.
Alcohol (e. g., 95% 20 ml. Alcohol (e. g., 95%) 10 ml.
Acetic acid (glacial) 50 ml. Distilled water 300 ml.
Distilled water 1000 ml. Solution No. 3
Mix, and agitate until dye is dissolved. Chrysoidin 1 or 2 g.
Hot water 300 ml.
Filter after dissolving
Dried films are stained 10 seconds in a mixture of 2 parts of Solution No. 1 and 1 part
of Solution No. 2. Wash. Stain 10 seconds in Solution No. 3. Wash briefly in water,
or not at all. Blot dry.
Bonder's Diphtheria Stain
Ponder (1912); Kinyoun (1915)
Original As modified
formula by Kinyoun
Toluidine blue 0.02 g. 0.1 g.
Azure I 0.01 g.
Methylene blue ■ 0.01 g.
Glacial acetic acid 1 ml. 1 ml.
Ethyl alcohol (see below) 2 ml. 5 ml.
Distilled water 100 ml. 120 ml.
Dissolve the dyes in the alcohol, add the water, then the acid and let stand 24 hours
before using. Do not filter. After prolonged standing, action may be intensified by
adding 1 or 2 drops of glacial acetic acid.
STAINING METHODS iV46-15
According to Kinyoun, smears are 6xed with heat, allowed to cool and stained 2-7
minutes.
In the source of the original formula above cited, alisolute alcohol is specified; Kin-
youn calls for 9.5% alcohol. On theoretical grounds, indeed, absolute alcohol is not
indicated and the 95% strength may well be substituted even in the original formifla.
Although the Committee has had no personal experience with either formula, informa-
tion is at hand indicating the superiority of the Kinyoun modification.
Flagella Staining — Recommended Procedures
Flagella staining is a difRcult technic and there have been numerous
methods proposed for the purpose. It has k)ng be^n reahzed that
flagella are actually below the visual limit in size; but of recent
years the electron microscope has given a definite idea how small
they really are — around 0.02 to 0.03 /x in diameter. Electron
micrographs, in fact, indicate that with many kinds of bacteria even
the best stained preparations give a very inadequate picture of the
actual number or length of the flagella attached to a cell. Were
the electron microscope more simple to use, it is possible that it might
supplant the light microscope entirely in the demonstration of flagella.
Since that is far from the case at present, one must do the best he can
with staining methods intended to make the flagella visible. This is
usually done by a preliminary mordanting which causes precipitation
on the flagella and increases their apparent size — a principle intro-
duced by Loeffler (1890).
A second difficulty in staining flagella is the ease with which bac-
teria shed these delicate appendages unless the cultures are properly
handled. To prevent this one ordinarily employs specially cleaned
slides and specially prepared smears on the slides.
Methods for 'preparing slides. Ordinary cleaning of glassware is
not sufficient for the purpose. Various methods have been proposed,
but the following directions seem to give as good results as any:
Use new slides if possible preferably of "Pyrex" glass or similar
heat resistant properties. (This is because under the drastic method
of cleaning to remove grease, old slides have a greater tendency to
break.) Clean first in a dichromate cleaning fluid, wash in water
and rinse in 95 per cent alcohol ; then wipe with a clean piece of cheese
cloth. (Wiping is not always necessary but is advisable unless fresh
alcohol is used after every few slides.) Pass each slide back and
forth through a flame for some time, ordinarily until the appearance
of an orange color in the flame; some experience is necessary before
the proper amount of heating can be accurately judged.
Unless heat-resistant slides are used, cool slides gradually in order
to minimize breakage. An ordinarily satisfactory method of doing
this is to place the flamed slides on a metal plate (flamed side up)
standing on a vessel of boiling water; and then to remove the flame
under the water so as to allow gradual cooling. (Too rapid cooling
may result in breakage, sometimes as long as two weeks after the
heating.)
Methods of handling cultures. Of various methods proposed, it
is not possible to recommend any one as unifc^rml}' the best. As any
laboratory worker becomes familiar with one particular method,
he soon finds he can get better results with that than with any other.
IV46-16 MANUAL OF METHODS FOR PURE CULTURE STUDY
The following method, however, can be given as one of the most
satisfactory, especially for students who have not had previous
experience- with some other method:
Use young and actively growing cultures (e.g. 18-22 hr. old) on
agar slants. Before proceding, check the culture for motility in
hanging drop. If motile, wash off the growth by gentle agitation with
2-3 ml. sterile distilled water. Transfer to a sterile test tube and
incubate at optimum temperature for 10 minutes (30 minutes for
those producinofslime). At this point, again check motility under
a microscope. Transfer a small drop from the top of the suspension
(where motile organisms are most numerous), by means of a capillary
pipette to one end of the slide prepared as above described. Tilt the
slide and allow the drop to run slowly to the other end. (Two or
three such streaks can be placed on a slide.) Place the slide in a
tilted position and allow it to dry in the air.
Staining 'procedure. Good results can be obtained with any of the
following methods, especially after familiarity has been obtained with
it. Special recommendation must be given to the last of the four
procedures (modified Bailey method). Although seeming a little
more complicated, on first reading, it has been found to give the
most uniformly satisfactory results in inexperienced hands.
casares-gil's flagella stain^
AS PUBLISHED BY PLIMMER AND PAINE (1921)
Mordant:
Tannic acid 10 g.
AlCls-eHoO 18 g.
ZnCls 10 g.
Basic fuchsin* 1-5 g.
Alcohol (60%) 40 ml.
The solids are dissolved in the alcohol by trituration in a mortar, adding 10 ml. of the
alcohol first, and the rest slowly. This alcoholic solution may be kept several years.
For use, mix with an equal quantity of water (Thatcher, 1926) or dilute with four
parts of water (Casares-Gil), filter off precipitate and collect filtrate on the slide.
Staining schedule:
1. Prepare smears of young cultures, on scrupulously cleaned
slides as above directed.
2. Filter mordant onto slide as above directed (preferably using
Thatcher's 1:1 dilution); allow to act for 60 sec. without heat-
ing.
3. Wash in tap water.
4. Flood slide with freshly filtered Ziehl's carbol fuchsin (p. 5),
and allow to stand 5 min. without heating.
5. Wash with tap water.
6. Air-dry and examine. Sometimes considerable search may be
needed before finding a satisfactorily stained part of the
smear.
Results: Fagella well stained (red) in the case of those bacteria (e.g.
3See Galli-Valerio (1915).
^The authors specify rosanilin hydrochloride. There are, however, other basic
fuchsins more universally available which ought to prove equally satisfactory.
STAINING METHODS IV46-17
colon-typhoid group, aerobic sporc-formcrs) that do not have
extremely delicate flagclla.
gray's flagella stain
Gray (1926)
Mordant: Solution A
KAl (804)2' 121120 (sat. aqu. solution) 5 ml.
Tannic acid {'■20% aqu. solution) 2 ml.
(A few drops of chloroform must be added to this if a large quantity is
made up)
HgCls (sat. aqu. solution) 2 ml.
Solution B
Basic fuchsin (sat. ale. solution) 0.4 ml.
Mix Solutions A and B less than twenty-four hours before using. Both solutions
separately may be kept indefinitely, but deteriorate rapidly after mixing.
Staining schedule:
1. Prepare smears from young cultures as above directed.
2. Flood slide with freshly filtered mordant and allow to act 8-10
min.
3. Wash with a gentle stream of distilled water, and follow steps
4-6 of above schedule (Casares-Gil's method).
Results: Same as with Casares-Gil method.
leifson's stain
Leifson (1930)
KA1(S04)2.12H20, or NH4A1(S04)2.12H20 (sat. aqu. solution) 20 ml.
Tannic acid (20% aqu. solution) 10 ml.
Distilled water 10 ml.
Ethyl alcohol, 95% 15 ml.
Basic fuchsin (sat. solution in 95% ethyl alcohol) 3 ml.
Mix ingredients in order named. Keep in tightly stoppered bottle and the stain
may be good for a week.
Staining schedule:
1. Prepare slides as for the preceding methods.
2. Flood slides with the above solution and allow to stand 10 min.
at room temperature in warm weather, or in an incubator in
cold weather.
3. Wash with tap water. (If a counterstain is desired, borax
methylene blue may be applied, without heat, followed
by another washing. See p. IV46-19).
4. Dry and examine.
Results: When no counterstain is used, same as with the two above
procedures; with methyelne blue counterstain, see under "Capsule
Stains", below.
BAILEY METHOD
Bailey (1929)
MODIFIED BY FISHER AND CONN (1942)
This method is specially recommended for bacteria on which
flagella are difficult to stain (as is frequently the case with soil and
IV46-18 MANUAL OF METHODS FOR PURE CULTURE STUDY
water non-spore-formers and with plant pathogens) because of slime
production, unusually fine flagella or flagella that are readily lost.
Mordant: Solution A
Tannic acid (10% aqu. solution) 18 ml.
FeClsGHjO (6% aqu. solution) 6 ml.
Solution B
Solution A 3.5 ml.
Basic fuchsin (0.5% in ethyl alcohol) 0.5 ml.
HCl, concentrated 0.5 ml.
Formalin 2.0 ml.
Staining schedule:
1. Prepare smears of young cultures, following carefully the
procedure recommended on p. 15 under "Methods of handling
cultures".
2. Filter the above Solution A onto the slide and allow it to remain
3}/2 min. without heating.
3. Pour off solution A, and without washing add solution B, also
through a filter, and allow it to stand 7 min. without heating.
4. Wash with distilled water.
5. Before the slide dries, cover with Ziehl's carbol fuchsin (p. 5),
allowing it to stand 1 min. on a hot plate heated just enough
for steam to be barely given off.
6. Wash in tap water.
7. Dry in the air and examine.
Results: Similar to the preceding methods; but the background pre-
cipitate is usually finer and less conspicuous, thus interfering less with
the demonstration of unusually fine, delicate flagella.
Staining flagella of anaerobes. O'Toole (19-12) calls attention to
certain difficulties in staining the flagella of anaerobes, and gives a
modification of the above Bailey stain which is intended to overcome
them. The method is not unlike that of Fisher and Conn who had
the O'Toole procedure in mind when working out their modifica-
tion. The O'Toole method does not seem to be as satisfactory as
the Fisher and Conn procedure for the above mentioned soil bacteria
and plant pathogens; but one must remember that it is particularly
recommended by its author for an entirely different type of organism.
Capsule Stains— Recommended Procedures
Bacterial capsules are more easily confused with artifacts than any
other structure pertaining to the organisms. Inasmuch as capsules
sometimes show merely as unstained areas around the cells, there is a
temptation to call any such surrounding area a capsule; very often,
however, they merely represent the tendency of a lightly stained sur-
rounding medium to retract from the cells on drying. For this
reason the best way to demonstrate capsules is actually to stain them
by some procedure which differentiates them from the cell itself.
Several of the flagella stains accomplish this, notably those of Bailey
and Leifson, given above. Much simpler is the procedure of Anthony
described below. The Anthony method can be recommended both
because of its simplicity and its dependability. Any of the other
STAINING METHODS iv«-19
methods which follow give satisfactory results. The student is
specially urged, however, not to pronounce any organism capsulated,
as a result of any of these staining procedures, until he has carefully
compared it with other organisms generally recognized as having
capsules.
LEIFSON METHOD
Leifson (1930)
This method is described in detail above (p. 17) and does not need
to be repeated here. The special methods of handling slides and
cultures, outlined for flagella staining, do not need to be observed,
but the following is essential:
After step 3;
4. Stain 5-10 min., without heating, in borax methylene blue
(methylene blue, 90% dye content, 0.1 g.; borax 1 g. ; distilled
water 100 ml.).
5. Wash in tap water.
6. Dry and examine.
Results: capsules red; cells, blue,
Anthony's method
WITH Tyler's modification
Anthony (1931)
Original formula Tyler's modification^
Crystal violet (85% dye content) 1 g. Crystal violet (85% dye con-
Distilled water 100 ml. tent) 0.1 g.
Glacial acetic acid 0.25 ml.
Distilled water 100 ml.
Staining schedule:
1. Prepare smears and dry them in the air.
2. Stain 2 min. in the above aqueous crystal violet; or according
to Tyler 4-7 min. in the above acetic crystal violet.
3. Wash with 20% aqueous CuS04-5H20.
4. Blot dry, and examine.
Results: capsules, blue violet; cells, dark blue.
hiss's method
Hiss (1905)
ORIGINAL STATEMENT OF FORMULA EMENDED FORMULA
Sat. ale. basic fuchsin or gentian Basic fuchsin (90% dye con-
violet 5-10 ml. tent) 0.15-0.3 g.
Water to make 100 ml. Distilled water 100 ml.
or
Crystal violet (85% dye con-
tent) 0.05-0.1 g.
Distilled water 100 ml.
Staining schedule:
1. Grow organisms in ascitic fluid or serum medium, or mix with
drop of serum and prepare smears from this mixture.
2. Dry smears in the air and fix with heat.
3. Stain with one of the above solutions a few seconds by gently
heating" until steam rises.
'See Park and Williams (1933), p. 84.
1V46-20 MANUAL OF METHODS FOR PURE CULTURE STUDY
4. Wash off with 20% aqueous CuS04-5H20.
5. Blot dry, and examine.
Results: capsules, faint blue; cells, dark purple.
Stains for Spirochaetes — Recommended Procedure
rONTANA STAIN
Preparation of ammoniacal silver nitrate:
Dissolve 5 g. AgNOs in 100 ml. distilled water. Remove a few
milliliters, and to the rest of the solution add drop by drop a con-
centrated ammonia solution until the sepia precipitate which forms
redissolves. Then add drop by drop enough more of the silver
nitrate solution to produce a slight cloud which persists after shaking.
It should remain in good condition for several months.
Staining schedule:
1. Prepare smear and fix with heat.
2. Pour on a solution of 5% tannic acid in 1% phenol and allow to
steam 30 sec.
3. Wash 30 sec. in running water.
4. Cover with a drop of the above ammoniacal silver nitrate, heat
gently over a flame and allow it to stand 20-30 sec. after
steaming begins.
5. Wash in tap water.
6. Blot dry, and examine.
Results : Spirochaetes, dark brown or black, in a dark maroon field.
Stains for Spirochaetes — Alternate Procedure
Tunnicliff's Stain
Tunnicliff has employed carbol gentian violet (3 to 4 seconds) followed by Lugol's
iodine (see p. IV46-8) for the same period in staining bacterial smears. With a slight
modification this proves a good spirochaete stain. The modification is:
Carbol crystal violet (1 vol. 10% ale. crystal violet to 10 vol. 1% aq. phenol)
30 seconds; wash with water; the Lugol-Gram iodine solution (see p. IV46-8) 30
seconds; wash with water; safranin 30 seconds; wash with water and dry.
Stain for Rickettsiae
macchiavello's method
Staining solution: 0.25 g. basic fuchsin (90% dye content) dis-
solved in 100 ml. distilled water, buffered to pH 7.2-7.4 with the
proper phosphate buffer mixture.
Staining schedule:
1. Smear a bit of tissue on a slide.
2. Dry in the air and fix with gentle heat.
3. Pour the above staining fluid onto the slide through a coarse
filter paper. Allow to stand 4 min.
4. Rinse very rapidly with 0.5% aqueous citric acid.
5. Wash quickly and thoroughly with tap water.
6. Counterstain about 10 sec. with 1% aqueous methylene blue.
7. Rinse in tap water.
8. Dry and examine.
Results: Rickettsiae, red; cell nuclei, deep blue; cytoplasm, light blue.
STAINING METHODS
IV46-21
Dye Solubilities at 2G°C.
Based on data obtained at the Color Laboratory of the U. S. Dept. of
Agriculture. From Biological Stains by II. ./. Conn, Jflh Ed.
Pub. by Biotech Publications, Geneva, N. Y., 19^0.
Note: These figures are ordinarily for recrystallized dyes. Commercial samples
are generally less soluble, often by as much as 30%.
Colour Index
Name of dye
Per cent soluble in
Number
Wilier
95% alcohol
1027
Alizarin
nil
0.125
1034
Alizarin red S
7.69
0.15
40
Alizarole orange G
0.40
0.57
36
Alizarole yellow GW
2.5.84
0.04
184
Amaranth
7.20
0.01
847
Amethyst violet
3.12
3.66
655
Auramin 0
0.74
4.49
12
Aurantia
nil
0.33
146
Azo acid yellow
2.17
0.81
88
Azo Bordeaux
3.83
0.19
448
Benzopiu'purin 4B
0.13
280
Biebrich scarlet
0.05
332
Bismarck brown R
1.10
0.98
331
Bismarck brown Y
1.36
1.08
252
Brilliant croceine
5.04
0.06
29
Chromotrope 2R
19.30
0.17
21
Chrysoidin 11
0.23
0.99
20
Chrysoidin Y
0.86
2.21
370
Congo red
— —
0.19
89
Crystal ponceau
0.80
0.06
681
Crystal violet (chloride) 1 gentian
Crystal violet (iodide) /violets
1.68
13.87
0.035
1.78
Cresyl violet (N. A. Co.)
0.38
0.25
715
Cyanole extra
1.38
0.44
771
Eosin B (Na salt)
39.11
0.75
768
Eosin Yf (Na salt)
44.20
2.18
Eosin Yf (Mg salt)
1.43
0.28
Eosin Yf (Ca salt)
0.24
0.09
Eosin Yf (Ba salt)
0.18
0.06
130
Erika B
0.04
0.17
254
Erythrin X
6.41
0.06
773
Erythrosinf (Na salt)
11.10
1.87
Erythrosinf (JNIg salt)
0.38
0.52
Erythrosinf (Ca salt)
0.15
0.35
Erythrosinf (Ba salt)
0.17
0.04
770
Ethyl eosin
0.03
1.13
Fast green FCF
16.04
0.35
176
Fast red A
1.67
0.42
16
Fast yellow
18.40
0.24
766
Fluorescein (color acid)
0.03
2.21
Fluorescein (Na salt)
50.20
7.19
Fluorescein (Mg salt)
4.51
0.35
Fluorescein (Ca salt)
1.13
0.41
Fluorescein (Ba salt)
6.54
0.56
Fuchsin, basic:
676
Pararosanilin (chloride)
0.26
5.93
Pararosanilin (acetate)
4.15
13.63
Rosanilin (chloride)
0.39
8.16
678
New fuchsin (chloride)
1.13
3.20
Gentian violet (see crystal or me-
thvl violet)
tThe color acids of these dyes (not listed here) are practically insoluble in water.
IV46-22
MANUAL OF METHODS FOR PURE CULTURE STUDY
Colour Index
Per cent soluble in
Name of dye
Number
Water
95% alcohol
666
Guinea green B
28.40*
7.30
1180
Indigo carmine
1.68
0.01
133
Janus green
5.18
1.12
670
Light green SF yellowish
20..S5
0.82
657
Malachite green (oxalate)
7.60
7.52
9
Martius yellow, Na salt
4.57
0.16
Martius yellow, Ca salt
0.05
1.90
138
Metanil yellow
5.36
1.45
142
Methyl orange
0.52
0.08
Methyl orange (acid)
0.015
0.015
680
Methyl violet (Gentian violet)
2.93
15.21*
922
Methylene blue (ZnCL double salt)
2.75
0.05
Methylene blue (chloride)
3.55
1.48
Methylene blue (iodide)
0.09
0.13
924
Methylene green
1.46
0.12
10
Naphthol yellow G
8.96
0.025
152
Narcein
10.02
0.06
825
Neutral red (chloride)
5.64
2.45
Neutral red (iodide)
0.15
0.16
826
Neutral violet
3.27
2.22
927
New methylene blue N
13.32*
1.65
728
New Victoria blue II
0.54
3.98
520
Niagara blue 4B
13.51
nil
914
Nile blue 2B
0.16
0.62
73
Oil red 0
nil
0.39
150
Orange I
5.17
0.64
151
Orange II
11.37
0.15
27
Orange G
10.86
0.22
714
Patent blue A
8.40
5.23
774
Phloxinet (Na salt)
50.90*
9.02
Phloxinef (Mg salt)
20.84
29.10
Phloxinet (Ca salt)
3.57
0.45
Phloxinef (Ba salt)
6.01
1.17
7
Picric acid
1.18
8.96
28
Ponceau 2G
1.75
0.21
186
Ponceau 6R
12.98
0.01
741
Pyronin B (iodide)
0.07
1.08
739
Pyronin Y
8.96
0.60
148
Resorcin yellow
0.37
0.19
749
Rhodamine B
0.78
1.47
750
Rhodamine G
1.34
6.31
779
Rose bengalf (Na salt)
36.25
7.53
Rose bengalf (Mg salt)
0.48
1.59
Rose bengalf (Ca salt)
0.20
0.07
Rose bengalf (Ba salt)
0.17
0.05
841
Safranin
5.45
3.41
689
Spirit blue
nil
1.10
24
Sudan I
nil
0.37
248
Sudan III
nil
0.15
258
Sudan IV
nil
0.09
920
Thionin
0.25
0.25
925
Toluidine blue 0
3.82
0.57
690
Victoria blue 4R
3.23
20.49
659
Victoria green 3B
0.04
2.24
8
Victoria yellow
1.66
1.18
*These figures are grams per hundred grams of saturated solution (the others
being grams per hundred milliliters).
tThe color acids of these dyes (not listed here) are practically insoluble in water.
STAINING METHODS iv„-23
REFERENCES
Albert, Henry. 1920. Diplithcria l)acilliis stains with a description of a "new"
one. Am. J. Pub. noalth, 10, ■iSi-7.
, 1921. Modification of stain for diphtheria bacilli. .1. .Vmer. Med.
Assn., 76, 240.
Anthony, E. E. 1931. A note on capsule staining. Science, 73, 319.
Bailey, H. D. 1929. A flagclla and capsule stain for bacteria. Troc. Soc. E.xp.
Biol. & Med., 27, 111-2.
Bartholomew, J. W., and Umbreit, W. W. lOl'l.. Riljonucleic acid and the Gram
stain. J. Bact., 48, 5(57-78.
Beni.'VNS, T. II. C. 1916. Relief staining for bacteria and spirochaetes. Brit. Med.
J. 1916 (2). 722.
Blumenthal, J. M. and Lipskerow, M. 1905. Vergleichcnde Bewertung der dif-
ferentiellen Methode zur Farbung des Diphtheriebacillus. Centbl. f. Bakt. I
Abt., Orig., 38, 359-66.
Burke, Victor. 1921. The Gram stain in the diagnosis of chronic gonorrhea.
J. Amer. Med. Assoc, 77, 1020-2.
Burke, Victor. 1922. Notes on the Gram stain with description of a new method.
J. Bact., 7, 159-82.
Conklin, Marie E. 1934. Mercurochrome as a bacteriological stain. J. Bact., 27,
30.
Conn, H. J. 1946. Biological Stains. 5th Ed. Biotech Publications, Geneva, N. Y.
Conn and Darrow. 1943-5. Staining Procedures. Biotech Publications, Geneva,
N. Y.
Cooper, F. B. 1926. A modification of the Ziehl-Neelsen staining method for
tubercle bacilli. Arch. Path. & Lab. Med., 2, 382-5.
Dorner, W. C. 1922. Ein neues Verfahren fur isolierte Sporenfarbung. Landw.
Jahrb. d. Schweiz., 36, 595-7.
Dorner, \V. C. 1926. Un procede simple pour la coloration des spores. Le Lait, 6,
8-12.
Dorner, W. C. 1930. The negative staining of bacteria. Stain Techn., 5, 25-7.
Fisher, P. J. and Conn, Jean E. 1942. A flagella staining technic for soil bacteria.
Stain Techn., 17, 117-21.
Fontana, Artur. 1912. Verfahren zur intensiver und raschen Farbung des Tre-
ponema pallidum und anderer Spirochaten. Derm. Wochnsch., 55, 1003-4.
Galli-Valerio, B. 1915. La methode de Casarcs-Gil pour la coloration des cils des
bacteries. Centbl. f. Bakt., I Abt. Orig., 76, 233-4.
Gray, P. H. H. 1926. A method of staining bacterial flagella. J. Bact., 12, 273-4.
Henry, H. and Stacey, M. 1943. Histochemistry of the Gram-staining reaction for
micro-organisms. Nature, 151, 671.
Hiss, P. H., Jr. 1905. A contribution to the physiological differentiation of Pneumo-
coccus and Streptococcus, and to methods of staining capsules. J. Exp. Med.,
6, 317-45.
Hucker, G. J. 1922. Comparison of various methods of Gram staining. (Pre-
liminary Report.) Abstr. Bact., 6, 2.
Hucker, G. J. and Conn, H. J. 1923. Methods of Gram staining. N. Y. S. Agr.
Exp. Sta., Tech. Bui. 129.
Hucker, G. J. and Conn, H. J. 1927. Further studies on the methods of Gram
staining. N. Y. S. Agr. Exp. Sta., Tech. Bui. 128.
Johnston, O. P., and Mack, W. B. 1904. A modification of existing methods for
staining flagella. American Medicine, 7, 754.
KiNYOUN, J. J. 1915. A modification of Ponder's stain for diphtheria. Am. J. Pub.
Health, 5, 246-7.
Kopeloff, N. and Beerman, P. 1922. Modified Gram stains. J. Inf. Dis., 31,
480-2.
Laybourn, R. L. 1924. A modification of Albert's stain for the diphtheria bacilli
J. Amer. Med. Assn., 83, 121.
Leifson, Ein.\r. 1930. A method of staining bacterial flagclla and capsules to-
gether with a study of the origin of flagella. J. Bact., 20, 203-11.
Loeffler, F. 1884. Untersuchungen tiber die Bedeutung der Mikroorganismen fiir
die Entstehung der Diphtheric beim Menschen, bei der Taube und beim
Kalbe. Mitt, a.d.k. Gcsundheitsamte, 2, 421-99. See p. 439.
IV46-24 MANUAL OF METHODS FOR PURE CULTURE STUDY
Mallory, F. B. and Wright, J. H. 1924. Pathological Technique. 8th Ed.
Saunders, Philadelphia.
Much, H. 1907. Uber die granulare, nach Teil nicht farbbare Form des Tuberku-
losevirus. Beitr. z. Klin. d. Tubercl., 8, 85-99.
Neelsen, F. 1883. Ein casuistischer Beitrag zur Lehre von der Tuberkulose.
Centbl. f. Med. Wissensch., 21, 497-501. (See p. 500.)
Neisser, M. 1903. Die Untersuchung auf Diphtheriebacillen in centralisierten
Untersuchungsstationen. Hyg. Rundsch., 13, 705-17.
NicOLLE, Ch. 1895. Pratique des colorations microbiennes. Ann. Inst. Past., 9,
664-70.
O'TooLE, Elizabeth. 1942. Flagella staining of anaerobic bacilli. Stain Techn., 17,
33-40.
Park, W. H. and Williams, Anna W. 1933. Pathogenic Microorganisms. 10th
Ed. Lea & Febiger, Philadelphia.
Plimmer, H. G., and Paine, S. G. 1921. A new method for the staining of bacterial
flagella. J. Path. & Bact., 24, 286-8.
Ponder, C. 1912. The examination of diphtheria specimens. A new technique in
staining with methylene blue. Lancet, 2, 22-3.
Richards, O. W. and Miller, D. K. 1941. An efiicient method for the identifica-
tion of M. tuberculosis with a simple fluorescence microscope. Amer. J. Clin.
Path., 11, 1-7.
Schaeffer, Alice B. and Fulton, McD. 1933. A simplified method of staining
endospores. Science, 77, 194.
Snyder, Marion A. 1934. A modification of the Dorner spore stain. Stain Techn.,
9,71.
Thatcher, Lida M. 1926. A modification of the Casares-Gil flagella stain. Stain
Techn., 1, 143.
TuNNicLiFF, Ruth. 1922. A simple method of staining Gram-negative organisms.
J. Am. Med. Assn., 78, 191.
Weiss, L. 1909. Zur Morphologic des Tuberkulosevirus unter besonderer Beriick-
sichtigung einer Doppelfarbung. Berl. Klin. Wochensch. 46, 1797-800.
WiRTZ, R. 1908. Ein einfache Art der Sporenfarbung. Centbl. f. Bakt., I Abt.
Orig., 46, 727-8.
ZiEHL, F. 1882. Zur Farbung des Tuberkelbacillus. Deut. Med. Wchnsch., 8, 451.
LEAFLET V
ROUTINE TESTS
FOR THE DESCRIPTIVE CHART
MORPHOLOGICAL AND BIOCHEMICAL
by
H. J. Conn
nth EDITION
Pure Culture Study of Bacteria, Vol. 17, No. 3-4
September, 1949
Revised with the assistance of
M. W. Jennison, L. S. McClung, C. A. Stuart and C. E. ZoBell
LEAFLET V
ROUTINE TESTS FOR THE DESCRIPTIVE CHART
MORPHOLOGICAL AND BIOCHEMICAL
Introduction
The Society of American Bacteriologists issues Descriptive Charts
for use in characterizing bacterial species. The Charts are blank forms
on which the characteristics of any culture under investigation are
to be recorded, at least one chart to be used for each culture studied.
The Manual of Methods for Pure Culture Study of Bacteria was
originally published to secure uniformity in the methods used for
determining these characteristics. At the present time the scope of
the Manual has become much broader than this, and practically all
the methods covered by the original Manual are now included in this
leaflet.
The methods described in this leaflet are intended primarily for
aerobic saprophytes, and cannot therefore be considered applicable
in general to obligate parasites or strict anaerobes. Leaflet III must
be consulted in studying the latter group; while Leaflet VII gives
methods specially applicable to animal pathogens. Special methods
for plant pathogens are given in Leaflet X. In the case of other
special groups, the investigator will often find the methods given
here to be unsatisfactory and will therefore be forced to modify
them or to use others more suited to the group in question.
The Descriptive Charts
There are two Descriptive Charts, each printed on 83^2 by 11 inch
sheets of heavy paper: the Standard Descriptive Chart, and the
Descriptive Chart for Instruction. The general plan of each is to
have the body of it consist, under various headings, of a series of
blanks to be completed and descriptive terms to be underlined, as
the various characteristics of the cultures are determined. In ad-
dition to this, there is a place on the margin for recording the most
important characteristics by a system of numerical notation.
The special feature of the Standard Descriptive Chart is that
all the most important characteristics of an organism may be re-
corded on the front of the sheet, partly in the margin, partly in the
larger section at the right, while the fermentative reactions are to be
V49-2
ROUTINE TESTS FOR THE DESCRIl'TIN E CHART V49-3
entered at the bottom. By the use of right-hand margin and bottom
edge, a long series of charts may be compared, one on top of the other,
by ghincing only at these two edges. The back of the Slaii(hird
Chart is now reserved hirgely for supplementary data, nearly all of
which is summarized on the front. (See first insert, following p. 12.)
The increasingly large number of tests called for in the study of
bacteria has resulted in making a somewhat complicated chart.
Although all these tests may be needed in some research work, they
plainly are not needed in the use of the chart for instruction pur-
poses. To meet the demand for a simpler chart for use in teaching,
a new form known as the Descriptive Chart for Instruction was
published in 1939. This chart is designed to fit a standard note-book
for 11 by 83^ inch sheets. (See second insert, p 12.) In numerous
research laboratories, also, this chart is proving more useful than the
Standard Chart, because of its flexibility and the amount of space
available for special tests.
Determining Optimum Conditions for Growth
Before beginning the study of any pure cidture, it is important to
know something about the growth requirements of the organism. If
the organism in question does not grow in ordinary media, either
because it is an obligate parasite or because it requires the complete
absence of oxygen or of organic matter, it ob\'iously cannot be studied
by the methods called for on the Descriptive Chart. For such organ-
isms the investigator must use his own methods of study, and may
record the results in the blank space at the bottom of the back of
the Chart. For those organisms that grow on ordinary media, methods
must be varied according to whether the organisms grow better in
liquid or in solid media and at high temperature or low temperature.
It is important, therefore, that before studying an unknown culture
which is able to grow in laboratory media, these two points in regard
to growth requirements be determined. (As pointed out in Leaflet
II, many such media are now available in dehydrated form.)
After these growth peculiarities are determined, it is possible to
proceed with the study of an organism under 0})tinium conditions.
Space is left on the Chart under all of the procedures listed where
the medium used and the temperature of incubation can be recorded.
So far as possible the same imiform set of conditions should be used
throughout the entire study of one organism. If, for example, one set
of tests is made on solid media at 25°C, the other tests should be
made likewise. Leaving out those organisms referred to above which
require special conditions of study, and other organisms of peculiar
growth requirements, such as the thermophilic bacteria, there are
four different sets of conditions that wall suit practically all bacteria.
V49-4 MANUAL OF METHODS FOR PURE CULTURE STUDY
namely: liquid media at 37°; solid media at 37°; liquid media at 21-
25°; and solid media at 21-25°.
Sj)ace is provided on the Standard Chart lor recording ojitimum
medium and temperature. This does not ordinarily mean that one
must determine the one best medium for the growth of the culture
nor the exact degree of temperature at which it grows most rapidly ^
In the first blank one may record such terms as "organic, solid,"
"organic, liquid," "inorganic, solid" etc., unless it be known that
there is one particular medium specially adapted to the organism in
question. Under the second blank one may record temperature in
general terms, as: "20-25°", "35-40°", "45-50°", or "over 55°."
It is also important to remember that certain organisms (frequently
facultative anaerobes) which do not grow in either solid or true liquid
media, will grow in a "semi-solid" medium (that is a nutrient solution
in which 0.05-0.1% of agar has been dissolved). It is of course im-
portant that such organisms be studied under optimum conditions;
and for their study the procedures given in this Manual should
ordinarily be modified by using media containing 0.05-0.1% agar
instead of the usual liquid or solid media.
Thermal death point, as called for under "Temperature Relations"
on the back of the Chart, is undoubtedly- best determined with the use
of capillary tubes. Short pieces of thin-walled tubing having an in-
ternal diameter of 1-1.5 mm. are filled with the culture (consisting
mostly of spores, if it is a spore-former) and are heated for varying
periods of time at the temperatures under investigation. After heat-
ing, each tube is broken into a tube of a medium in which the organ-
isms grow well. A tabulation of results gives a good idea of the ther-
mal death point. This procedure requires careful attention to detail;
and one should consult the description of it by Magoon (1926).
Results are most valuable if the length of time before death is re-
corded: in which case, this becomes a test for Thermal death time.
Incubation
Cultures should be incubated at or near the optimum temperature
of the organism or organisms under investigation. As a rule it is not
necessary, however, to know the exact optimum temperature of each
organism. If the laboratory is equipped with a series of incubators
running at 20°, 25°, 30°, and 37°C, the tem])erature requirements of
practically all bacteria except the thermophilic forms can be very
satisfactorily met. Room temperature is sometimes used in place of 25°;
but is not to be recommended because of its uncontrollable variations.
Length of incubation varies and is specified on the Chart under
many of the tests. In cases where it is not specified one should
observe the following general rule: On the day when good growth
ROUTINE TESTS FOR THE DESCRIPTIVE CHART v«-5
first appears the proper descriptive terms on tlie Chart shouhl be
underhiied. Any changes occurring and noted in subsequent study
should also be recorded on the Chart. The meaning of the terms
given in this section of the Chart will in general be made clear by
consulting the glossary included in Leaflet I.
Variation
In using these methods it must be remembered that among bac-
teria, the individual members of any species may differ from each
other in respect to both physiology and morphology, thus making it
difficult to define the limits of the species; also that any individual
culture in repeated examinations may produce variable results in
connection with some test even when studied under apparently con-
stant conditions. For these reasons it is important that single deter-
minations shall never be used for characterizing any culture that has
been studied, or much less for characterizing any species or type
that is being described. Determinations must be repeated at differ-
ent times and under different conditions in order to learn definitely
the physiological characteristics of a culture. Whenever possible, an
effort should be made to correlate the variations in physiology and
serology with colony typo and to list sej)arately the ])hysiological
characteristics of the "smooth", "rough", "mucoid", "opaque",
"translucent" strains, etc. When an organism shows any tendency
to "dissociate" into "phase variants", its description is incomplete if
it applies to only one phase or to a culture containing a mixture of
two phases or more. In such case the phase variants should be
separated by plating methods, or otherwise, and a separate chart
should be used for each individual strain studied. The individual
charts may be filed for the investigator's information; but it must be
insisted that results of such work should not be published for the use
of other bacteriologists until repeated determinations have l)een made
and, if possible, have been shown to bear some relation to the j)hase
indicated by colony type.
Cultural Characteristics
Space is provided on both Charts for recording appearance of
colonies, growth on agar stroke, in broth and gelatin stab. In ad-
dition to the space i)rovided for sketches, various terms are listed in
order that those which apply may be underlined. The meaning of
all the terms is given in the Glossary in Leaflet I.
As some of the terms, especially in regard to shape and struct ur(> of
colonies, are more easily described graphically than verbally the
diagram on page 7 (also published separately) is included here to
assist the student in understanding the appropriate terms.
V49-6 MANUAL OF METHODS FOR PURE CULTURE STUDY
Study of Cell Morphology
The routine study of morphology should include examinations of
stained dried preparations and of unstained organisms in hanging
drop. Stained preparations to show the vegetative cells should be
made, preferably from agar slant cultures, from a few hours to two
days old, according to the rapidity of growth. The medium and
temperature used and the age of the culture should be recorded. The
examination of unstained organisms in hanging drop is a useful sup-
plementary procedure too often neglected.
Motility. Hanging-drop preparations of young broth or agar cul-
tures should be examined for motility. Before drawing definite con-
clusions, cultures grown at several temperatures between 20° and
37°C. should be examined. It is important not to confuse Brownian
or molecular movement with true motility. The former consists of a
"to and fro" motion without change in position, except as influenced
by currents in the fluid. A phase microscope can prove useful in
studying motility.
When interpreting results it is important to remember that whereas
definite motility in a hanging drop preparation is conclusive, weak
motility or none has little significance, and other means of confirma-
tion, such as those that follow, must be undertaken. In particular,
an increasing number of cases are found of organisms fully flagellated
as shown by staining methods and serology, but absolutely non-
motile by any other method — bacteria with so-called "paralyzed
flagella".
Tittsler and Sandholzer (1936) have, in fact, proposed the use of
stabs in a semi-solid agar (meat extract 0.3%, peptone 0.5%, agar
0.5%). Motile organisms show a diffuse zone of growth spreading
from the line of inoculation; non-motile cultures do not. For this
test, incubation should be for 6 days at 30°C. unless positive results
are secured sooner. For Gram-negative non-spore-formers, 12-18
hour incubation gives more clear-cut results. This test is a good
check on the hanging drop method, but is slow and requires some
experience before one can be certain how to interpret results.
For this reason Conn and Wolfe (1938) have recommended a
flagella stain even on cultures that do not appear motile upon exami-
nation in hanging drop. The modification of the Bailey flagella stain
given in Leaflet IV is simple and quick enough to be employed for
routine examinations; positive results cannot be misinterpreted, and
show the arrangement of flagella as well as the mere presence or
absence of motility. A few further refinements of the method, mak-
ROUTINE TESTS FOR THE DESCRIPTIVE CHART v„-7
CULTURAL CHARACTERISTICS OF BACTERIA
COLONIES
PUNCTIFORM CIRCULAR FILAMENTOUS IRREGULAR RHIZOID SPINDLE
ELEVATION
FLAT RAISED CONVEX PULVINATE UMBONATE
^ t^ e^ [:i
ENTIRE UNDULATE LOBATE CROSE ruAMENTOUf CURLED
AGAR STROKE - FORM OF GROWTH
vy
1
\y
vy
ECMINULATC BZADt^ EFF'USE. AflBORCSC^N
GELATIN STAB
LINE OF PUNCTURE
LIQUEFACTION
TTT
W vzJ vL*
vjy
w
v|y
yMJ
FILIFORM- BEADED PAPILLATE- VIILOUSARBORESCENT- -CRATERIFOBHNAPIFORMINFUNOIBUIE- SACCATE STRATIFORH'
NUTRIENT BROTH - SURFACE GROWTH
^ A
PELLICLE MfMBRANOUS
{Copies of this chart on sale by Biotech Publications, Geneva, N. Y.)
V49-8 MANUAL OF METHODS FOR PURE CULTURE STUDY
ing it more adaptable to routine use on bacteria of various types, pub-
lished by Fisher and Conn (1942), is also given in Leaflet IV.
Presence of endospores. Routine examinations should be made on
agar slant cultures a week old, employing methylene blue or dilute
crystal violet, to stain the vegetative rods and leave spores unstained.
If spore-like bodies are present whose exact nature is uncertain, one of
the spore stains recommended in Leaflet IV should be employed.
In most cases there is little trouble in finding spores if the organism
produces them. All rather large rods, however, (0.8 micron or more
in diameter) should be regarded as possible spore-producers even if
microscopic examination does not show spores. Such bacteria
should be mixed with sterile broth or physiological saline solution and
heated to 85°C. for ten minutes; if still alive, endospores may be re-
garded as probably present. One should also make repeated trans-
fers of the culture onto agar and examine at various ages. A culture
of a large rod should not be recorded as a non-spore-former unless all
these tests are negative.
Acid-fast staining. Various methods have been proposed for de-
termining whether an organism is "acid fast." They are all essen-
tially modifications of the same general procedure, and are similar
to the spore stains of Moeller (1891) and Foth (1892). The Commit-
tee is as yet unprepared to recommend any one of them in particular.
Several are listed in Leaflet IV.
Capsules. An organism should not be recorded as having capsules
unless they have been actually stained by one of the methods of cap-
sule staining described in bacteriological text books. Four of the com-
mon methods of capsule staining, namely those of Anthony, of Hiss,
of Huntoon, and of Churchman, are given in Leaflet IV. The Com-
mittee has obtained good results with Anthony's and Hiss' methods.
Capsules do not appear in all media; the medium of choice should be
milk serum slants, or exudates from infected animals.
Irregular forms. Forms that differ from the typical shape for the
organism, such as branching forms, clubs, spindles, or filaments should
be noted and sketched. Simple observation is enough to show
that these irregular forms occur quite uniformly in certain cultures,
hence their existence must not be ignored; the interpretation of these
forms is at present under dispute and the decision as to their signifi-
cance must be awaited. The Committee recommends that the
microscopic study of any culture include an examination of the
growth on various media and at various ages upon each medium,
with sketches of all the shapes that occur.
ROUTINE TESTS FOR THE DESCRIPTIVE CHART V49-9
Gram stain. The Gram stain was until recently an entirely empiri-
cal procedure for distinguishing between two groups of organisms, the
actual significance of which was not understood. Since 1940, how-
over, the work of Henry and Stacey (194'3), of Bartholomew and Um-
breit (1944) and others has shown that a positive reaction is de-
l)endent upon the presence of ribonucleic acid in the outer layers of
the cells, which can be removed by treatment with ribonuclease and
replated on them by treatment with magnesium ribonucleate. Thus
Gram-positive organisms can be artificially converted to Gram-
negative ones and then restored to their Gram-positive state.
In addition to this fact, it is also true that many bacteria are
neither definitely positive nor negative; some organisms are Gram-
variable and may appear either negative or positive according to
conditions. Other organisms contain granules which resist de-
colorization and may cause misinterpretation. The importance of
taking such variations into account has been emphasized in all
previous editions of this Leaflet. (Also see Committee Report, 1927.)
Such organisms should be recorded as Gram-variable rather than
made to appear either positive or negative by some modification of
technic. To determine whether an organism belongs to this variable
group, it is necessary that it be stained at two or three different ages
by more than one procedure. If an organism changes from positive
to negative or vice versa during its life history, this change should be
recorded, with a statement as to the age of the culture when the
change was first observed. It is often practical to record such an
organism as prevailingly positive or prevailingly negative; obviously,
however, this cannot be done without a very considerable series of
determinations. Tests must therefore be made after 1 day's and 2
days' incubation, sometimes also in even older cultures. It must,
moreover, be recognized that Gram-variable organisms are not neces-
sarily ones that show uneven Gram staining; the latter should be
recorded as staining unevenly, not as Gram-variable.
The two methods at present recommended are the ammonium ox-
alate method (Hucker) and KopelofI and Beerman's modification of
the Burke technic. In the former the manipulation is more simple;
but the latter is understood to give better results if the organism is
growing in a medium that may be of acid reaction, and is claimed to
distinguish better between true and false positive reactions. These
two procedures are given in Leaflet IV.
V49-10 MANUAL OF METHODS FOR PURE CULTURE STUDY
Relation to Free Oxygen
In relation to free oxygen, organisms are generally classified as strict
aerobes, facultative anaerobes, or strict anaerobes. A fourth group
of microaerophiles may also be recognized. None of these distinc-
tions is clear-cut; but the following method gives a rough grouping of
bacteria in regard to their oxygen requirements.
Agar shake culture affords a good routine method of determining
the oxygen requirements of an organism. A tube of deep agar medium
containing glucose or some other available carbon source, is inoculated
while in fluid condition at 45°C. with an inoculum not too heavy to
permit discrete colonies, rotated to mix the inoculum with the medium,
and cooled. Some bacteriologists prefer to pour or pipet the inocu-
lated medium into another sterile tube to insure thorough mixing.
Upon incubation, strict aerobes will be found to grow upon the
surface and in the upper layers only; microaerophiles will grow best
just a few millimeters below the surface; facultative anaerobes will
grow throughout the medium; and strict anaerobes will grow only
in the depths, if at all.
Action on Nitrates
Nitrate reduction should be indicated by complete or partial dis-
appearance of nitrate accompanied by appearance of nitrite, am-
monia, or free nitrogen. As quantitative nitrate tests are too time-
consuming for routine pure culture work, one must ordinarily be
satisfied with tests for the end-products only.
The following routine procedure is recommended: Inoculate into
nitrate broth and onto slants of nitrate agar (containing 0.1% KNO3
plus beef extract and peptone as usual). Test the cultures on various
days as indicated on the Chart. On these days examine first for gas
as shown by foam on the broth or by cracks in the agar. Then test
for nitrite with the following reagents.
1. Dissolve 8 grams sulphanilic acid in 1 liter of 5 N acetic acid (1 part glacial
acetic acid to 2.5 parts of water), or in 1 liter of dilute sulphuric acid (1 part concen-
trated acid to 20 parts water).
2. Dissolve 5 grams a-naphthylamine in 1 liter of 5 N acetic acid or of very dilute
sulphuric acid (1 part concentrated acid to 125 parts water). Or dissolve 6 ml. of
dimetliyl-a-naphthylamine in 1 liter of 5 N acetic acid. This latter reagent has re-
cently been recommended by Wallace and Neave (1927), and by Tittsler (1930) as it
gives a permanent red color in the presence of high concentrations of nitrite.
Put a few drops of each of these reagents in each broth culture to
be tested, and on the surface of each agar slant. A distinct pink or
red in the broth or agar indicates the presence of nitrite. It is well
ROUTINE TESTS FOR THE DESCRIPTIVE CHART V49-II
to test a sterile control which has been kept under the same condition
to guard against errors due to absorption of nitrous acid from the air.
Presence of nitrite shows the nitrate to have been reduced, and the
presence of gas is a strong indication that reduction has taken place.
A negative result does not prove that the organism is unable to
reduce nitrates; in such a case further study is necessary as follows:
In case the fault seems to lie in poor growth, search should be made for a nitrate
medium in which the organism in question docs make good growth by means of the
following modifications: increasing or decreasing the amount of peptone; changing the
amount of nitrate; altering the reaction; adding some readily available carbohydrate;
adding 0.1-0.5% agar to a liquid medium to furnish a semi-solid substrate. The ap-
pearance of nitrite in any nitrate medium whatever (while it is absent in a sterile con-
trol) should be recorded as nitrate reduction.
Absence of nitrite in the presence of good growth may indicate complete consump-
tion of nitrate or its decomposition beyond the nitrite stage as well as no reduction at
all. Test, therefore, for nitrate by adding a pinch of zinc dust to the tube to which the
nitrite reagents have been introduced and allowing it to stand a few minutes. If
nitrate is present it will be reduced to nitrite and show the characteristic pink color.
Confirmation of the test may be obtained by placing a crystal of diphenylamine in a
drop of concentrated sulfuric acid in a depression in a porcelain spot plate and touching
with a drop of the culture (or of the liquid at the base of the slant if agar cultures are
used). The test will be more delicate if the culture is first mixed with concentrated
sulfuric acid and allowed to cool. A blue color indicates presence of nitrate, provided
nitrite is absent; but as nitrite gives the same color with diphenylamine, this test must
not be used when nitrite is present in the same or greater order of magnitude.
If none of these tests indicate utilization of the nitrate, the organ-
ism probably does not reduce nitrate, but to be certain of the fact
further investigation is necessary as outlined in Leaflet VI. It must
be understood, however, that for routine diagnostic work a determi-
nation of nitrite on standard nitrate broth or agar is ordinarily suffi-
cient; this is because most descriptions in the literature containing the
words "Nitrates not reduced" merely mean that no nitrite is pro-
duced on this medium. But in recording such results the student
should be careful to state only the observed fact, i.e. that nitrite is or is not
found in the nitrate medium employed.
Chbomogenesis
Color production should be recorded if observed in broth, on beef-
extract agar, gelatin or potato, or if noticed to a striking extent on
any other medium (e.g., starch media). In the margin the space de-
voted to chromogenesis refers to the color j)roduced on beef extract
agar. Note difTerences, if any, in pigmentation of growth exposed to
air and shielded from air, or in presence or absence of light. Fre-
quently it is well to note the final H-ion concentration of the culture,
as some pigments act as H-ion indicators.
V49-12 MANUAL OF METHODS FOR PURE CULTURE STUDY
Indole Production
During the last 40 years, results of investigations on the indole test
have been published by Zipfel (1912), Frieber (1921), Fellers and
Clough (1925), Gore (1921), Holman and Gonzales (1923), Kulp
(1925), Koser and Gait (1926) and Kovacs (1928). The two im-
portant points brought out in these papers are: that the medium be
of correct composition; and that the test used be specific for indole.
The important consideration in regard to the medium is that a
peptone be employed containing tryptophane, which is not always
present in bacteriologic peptones. Peptones are ordinarily digests of
lean meat; but for the indole test a casein digest which contains tryp-
tophane is apparently more satisfactory.
The medium used should, therefore, contain 1.0% of casein digest.
No other ingredients need be added if the organism under investi-
gation will grow in a solution of it alone. If the organism is not able
to grow in such a medium, add such ingredients as are needed to
assure its growth. K necessary, add agar and perform the test on
agar slants.
If the organism produces good growth, 1-2 'days' incubation is
ordinarily sufficient. In fact, with rapid-growing organisms, the
reaction may be positive in 24 hours, but negative the following day.
Therefore both 24-hour and 48-hour tests are recommended. The
test for indole may be performed by the technic of Ehrlich-Bohme, by
either the Gore or the Kovacs modification of the same, or by the
Gnezda technic. The Kovdcs method is especially simple and con-
venient. These procedures are as follows:
Bohme (1905) called for the following solutions:
Solution 1
Para-dimethyl-amino-benzaldehyde 1 g.
Ethyl alcohol (95%) 95 ml.
Hydrochloric acid, concentrated 20 ml.
Solution 2
Saturated aqueous solution of potassium persulfate (KjSaOg).
To about 10 ml. of the culture fluid add 5 ml. of solution No. 1, then 5 ml. of solution
No. 2, and shake; a red color appearing in five minutes indicates a, positive reaction.
This test may also be performed (and sometimes more satisfactorily) by first shaking
up the culture with ether and adding solution No. 1 (Ehrlich's reagent) dropping down
the side of the tube so that it spreads out as a layer between the ether and the culture
fluid. After this method of applying, solution No. 2 seems to be unnecessary.
The Got6 (1921) test uses these same solutions, but the method of application is as
follows: Remove the plug of the culture tube (which must be of white absorbent
cotton), moisten it first with four to six drops of solution No. 2, then with the same
STANDARD DESCRIPTIVE CHART
,; ine of organism Source Studied by
,10 of isolation Habitat Optimum conditions: Media
phase variation observed? Phase on this Chart: S, R, M, G (smooth, rough, mucoid, gonidial) Phases recorded on other charts:
..Culture No...
Temp...
Jerscore required terms.
..Age
xTATivE Cells: Medium used
Reaction (pH) Temp
: eof Majority
tiids. rounded, truncate, concave, tapering
yiLiTY: In broth On agar
f;ANGiA and Endospores: present, absent.
Medium used pH Temp Age c
Endospore Form: spherical, ellipsoid, cylindrical
ji-rLAR Forms:
P'rsent on in days at "C
:,:R Colonies; Temperature "C. Age d
r -rm, punctiform (i. e. under i m.m. diam.), circular (i. e. ove\
J m.m. diam.) , filamentous, irregular, rhizoid.
^' irface, smooth, rough, concentrically ringed, radiately ridged
^jj^c. entire, undulate, lobate, erose, filamentous, curled.
r;. vation of growth, effuse, fiat, raised, convex.
Optical Characters, opaque, translucent, opalescent, iridescent
Gelatin Colonies: Temperature °C. Age d
Form, punctiform, circular, irregular, filamentous.
E evation, fiat, raised, convex, pulvinate, crateriform (Hque-
fyttig).
E'ige, entire, undulate, lobate, erose, filamentous, curled.
ijnuefaction, cttP, saucer, spreading.
rtace, smooth, contoured, rugose.
:iticai Characters, opaque, translucent, opalescent, iridescent.
; Stroke:
Temperature °C. Age d,
I . wth, scanty, moderate, abundant, none.
n of growth, filiform, echinulate, beaded, spreading,
.:, horescent, rhizoid. ^— ii^
. ire, glistening, dull.
iromogenesis photogenic, fluorescent.
I Mr, absent, decided, resembling..
( nsistency, butyrous, viscid, membranous, brittle.
Medium, grayed, browned, reddened, blued, greened, unchanged.
NiTRiENT Broth: Temperature "C. Age c
Surface growth, ring, pellicle, fhcculent, membranous, none.
Clouding, slight, moderate, strong, transient, persistent, nofie,
Jlutd turbid, granular growth.
<Jd ir, abse7it, decided, resembling
S-Jiment, compact, fiocculent, granular, flaky, viscid.
.'vri.ount of sediment, abundant, scanty, none.
jELATiN Ptab: Temperature °C. Age d.
Growth, uniform, best at top, best at bottom.
Line of puncture, ^ii/orjn, beaded, papillate .villous , arbor escent .
Liquefaction, none, crateriform, infundibuHform, napiform,
saccate, stratiform: begins in d. complete in d.
Degree of liquefaction in days
Method used
^Medium, fluorescent, browned, unchanged.
Surface Colonies
Surface Colonies
yy
Medium:
Teraperatui
Age
Medium:
Temperatu:
Medium:
Temperatun
FERMENTATION
Temperature °C.
Medium
staining..
.and:
J^^^mcntation liibe
'•""• CO, in Eldredge tube
^Mion {pH) after d.
^'•'"bU acidity in
Jii::: NaOH
Monosaccharides
Disaccharides
Polysaccharides
Alcohols
Glucosides
BRIEF CHARACTERIZATION
As each of the following characteristics is determined, indicate in proper marginal
square by means of figure, as desi^ated below. In case any of these characteristics are
doubtful or have not been determined, indicate with the letters U, V, and X according
to the following code:
U. undetermined; V, variable; X. doubtful.
Form & arrangement: 1, streptococci; 2, diplococci; 3, micrococci;
4. sarcinae; 5, rods; 6, commas; 7, spirals; 8, branched rods; 9, filamentous
Diameter: 1, under 0.5/i; 2, between 0.5)u and 1/*; 3,
Gram stain: 0, negative; 1, positive
i:0, absent; 1, peritrichic; 2, polar; 3, present but undetermined
Capsules: 0, absent; 1, present
Chains (4 or more cells) : 0. absent; 1, present
Sporangia: 0, absent; 1 , elliptical ; 2, short rods : 3, spindled ; 4, clavate; 5, drumsticks
Endospores: 0, absent; 1, central to excentric; 2, subterminal; 3, terminal
Growth: 0, absent; 1, abundant; 2, moderate; 3, scanty
Lustre: 1, glistening; 2, dull
Form: 1, punctiform; 2. circular (over 1 mm. diameter);
3, rhizoid; 4, filamentous; 5, curled; 6, irregular
Surface: 1, smooth; 2, contoured; 3. rugose
Form: 1, punctiform; 2, circular (o
1.); 3, irregular; 4, filamentous
Surface: 1, smooth; 2, contoured; 3, rugose
Biologic relationships: 1, pathogenic for man; 2, for animals but not for n
3, for plants; 4, parasitic but not pathogenic; 5, saprophytic; 6, autotrophi(
Relation to free oxygen: 1, strict aerobe; 2, facultative anaerobe; 3, strict
anaerobe; 4, microaerophile
In nitrate media : 0, neither nitrite nor gas; 1 , both nitrite and gas; 2, nitrite but
no gas; 3, gas but no nitrite
Chromogenesis: 0, none; 1, pink; 2, violet; 3, blue; 4, green; 5, yellow;
6, orange; 7. red; 8. brown; 9, black
Other photic characters: 0,
; 1, photogenic; 2, fluorescent; 3, iridescent
Indole: 0, negative; 1, positive
Hydrogen sulfide: 0, negative; 1, positive
Hemolysis: 0, negative; 1, positive
Methemoglobin: 0, negative; 1, positive
Protein
Liquefac-
tion or
Digestion
Gelatin: 0, negative; 1, positii
Casein :0, negative; 1, positive
Egg albumin : 0, negative ; 1 , positive
Blood serum: 0, negative; 1, positive
Indicator
Reduc-
tion
Litmus : 0. negative ; 1 , positive
Methylene blue: 0, negative; 1. positive
Janus green: 0, negative; 1, positive
Rennet production: 0, negative; 1, positive
SUPPLEMENTARY DATA
TEMPERATURE RELATIONS
Medium PH
Optimum temperature for growth ^•
Maximum temperature for growth °C.
Minimum temperature for growth °C.
Thermal death point: Time 10 minutes: °C.
Medium pH
Thermal death time:
Medium "• PH
Temp.
Time
...min.
Temp.
"C.
"C.
Time
...min
...min
CHROMOGENESIS
Gelatin
Agar
Potato
OTHER PHOTIC CHARACTERS
Photogenesis on
Iridescence on
Fluorescence in
RELATION TO REACTION (pH) OF MEDIUM
Medium
Optimum for growth : oioM/ />if
Limits for growth :/i-om pH to
RELATION TO FREE OXYGEN
Method
Medium Temp °C.
Aerobic growth: absent, present, better than anaerobic growth,
micro-aerophilic
Anaerobic growth: absent, occurs in presence of glucose, of
sucrose, of lactose, of nitrate; better than aerobic growth
Additional data:
Reaction:
Acid curd:
Rennet curd:
Peptonization:
Reaction: d. .
Acid curd: d. .
Rennet curd: d. .
Peptonization: d. .
Reduction of litmus beg]
LITMUS MILK
Temperature *^C.
d ; d ; d
..days, ends in days
ACTION ON ERYTHROCYTES
Cells:
Method: plate, broth, filtrate
Hemolysis: negative, positive
Methemoglobin: negative, positive
PRODUCTION OF INDOLE
Medium
Test used
Indole absent, present in days
PRODUCTION OF HYDROGEN SULFIDE
Medium
Test used
H.S absent, present in days
ACTION ON NITRATES
Medium Temp °C.
Nitrite: d ; d ; d ; d
Gas(N.): d ; d ; d ; d
Medium Temp °C.
Nitrite: d : d ; d ; d
Gas(N.): d ; d ; d : d
Ammonia production (in amino-N-free nitrate medium):
negative, positive
Complete disappearance of nitrate in medium:
negative, positive
Disappearance of 2 p. p.m. nitrite in medium:
negative, positive
REDUCTION OF INDICATORS
Medium pH Temp ^
Indicator Cone. Reduction:
% hr ; hr.....
% hr ; hi. "
% hr ; hi."
% hr ; hr. '
Gram: d. ...
Method
Spores: Method
Capsules: Method
Medium
Flagella: Method..
Special Stains:
STAINING REACTIONS
; d ; d. ...
ADDITIONAL TESTS
Methyl red: negative, positive
Voges-Proskauer: negative, positive
Growth in sodium citrate: absent, present
Growth in uric acid : absent, present
Hydrolysis of starch: complete (iodine colorless): iirU
(iodine reddish-brown): none (iodine blue)
Nitrogen obtained from the following compounds: __
PATHOLOGY
..C..4
ANIMAL INOCULATION
Aoonf ri.1ti,ro Atnniint
■^
Whole culture
Cells
Filtrate
Animal
Subcutaneous
*
o o
Intraperitoneal
II
Intravenous
fr>^
Per OS
*In each instance where pathogenicity is observed, indicate location of lesion, and type, e. g. edema, histolysis, gas,
hemorrhage, ulcer, diphtheritic, etc.
ANTIGENIC ACTION
Ammal Medium used Age of culture
Type injection Number of injections
Culture causes production of cytolysins, aggulutinins, precipitins, antitoxin.
Specificity: Antibodies produced effective against other antigens as follows
Immune sera from..
..efEective against this organism as antigen
SPECIAL TESTS
This Descriptive Chart presented at the annual meeting of the Society of American Bacteriologists, Dec. 28, 1934. by the Committee on Bacteriological Technic.
Prepared by a sub-committee consisting of M. W. Jennison and H. J. Conn.
DESCRIPTIVE CHART FOR INSTRUCTION
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Medium:
Carbohydrate: %
Indicator:
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ROUTINE TESTS FOR THE DESCRIPTIVE CHART V4,-13
amount of solution No. 1. Replace the plug and push down until an inch or an inch and
a half above the surface of the culture. Place the tube upright in a boiling water-bath
and heat for 15 minutes without letting the culture solution come in contact with the
plug. The appearance of a red color on the plug indicates the presence of indole.
The Kovacs (19£8) test is a simplification of that of Bohme, using only one solution;
it is now the method of choice in many laboratories:
Para-dimethyl-amino-benzaldehyde 5 g.
Amyl or butyl alcohol 75 ml.
Hydrochloric acid, concentrated 25 ml.
This reagent may be used as in the Bohme test, but no solution 2 is required.
The Gnezda (1899) oxalic acid test is made as follows: dip a strip of filter paper in a
warm saturated solution of oxalic acid; on cooling, this is covered with crystals of the
acid. Dry the strip of paper thoroughly (sterilization by heat seems unnecessary),
and insert into the culture tube under aseptic conditions, bent at such an angle that it
presses against the side of the tube and remains near the mouth. Reinsert the plug
and incubate the culture. If indole is formed, the oxalic acid crystals take on a pink
color.
It is recommended that the Gore or the Kovdcs test be used in a
routine way. In interj^reting the results obtained it must be re-
membered that when the reagents are added directly to the medium
they react with alpha-methyl-indole as well as with indole itself; but
as the former compound is non-volatile it cannot react to the Gore or
Gnezda tests. Hence the Ehrlich test unmodified is less specific for
indole than the Gore modification or the Gnezda test.
Some samples of para-dimethyl-amino-benzaldehyde and of amyl
and butyl alcohol have been found unsatisfactory for the indole test.
It is well, therefore, to check new supplies of these chemicals against
samples known to be satisfactory.
In early editions of this section of the ^lanual, the vanillin, or Steensma, test was
also described. It is now omitted, as it is regarded as unreliable; Fellers and Clough
(1925), for instance, have shown it to give too high a percentage of positive reactions
unconfirmable by any other test.
The Production of Hydrogen Sulfide
Hydrogen sulfide is generally detected in bacterial cultures by
observing the blackening which it produces in the presence of salts
of certain metals, such as lead, iron or bismuth, due to the dark color
of the sulfide of these metals. Two methods have been utilized for
employing these tests: one by incorporating the metallic salt in the
medium, and the other by using a test strip of filter paper impreg-
nated with the metallic salt in question.
In early editions of this Manual four media containing either lead or
iron salts were given. The lead salt media, however, were discredited
V49-U MANUAL OF METHODS FOR PURE CULTURE STUDY
some time ago because of the toxic properties of these salts; and
Hunter and CreceHus (1938) show the superiority of bismuth media
over iron media. ZoBell and Feltham (1934), moreover, have
shown distinct advantages from the use of lead acetate test strips,
without any of these metallic salts in the media. The advantage of
the test strip technic is that it is more sensitive and does not intro-
duce the possibility of inhibiting the bacterial growth if the con-
centration of metallic salt in the medium is too great. It is important,
as emphasized by Hunter and Crecelius, that the indicator and
method employed be stated when results are given. Untermohlen
and Georgi (1940) suggest use of nickel or cobalt salts, but specially
emphasize the variations in results with different media and indica-
tors.
When using the test strip technic the bacteria may be grown in ordinary broth,
peptone sokition alone, or a peptone agar suitable to the organism in question. One
must be certain that the peptone contains available sulphur compounds. This can be
determined by running a check tube inoculated with a slow hydrogen sulfide producer.
For this procedure the test strip should be prepared by cutting white filter paper
into strips approximately 5 x 50 mm., soaking them in a saturated solution of lead
acetate, sterilizing them in plugged test tubes and drying in an oven at 120°C. One
of these strips should be placed in the mouth of the culture tube before incubation in
such a position that one-quarter to one-half of the strip projects below the cotton plug.
These tubes should be incubated at about the optimum temperature of the organism
under investigation and examined daily to notice whether or not blackening of the test
strip has occurred.
Because of the inconvenience of the test strip technic, media in
which iron salts are incorporated are now generally preferred. A
dehydrated medium of such composition is available and has been
found quite satisfactory.
Quantitative methods for determining hydrogen sulfide produc-
tion are given in Leaflet VI.
Liquefaction of Gelatin
The conventional method of determining liquefaction, which has
been given with but slight modification in all the reports on methods
is as follows:
Make a gelatin stab (plain 12% gelatin) and incubate 6 weeks at
20°C., provided the organism under investigation will grow at that
temperature. Care must be taken to observe whether the organisms
produce rapid and progressive liquefaction or merely slow liquefaction
not extending far from the point of inoculation. In the latter case
the liquefaction may be due merely to endo-enzymes that are re-
leased from the cell after death and may not be what is generally
called "true liquefaction" (that is, the process resulting from the
ROUTINE TESTS FOR THE DESCRIPTIVE CHART V49-I5
action of enzymes diffusing out of actively growing cells). Some
slow liquefiers are true liquefiers, however; and the distinction be-
tween slow and rapid liquefaction must be regarded as very artificial.
In early editions of this Leaflet the Frazier (192()) method was given, but it was
omitted from hiter editions as not proving practicable. A recent modification of it
by Smith (19-i(i), however, proves useful, and has two advantages over the gelatin
stab method: (1) it does not require low temperature incubation; (2) it is more sensi-
tive in the case of weak liquefiers. The procedure is as follows: Streak culture on a
plate of nutrient agar containing 0.4% of gelatin. Incubate at 28°C for 2-14 days
according to rate of growth. Cover plate with 8-10 ml. of a solution of 15 g. of HgCl2
in 100 ml. distilled water and 20 ml. concentrated HCl. This reagent forms a white
opaque precipitate with the unchanged gelatin, but a liquefier is surrounded bj- a clear
zone.
There is another method recommended for organisms that do not grow at 20°C. By
this technic an inoculated tube of gelatin is incubated at 37°C., or whatever tempera-
ture may be the optimum, and then after incubation the tubes are placed in a cold
water bath or in a refrigerator to determine whether or not the gelatin is still capable
of solidifying. Suitable uninoculated controls must always be run in parallel, especi-
ally if the optimum growth conditions for the organism necessitate prolonged ex-
posure of the gelatin to hydrolysis by mild acid, alkali or heat. In addition, pre-
cautions should always be taken to prevent evaporation of moisture which might
conceivably tend to obscure a slow liquefaction. This method has the advantage of
rarely giving positive results except in case of "true liquefaction". On the otlier
hand, it may well fail to detect cases of real liquefaction that have proceeded so slowly
that the gelatin can still set even after several weeks's incubation. The significance
of this test can be increased by using weaker than normal gelatin, — 4% gelatin, for ex-
ample, or even less.
Other methods designed to give more technical information on the
subject are given in Leaflet VI.
Cleavage of Sugars, Alcohols, and Glucosides
Fermentable substance to employ. Quite a wide range of pure alcohols
and carbohydrates is available for use in fermentation tests. In
routine work the choice is often limited to the more common and
less expensive substances; but in special research work economy
is of less importance. The three sugars, glucose, sucrose, and lactose,
and the alcohols, glycerol and mannitol, are most widely employed
because they are readily available. Whether these compounds give
valuable information depends upon the group of organisms being
studied. If the group, like the colon group, is capable of fermenting
nearly all these substances, these readily fermented sugars and
alcohols may have very little value in separating the species one from
another; one must then employ one or more of the rarer compounds
In other words the selection is based upon the group of bacteria
under investigation.
V49-16 MANUAL OF METHODS FOR PURE CULTURE STUDY
The list of fermentable substances often used in such work is given
in Leaflet VI.
Basal Medium. The compound to be tested must be added to some
basal medium suited to the group of organisms under investigation.
For routine work it is best to employ two such basal media; namely,
beef extract peptone broth and beef extract peptone agar, selecting
one or the other according to whether the organisms under investi-
gation grow better in liquid or solid media. These media should be
prepared as directed in Leaflet II. It should be noted that some
commercial peptones contain fermentable sugars (Vera, 1949) ; hence
care must be exercised in regard to the peptone selected, and controls
must be run.
Another important basal medium is the synthetic formula (Ayers,
Rupp and Johnson) given on p. II44-I4 of Leaflet II. This can be
used only for organisms that utilize ammonium salts as a source of
nitrogen; but is valuable for organisms that cause misleading changes
in reaction from proteins or which produce so little acid that it does
not become evident in a highly buffered medium.
One should notice particularly whether or not good growth is ob-
tained in any or all of these media after adding the fermentable
substance under investigation. If poor growth or none is obtained
in the broth and on the agar, one should vary the basal medium em-
ployed, following the suggestions given in Leaflet VI.
If a culture is to be studied in liquid, the media should be sterilized
in fermentation tubes; if on solid media, agar slants should be used —
see Conn and Hucker (1920). Agar slants may be inoculated either on
the surface alone or partly on the surface and partly iii a stab at the
base. It has been found in practice that if much gas is produced it
may occur at the very base of the column of agar even when all the
growth seems to occur on the surface; but if there is reason to suspect
that gas production is being overlooked, shake cultures may be used
in addition to the agar slant.
Demonstration of Cleavage. Utilization of the sugar (or other
fermentable substance) may be indicated by a chemical determination
showing its partial or complete disappearance, or by the demonstra-
tion of the end-products of fermentation. These end-products are
generally organic acids, sometimes accompanied with the evolution
of gases, e. g., free hydrogen, carbon dioxide, or occasionally methane.
Determinations of the amount of sugar remaining or of the nature of
the organic acids produced are very valuable in discriminating investi-
gations, but require time-consuming chemical work that is difficult
to employ in the routine examination of large numbers of cultures.
ROUTINE TESTS FOR THE DESCRIPTIVE CHART V49-I7
These chemical methods are referred to in more detail elsewhere
(Leaflet VI). In many instances, however, a sufficient amount of in-
formation is obtained merely by demonstrating an increase in acid or
the presence of gas.
For routine work in the case of organisms concerning which little
advance information is at hand, the use of indicators is especially
valuable in determining whether or not production of acid has oc-
curred. It must be remembered, however, that in many instances
more useful and significant information can be obtained by means of
titration. (See Leaflet VI.)
When the indicator method is employed, the indicators may be
incorporated with the media in the first place or may be added subse-
quently when the final reaction is being determined. If they are
added when determining final reaction, the color obtained should be
compared with color standards (see Leaflet IX) in order to secure
accuracy. The use of indicator media is less accurate, but is a much
more rapid procedure; when the cultures are growing on agar, more-
over, it is the only satisfactory procedure.
When using indicator media, make them up according to the directions given on
pp. 1I44-7, 8, of Leaflet II. The indicator most commonly added is brom cresol
purple; but with organisms producing considerable acid, brom cresol green or even
brom phenol blue may be employed. When studying a series of unknown organisms
it is often advisable to inoculate all onto the prescribed sugar medium with brom cresol
purple; later those that show acid may be reinoculated onto the same medium with
brom cresol green; and subsequently those positive to this indicator upon the same
medium with brom phenol blue. If it is decided to observe the production of alkalinity
as well as acidity, one may employ brom thymol blue or better a mixture of brom cresol
purple with cresol red, making up the medium as directed on p. 1I44-8 of Leaflet II; in
a solid medium this practice is often of value as it may show the production of acid
in one part of the tube, and of alkalinity in another.
TABLE 1
THE SENSITIVE IL\NGES OF THE THREE INDICATORS RECOMMENDED FOR USE IN
INDICATOR MEDIA
i)H: 7.0 6.0 5.5 5.0 4.0 3.0
Br. Cres. Purple:
Br Cres. Green:
Br. Phenol Hhie:
Purple] ^^Sensitive range->-| Yellow
• • Blue! -^Sensitive range-^^ I Yellow
Bluel -^-Sensitive range^^l Yellow
With indicator media it is difficult to learn the exact reaction by
reference to color standards, but a good estimate as to hydrogen-ion
concentration can be obtained by inspection, particularly when three
tubes are used, one with each of the three indicators recommended
above. For this purpose Table 1, showing the relation of the ranges
of these three indicators to each other, will be found useful.
V49-18
MANUAL OF METHODS FOR PURE CULTURE STUDY
After some experience a bacteriologist can usually devise some method for recording
on the Chart, by a system of numerals or + signs, the strength of reaction observed
with each indicator employed; such a system often proves practical for comparative
purposes, l)ut gives no very definite information as to final H-ion concentration.
Gas production in liquid media
is ordinarily measured in percent-
age of gas in the closed arm of the
Smith or the Durham fermenta-
tion tube. The Durham tube
consists of small test tube (e. g. 75 x
10 mm.) inverted in a large tube
(e. g. 150 X 18 mm.). In the case
of solid media it is recorded as
present or absent according to
whether or not bubbles or cracks
are present in the agar. This test
is especially valuable if the organ-
ism is tested in a shake culture;
but the presence of gas can usually
be detected in an ordinary agar
slant. These tests for gas produc-
tion are chiefly useful if the organ-
ism produces primarily hydrogen;
if the gas is all carbon dioxide little
or none will accumulate in the fer-
mentation tube because of the
great solubility and rapid diffu-
sion into the air. A convenient, simple method that has been pro-
posed for the accurate determination of carbon dioxide is that of
Eldredge and Rogers (1914). (See Leaflet VI.)
Interpretation of Results. In case an organism produces gas or con-
siderable increase in acidity in either broth or beef extract peptone
agar in the presence of some fermentable substance, and this does not
occur in the basal medium without the addition of the fermentable
substance, it may safely be concluded that cleavage of this sub-
stance has occurred. Very often for routine diagnostic purposes
such information is enough. To understand the true action of the
organism on any carbon compound, however, much more investiga-
tion must be made as explained elsewhere. (See Leaflet VI) . This is par-
ticularly necessary in the case of organisms that produce a small amount
of acid in some tubes but not in others containing the same carbon
source, and in cases where the addition of some carbon source results
in a distinctly improved growth without the appearance of demon-
FlG. 1.
The Smith Fermentation
Tube
ROUTINE TESTS P^OR THE DESCRIPTIVE CHART v,,,-19
strable acid or lijas. In routine work, accordini^ly, one should record
as positive only those organisms that produce considerable acid or
gas from a given compound and as negative only those that con-
sistently fail to show any acid or gas, nor any increase of growth
when supplied with the carbon compound under investigation. All
others should be regarded as border-line cultures, calling for further
investigation as given in Leaflet VI.
Hydrolysis of Starch
The breaking down of starch is rather more complicated than that
of sugars because of the extensive hydrolysis that is necessary be-
fore it can be utilized by the bacteria. The first stage of this process
is generally known as diastatic action because of the similarity to
that brought about by the enzyme diastase. The final end result is
usually an increase in acid, so one may obtain good evidence as to the
utilization of starch by substituting it for sugar in the above methods
(pp. V49I5-I7) and determining acid produced or increase in H-ion
concentration. It is often desirable, however, to secure evidence as to
the intermediate products and as to whether the starch has been
entirely consumed or not; and various methods have been proposed
for this purpose.
This test may be made on raw starch, dissolved by boiling, or on
the so-called "soluble starch." The latter is a partly hydrolyzed
product; but it is often used as "starch" in this test because its
iodine reaction is like that of true starch and different from that given
by typical dextrins. If soluble starch is used, its true nature must
be taken into account; but at the same time it must be remembered
that true starch is partly hydrolyzed when sterilized in culture media,
and even cultures growing in such a substratum are not furnished
with raw starch as the sole carbohydrate. When such media are
filtered, possibly "soluble starch" is all that remains.
A satisfactory method has been proposed by Eckford (1927) for
learning the type of action on starch brought about by organisms
capable of making good growth in broth. The same method may be
adapted to organisms which prefer some other liquid medium by
substituting it for broth in Eckford's method. The procedure,
however, is not well adapted to those bacteria that fail to grow well
in liquid medium. The technic is as follows:
Add 0.2% soluble starch to broth and incubate cultures a week to ten days. Ex-
amine on 2nd, 4th, 7th and 10th days for hydrolysis of starch, production of acid, and
reduction of Fehling's solution. For this test a drop is placed in a depression on a
porcelain plate and a larger quantity in a serological test tube. The latter is tested
for acid production with an indicator of the proper pll-range. To the drop on the
V49-20 MANUAL OF METHODS FOR PURE CULTURE STUDY
plate add a drop of dilute iodine solution and read reaction as follows: if blue, no
hydrolysis; if reddish brown, partial hydrolysis with production of erythrodextrin; if
clear, hydrolysis complete, with production of dextrin or perhaps glucose. The tubes
showing complete hydrolysis may be tested for reducing sugar with Fehling's solution.
For bacteria that do not grow well in liquid media, no better
method has yet been proposed than the plate technic given in all
previous editions of the Manual with little modification. This
method has its disadv^antages, but is often useful; it is as follows:
Use beef -extract agar containing 0.2% of soluble starch. Pour it into a Petri dish,
and after hardening make a streak inoculation on its surface. Incubate at optimum
temperature for the organism under investigation. Observations are to be made on
the second day for rapidly growing organisms but not until the 7th day for the more
slowly growing ones. To make the test, flood the surface of the Petri dishes with
Liigol's iodine or with a saturated solution of iodine in 50% alcohol. The breadth
of the clear zone outside of the area of growth indicates the extent of starch
destruction. By means of a simultaneous inoculation on another plate containing
the same medium with brom cresol purple as an indicator one may at the same time
learn whether or not acid is produced as an end-product.
THE METHYL RED AND VOGES-PROSKAUER TESTS
Special tests as to cleavage of glucose are commonlj^ made in the
differentiation of the organisms of the colon-aerogenes group. The
medium ordinarily employed is as follows: 5 g. proteose peptone
(Difco, Witte's, or some brand recognized as equivalent), 5 g.
C. P. glucose, 5 g. K2HPO4 in 1000 ml. distilled water. The dry
potassium phosphate slioidd be tested before using in dilute solution
to see that it gives a distinct pink color with phenolphthalein. Accord-
ing to Smith (1940), the K2HPO4 in this medium should be replaced
with the same amount of NaCl, if the tests are to be carried out on
aerobic spore-formers. Tubes should be filled with 5 ml. each and
each culture should be inoculated into duplicate (or triplicate) tubes
for each of the two tests. Incubation should be at optimum tempera-
ture of the organism under investigation, and tubes shoidd be in-
cubated 2-7 days, according to the rate of growth of the organism in
question. Although the same medium is used for both the methyl
red and the Voges-Proskauer tests, they must l^e performed in
separate tubes. The latter test depends upon the production of
acetyl-methyl-carbinol from the glucose; see Leaflet \T.
A positive methyl red reaction is regarded as being present when
the culture is sufficiently acid to turn the methyl red (0.1 g. dissolved
in 300 ml. 95% ethyl alcohol and diluted to 500 ml. with distilled
water) a distinct red; a yellow color with the methyl red indicator is
regarded as a negative reaction, while intermediate shades should be
considered doubtful.
ROUTINE TESTS FOR THE DESCRIPTIVE CHART v«-21
For the Vogcs-Proskaucr reaction, according to the "Standard
Methods" of the A. P. H. A. (1946), to 1 ml. of culture add 0.6 ml.
of 5% a-naphthol in absolute alcohol and 0.^2 ml. of 40% KOII. The
development of a crimson to ruby color in the mixture from 2 to 4
hours after adding the reagents constitutes a positive test for acetyl-
methyl-carbinol. Itesults should be read not later than 4 hours
after addition of the reagents.
Various other tests have been suggested for this reaction, both to obtain results
more quickly and because some organisms apparently give different results with dif-
ferent tests. In any case, weakly positive reactions may be obscured by the color of
the reagent. A procedure which has given excellent results with many thousand cul-
tures run by a member of the committee (C.A.S.) is the creatine test of O'Meara, as
modified by Levine, Epstein and Vaughn (1934). In this procedure the test reagent
added to the culture is 0.3% creatine in 40% KOH. This reagent deteriorates rapidly
at temperatures over 50°C. but maybe kept 2 weeks at room temperature (22-25°C.)
or for 4 to 6 weeks in a refrigerator.
A recent modification of Coblentz (1943) is similar to the A. P. H. A. method, but
uses a massive inoculum in broth from an infusion-agar slant culture, followed by
incubation of the broth for 6 hours. Also, the 40% KOH has 0.3% creatine added to
it to intensify the reaction. After addition of the reagents (a-naphthol and KOH-
creatine) the culture is shaken vigorously for one minute; a positive reaction is charac-
terized by an intense rose-pink color developing in a few seconds to ten minutes.
A more detailed and accurate procedure for determining acetyl-
methyl-carbinol is given in Leaflet VI.
Acid Production in Milk
Acid production in milk may be determined very simply; but
the opacity of the milk must be taken into account if accurate de-
terminations are desired. The milk must be considerably diluted
before adding indicator for comparison with a buffer standard.
Indicator milk is often useful. Litmus has been used most fre-
quently, as it indicates reduction as well as pH changes (although
roughly). Neutral litmus milk (about pH 6.8) has a lavender color,
which becomes red with acid production or blue with production of
alkalinity. Reduction is indicated by a partial or complete fading
of the color. The use of litmus milk has been seriously criticized
because of the inaccurate nature of litmus as a pll indicator; never-
theless the differences it brings out have enough practical value so
that it has not yet been superceded by any other indicator in milk.
The use of brom cresol puri:)le, as was reconmien<led by
Clark and Lubs (1917) does not show changes in 0-R potential.
V49 22 MANUAL OF METHODS FOR PURE CULTURE STUDY
TABLE 2
DEGREES OF ACIDITY EASILY RECOGNIZED IN MILK
'Neutral"
'Weak"
'Moderate"... .
'Strong"
'Very strong" .
INDICATOR. REACTION, ETC.
Same color with brom cresol purple as sterile inilk-
i. e. blue to gray-green
Color with brom cresol purple lighter than in steril
milk — i. e. gray-green to greenish yellow
Yellow with brom cresol purple. Not curdled
Curdled. Blue or green to brom phenol blue
Yellow to brom phenol blue
approximate-
pH-value
6.2-6 8
5.2-6.0
4.7-60
3.4-4 6
Under 3.4
During the second World War, stimulated by the unavailability of
litmus, Ulricli (1944) proposed using instead of litmus a mixture
of methylene blue and chlor phenol red. This combination added
to milk shows, for many species, all that litmus does and in addition
shows a distinction between acid reduction and alkaline reduction;
but in using it one must accustom himself to alkalinity being indicated
by red, acid by yellow or green. When using litmus or the Ulrich
combination, one must distinguish between reduction before and
after coagulation, as the latter is often of little significance.
It is possible to recognize the five degrees of acidity listed in Table
'i by the use of brom cresol purple (either in the milk before inocu-
lation or added after incubation), the subsequent addition of brom
phenol blue, and observation as to the presence of curdling. This is
only a rough method of measurement; but in the routine study of
milk cultures it will often be found valuable.
H. C. Brown (1922) proposed condensed milk diluted with 4 parts water containing
phenol red. The reaction is adjusted by addition of alkali until first appearance of a
brick red. Subsequent changes of reaction in either direction can be observed.
Rennet Production
The production of the enzyme, rennet (lab), can sometimes be recog-
nized in litmus milk by noticing the occurrence of coagulation with-
out the appearance of acid. It is often obscured by simultaneous di-
gestion, however, and two other methods have been proposed which
often show rennet production with cultures that fail to show it when
inoculated directly into milk.
Conn (1922) grows bacteria in milk sterilized in the usual manner; after the appear-
ance of whey or peptonized milk, 0.5 ml. is transferred to 10 ml. of unsterilized milk
and placed in a 37° incubator. Examinations are made every 5 minutes for the first half
hour, and at less frequent periods thereafter for a few hours longer. First appearance of
coagulation is noted.
Gorini (1932) obtains vigorous growth on an agar slant, then covers the growth with
ROUTINE TESTS FOR THE DESCRIPTIVE CHART v49-23
milk, fractionally sterilized at temperatures not over 100° so as not to alter the color of
the milk. The growth is mixed with the milk by use of a platinum needle, and the tube
is incubated at 37° until coagulation occurs.
Although the Committee is not prepared to recommend cither
method, it is felt that by a combination of the two a good indication
of rennet production can be obtained.
REFERENCES
Amer. Public Health Assn. 1946. Standard Methods for the Examination of
Water and Sewage. 9th Ed. Published by the Association, New York.
B.\RTHOLOMEW, J. W. and Umbreit, W. W. 194-1. Ribonucleic acid and the Gram
stain. J. Bact., 48, 567-78.
BOHME, A. 1905. Die Anwendung der Ehrlichschen Indolreaktion fur bacteriologische
Zwecke. Centbl. f. Bakt., I Abt. Orig., 40, 129-133.
Brown, H. C. 1922. Use of phenol red as an indicator for milk and sugar media.
Lancet, 202, 842.
Clark, W. M., and Lubs, H. A. 1917. A substitute for litmus for use in milk cultures.
J. Agric. Research, 10, 105-111
CoBLENTZ, J. M. 1943. A rapid test for acetyl methyl carbinol production. Amer.
J. Pub. Hlth., 33, 815.
Committee on Bact. Technic. 1927. Variability of the Gram reaction. Stain Tech-
nology, 2, 80-87.
Conn, H. J. 1922. A method of detecting rennet production by bacteria. J. Bact.,
7, 447-8.
Conn, H. J., and Hucker, G. J. 1920. The use of agar slants in detecting fermen-
tation. J. Bact., 5, 433-435.
Conn, H. J., and Wolfe, Gladys E. 1938. Flagella staining as a routine test for
bacteria. J. Bact., 36, 517-20.
Eckford, Marth.\ O. 1927. Thermophilic bacteria in milk. Amer. J. of Hyg-, 7,
201-221. (Seep. 208.)
Eldredge, E. E., and Rog^s, L. A. 1914. The bacteriology of cheese of the Em-
mental type. Centbl. f. Bakt., II Abt., 40, 5-21. (See p. 13.)
Fellers, C. R., and Clough, R. W. 1925. Indol and skatol determination in bacte-
rial cultures. J. Bact., 10, 105-133.
Fisher, P. J. and Conn, Jean E. 1942. A flagella staining technic for soil bacteria.
Stain Technology, 17, 117-121.
FoTH, 1892. Zur Frage der Sporenfarbung. Centbl. f. Bakt. 11, 272-278.
Frazier, W. C. 1926. A method for the detection of changes in gelatin due to
bacteria. J. Inf. Dis., 39, 302-9.
Frieber, W. 1921. Beitrage zur Frage der Indolbildung und der Indolreacktionen
sowie zur Kenntnis des Verhaltens indolnegativer Bacterien. Centbl. f . Bakt.,
I Abt. Orig., 87, 254-277.
Gnezda, J. 1899. Sur les reactions nouvelles des bases indoliques et des corps al-
buminoides. Com. Rend., .\cad. Sci., 128, 1584.
Gore, S. N. 1921. The cotton-wool plug test for indole. Indian J. of Med. Res., 8,
505-507.
GoRiNi, C. 1932. The coagulation of milk by B. typhosus smd other bacteria considered
inactive on milk. J. Path, and Bact., 35, 637.
Henry, H. and Stacey, M. 1943. Histochemistry of the Gram-staining reaction for
microorganisms. Nature, 151, 671.
V49-24 MANUAL OF METHODS FOR PURE CULTURE STUDY
HoLMAN, W. L., and Gonzales, F. L. 1923. A test for indol based on the oxalic acid
reaction of Gnezda. J. Bact., 8, 577-583.
Hunter, C. A., and Crecelius, H. G. 1938, Hydrogen sulphide studies. I Detec-
tion of hydrogen sulphide in cultures. J. Bact. 35, 185-196.
Koser, S. A., and Galt, R. H. 1926. The oxalic acid test for indol. J. Bact., 11, 293-
303.
KovAcs, N. 1928. Eine vereinfachte Methode zum Nachweis der Indolbildung
durch Bakterien. Zts. f. Immunitats. 55, 311-15.
Levine, Max, Epstein, S. S. and Vaughn, R. H. 1934. DifiFerential reactions
in the colon group of bacteria. Amer. J. Pub. Hlth. 24, 505-10.
Magoon, C. a. 1926. Studies upon bacterial spores. J. Bact., 11, 253-83. (See p.
261-4.)
MoELLER, H. 1891. Uber eine neue Methode der Sporenfarbung. Centbl. f. Bakt.
10, 273-277.
Smith, N. R. 1940. Factors influencing the production of acetyl-methyl-carbinol
by the aerobic spore-formers. J. Bact. 39, 575.
Smith, N. R. 1946. Aerobic mesophilic sporeforming bacteria. U. S. Dept. of Agric,
Misc. Publ. No. 559.
TiTTSLER, R. P. 1930. The reduction of nitrates to nitrites by Salmonella pullorum
and Salmonella gallinarum, J. Bact., 19, 261-267.
TiTTSLER, R. p., and Sandholzer, L. A. 1936. The use of semi-solid agar for the
detection of bacterial motility. J. Bact. 31, 575-80.
Ulrich, J. A. 1944. New indicators to replace litmus in milk. Sci., 99, 352.
Untermohlen, W. p. and Georgi, C. E. 1940 A comparison of cobalt and nickel
salts with other agents for the detection of hydrogen sulfide in bacterial
cultures. J. Bact. 40, 449-59.
Vera, H. D. 1949. Accuracy and sensitivity of fermentation tests. Abs. of
Papers, Soc. Amer. Bact., 49th Gen. Meeting, p. 6.
Wallace, G. I., and Neave, S. L. 1927. The nitrite test as applied to bacterial
cultures. J. Bact., 14, 377-384.
Zipfel, H. 1912. Zur Kenntnis der Indolreaktion. Centbl. f. Bakt., I Abt. Orig., 64,
65-80. ^
Zo Bell, C. A., and Felth.am, Catherine B. 1934. A comparison of lead, bismuth,
and iron as detectors of hydrogen sulphide produced by bacteria. J. Bact.
28. 169-178
LEAFLET VI
FURTHER BIOCHEMICAL METHODS
9th EDITION
Pure Culture Study of Bacteria. Vol. 10, No. 4
November, 1942
Revised by
C. H. Werkman
Committee members assisting in the revision:
Barnett Cohen, W. W. Jennison and J. A. Kennedy
LEAFLET VI
FURTHER BIOCHEMICAL METHODS
Introduction
Leaflet V dealing with routine tests for the Descriptive Chart
describes certain of the simpler biochemical tests used quite generally
in the study of bacteria. There are, however, a considerable number of
biochemical tests which are in fairly common use in the pure culture
study of bacteria but which are not included in Leaflet V for one or
the other of two reasons: they either apply only to certain special
groups of bacteria or they involve such intensive chemical study that
they cannot easily be used in routine work. The methods given in this
Leaflet, therefore, are to be used primarily in the study of special
groups of bacteria after a preliminary survey has established most of
their general morphological and physiological characteristics. In such
cases it is very often desired to make a more careful physiological
study of a few strains, and the routine tests given in Leaflet V or in
previous editions of Leaflet VI are entirely inadequate for any de-
tailed biochemical investigations.
The sixth (1935) edition of Leaflet VI was the first to deal with any
but routine biochemical tests, and accordingly its title was then
changed to show the new field covered by it. The first editions of the
Leaflet under its new title are necessarily incomplete. The object of this
Manual has always been to list methods that have actually been used
by members of the Committee and have been found practical in pure
culture study of bacteria. Inasmuch as the new field now covered by
Leaflet VI is a very broad one, the present Committee members have
not had experience with procedures in all the lines that should be in-
cluded. Accordingly, it is planned to make the first editions quite
brief, with the intention of revising this text and adding new material
with each successive edition until the field is more adequately covered.
Assistance from users loill he greatly appreciated in making suggestions
as to what should be covered in future editions. It is hoped that the
present edition will be of value in pointing the way to methods for those
who are confused by the multiplicity of procedures in the literature.
In making a physiological study of any kind of bacteria, special
consideration should be given to the question of variation as discussed
at the beginning of Leaflet V. Strain variations, in fact, are more likely
to affect biochemical reactions than matters of morphology. It is, ac-
cordingly, important that no conclusions be based upon single deter-
minations, nor even upon several determinations when all are made
upon a single strain. It cannot be overemphasized that a physiological
study of any type of bacteria should always be based upon repeated
VI42-2
FURTHER BIOCHEMICAL METHODS vi^.-S
determinations with several strains believed to be of the same species
or at least very closely related one to another.
Each fermentation is a problem of its own, and the choice of analyt-
ical methods must vary with the group of bacteria under investiga-
tion. To give specific directions here for even the most common con-
tingencies would consume an inordinate amount of space. Except in
one instance (action on nitrates) which seems nowhere to have been
treated adequately, only the main features will be considered here. Of
the various compilations of methods, the three following may be parti-
cularly useful for purposes of reference: A.O.A.C., Official and Tenta-
tive Methods of Analysis, 5th Ed. 1940; Abderhalden, E., Handbuch
der biologischen Arbeitsmethoden (Urban, Berlin); and Peters and
Van Slyke (1931, 1932).
Preparation of Bacterial Juices
Cell-free juices prepared from bacteria are receiving increasing use
in physiological studies and are serving in the elucidation of problems
dealing with mechanism of bacterial action on substrates. Juices are
obtained usually by one of the following methods: (a) Extraction of
juice, (b) press juice, (c) filtrates, (d) grinding, or a combination of
methods. The Booth-Green (1938) mill has been used to good ad-
vantage; in the United States, the powdered glass-grinding-extraction
technic has given good results. The Booth-Green mill is unobtainable
at present; it has been used especially by the English workers. In
general the technic of grinding with powdered glass, followed by ex-
traction, has certain advantages both in cost of equipment and
breadth of application. Bacteria are grown in liquid culture, centri-
fuged in a Sharpies super-centrifuge at 30,000 r.p.m. and the resulting
paste mixed with a quantity of powdered glass (generally two parts
paste: 1 part glass) with a particle size of about 2 /i. The powdered
glass is prepared by grinding clean pyrex in a ball mill with steel balls
for one hour. A mask should be worn. The bacteria-glass mixture is
forced through a grinding apparatus comprising two glass cones, one
turning within the other. The bacteria are cut by the fine glass
particles. See: Wiggert, et al (1940); Werkman and Wood (1940).
The mixture is extracted with water or a buffer solution, and after
that is centrifuged to throw down the glass. The extract then may be
centrifuged in a Beams air-driven centrifuge until clear. A differential
separation of enzymes may be accomplished by the Beams centri-
fugation. The supernatant liquid may be dialyzed through collodion or
cellophane membranes to remove coenzymes and inorganic ions.
Juices are desirable when separate enzyme systems are under in-
vestigation; also when the cell wall is impermeable to a substrate.
VI4.-4 MANUAL OF METHODS FOR PURE CULTURE STUDY
particularly in the case of an intermediate product which is formed
within the cell.
Relation to Free Oxygen
A section of Leaflet V having the same heading as this describes
methods for distinguishing roughly between aerobes and anaerobes.
For a careful physiological study of any organism one must realize,
however, that such determinations as those mentioned in Leaflet V
are quite incomplete. It is especially to be observed that the rough
methods given there do not distinguish between strict anaerobes and
microaerophilic organisms. For a more adequate study of the relation
of an organism to free oxygen, there are two points in particular
which require careful investigation: first, the optimum oxygen tension
(which may be considered in the case of anaerobes from the stand-
point of oxygen tolerance); second, the respiratory quotient.
Optimum Oxygen Tension. Vessels large enough to furnish an ade-
quate oxygen supply must be used. Probably the best method is to
place the cultures growing on liquid or agar as desired, in a Novy jar,
to evacuate and to replace the air with a mixture of gases containing a
known percentage of oxygen. Such a method is well adapted to deter-
mining oxygen tolerance of microaerophilic organisms. It should be
particularly remarked that the 'absence' of oxygen in the gas space
over the bacterial culture should be tested for directly by employ-
ment of a suitable indicator (e.g., solution of reduced methylene blue
or indigo disulfonate) properly applied to the gas phase. Use of the
indicator within the medium is of uncertain value.
Respiratory Quotient. A similar apparatus may be used if provided
with stop-cocks to allow the removal of samples of gas for analysis. A
manometer should always be present on such a system to show
changes in gas pressure. Inthe sample of gas removed, the carbon
dioxide may be determined by absorption with standard alkali, after
which the oxygen may be removed by alkaline pyrogallol. The respir-
atory quotient is obtained by dividing the volume of carbon dioxide
produced by that of the oxygen consumed.
Details of these methods are not given here and must of necessity
be varied with the organisms under investigation. A useful set-up for
determining the points above mentioned is described by Soule (1928).
Attention should also be called to the manometric tcchnic for physio-
logical studies on microorganisms. This technic provides a powerful
method of attack and should find wide use in bacteriological research.
It offers the most convenient and accurate method available for fol-
lowing reactions in which gas is evolved or taken up, and has been
extended to include chemical determination of products. The
manometric technic has been used successfully in studies determining
rates of reaction (especially when CO2 is evolved, O2 taken up, or
acids formed), vitamin or growth factor requirements, CO2 utiliza-
tion, and efficacy of disinfectants. Manometric methods can be
adapted to a wide variety of uses. For general purposes in physio-
logical bacteriology, the Warburg type of manometer is used. This
FURTHER BIOCHEMICAL METHODS vi,,-5
is a constant volume type in which the reaction flask is attaclied to a
U-shaped manomctric tube. The change in pressure on the Hquid in
the tube is read, from which the Oi-uptakc and CO2 evolved are
easily calculated. Anacrobically, CO2 and II 2 arc readily deter-
mined. The manual by Dixon (1934) may be consulted for theoretical
and manipulative details.
Cleavage of Carbohydrates, Alcohols, and Glucosides
Under this heading in Leaflet V are given the most common rou-
tine tests, designed merely to show whether or not an organism pro-
duces acid or gas in certain standard media. Such tests are valuable,
but do not give a sufficient idea as to the action of the organism on the
carbon compound under investigation. In a comprehensive physiologi-
cal study, various more detailed methods are necessary. The present
leaflet is designed to indicate a few of these methods.
Choice of Carbon Compounds. The carbon compounds employed in a
study of this sort should be of the utmost purity. A considerable
variety of such compounds is now available. It is not always necessary
to use all of them; but for many groups of bacteria it will be known in
advance which may be expected to give the most useful information.
The following list gives the compounds most frequently used in fer-
mentation studies:
Monosaccharides: Pentoses: 1-arabinose, xylose, rhamnose
Hexoses: glucose, fructose, mannose, galactose
Disaccharides: Sucrose, maltose, lactose, trehalose, cellobiose, melibiose
Trisaccharides: Raffinose, melezitose
Polysaccharides: Starch, inulin, dextrin, glycogen
Alcohols : Trihydric : glycerol
Tetrahydric: erythritol
Pentahydric: adonitol, arabitol
Hexahydric: mannitol, dulcitol, sorbitol
Glucosides : Salicin, coniferin, aesculin
Several of these compounds are hydrolyzed or otherwise decom-
posed at the temperature necessary for sterilization. For careful
work, therefore, such compounds must be sterilized separately, by
Berkefeld filtration or by autoclaving in concentrated (ordinarily
20%, unless the viscosity is too great), slightly acid (pll().8) aqueous
solution, and added aseptically to the basal medium. In the latter
case, autoclaving for 15 minutes at 15 pounds pressure and plunging
into cold water has proved useful. Sugars are particularly subject to
chemical change in the presence of phosphates or in alkaline solution.
Ordinarily a concentration of 1% in the medium is satisfactory;
but one can often economize (in the case of expensive compounds) by
employing low'er concentrations.
Choice of a Basal Medium. There are many bacteria that will not
grow in beef extract agar or broth, and modifications are necessary in
order to secure sufficient urowth to determine whether or not utiliza-
VI42-6 MANUAL OF METHODS FOR PURE CULTURE STUDY
tion of the added carbon compound can occur. Often the poor growth
may be due to the lack of necessary inorganic salts or to some un-
known organic factor in the peptone which is required by many
bacteria. Probably the most satisfactory way to supply the latter fac-
tor is thru the use of yeast extract. (See yeast extract broth, p. Uu-5,
Leaflet II.) This furnishes a satisfactory basal medium in studying
propionic acid bacteria, streptococci or lactobacilli. In the case of
some microaerophiles better growth may be secured by employing a
semisolid agar as a basal medium (see p. iii4-5). Some bacteria, on
the other hand, fail to grow on standard broth or agar because of the
presence of too much organic matter. For them the ammonium
phosphate medium (liquid or agar) given on page iiii-15 will often give
satisfactory results. This synthetic medium must be used with a little
caution, however, as it is poorly buffered and quite a high final H-ion
concentration (e.g., pH 5) may not necessarily mean acid production
from the carbohydrate (see discussion three paragraphs below).
It is often necessary to prevent an appreciable rise in H-ion con-
centration. This is ordinarily accomplished by adding an excess of
sterilized CaCOa to each culture tube or flask, or by suitable buffering
of the medium.
If calcium carbonate is used, it should be a fine powder so as to
provide great surface for neutralization of the acids formed. In addi-
tion the carbonate should be suspended throughout the medium by
adequate agitation, otherwise calcium carbonate is not a very effec-
tive neutralizing agent.
Analytical Methods. In a study of fermentation, the following deter-
minations are commonly made: Final H-ion concentration, residual
sugar, kinds and quantities of organic acids, neutral solvents, carbon
dioxide. The choice as to which of these determinations to make and
sometimes as to what methods to employ must often depend on the
organism or group of organisms under investigation. In a complete
study it is necessary to account for the carbon originally present in
the substrate (usually a carbohydrate). This carbon should theoreti-
cally be accounted for among the products of fermentation. Likewise
the state of oxidation of the products should equal that of the sub-
strate, indicated by the redox index. The use of the redox index is
extremely useful in careful fermentation studies as a measure of the
accuracy of results. For a discussion see Johnson, Peterson and Fred
(1931).
Final H-ion Concentration. This may be determined colorimetrically
or electrometrically according to the accuracy desired and the appli-
cability of the method to the conditions of the experiment. The color-
imetric method is given in Leaflet IX. Standard texts, like Clark's
"The Determination of Hydrogen Ions" 3rd Ed., should be consulted
for the electrometric method; the use of the glass electrode has
recently found marked favor (see Leaflet IX, p. iXi5-7.)
In interpreting results, the buffer content of the medium must be
taken into consideration. The final reaction is the resultant of various
factors including the following: production of fatty acids, of COo, of
ammonia (or other basic substances) from nitrogenous matter pres-
ent; withdrawal of either cation or anion from mineral salts with con-
FURTHER BIOCHEMICAL METHODS vi4,-7
sequent freeing of acid or base. Accordingly, direct comparisons be-
tween results in different basal media should not be made.
Residual Sugar. Determination of sugar in cultures and in uninocu-
lated controls may be made by the method of Shaffer and Hartmann
(1921) or its modification by Stiles, Peterson, and Fred (1926). Both
are iodometric modifications of the Fehling procedure. In using this
analytical method it is important that the medium contain only a
little more sugar than the bacteria can use. The method has its great-
est accuracy only within certain limits, so it is important that wher-
ever possible the amount of reducing sugar in the aliquot lie within
those limits. Accordingly, preliminary determinations with varying
percentages of sugar are often necessary before deciding on the most
suitable concentration or the most satisfactory volume to employ
for an aliquot.
It is understood that the method is not as accurate in media con-
taining beef broth as in solutions that are free from it. It cannot be
used in the presence of nitrites; but these may first be removed by
heating in the presence of urea and acid.
Quantity of Acid Produced {Titratable Acidity). Titration of an ali-
quot sample of a culture with standard alkali to an arbitrarily chosen
end-point (usually phenolphthalein or phenol red) is often employed
(after deduction of corresponding blank titration value) as a measure
of the quantity of acid products present. The sample may be boiled
before titration if it is desired to exclude COifrom the determination.
The results are most directly expressed in terms of normal acid, or as
milliliters of N /lO acid per 100 ml. of culture. They are sometimes ex-
pressed presumptively in terms of the predominant organic acid (e.g.,
lactic acid) assumed to be produced by the bacteria.
Nature of Acids Produced. To neutralize the acids produced, an
excess of CaCOa may be added to the medium (see p. VI42-6). Or if it
is not desirable to have carbonate present an indicator may be added
and sterile NaOH introduced aseptically from time to time from a
container sterilized with the culture flask. Incubation should con-
tinue to completion.
The acids most frequently present are: (1) the volatile fatty acids,
formic, acetic, propionic and butyric; (2) the non-volatile acids,
lactic and succinic. Separation of the volatile acids is ordinarily
effected by steam distillation after acidification with H2SO4 to pH 2.0
to liberate the acids. It is necessary to collect twelve volumes of dis-
tillate; e.g., 300 ml. from 25 ml. of medium, in order to remove the
volatile acids quantitatively. The non-volatile acids are recovered
from the residue of the steam distillation by continuous extraction
with ether for 48 hours.
Lactic acid may be determined in the extract by oxidation with
permanganate to acetaldehyde. The aldehyde is bound in bisulfite
and the bound bisulfite determined iodomctrically (Friedemann and
Graeser, 1933). The succinic acid may be precipitated as the silver
salt and weighed, or the silver of the salt determined volumetrically
(Moyle, 1924).
The volatile fatty acids frequently consist of formic and acetic
VI4 2-8 MANUAL OF METHODS FOR PURE CULTURE STUDY
acids. In this case the total volatile acid in the distillate may be
determined by titration, and the acetic acid calculated by difference.
The formic acid may be determined by oxidation with HgCl2 and
the resulting HgCl weighed (Auerbach and Zeglin, 192l2). The
Duclaux distillation method as modified by Gillespie and Walters
(1917), or Virtanen and Pulkki (1928), or the partition method of
Osburn, Wood and Werkman (1933), (1936), may be used for quanti-
tative determination of more complex mixtures.
The partition method is applicable to the quantitative estimation
of mixtures of formic, acetic, propionic and butyric acids, and the
qualitative detection of other acids. The basis of the method is the
characteristic distribution of an acid between water and an immiscible
solvent, such as ethyl ether, when the two are vigorously shaken
together.
Pyruvic Acid. A qualitative test is finding increasing use. The test
is not absolutely specific for pyruvic acid but under the conditions
used in bacteriology is of qualitative significance.
Pyruvic acid may be determined qualitatively by a blue color pro-
duced with Na nitroprusside (Simon and Piaux, 1924). Two milli-
liters of the solution containing pyruvic acid are saturated with
(NH4)2S04, 4 drops of a 2% nitroprusside solution are added, plus
1 ml. cone. NH4OH. After a few minutes, a blue color is produced,
specific for pyruvic acid (and acetophenone). Other ketone com-
pounds, such as acetone, acetoacetic ester, acetoacetic acid, creati-
nine, and glutathione give color reactions varying from orange to
red to purple.
Pyruvic acid may be determined quantitatively either by the re-
action with eerie sulfate, or salicylaldehyde. With the eerie sulfate
method, pyruvic acid is oxidized to acetic acid and COo. The CO2 re-
leased can be determined manometrically, or the excess of Ce++++
can be determined titrimetrically with FeS04. Lactic acid will inter-
fere when present in large amounts. The reaction is specific for alpha
keto acids. (Fromageot and Desnuelle, 1935.)
Pyruvic acid may be determined colorimetrically by reaction with
salicylaldehyde plus strong KOH. Oxalacetic acid will not interfere.
(Straub, 1936.) Less than 0.1 mg. of pyruvic acid can be determined
accurately by this method. Acetaldehyde and acetone also give a
color reaction.
Succinic Acid may be precipitated as the silver salt and weighed,
or the silver of the salt may be determined volumetrically (Moyle,
1924.) The acid may also be determined quantitatively by the use of
an enzyme obtained from beef heart. Succinic acid is extracted from
solution with ether and determined by measuring the oxygen neces-
sary for oxidation of succinate to fumarate in the presence of the
enzyme. (Gozsy, 1935). One mole of O2 taken up is equivalent to two
moles of succinic acid. The preparation of the enzyme is described by
Weil-Malhcrbe (1937) and Krebs (1937). Potter and Elvehjem (1936)
describe a simple mechanical modification to replace grinding with
sand.
Substances which will be oxidized by this enzyme preparation are
FURTHER BIOCHEMICAL METHODS vi^.-Q
succinate, methyl succinate (Thunberg, 1933), a-glycerophosphate
(Green, 1936) and d-glutaric acid (Wcil-Malherbe, 1937). Methyl
succinate has not been found in biological material and a-glycero-
phosphate and d-glutamate are not extracted with ether, therefore,
this method is highly specific for succinic acid (Krebs, 1937).
Neutral Solvents {acetone and ethyl, butyl and isopropyl alcohols).
These solvents are best distilled from a neutral or slightly alkaline
fermentation liquor. Acetone, in an aliquot of the distillate, is oxidized
with iodine in alkali and excess of the iodine back-titrated with thio-
sulfate. (Goodwin 19'-20). The other solvents are not oxidized under
these conditions. Isopropyl alcohol can be oxidized by dichromate
and orthophosphoric acid to acetone and the latter distilled off and
determined as above. Stahly, Osburn and Werkman (1934) show that
94% of the acetone is recovered in the distillation. The analytical re-
sults should, therefore, be corrected accordingly. These authors state
that ethyl alcohol can be entirely oxidized by dichromate to acetic
acid, while in the case of butyl alcohol, 89.6% is oxidized to butyric
acid and 10.4% to acetic acid. The two acids may then be deter-
mined by distillation or by partition. For small quantities of ethyl or
butyl alcohol the method of Johnson (1932) may be used.
Carbon Dioxide. Large quantities of CO2 may be detected by the
appearance of gas and its characteristic reactions. Because of its great
solubility, however, it can seldom be thus detected, and a measure of
the gas evolved is never an accurate determination of the quantity of
CO2 produced.
For accurate results, use should be made of an aeration train of
which the essential elements are: a wash tower containing alkali to
remove CO2 from incoming air; a flask or other container for the cul-
ture; an absorption tower containing a measured amount of standard
alkali with beads or other device to break up the stream of air; and an
aspirator or pump to force or to pull the air thru the train. When
using this method special precautions to avoid contamination should
be observed; and no reliance should be placed on results unless tests
at the end of the experiment show that the original organism is
present in pure culture.
When a considerable number of cidtures are to be studied simul-
taneously, the Eldredge tube (Fig. 1) can be used more conveniently
and often with sufficiently accurate results. A satisfactory sized tube
is one having a capacity of about 60 ml. in each arm. (These tubes are
not as yet listed by supply houses, but arrangements to handle them
have been made with the W ill Corp., Rochester, N. Y. and Macalaster
Bicknell Co., Washington and Moore Sts., Cambridge, Mass.)
In using the Eldredge tube, place 20 ml. of the medium in one of the horizontal arms
and sterilize. Inoculate and then place in the other arm a measured quantity (usually 15
to 25 ml., depending upon the amount of COj expected) of a freshly prepared X/10
barium hydroxide sohition. (One may use NaOII or KOII, but the insohdjility of the
BaCOj formed makes I5a(OH)j more satisfactory in giving a visual indication of CO3
production.) Immediately after inserting the alkali, push the cotton i)lugs down in the
tubes and seal, .\fter at least two weeks incubation titrate the barium hydroxide with
N/10 HCl or preferably H2SO4. using i)henolphthalein as an indicator. Compute the
amount of CO 2 produced from the equation: ml. of Ba(OH)2Xnormality of Ba(0H)2
vi4^10 MANUAL OF METHODS FOR PURE CULTURE STUDY
X0.022 = grams of CO2 (i.e., 1 ml. of N/10 Ba(0H)2 converted into the carbonate
represents 0.0022 g. CO2.)
The contents of the culture arm of the Eldredge tube may be ana-
lyzed, if desired, to show the amount of sugar remaining, by the
methods given above (p. VI42-7). One can strike a balance between the
CO2 given off and the sugar-carbon consumed, and thus decide wheth-
er to look for other end-products. This makes the Eldredge tube
method a useful preliminary in some cases for a more extensive study
of the fermentation.
Hydrogen. Hydrogen is usually determined in one of two ways:
by measurement of volume; by combustion to water and determina-
tion of the water by weight. In the first procedure the gas produced
Fig. 1. The Eldredge Tube
Height 4", width 3>^", length horizontal tubes, 4^".
in the fermentation may be collected over alkali, and in the absence
of other gases such as methane, the hydrogen measured directly.
Also the gas may be exploded in a Hempel pipette, and the decrease
in volume of gas measured. When methane is present, the CO2
formed by its combustion must be mea^ared and a correction ap-
plied (cf. Gas Chemists' Handbook, 1929; McCulloch, 1938).
The combustion of hydrogen to water may be brought about by
CuO at 250° C. Methane is not oxidized under these conditions. The
water is collected in a suitable train and weighed.
Acetyl-methyl-carhinol. A minor by-product, which has come into
prominence because of its detection in the Voges-Proskauer test for
FURTHER BIOCHEMICAL METHODS VI4.-I]
distinguishing between the members of the colon group, is acetyl-
methyl-carbinol (CHs'CO-CHOII-CHa). The Voges-Proskauer test is
described in Leaflet V (p. V44-2O) . A method for accurately detecting this
compound, originally i)roposed l)y Lemoigne (191 '3) has been im-
proved and described in detail by Kluyver, et al (19'-2.5). The pro-
cedure depends u])on oxidation to diacetyl (CHsCOCOCHs), dis-
tillation and precipitation in the form of nickel dimethylglyoxime,
which shows as characteristic reddish crystals. Stahly and Werkman
(1936) show that approximately 84% of the acetyl-methyl-carbinol
may be thus determined.
2,3-Butiilcne Glycol. A further common by-product in the case of
organisms of the groups that frequently show the presence of acetyl-
methyl-carbinol is 2,3-butylene glycol. A method of determining this,
depending upon oxidation to acetaldehyde and subsequent titration
of the HCl formed by the reaction between the acetaldehyde and
hydroxylamine hydrochloride, has been developed by Brockmann
and Werkman (1933). The following method is a modification of that
of Brockmann and Werkman.
Sugar interferes in the alkaline distillation, and if present must be
removed prior to analysis. This is accomplished by the CuS04-lime
method of Hewitt. (Hewitt, 1932). The liquor to which has been
added the copper-lime reagent is brought to definite volume and
centrifuged. The supernatant is decanted and filtered. This method
also removes citric acid.
An aliquot of the sample is made alkaline to phenolphthalein and
anhydrous Na2S04 added. (10 g. Na2S04 for 50 ml. aliquot). The
solution is directly distilled (in a Kjeldahl flask of convenient volume)
to saturation (20 ml.), and 14 volumes (280 ml.) removed by steam
distillation. The distillate is made up to definite volume, and an
aliquot, containing not more than 0.6 mM of glycol, removed, and
6 ml. of a potassium periodate reagent (5.75 g. KIO4 dissolved in
100 ml. 3.6 N H2SO4) is added.
Distill into 10 ml. fresh 1% NaHSOa, with the end of the adapter
beneath the surface of the NaHSOs solution. Destroy excess bisulfite
by adding 0.25 A^ I2, with starch indicator. Destroy aldehyde-bisulfite
complex by adding excess NaHCOs, (0.5-1 g.) and titrate the
liberated bisulfite with weak (0.()5A0 I2, using starch indicator
(Friedemann and Graeser, 1933). Compute the amount of 2,3-
butylene glycol from the equation: ml. of I2X normality of l2^4 =
cone, of butylene glycol in millimols; (i.e. 1 ml. 0.05 A' 12 = 0.00112 g.
butylene glycol).
If acetyl-methyl-carbinol is present, an abnormally high glycol
value results. Acetyl-methyl-carbinol must be determined in the
distillate, and one-half the value obtained subtracted from the un-
corrected glycol value. (Stahly and Werkman, 1936).
Interfering Reactions. Many of the methods of analysis may result
in serious error, owing to their lack of specificity. Each type of fermen-
tation requires a careful selection of methods. The following are a
few examples of interference.
VI42-12 MANUAL OF METHODS FOR PURE CULTURE STUDY
Acetyl-methyl-carbinol is oxidized by CuSOi in the determination
of reducing sugars (cf. Stahly and Werkman, 1936, and Langlykke
and Peterson, 1937, for correction factors).
Approximately 5% of the lactic acid volatilizes during steam dis-
tillation of the volatile acids. When lactic acid is present in large
amounts, the volatile acids should be neutralized, evaporated to a
small volume (25-50 ml.), acidified with H2SO4, and again steam
distilled. This procedure eliminates most of the lactic acid from the
distillate. Thirty per cent of pyruvic acid volatilizes; usually three dis-
tillations are necessary to eliminate this acid from the distillate.
It is, perhaps, better to determine the volatilized pyruvic acid by
eerie sulfate oxidation (Fromageot and Desnuelle, 1935) or by the
iodoform reaction (Wendel, 1932) and apply a correction for this acid.
Acetone is usually determined by the iodoform reaction. Any other
neutral volatile compound which gives the iodoform reaction will,
of course, interfere with this method, particularly acetyl-methyl-
carbinol, nearly 60% of which volatilizes during a half volume dis-
tillation. The acetyl-methyl-carbinol in the distillate can be deter-
mined as nickel dimethyl-glyoximate and a correction applied, or
the procedure of Langlykke and Peterson (1937) may be used.
Acetyl-methyl-carbinol and 2,3-butylene glycol interfere in the
determination of lactic acid. They may be removed by alkaline
steam distillation (14 volumes) from a solution saturated with
Na2S04. The lactic acid is determined on the residue of distillation.
When sugars are present, alkaline distillation causes caramelization
and consequently, interference with both the glycol and lactic acid
determinations. Separation of the glycol from the sugar and lactic
acid may be accomplished by extraction of an alkaline solution with
ether continuously for 72 hours. The glycol is recovered in the extract.
The interference of sugar may also be avoided, without extraction, by
removing the sugar by copper-lime treatment (Hewitt, 1932) and
then making an alkaline distillation.
Determination of Dehydrogenases
The determination of the presence of a specific dehydrogenase may
be made by the Thunberg technique (methylene blue reduction).
There are many modifications of this procedure (e.g. Hopkins and
Dixon, 1922; Yudkin, 1933). These modifications are concerned
with methods of obtaining anaerobic conditions and amounts of
reactants.
The essential points of the procedure are:
1. To have a glass tube with a side arm or hollow stopper in which
anaerobic conditions can be maintained.
2. A constant temperature water-bath.
3. An adequate buffer.
4. An accurate control.
Anaerobic conditions may be obtained by vacuum, vacuum fol-
lowed by oxygen-free nitrogen, or by oxygen-free nitrogen alone. If
the latter is employed, the apparatus should be arranged to allow
bubbling of nitrogen through the reagents for a few minutes.
FURTHER BIOCHEMICAL METHODS VI4.-I3
A constant temperature water-bath is essential to bring the re-
actants quickly to the desired temperature and to maintain that
temperature throughout the experiment.
The buffer must be carefully selected as to type of buffer and con-
centration. One must have sufficient buffer to maintain the desired
pH throughout the duration of the experiment.
The standard (90% reduction) may be prepared by substituting
distilled water for the substrate and by adding 0.1 the regular amount
of methylene blue and leaving it open to the air.
The reactants may consist of 1 ml. each of buffer, substrate (N/10),
methylene blue solution (1/5,000) and the bacterial suspension. The
buffer, substrate and methylene blue are mixed together. The sus-
pension is placed in the side arm or in the hollow stopper. The system
is made anaerobic and placed in the water-bath at a predetermined
temperature, usually 30°, 37° or 40°C. When the temperature has
reached that of the water-bath, the suspension is mixed with the
other substances and the time recorded. The time required by the
substrate (Ho-donator) to reduce the methylene blue, until the color
matches that of the standard, is compared to the endogenous reduc-
tion time; the latter is the time required by the suspension to reduce
the methylene blue in the absence of the substrate.
A dehydrogenase is considered present when the reduction time
in the presence of substrate is less than the endogenous time.
Cleavage of Proteins and Their Products
The liquefaction of insoluble nitrogenous organic material such as
gelatin, coagulated casein or blood serum is one criterion of the cleav-
age of these substances. As the process continues, progressive changes
occur in the biuret reaction and in the number of "free" amino and
carboxyl groups. In addition, there appear certain more or less
characteristic end-products, such as ammonia, hydrogen sulfide,
mercaptans, and tyrosine (depending on the constitution of the nitrog-
enous substrate) which are often readily perceptible.
The Biuret Reaciion. Proteins form colored complexes with cup-
ric ions in alkaline solution. This is one of a general type of reactions
by ammonia or substituted ammonias. The color of the complex is
violet with the more complex polypeptides and proteins, and pinkish
lavender with peptones.
The test is carried out by making the culture solution alkaline (about molar) with
iVaOH and then adding 0.1% CUSO4 dropwise imtil the minimum amovint has been
added to produce the pink to violet color. Ammonium salts interfere and, if present,
should be removed before testing.
Amino Nitrogen. The commonly employed measures of amino com-
pounds are the Sorensen formol titration and the well-known Van
Slyke procedure.
The Formol Titration: This method depends on the increase in
acidity brought about when neutralized formaldehyde is added to a
solution containing ammonia, primary amines, amino acids or poly-
peptides. A practical procedure is given by Brown (1923).
vi4^-14 MANUAL OF METHODS FOR PURE CULTURE STUDY
To 1 volume of the culture fluid add 9 volumes of water and bring the reaction to pH
8 by the addition of N/20 NaOH or HCl. Add 8 volumes of formaldehyde solution
(approximately 40% formaldehyde). Immediately titrate the mixture to pH 8 with
N/20 NaOH. Determine the amount of N/20 NaOH required to bring 8 volumes of
the formaldehyde solution to pH 8 and subtract this from the titration of the mixture.
The result is the formol titration expressed as ml. of N/20 NaOH per 100 ml. of the
culture fluid.
Deductions as to the amino-N content of the fluid should take into
account the ammonia inchided in the determination.
The Van Slyke (1913) Amino-N Method: This procedure depends
upon the production of gaseous nitrogen when nitrous acid acts on an
aHphatic amine. Special apparatus is required. The recently intro-
duced (Van Slyke, 1929) manometric method is more generally useful
than the older volumetric. For details of the procedure consult the
original references (also, Peters and Van Slyke, 1932, 385).
Ammonia. The quantitative determination of ammonia must be
carried out by a procedure which will not decompose potential am-
monia-producing compounds. From this standpoint, the Folin (1902)
aeration method as modified by Van Slyke and Cullen (1916) is prob-
ably safer than the usual distillation from a solution treated with
MgO. The procedure involves a cautious aeration of the alkalinized
solution with ammonia-free air into standard acid which is subse-
quently titrated.
HoS and Volatile Mercaptans. Aeration of the acidified culture fluid
with HoS-free air and absorption of the volatile sulfides in a solution of
zinc or lead acetate would be the first step. Oxidation of the sulfide
with NaoOo would produce sulfate which is precipitable as BaS04;
oxidation of the mercaptans woidd produce sulfonic acid which is not
precipitable as lJaS04. Intensive oxidation in the presence of nitrate
and chlorate would convert all of the sulfur to sulfate. These are the
general principles upon which a method of analysis can be based.
Action on Inorganic Nitrogenous Compounds
There are many bacteria that are capable of utilizing inorganic
sources of nitrogen, such as nitrates or ammonium salts. Some utilize
such nitrogen sources in preference to organic forms, others in addi-
tion to the latter. When action on such compounds occurs it is of value
to make more of a study of it than is given in Leaflet V under "Action
on Nitrates".
Action on Ammonium Salts. There are a few bacteria that utilize
ammonium salts when furnished with no other source of nitrogen. In
such cases it is frequently of value to determine what percentage of
the ammonia furnished is used by the organisms. For such purposes
the ammonia can best be determined by distillation with magnesium
oxide and collection of the ammonia in standard acid solution, in
which it can be determined by titration.
Action on Nitrates. The most common action of bacteria on nitrates
is one of reduction to nitrite, to ammonia, or to free nitrogen, or pos-
FURTHER BIOCHEMICAL METHODS vi,.-15
sibly to all three. Apparently reduction often accompanies or is pre-
liminary to utilization of nitrate by bacteria, and confusion often
arises in not distinguishing between the two processes. Thus, if an
organism is furnished with either nitrate or nitrite and that com|)ound
disappears, one sometimes finds the statement made that it has been
reduced, when it may well have been utilized without reduction.
Before beginning the study of any organism in regard to this point,
one must first decide the object of his study — whether he merely
wishes to make a test for diagnostic purpo.ses in identifying his cul-
ture with some published description, or desires to know just what
the organism actually does to nitrate. In the former case he must
remember that if an organism has been described in the literature as
reducing or not reducing nitrate, such a statement ordinarily means
that it does or does not produce nitrite in a nitrate medium. For
diagnostic purposes, therefore, one can ordinarily get along with a
nitrite determination alone, if the test is made under the proper con-
ditions and is properly interpreted. This use of the nitrate reduction
test for diagnostic purposes alone is discussed in Leaflet V (page V44-9).
If, on the other hand, the investigator desires to know the real
action of his culture on nitrate, a series of tests is often needed, since a
negative result is meaningless unless supported by evidence from
other tests. In case of a negative nitrite test, several possibilities are
to be considered: 1) nitrite may be demonstrable if some other nitrate
medium is used^; 2) nitrate may be utilized by the bacteria without
reduction; 3) nitrates may be reduced to ammonia or free nitrogen
without accumulation of nitrite in detectable quantit}^; 4) no action
on the nitrate may have occurred. Methods for determining which of
these explanations applies have been recently discussed by Conn
(1936). To make this determination often means a small research
problem in the case of any organism under investigation. Tests called
for in such an investigation are as follows:
Qualitative tests for nitrate: (good only in the absence of nitrite.)
Zinc dust test: See Leaflet V, p. V44-IO.
Diphenylamine test: See idem.
Quantitative test for nitrate: This is necessary if it is desired to
know whether the nitrate has been partially consumed even tho no
end-products can be detected. For details of procedure see Methods
of Analysis of the A.O.A.C. (1934) Chapter XXXVH, Sec. 16 and 17.
Qualitative test for ammonia: (Significant only if the organism has
been growing on a synthetic medium with no nitrogen source other
than the nitrate.) The Thomas test as employed by Hucker and Wall
(1922) may be employed; but the modification of this test described
by Hansen (1930), using hypobromite instead of hypochlorite, seems
to be more reliable.
Test for nitrite consumption: In instances when it is suspected
that nitrite may be consumed as rapidly as it is formed from the
nitrate, Bronfenbrenner and Schlesinger (1920) inoculate the organ-
isms in question into a medium containing only 2 p. p.m. of potassium
'Thus ZoBell (1932) finds semi-solid agar (0.3%) invaluable in studying nitrate re-
duction of the Brucella and Salmonella groups.
VI42-16 MANUAL OF METHODS FOR PURE CULTURE STUDY
nitrite and after incubation apply the regular nitrite test. It is as-
sumed that an organism not able to destroy this small amount of
nitrite cannot destroy or consume it as rapidly as it may be produced
from nitrate.
The matter of action on nitrates can well be summarized by listing
a series of questions that must be answered before this action can be
thoroly understood. These questions are:
1) Does the organism utilize completely 1% of KNO3? In what
media?
2) If not, does it utilize part of the KNO3 furnished? What per-
centage?
3) If either of these qviestions are answered in the affirmative, does
the organism actually reduce nitrate? Before answering this question
the following subordinate questions must be answered:
a) Does it produce nitrite or gas in a nitrate medium but not in
the same medium without nitrate?
b) Does it produce ammonia in a nitrate medium containing no
source of nitrogen other than the nitrate?
c) If both these questions are answered in the negative, is the
organism able to destroy 2 p. p.m. KNO2 in a medium in which it is
being studied? If so, it may still be a nitrate-reducer, the accumu-
lation of nitrite being prevented by its action on nitrite. It must not
however, be assumed to reduce nitrite as it may utilize it as a source
of nitrogen without reduction, or may conceivably convert it to
nitrate.
Recommendations: It must be distinctly understood that the Com-
mittee does not recommend making such a study as the above to
determine the action on nitrates in securing data for routine descrip-
tions of organisms. For such routine purposes the tests given in Leaf-
let V are ordinarily sufficient; but it is emphasized that in recording or
publishing negative results of such tests one must not make the state-
ment "nitrates not reduced". A negative nitrite test should merely be
recorded "no nitrite produced from nitrate under the conditions of
the experiment".
The further tests outlined in this Leaflet are to be regarded as re-
search methods to be employed when information is desired concern-
ing the true action on nitrate of any organism under investigation.
Action on Erythrocytes
Certain organisms during their growth cause a number of changes
in the pigment of red blood corpuscles. Some organisms break down
the corpuscles, liberating the hemoglobin, due to the action of hemo-
lytic substances. Some of these substances are analogous to exotoxins
and can be found in the filtrate of broth cultures. Other organisms
change the hemoglobin in the cells to methemoglobin or sulfhemo-
globin, producing a greenish coloration. While these organisms are
FURTHER BIOCHEMICAL METHODS vi,.-17
intact, the erythrocytes are not hemolyzed. Later, when the bacteria
break down, substances are Hberated which have a more or less
pronounced hemolytic action. A third group of organisms are
"indifferent," producing no visible change in the hemoglobin or
erythrocytes. The production of hemolysins and changes occurring
in the hemoglobin under bacterial action are important in the dif-
ferentiation of streptococci, pneumococci and other bacteria. Strepto-
coccus pyogenes is the type of organism which produces an exohemo-
lysin; pneumococci and streptococci of the viridans group, are types
of organisms which produce methemoglobin.
Method I. Blood Agar Plate Method. Either streak cultures on blood
agar plates or poured i)lates of blood agar mixed with bacteria can be
used for this purpose. The sharpest results are obtained with poured
plates. For the streak method, prepare blood agar plates by melting
100 ml. of 2% meat infusion agar, cooling the agar to 45° C, adding
5-10 ml. of sterile defibrinated blood (sheep, rabbit or horse blood)
and pouring this blood agar into Petri dishes. After the agar has
hardened, streak the surface with the organism. Incubate the plate for
24 hours or longer at 37° C. Also incubate uninoculated plates as
checks against contamination. A clear area under and beyond the
edge of the growth (beta hemolysis) indicates laking of the red cells
due to an hemolysin elaborated by the organism. Organisms which
produce methemoglobin cause a greenish coloration (alpha hemolysis)
in the blood adjacent to the growth. In using the poured plate
method, the blood agar is prepared in a tube or flask and inoculated
with a suspension of the organisms that will give 25 to 50 colonies
per plate. It is important that no sugar be added to the agar. The
temperature at the time of mixing the organisms with agar should
be approximately 45° C. The inoculated blood agar is poured into
Petri dishes, allowed to harden and incubated. After incubation,
clear areas, having varied significant characteristics, appear around
the colonies which produce hemolysin (beta). The colonies of "green
producing" streptococci and pneumococci appear surrounded by a
greenish zone of erythrocytes containing methemoglobin (alpha).
After continued incubation of this type of culture, a zone of hemolysis
occurs beyond the zone of greenish cells, and at times several rings of
alternate hemolysis and methemoglobin formation may be observed.
Method II. Blood Broth Mixtures. To 0.5 ml. of a sterile 5% suspen-
sion of washed rabbit, sheep or horse blood cells in 0.85% NaCl
solution, add 0.5 ml. of a 12 to 18 hour sugar-free broth culture of the
organism to be tested. Incubate this mixture for 2 hours, at 37° C,
preferably in a water bath. The production of an hemolysin is shown
by the laking of the cells, giving a clear solution. Organisms which
form methemoglobin produce darkening of the cells, and do not
hemolyze them in this test. A tube containing 0.5 ml. each of the
blood suspension and of sterile broth should be inoculated as a
control. The corpuscles of rabbits blood are removed by centrifuging
and washed as described on p. viii4o-15 of Leaflet VIII.
VI42-18 MANUAL OF METHODS FOR PURE CULTURE STUDY
For hemolytic streptococci, the addition of serum to the broth
enhances hemolysin production.
Method III. Filtrates. The hemolysin produced by some bacteria oc-
curs free in the broth in which the organism has been growing. Its
presence can be demonstrated by adding a sterile filtrate (Berkfeld or
Seitz filtrate) of the 12 to 18 hour culture to a 5% suspension of the
sterile blood or of suitable washed erythrocytes.
Excessive exposure to air may inhibit or destroy the hemolytic
activity of the filtrate. (See Shwachman, Hellerman, and Cohen,
1934.)
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FURTHER BIOCHEMICAL METHODS VI4.-I9
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OsBURN, O. L., Wood, H. G. and Werkman, C. H. 1933. Determination of formic,
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OsBURN, O. L., Wood, H. G. and Wekkman, C. H. 1936. The determination of
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VI42-20 MANUAL OF METHODS FOR PURE CULTURE STUDY
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LEAFLET VII
DETERMINATION OF THE PATHOGENICITY
OF AEROBES
Revised by
George H. Chapman
Committeeman on Pathologic Methods
5th EDITION
Pure Culture Study of Bacteria. Vol. 16, No. 1-2
March, 1948
VII48-4 MANUAL OF METHODS FOR PURE CULTURE STUDY
General
Koch's postulates. Koch's postulates constitute the accepted
standard for demonstrating the relation of a microorganism to disease.
They are: (1) the organism must always be present where the disease
occurs; (2) the organism must be obtained in pure culture from
pathological tissue; (3) this pure culture must cause the disease w^hen
injected into a favorable region or tissue of a normal susceptible ani-
mal; and (4) the organism must be recovered from the latter.
Rivers (1937) pointed out that strict adherence to Koch's postu-
lates may hinder the study of pathogenicity, particularly with regard
to viruses and to the synergistic effect of two organisms. He stated
that error may result even when Koch's postulates apparently have
been fulfilled and that fulfillment is not always essential or desirable.
Koch's postulates are inapplicable to certain microorganisms for
the reasons mentioned in the introduction and because it may be
difficult to establish the presence of the pathogen in the diseased tis-
sue even though it may be present in large numbers. (See, e.g.. Chap-
man, 1945). In these instances, strong circumstantial evidence
may be presented as "proof" of pathogenicity. However, extreme
caution must be observed in drawing any such conclusions for the
following reasons: about 15% of animals purchased through usual
channels die from causes unconnected with the injections (Chapman,
unpublished studies); infections are common in laboratory animals
(see, e.g., Farris et al, 1945); the pathologic effects may be caused
by some ingredient of the culture medium (Rigdon, 1938) and patho-
genicity for one animal may not be comparable with pathogenicity
for another.
Bacterial pathogenicity. Every organism that lives the normal
length of life for the species passes through a life cycle. There is con-
siderable difference of opinion among bacteriologists concerning
physiological and pathogenic relationships to different stages of
growth but regardless of what one considers a "life cycle" and whether
"microbic dissociation" and filterable forms are included in it, there
is evidence that in some pathogenic bacteria at least, differences in
the growth phase are associated with differences in pathogenicity.
(See, e.g., Dubos, 1945).
The pathogenicity of a microorganism can be maintained by growth
in a suitable medium, animal passage, maintenance at low oxygen
tension, low temperature, frequent subculturing, and drying in
animal organs. Increase in pathogenic properties by animal passage
is limited by exposure and selection (Zinsser and Wilson, 1932).
Different pathogenic properties. "Virulence" is used loosely to
signify pathogenicity but, since different types of pathological effects
are caused by different agents it is desirable to use more precise terms
which designate, for example, the specific toxic power, ability to
multiply rapidly in the body, etc.
Bacterial toxins. It should be determined whether the pathogenic
principle is associated with intra- or extra-cellular products or is
intimately connected with the bacterial cell wall. Injection of
filtrates differentiates the first two from the last. Many factors, such
DETERMINATION OF THE PATHOGENICITY OF AEROBES viiis-S
as peculiarities of the organism, the cultural conditions, the age of the
culture and the nature of the filtering agent must be considered when
testing the toxic properties of bacterial filtrates. As a rule, exotoxins
are heat labile and deteriorate on standing. Scarlet fever is the most
heat stable of the exotoxins and approaches the endotoxins in this
I'espect. Heat stability may assist in diflferentiating the two types,
but the final criterion of a true exotoxin is its ability to stimulate the
production of a specific antitoxin when injected into a suitable animal.
The exotoxin in a filtrate may be neutralized by the addition of im-
mune serum and any residual toxic action may then be assumed to be
due to other toxic principles. The different organs affected and the
type of tissue damage should be recorded.
The Use of Laboratory Animals
For a general discussion of the care and use of laboratory animals
see, e.g., Meyer (1932), Farris et al (1945), Gumming (1947) and
Wadsworth (1947). Animals are necessary, not only for determining
the etiology of specific infectious diseases and the pathogenicity of
particular cultures of bacteria, but they are also utilized as a means of
isolation, to determine specific pathogenic properties, to maintain
organisms that grow only in vivo, to increase pathogenicity and to
produce antibodies and other agents used in the growth and identi-
fication of microorganisms and in the diagnosis and therapy of disease.
The choice of an experimental animal and the method of injection
and recovery of the organism depend upon the bacterial species and
the property to be studied. The human animal would be most
satisfactory in dealing with diseases of man but he is not available
except on rare occasions. This limits the application of Koch's
postulates in the case of man, but natural infections and accidental
infection of laboratory workers are useful in supplying circumstantial
evidence as to the pathogenicity of certain bacteria for man.
Healthy, previously unused animals should be employed. Several
days of observation prior to injection are necessary to insure that the
animals are in good condition and to provide a period of acclimati-
zation. Following injection the animals should be observed daily
for gross abnormalities and symptoms of disease and in certain cases
it may be necessary to take daily temperature, pulse, respiration
changes, hematology, etc. Large animals may be marked with
metal tags in the ears, and the ears of small animals may be tattooed
or marked with an indelible pencil.
Pathogenic bacteria produce different types of lesions in animals
which may be specific and equally as important as immunological,
serological and biochemical properties. To recognize them the stu-
dent should be trained in pathologic technic and should be familiar
with the gross and microscopic appearance of normal and diseased
tissue.
Methods of Injection
Bacteria or their products which cause disease when injected
parenterally may fail to do so when placed on the skin or when intro-
duced by insufflation or by mouth. Hence the importance of differ-
ent routes of injection.
VII48-6 MANUAL OF METHODS FOR PURE CULTURE STUDY
The required amount of material is drawn into a sterile syringe;
with the needle held up, air and any excess material is expelled onto
cotton moistened with a suitable disinfectant, which should be kept
away from the tip of the needle. Any undesirable disinfectant may
be removed with cotton moistened with alcohol. The following types
of injection are used: —
Cutaneous. This is a rather loose term and includes rubbing into,
or scratching the skin or placing the inoculum under an adhesive
patch. The precise method is determined by the object to be at-
tained. If it is desired to determine whether an organism can pene-
trate the normal skin, the material should be spread over the skin.
Irritation from shaving or depilation should be avoided. The skin
should be cleansed and sterilized with an antiseptic that has brief
action. The inoculated area may be covered with sterile gauze pro-
vided the adhesive does not aflfect the skin. Coating the skin with
collodion excludes air and may make the conditions abnormal and
affect the skin-penetrating power of the organism. It is common
practice in cutaneous inoculation to abrade the epidermis by scratch-
ing or scraping with a sharp instrument. This aids penetration by
removing the outer defensive layer and is similar to intracutaneous
injection.
Intracutaneous. By intracutaneous injection is meant the intro-
duction of material between the intradermal layers. The formation
of a bleb indicates successful injection. It is advisable to use animals
with unpigmented skin and rabbits should not be in moult. A 27-
gage needle is best. Shaving and the application of antiseptics,
particularly those that penetrate the skin, may interfere with the
test and should be used judiciously.
Subcutaneous. The skin may be shaved or the hair clipped with-
out interfering with the test. The point of puncture before injection
and the puncture after inoculation should be disinfected with a non-
irritating disinfectant such as tincture of zephiran chloride, alcohol,
merthiolate or, best of all, green soap and water. The area may be
marked with an indelible pencil. Material should be injected into
the subcutaneous tissue, with care not to puncture the peritoneal
wall when done in the abdomen.
If the material will not pass through the needle, the skin may be
sterilized, after removing hair, and a V-shaped opening cut in the skin
with sterile scissors. The flap is then lifted up and loosened until a
pocket is formed and the material to be tested is inserted. The flap
is replaced, sterilized and covered with collodion, or sutured asepti-
cally.
Intramuscular. The skin is treated as for subcutaneous injection
and the culture injected deep into the muscles.
Intravenous. The choice of a vein is mainly a matter of con-
venience and varies with the experimental animal. Rabbits usually
are injected in the marginal ear vein, mice and rats in the tail veins,
guinea pigs in the ear vein or jugular vein, horses and cows in the
jugular vein, swine in the ear, dogs and cats in the jugular or the vein
crossing the inner surface of the thigh and fowl in the radial vein that
DETERMINATION OF THE PATHOGENICITY OF AEROBES vii48-7
crosses the elbow joint. If the material is considerably acid or alka-
line it is adjusted to pH 7.3. The coarse particles are removed.
Veins may be enlarged by rubbing with xylene or immersing them in
warm water; but xylene should be avoided if the resulting reaction
interferes with the test. They are washed off with alcohol before and
after the injection. HgCl2 should be used when working with highly
pathogenic cultures. The previously warmed material free from air
bubbles is slowly injected. Alcohol saturated cotton is then pressed
over the puncture until the bleeding stops.
Intraperito7ieal. The disinfectant is applied as with subcutaneous
injection. The needle is passed through the skin and then through
the abdominal wall with a short stab. Caution: Avoid puncturing
the intestines and liver, the latter by injecting in a lower quadrant.
Intrapleural. The procedure is the same as with intraperitoneal
injection except that one injects into the pleural cavity anterior to the
diaphragm, the point depending upon the experimental animal.
Caution: Avoid puncturing the lungs and pericardial sac.
Per OS. Introduction of the material into the stomach or intestines
may be accomplished by a catheter or capsules or by mixing the
material with food or drink. To avoid exposure to the acid of the
stomach the material may be enclosed in enteric coated capsules.
Liquids may be mixed with starch and made into pills which are
digested in the intestines. Peristalsis can be controlled with mor-
phine.
Per Rectum.
Inhalation. Material for inhalation should be atomized in a
closed space about the head of the animal. (See Rosebury, 1947,
for complete details of inhalation technics).
Insufflation. Light anaesthesia is necessary to quiet the animal
for insufflation. The material is blown into the trachea or bronchial
tubes through a tube introduced into the larynx. Liquid may be
passed into the trachea and then blown into the bronchia. In some
instances the material is dropped into the nostrils and the animal is
allowed to insufflate, or the material is sprayed onto the membranes
of the nose and throat. The use of force and anaesthesia may reduce
the resistance of the membranes. The results obtained vary with
the method used, which should be reported in detail.
Intratracheal, Material may be introduced into the trachea
through a tube introduced into the larynx or by means of a syringe
through the side of the neck. In the latter method the skin may be
incised after shaving and sterilizing it.
Ophthalmic. Material is dropped into one eye, the other serving
as a control. It may also be inoculated upon the scarified bulbar
conjunctiva or injected subconjunctivally.
Intracranial. Injections are made into the brain through the skull.
Intracerebral. The method varies with different species of animals
depending on the material and the desired location for the inoculum.
In most instances the material is deposited into one of the frontal
lobes. Caution: Do not use enough to cause pressure. For large
VII48-8 MANUAL OF METHODS FOR PURE CULTURE STUDY
animals (large rabbits and monkeys) use about 0.5 to 1.0 ml.; for
medium size animals (e.g., guinea pigs) use 0.1 to 0.25 ml.; and for
small animals, such as mice, use 0.02 to 0.03 ml.
Cisternal 'puncture. The skin is shaved and sterilized at the base
of the skull over the cisterna magna. Withdraw as much fluid as is
to be injected and then introduce the material with a syringe and
needle, taking care not to injure nerve tissue.
Intraspinal. Injection is made between the lumbar vertebrae into
the spinal canal after withdrawing an equivalent amount of fluid.
Infracardial. Attempts to inject intracardially frequently fail
and numerous attempts are inadvisable. The animal should be
anaesthetized, the hair clipped over the cardiac region, the skin
shaved and disinfected. One should palpate for the point of maxi-
mum pulsation, insert a sharp needle (the size depending upon the
animal) and feel again for the heart with the needle. When it is
located, the heart beat will pulsate the needle and syringe. The
needle can be easily plunged into the heart. Its entrance will be
indicated by the appearance of blood in the syringe. The material
must be injected slowly.
Recovery of Organisms from Blood Culture
The following factors affect the accuracy of blood cultures: —
Bacteremia. The isolation of bacteria from the blood of apparently
normal animals is not related to sepsis nor to the pathogenicity of the
organism. In localized infection, showers of organisms may be
thrown into the blood stream at irregular intervals, necessitating
repeated cultures to demonstrate them. The sequence of organisms
in the blood is related to the stage of the infection, the rise in tempera-
ture and the ingestion of food. In typhoid fever the organisms are
more likely to be present in the blood during the first week or 10 days,
but in many other infections the best time is during the rise in fever.
Organisms invade the blood stream when resistance is low.
Contamination. It is particularly important to prevent contami-
nation when studying diseases of unknown etiology. The Keidel
blood culture tube is of distinct advantage although Feder (1937)
recommended a blood culture technic which he claimed had advan-
tages over the Keidel tube. A special apparatus with the advantages
of the Keidel tube and with facilities for subculturing was described
by McLeod and Bevan-Brown (1918). Recently, a "Vacutainer"*
appeared on the market.
The skin should be shaved and treated with a disinfectant such as
mentioned for subcutaneous injections. The particular culture
medium depends on the organisms expected. The antibacterial
action of complement can be overcome by using the culture medium
of Kracke and Teasley (1930) (See Leaflet II, p. II44-IO) or by using
a large volume of culture medium such as 200 ml. of broth. The
smaller the amount of blood used the less chance of non-pathogenic
*Becton, Dickinson & Co.
DETERMINATION OF THE PATHOGENICITY OF AEROBES vir4s-9
organisms contaminating the cultures (Heith, 1926). Elliott (1938)
claimed that lysis of erythrocytes and leucocytes by saponin in-
creases the number of positive cultures when few bacteria are present.
The presence and type of antibodies for the organism recovered
should be determined, particularly if the animal does not die. Recov-
ery without the development of antibodies suggests that the organism
recovered may not have been the cause of the infection but may have
been a temporary invader which disappeared without stimulating
much antibody production. If the animal dies, antibodies probably
will not be present to any extent but if at all will be most intense just
before death. Therefore, blood should be drawn immediately after
death. Antibodies do not indicate pathogenicity, but they are sup-
porting evidence.
Autopsy
The following should be determined at autopsy The cause of
death; the type and distribution of the lesions; any cellular changes;
distribution of the infecting organism; changes that may have taken
place in the microorganism; and whether antibodies are present.
Natural infection may interfere wdtli animal experimentation;
hence, the autopsy should be made immediately after death to reduce
terminal invasion. If the autopsy cannot be made promptly the
body should be kept in the refrigerator. The autopsy should be
done in a good light with instruments that have been sterilized by
dry heat or in the autoclave.
The animal should be prepared by wetting the hair with a disin-
fectant that penetrates to the skin. Wetting with alcohol first helps
Examine the area of the injection. Open the animal down the
median ventral line and pull the skin back. Cover all but the
exposed area with towels moistened with the antiseptic. Search for
gross lesions, remove suspicious glands, tissues, etc. and place them in
Petri dishes for culture and histologic examination. Moisten the
exposed surfaces with alcohol and ignite.
Open the pleural cavity with a fresh set of instruments, taking care
not to cut the diaphragm or pierce the lungs. If desired, seal a
sample of the pleural fluid in a capillary tube and store it in the
refrigerator for cytological and cultural study. Make smears and
cultures of exudates. If the animal died from an infection, the
organism will be abundant in most of the body fluids, and a small
amount, such as a loopful, of each will lessen the chance of recovering
contaminants.
Open the pericardium and sear the surface of the heart. Make an
incision with a sterile instrument and proceed as with the pleural
exudate.
The lungs may then be examined and any cultures or sections made.
Peripheral blood may be compared with the heart blood. The blood
and other body fluids may be tested for antibodies, but if the infection
was of short duration they may not be detected. A high titer of
antibodies for the organisms recovered suggests that they may not
have caused death but this is not necessarily so because in diphtheria,
e.g., the appearance of antibodies may be followed by improvement
and yet the animal may die from liberated cardiotoxins.
VII48-10 MANUAL OF METHODS FOR PURE CULTURE STUDY
Open the peritoneal cavity with a new set of instruments. Treat
the peritoneal exudate like the pleural exudate. Sear the surface of
the liver, spleen, kidney, etc. for cultures and store pieces for patholo-
gical study where indicated.
Examine all the organs, joints and cavities and make cultures
where indicated. In cultures of the brain take samples from different
regions to determine the distribution.
Smears made at autopsy should be stained for Gram reaction,
capsules and spores.
Factors Interfering with the Determination of Pathogenicity
Factors interfering with the determination of pathogenicity were
described by Teale (1933). Unless they are taken into considera-
tion, they may lead to erroneous conclusions. An organism or its
products may affect only one part of the body, and this in a specific
manner, while other organisms may attack any part of the body
and produce a variety of disease conditions. Different organisms
may attack the same part and produce similar changes.
A pathological change in the animal tissues produced by the injec-
tion of an organism or its products indicates pathogenicity but con-
trols must be used to exclude other factors. The ability to grow in
or upon animal tissues or fluids is not of itself evidence of pathogenic-
ity. Finally, non-pathogenic organisms may produce serological
and other changes.
Variations in the resistance of individual animals or strains must
also be taken into account. (As by Gumming, 1943). Infection
may occur when an individual of low resistance is injected even with a
normally non-pathogenic strain. Hence, several animals should
always be used in tests of pathogenicity.
The following factors also interfere with the determination of
pathogenicity :
Variations in the bacterial mass. Bacterial cells, like other biologi-
cal units, vary around a mean because the transmission of different
characters is imperfect. To reduce errors from this source it is
desirable to use a culture prepared from several colonies. The cells
vary with age, both naturally and in response to the environment,
the latter as temporary adaptations or non-adaptative changes
which may be transmitted through successive generations and then
disappear. The changes rarely result in mutations. Holman and
Garson (1935) discussed precautions that must be observed in the
study of bacterial variation.
Natural variations. Natural or normal variations include varia-
tions of individual cells around the mean and variations resulting
from the life cycle which may vary in all the morphological, physio-
logical and pathogenic properties of the culture. Selective cultiva-
tion and animal passage of cultures that have lost pathogenicity
may lead to development of pathogenic cells in the culture. Some
non-pathogenic cultures may contain pathogenic variants, parti-
cularly if the culture was associated with a disease process. Hence,
the advisability of testing a number of colonies separately.
DETERMINATION OF THE PATHOGENICITY OF AEROBES viijs-ll
If, as some still believe, the normal life cycle of a bacterial cell
consists mainly of an increase in size with age, with minor morphologi-
cal and physiological changes, the relationship of pathogenicity to
the life cycle has little significance. If, however, the life cycle is
represented by complicated ontogenetic changes (dissociations),
each phase or stage representing distinct characteristics and varying
in stability and in response to the environment, the relationship of
pathogenicity assumes considerable importance. This problem
concerns Leaflet VII only in so far as the variations affect the study of
pathogenicity.
As regards pathogenicity, dissociation may occur as readily in this
property as do morphologic and physiological changes in the cell or
colony and may be associated with one or more of these latter changes.
The relationship of pathogenicity to R, S, M and G colony types and
to the morphology, size and age of the cell may have to be determined
for each culture. With some organisms, e.g.. Salmonella typhosa and
Corynebacteriiim diphtheriae, the smooth colony type is the most
pathogenic, whereas the mucoid phase of Diplococcus pneumoniae and
Klebsiella pneumoniae and the rough phase of other organisms, such as
Bacillus anthracis, are the most pathogenic. The relation of the G
phase to pathogenicity has not been clearly established but in staphy-
lococci, e.g., it appears to be non-pathogenic.
Acquired variations. Acquired or new variation represents
changes in the average cell in response to environmental changes.
When they are favorable to survival of the organism they are adapta-
tions.
Organisms not ordinarily pathogenic may acquire some degree of
pathogenicity in animal passage but they are not strictly pathogenic.
Consequently, the history of an organism in vivo should always be
reported. Organisms grown in immune serum may increase in
pathogenicity and resist agglutinating and other antibodies.
Some organisms lose pathogenicity quickly, particularly when
grown on artificial culture media. They are usually most pathogenic
in the late logarithmic phase. To reduce this tendency to lose patho-
genicity the culture medium and incubation temperature should favor
optimum growth and should be similar to conditions existing in body
fluids and tissues (See Felton, 1932). Tissue culture or fresh blood,
either unheated or inactivated at 57°C for 1 hour to destroy transient
organisms, used alone or added to the culture medium are valuable
in maintaining pathogenicity. Transferring from one animal to
another should be done quickly.
Antigenic variations. In addition to those changes in antigenic
specificity associated with different phases, there is some evidence
that bacteria may adsorb antigen from the environment with result-
ing change in antigenicity (See, e.g., Burky, 1934 and Rosenow,
1945). False serologic reactions have resulted from foreign antigen,
such as agar. Two different organisms may have a common anti-
gen from being grown on the same medium. Thus, a common anti-
gen may not necessarily indicate a natural relationship (See, e.g.,
Dubos, 1945). Extraneous or unnatural antigens or their antibodies
VII48-12 MANUAL OF METHODS FOR PURE CULTURE STUDY
may sometimes be eliminated by growing the organisms on different
culture media. This would be simpler than by adsorbing immune
sera with a common antigen.
Distinction should be made between adsorption of a foreign
antigen, change in the bacterial antigen and physical mixture of a
foreign antigen. The latter can be removed by thorough washing
with saline. In working with obligate parasites, particularly filter-
able forms, the difficulties are increased due to antigens present in
tissues.
The occurrence of heterophile or non-specific antigens and anti-
bodies complicates the study of pathogens. Yeast and Klebsiella
fneiimoniae stimulate immunity to Type II pneumococci, injection of
sheep cells produces immunity to anthrax (Rockwell, 1933) and in-
jection of Salmonella pulloruvi stimulates antibodies against S.
schottmulleri, S. paratyphi, S. (Eberthella) typJiosa and Shigella dysen-
teriae. Therefore, the presence in the blood of antibodies for a par-
ticular organism is not of itself convincing evidence that the organ-
ism caused the infection or that it acted as the antigen.
The number, nature and natural occurrence of non-specific anti-
gens, their relationship to phase variations and their distribution
should be determined. Methods for studying non-specific antigens
concern Leaflet VIIL
Obligate parasites. Still greater difficulties are encountered in
determining the pathogenicity of obligate parasites. The direct
transfer of body fluids or tissues involves the objections just dis-
cussed. Also, two organisms or non-specific antigens may be present,
as in typhus fever, and the immune sera produced when the animal
tissues or fluids are used as antigens may contain antibodies for both
organisms, making serological evidence inconclusive.
The presence of organisms in tissue or in the blood stream is not
necessarily evidence of pathogenicity or parasitism. Organisms
from different sources are continually entering the blood stream and
dead tissues may be present in living animals permitting non-patho-
genic saprophytes to flourish. For these reasons, the only indication
of pathogenicity in the case of obligate parasites consists of an ac-
cumulation of circumstantial evidence. Improvements in tissue
culture technic may provide a solution.
Specificity. The pathogenicity of an organism may be confined to
a single species of animals, which stresses the importance of the
proper selection of an experimental animal. The designation of an
organism as pathogenic or non-pathogenic, etc. refers solely to the
animal and method used.
Passage through one animal may result in reduced pathogenicity
for another. The pathogenicity of an organism for a different species
of animal should be tested w4th cultures grown on artificial culture
media for some time as well as with freshly isolated cultures.
Synergism. Occasionally two organisms may grow together and
produce a pathologic condition whereas neither can do so alone.
Both organisms do not necessarily produce toxins and it is possible
that one of them may in no way contribute directly to the disease
DETERMINATION OF THE PATHOGENICITY OF AEROBES vii^s-lS
even though it may be universally present. One may be a harmless
invader, constantly associated with the disease but not contributing
to it. Even when both organisms are essential to produce the disease,
one may be a saprophyte in dead tissues and may contribute to the
infectious process only by providing conditions essential to the growth
of the pathogen. Unless the organisms are also associated in other
diseases, serologic tests may be of differential value. Bacterial
antagonism also plays a role in pathogenicity in some instances.
It should be determined whether the bacterial product causes
disease by its direct action on the tissues or by sensitizing them to it.
If the latter, then other organisms that produce a similar antigen
or a similar non-specific antigen may also account for the pathology.
A pathogenic organism can be differentiated because it grows in
the animal and produces sensitization (Hanger, 1928).
Cultural co)isideratio7is. The cultivation of pathogenic bacteria
may not always be favorable for producing the pathogenic factors.
Corynebacterium diphtheriae and streptococci, e.g., grow luxuriantly
under certain conditions without producing toxin. Certain bacteria
require oxygen for toxin production. The toxin also may be pro-
duced and then disappear in a culture or may be destroyed by unfa-
vorable manipulation. Finally, an early toxin and a late one may
have different properties.
Most pathogenic aerobes are facultative anaerobes or facultative
microaerophiles. Parasitic species may prefer tissues or cavities with
low oxygen tension.
The Use of Biochemical Methods in Lieu of Animal
Inoculation Tests to Study Certain Pathogenic
Properties
Because they give results parallel with certain pathogenic effects,
tests have been proposed, based on biochemical properties, that ap-
pear to be satisfactory as substitutes for animal inoculation experi-
ments, e.g., when a large number of cultures are to be tested as in
clinical work, when animal inoculation experiments are inconclusive,
as in non-hemolytic streptococci, or when animal inoculation experi-
ments involve considerable danger (see, e.g., Dozois and Rauss, 1935;
and De Angelis, 1937). For example, power to clot plasma is now
recognized as an excellent in vitro method for differentiating patho-
genic from non-pathogenic staphylococci. For a summary of recent
biochemical methods for staphylococci, see Chapman (1946).
Resistance of streptococci to the bactericidal power of fresh,
diluted, defibrinated guinea pig blood and to different chemicals
is an excellent indicator of pathogenicity (probably toxicity). A
complete up-to-date discussion of this work will be found in Chapman
(1947).
Although the writer is enthusiastic about carefully applied bio-
chemical tests of such organisms as staphylococci and streptococci
as substitutes for animal inoculation tests, he is aware of their
shortcomings and is not in favor of universal acceptance at the present
time. So many technical considerations enter into the reliability
VII48-14 MANUAL OF METHODS FOR PURE CULTURE STUDY
of the methods that few bacteriologists possess the technical knowl-
edge or skill to apply them satisfactorily.
Summary
It is obvious that suitable technic, skilfully applied, and extreme
caution in interpretation of the results are necessary to determine the
pathogenic properties of microorganisms. Indirect evidence is ac-
ceptable as a substitute when Koch's postulates are inapplicable or
when animal inoculation experiments are not entirely satisfactory;
but such evidence is rarely sufficiently conclusive except as a working
hypothesis. In the absence of conclusive evidence, the organism
should be considered pathogenic only so far as the experiments
indicate.
To facilitate study of an investigation by others the methods used
should be reported punctiliously.
REFERENCES
BuRKY, Earl L. 1934. Production of lens sensitivity in rabbits by the action of
staphylococcus toxin. Proc. Soc. Exp. Biol. & Med., 31, 445.
Chapman, George H. 1945. Staphylococci in gastroenterology. Am. J. Digestive
Dis., 12, 399.
Ch.-vpman, George H. 1946. The staphylococci. Trans. N. Y. Acad. Sci., 9, 52.
Chapman, George H. 1947. Relationships of nonhemolytic and viridans strepto-
cocci in man. Trans. N. Y. Acad. Sci., 10, 45.
Gumming, C. N. Wentworth. 1943. The importance of Mus musculus in research:
A discussion of its genetic aspects. Presented before the Am. Public Health
Assn., New York, Oct. 11-14.
Cumming, C. N. Wentworth. 1947. Modern mass production in animal breeding
for experimental research. In press. Read before the Fourth International
Cancer Research Congress.
De Angelis, Eugene. 1937. A reaction with iron compounds for the determination
of B. anthracis and of its pathogenicity. J. Bact., 33, 197.
Dozois, K. Pierre and Rauss, K. F. 1935. Relationship between electrophoretic
migration velocities, the virulence and the types of the diphtheria and
diphtheria-like bacilli. Am. J. Pub. Health, 25, 1099.
DuBOS, Rene J. 1945. The Bacterial Cell In Its Relation to Problems of Virulence,
Immunity and Chemotherapy. Harvard Univ. Press.
Elliott, S. D. 1938. The use of saponin in blood culture media, with special refer-
ence to blood cultures in subacute bacterial endocarditis. J. Path. & Bact.,
46, 121.
Farris, Edmond J., Carnochan, F. G., Cumming, C. N. W., Farber, Sidney, Hart-
man, Carl G., Hutt, Frederick B., Loosli, J. K., Mills, Clarence A. and
Ratcliffe, Herbert L. 1945. Animal colony maintenance. Ann. New
York Acad. Sci., 46, (Art. 1) 1.
Feder, F. M. 1937. A new and simplified blood culture technic. J. Lab. & Clin.
Med., 22, 846.
Felton, Lloyd D. 1932. Studies on Virulence: Influence on virulence of pneumo-
cocci of growth on various media. J. Exp. Med., 56, 13.
Fulton, F. 1943. Staphylococcal enterotoxin — with special reference to the kitten
test. Brit. J. Exp. Path., 24, 65.
Hanger, Jr., Franklin M. 1928. Effect of intravenous bacterial filtrates on skin
tests and local infections. Proc. Soc. Exp. Biol. & Med., 25, 775.
Holman, W. L. and Carson, Arline E. 1935. Technical errors in studies of bac-
terial variations. J. Inf. Dis., 56, 165.
Kracke, Roy R. and Teasley, Harry E. 1930. The efficiency of blood cultures.
J. Lab. & Clin. Med., 16, 169.
McLeod, J. W. and Bevan-Brown, R. E. 1918. The technique of blood culture.
J. Path. & Bact., 22, 74.
DETERMINATION OF THE PATHOGENICITY OF AEROBES vii48-15
Meyer, K. F. 1932. Use of animals in routine diagnostic work. J. Lab. & Clin.
Med., 17, 510.
Reith, Allan F. 1926. Bacteria in the muscular tissues and blood of apparently
normal animals. J. Bact., 12, 367.
RiGDON, R. H. 1938. Observations on Dolman's test for determining the presence of
staphylococcal enterotoxin. Proc. Soc. Exp. Biol. & Med., 38, 82.
Rivers, Thomas M. 1937. Viruses and Koch's postulates. J. Bact., 33, 1.
Rockwell, George E. 1933. Active immunization to anthrax by means of hetero-
phile antigen. Science, 77, 612.
Rosebury, Theodor. 1947. Experimental Air-Borne Infection. Williams and
Wilkins, Baltimore, Md.
Rosenow, Edward C. 1945. Production in vitro of substances resembling anti-
bodies from bacteria. J. Inf. Dis., 76, 163.
Teale, F. H. 1933. Factors influencing the pathogenicity of bacteria. J. Path. &
Bact., 37, 185.
Wadsworth, a. B. 1947. Standard methods of the Division of Laboratories and
Research of the New York State Department of Health. Williams and
Wilkins, Baltimore, Md.
Zinsser, H.\ns and Wilson, E. B. 1932. Bacterial dissociation and a theory of the
rise and decline of epidemic waves. J. Prev. Med., 6, 497.
LEAFLET VIII
SEROLOGICAL METHODS
6th edition
Pure Culture Study of Bacteria, Vol. 15, No. 3-4
November, 1947
Revised by
C. A. Stuart and K. M. Wheeler
LEAFLET VIII
SEROLOGICAL METHODS
THE USE OF SEROLOGY IN PURE CULTURE STUDY
In the study of bacteria the facts that may be estabhshed with
the agency of serological reactions often have peculiar value, not
as substitutes for those to be gained from morphological, cultural,
or biochemical means, but as supplemental to them. This is espe-
cially true in so-called "pure culture" investigations. Thus, serologi-
cal studies may show that a group of organisms, apparently alike
morphologically and physiologically, in reality consists of different
sub-groups, which cannot be distinguished by other means. This
leaflet is intended to make readily available such serological pro-
cedures as are useful in pure culture studies. No implication is
made that these procedures are necessarily the best among the great
variety of serological methods now in use. They have, however,
proved practical in pure culture studies, and they are given herewith
the hope that they will be helpful to users of this Manual.
The field designated "serology," as applied to pure culture study
of bacteria, deals with the reactions of the blood sera of animals
that have been injected with micro-organisms or their products.
Such substances, acting as antigens when injected into an animal,
stimulate the appearance of antibodies in its blood serum. This
serum when mixed with suspensions of unknown bacteria or their
products, gives a positive reaction only if the bacteria or their pro-
ducts are of the same type as those introduced or else are related
to them. This specificity is not absolute but may vary within certain
limits both quantitatively and qualitatively. It is often possible, how-
ever, by means of these relatively specific reactions to identify an
unknown organism, to group or sub-group closely allied forms, and
to study the relationship between the groups, sub-groups, and
strains. Divisions so established may or may not agree with previous
groupings based upon morphological, cultural or biochemical obser-
vations. As a rule the methods will be found supplemental to each
other and more often than not their results will coincide. Frequently,
when other procedures fail to show differences, serological reactions
will reveal them and varieties of a given species may be differentiated
by their antigenic properties.
Immune sera can yield information in two ways: either a known
serum may be employed as the test agent for determining an unknown
antigen; or a known antigen may be used as a test agent to denote
the presence or absence of a specific antibody. Serological pro-
cedures that may be useful in the identification of pure cultures are:
1) Agglutination: agglutinogen (antigen) -agglutinin reactions; ag-
glutinin absorptions.
2) Precipitation: precipitinogen (antigen)-precipitin reactions.
3) Complement fixation : antigen-antibody-complement-hemolysin-
RBC reactions.
V11147-3
VIII47-4 MANUAL OF METHODS FOR PURE CULTURE STUDY
4) Toxin neutralization: toxin-antitoxin reactions.
5) Hemolysis: hemolysin reactions. (See Leaflet VI, pages VI40-I6-
18.)
The most frequently employed serological reactions for the identi-
fication of bacteria are those of agglutination and complement
fixation. The agglutination method is especially rapid and reliable.
Adequate controls on all reagents are essential for each procedure.
DEFINITION OF TERMS
An aniige7i is defined as a substance which, when introduced parenterally into an
animal body, stimulates the animal to produce specific bodies that react or unite with
the substance introduced. In this outline of methods the term will be limited to sus-
pensions of living or killed bacteria or their products. Agglutinogen, 'precipitinogen,
toxin, and toxoid are some of the names applied to antigens employed in the various
serological procedures.
An antibody is the specific body above mentioned, produced by the animal in re-
sponse to the introduction of an antigen. These antibodies under the right conditions
may act as one of the principal factors in jireventing any injurious action which the
antigen might otherwise exert. For u.se in practical serology, antibodies are obtained
from the blood serum and appear in the globulin fraction. Agglutinin, precipitin, and
antitoxin are designations in common use.
Complement is a third substance which may take part in serological reactions. It is
present in varying degree in the normal serum of all animals, combines with the anti-
gen-antibody union and may bring about lysis of the bacteria, is non-specific and is
not increased during immunization, and in contrast to bacterial antibodies which are
relatively heat stable, is inactivated by exposure at 56°C for 30 minutes and deterio-
rates in a few days at refrigerator temperature.
A hapten or partial antigen is that portion of an antigen which contains the chemical
grouping upon which the specificity depends. The hapten reacts specifically with the
corresponding antibody, but by itself, when separated from the carrier molecule, is
incapable of stimulating the formation of antibodies in vivo.
Heterophile antigen is the term applied to common antigens which may occur in the
tissues of animals which are not closely related. Several have been described of which
the Forssman heterophile antigen is an example. When guinea pig kidney emulsion
is injected into rabbits an antibody (Forssman antibody) is formed which reacts with
sheep erythrocytes. The Forssman antigen has been found in several species of
bacteria. Among bacteria, common antigens may be found in groups that are widely
divergent in morphological and biochemical characters.
BACTERIAL DISSOCIATION
Bacteria may produce variants which differ from the parent in one or more charac-
ters such as colony form, morphology, virulence, biochemical activity or antigenic
composition. Variation in serological reactivity may or may not be correlated with
other variation, may occur naturally or be induced, and may or may not be a stable
change. There is, for example, change from smooth (S) to rough (R) with intermedi-
ate phases, variation in motility or presence oi flagellar (H) antigens, form variation or
change of somatic (O) antigen, pliase variation of flagellar antigen involving change in
serological reactivity of motile forms. These and other variations in the antigenic con-
stitution of bacteria must be considered in pure culture study by serological methods,
and these variations arc often the explanation of anomalies observed in the results of
serological tests.
AGGLUTINATION
The antibodies in the blood scrum of immunized animals that cause
clumping or agglutination of bacteria are called agglutinins. Agglu-
tinins may occur naturally, but if present are usually weak.
The agglutination of a suspension of bacteria by its homologous
immune serum may be observed either microscopically or macroscopi-
SEROLOGICAL METHODS viii,,-5
cally. The macroscopic tube test is probably the most accurate and
is a convenient method. The macroscopic slide test has been used
extensively for typing enteric bacteria. The phenomenon of agglu-
tination is evidenced by the appearance of granulation in the bacterial
suspension. Granulation may be extremely fine, or clump size may
range to very coarse. Clumps may be compact and dense as in the
case of somatic agglutination, may be light and cottony with flagel-
lar reactions, or may be stringy and thread-like with some mucoid
organisms. Conditions of optimal incubation time and tempera-
ture vary considerably depending on the organisms tested.
PREPARATION OF IMMUNE SERUM
The rabbit is the most satisfactorj' animal for the production of agglutinating
serum. The technics employed for inuuunization are many, and vary widely with dif-
ferent workers. The particular properties of the bacteria under examination are the
factors determining the method chosen.
A healthy, well-developed rabbit is selected for immunization. It may conven-
iently be held in a squatting position by an attendant or locked in a special box in
such a manner that only the head protrudes. The hair is removed from around a
marginal ear vein and along the edge of tlie ear by shaving. The ear is then cleansed
with 70% ethyl alcohol. Dilation of the vein is promoted Ijy rubbing or patting, by
heat, or by applying xylol. Any sharp sterile instrument will serve to open the vein,
a clean wound favoring the escape of blood. About 5 ml. of blood are collected in a
sterile test tube to provide serum for determining the presence or absence of natural
agglutinins in the blood of the rabbit selected. The technic is described in the next
section. Having determined the absence of natural agglutinins the immunization
procedure may be undertaken.
Immune Serum: Immunize the rabbit by repeated subcutaneous, intraperitoneal, or
intravenous injections of saline suspensions of young (18-24 hour) cultures of the
organism to be studied. Growth can be taken from agar or from silica gel medium or
in the case of flagellar antigens young broth cultures may be used. A convenient sus-
pension is one containing about 500 million organisms per milliliter, although suspen-
sions containing more or less than this number of organisms can be used, depending
upon the toxicity of the cidture. The number of organisms in the suspension can be
c}uickly and roughly determined by various methods outlined in any treatise on sero-
logical methods. If the organism is non-pathogenic for rabbits, suspensions of living
organisms can be injected. As a rule, however, the organisms are killed Ijefore injec-
tion by heating the suspension in a water bath at (JO°C. for 1 hour, or by mild chemical
treatment such as 0.3% formalin. Inject subcutaneously, intrapcritoneally, or intra-
venously into a rabbit at intervals of 5-7 days starting with a dose of 0.5 ml. and in-
creasing each dose by 0.5 ml. After the third injection a test bleeding m;iy be made to
determine titer, and the rabbit bled out or reinjected as necessary. Titrations of the
serum should be made following each subsequent injection, and immunization con-
tinued until a satisfactory titer is attained or until no further increase occurs. Six to
eight injections are usually required to produce agglutinins of sufficient titer. The
method of immunization can be varied to meet the needs of special cases. The shorter
the period of immunization, the more specific is the imtnune scrum. Long immunization
increases the content of group agglutinins. To test the titer of the serum, draw 1-2 ml.
of blood from the marginal vein of the ear of the rabbit 5-7 days after the last injection;
collect the serum and carry out an agglutination test with it, as described below. If the
titer is sufficiently high, bleed the rabbit from the heart or an artery to obtain as much
blood as possible. Allow the blood to clot, collect the serum aseptically, and add 0.5%
phenol or 0.3% tricresol to the serum as a preservative. Place the serum in ampules
or bottles and store in the refrigerator.
Bleeding of Rabbit: Rabiiits are more easily and readily bled from the heart when
large amounts (25-100 ml.) of blood are desired. Etherize the rabbit, clip the hair over
the region of the heart, and shave. .\n added precaution to prevent contamination is
to wet the hair of the rabbit thoroughly over the entire left side. With the rabbit
lying on its right side paint the shaved area with tincture of iodine. Determine the
point of maximum pulsation. Using a sterile 50 ml. Luer syringe and a needle of 17 or
18 gauge (2-2)^2 inches), insert the needle at the point of maximum pulsation. The
viii„-6 MANUAL OF METHODS FOR PURE CULTURE STUDY
heart can now be located with the needle which will be moved by the heart beat. Force
the needle into the heart. When it is in the heart, blood will flow into the syringe.
Slowly withdraw 50 ml. Quickly withdraw the needle and eject the blood into a 500
ml. Erlenmeycr flask, or into a large test tube and allow it to clot. The serum may be
obtained free of clot and cells by centrifugation. Five to six pound rabbits can be
bled monthly in this way. If the rabbit is to be sacrificed, another 50 ml. portion of
blood can be obtained in a similar manner, but preferably with another clean sterile
syringe and needle in order to avoid clotting in the syringe.
PROCEDURE
Procedure for Microscopic Agglutination Test: Dilutions of the
serum are prepared by diluting the immune serum with saline solu-
tion, care being taken to keep the serum twice the strength of the
final dilution desired, since the addition of an equal volume of the
antigen doubles the dilution of the serum on each cover slip. Upon
separate clean cover slips is placed a loop of the diluted serum. A
loop of the suspension of the organism is placed beside each drop of
diluted serum and the two mixed with a platinum wire. The cover
slips are then suspended over hollow ground slides as noted in the
technic for preparing a hanging drop preparation. The slides may
be held at room temperature for a short time, usually less than one
hour, and examined under a magnification of approximately 500
diameters.
Some experience is necessary to discriminate between normal
reactions and false dumpings. In the true reaction all the organisms
in the field will be gathered into a few clumps and no organisms will
be found around the edges of the drop. In pseudo-reactions the
organisms may collect around small foreign particles, around the
edge of the drop, and in many small clumps containing a relatively
small number of cells. The beginner generally uses too heavy sus-
pensions. Much sharper readings can be made w^ith a very light
suspension of the organism being studied.
Macroscopic Agglutination Test. Antigen: Wash off in saline the
growth from a 24-hour agar slant culture of the organism to be tested.
An emulsion which is too thick obscures the agglutination, while one
which is too thin does not provide enough bacteria for macroscopic
comparisons. The density of the emulsion of bacteria must be ad-
justed to meet the requirements of special conditions and to assure
constancy in the results. This adjustment can be made on the basis
of an actual count of the number of bacteria per ml. or by compari-
son with a standardized suspension of insoluble particles. The
latter method is usually more convenient, using the McFarland (1907)
nephelometer. A density of 0.5 on the McFarland nephelometer
scale is satisfactory for most purposes. The suspension should
be homogeneous, "smooth", and entirely free from particles. The
bacteria in the suspension may be killed by heat at 60°C. for 1 hour, or
living bacteria maybe used. Satisfactory preservatives for a suspen-
sion for the agglutination test are 0.5% phenol or 0.3% formalin.
Some suspensions of bacteria tend to flocculate spontaneously,
necessitating as a control a suspension of the bacteria in saline which
is carried through the incubation period of the test. Spontaneous
agglutination may be due to many factors, such as surface tension,
electrical charges upon the surfaces of the bacteria and other un-
SEROLOGICAL METHODS vin„-7
known conditions associated Avitli the composition of the bacterial
cell. Spontaneous flocculation can at times be avoided l)y proper re-
gard to the pH of the suspending fluid with the use of buft'er mixtures,
by passing the organism through several transfers immediately before
the final culture to be used in making the suspensions, and by the
growth of the organisms in media which favor diffuse growth. Wash-
ing the organisms in distilled water, ether, and chloroform, and taking
the supernatant fluid from heavy suspensions which have been
allowed to sediment are procedures which may make it possible
to obtain a smooth suspension of an organism which originally
flocculated spontaneously in saline.
Procedure for Macroscopic Agglutination Test: The test is per-
formed by mixing a constant amount of the bacterial suspension
(antigen) with decreasing amounts of the antiserum, according to
the protocol in Table 1.
TABLE 1
COMPLETE AGGLUTINATION TEST WITH RESULTS IN A TYPICAL INSTANCE
Tube
No.
0.85%
aque-
ous
NaCl
Immune serum:
preparation of dilution
Dilu-
tion
Antigen
(bacterial
suspen-
sion)
Final
dilu-
tion
d
>o
1
o
m
u
^ i2
■Eg
■° u
go
Aggluti-
nation
observed
in a typi-
cal in-
stance
1
2
3
4
5
6
7
8
9
10
0.9 ml.
0.5 ml.
0.5 ml.
0.5 ml.
0.5 ml.
0.5 ml.
0.5 ml.
0.5 ml.
0.5 ml.
0.5 ml.
0.1 ml. of immvme serum*
0.5 ml. from tube No. 1
0.5 ml. from tube No. 2
0.5 ml. from tube No. 3
0.5 ml. from tube No. 4
0.5 ml. from tube No. 5
0.5 ml. from tube No. 6
0.5 ml. from tube No. 7
0.5 ml. from tube No. 8
1:10
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
1:2560
0.5 ml.
0.5 ml.
0.5 ml.
0.5 ml.
0.5 ml.
0.5 ml.
0.5 ml.
0.5 ml.
0.5 ml.
0.5 ml.
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
1 :2560
1:5120
complete
complete
complete
complete
complete
complete
complete
partial
none
1
*The contents of each tube should be thoroughly mixed by sucking up the fluid in the
pipette and blowing it back into the tube several times before transferring the 0.5 ml.
to the next tube. After mixing, 0.5 ml. is discarded from tube No. 9. A one jnl. pipette
graduated to tip is the most convenient size. Tubes of about 10 mm. inside diameter
are suitable for this volume of fluid.
Starting with the 1 :10 dilution of this antiserum, the series of dilu-
tions can be made readily in the same tubes in which the test is to
be done. Tube 10 is used as a control for the smoothness of the
bacterial suspension. It should be free from clumps. After the
antigen is added, shake well and incubate for 2-4 hours at .50 to 52°C.^
After this period of incubation readings may be taken at once, or
the tubes may be allowed to stand overnight at room temperature
or preferably in the refrigerator.
'The time and temperature of incubation is not the same for all bacteria. Agglutina-
tion proceeds more rapidly witli motile than with non-moti!e bacteria. Agglutination
of non-motile bacteria may be accelerated by shaking or by taking advantage of the
convection currents set up in the tubes where the level of the water is below the level
of the liquid in the tubes.
VIII47-8 MAN UAL OF METHODS FOR PURE CULTURE STUDY
Readings and Residfs: At the end of the period of incubation,
for the test to be satisfactory, the control tube should show a uni-
form cloudiness without sedimentation or flaking. A positive reac-
tion will vary in appearance with the tyipe of agglutination which
has taken place. With progressive dilutions the reduction in the
quantity of agglutinins is accompanied by less and less complete
agglutination. This is observed in the tube as decreased amounts
of sediment and less marked granulation or clumping. Conversely,
it is associated w^ith correspondingly increased turbidity of the
supernatant fluid and closer and closer approximation to the ap-
pearance of the control tube. The titer of the agglutinin is taken
as the highest dilution in which agglutination takes place. Certain
immune sera agglutinate only in the higher dilutions. The failure
of relatively concentrated serum to cause agglutination has been
designated by such terms as "prezone," "prozone", and "zone of
inhibition." Example: If in Table 1 (it is to be empJiasized that the
residts set down in this table are arbitrarily chosen to serve as an example
only) no agglutination resulted in tubes Nos. 1 to 3, partial clumping
in tube No. 4, complete agglutination in tubes Nos. 5 to 7, while in
the succeeding tubes the reactions were less and less complete, then a
zone of inhibition would be indicated in the concentrations of the
sera employed in tubes Nos. 1 to 3. When absence of clumping is
seen in one or more tubes other than at the beginning of a series it is
usually due to an error in technic. Zones of inhibition should always
be guarded against by using a sufficient range of dilution of the
antiserum, lest a false negative result appear. Great care in carrying
out the steps in agglutination technics is essential if accurate results
are to be obtained by such methods.
The macroscopic slide agglutination test is performed on a glass
slide using a drop of serum dilution plus a drop of heavy bacterial
suspension (density of McFarland 7-8). Serum and antigen are
mixed over a surface of about 1 cm. diameter and mixing is continued
by rocking the slide. The degree of clumping is read after about 2
minutes. While the slide technic has advantages of simplicity and
speed, the macroscopic tube test provides a more reliable and
adaptable technic for pure culture study.
For the complete identification of a bacterial strain, agglutination
to titer should be secured with an antiserum produced with organ-
isms of known type; and, furthermore, the organism in question,
if used in sufficient quantity, should absorb all of the agglutinins from
such an antiserum, thus leaving the antiserum devoid of agglutinating
power against both the organism in question and the organism used
to produce the antiserum. Partial agglutinin absorption may indicate
a degree of relationship. In order to establish the identity of two
bacterial strains complete cross-agglutination and cross-absorption
should take place between the two organisms and the two antisera.
Attention may be directed here to the phenomenon of "group
agglutination" which results from common agglutinins acting on
bacterial species which are closely allied to each other. An example
is to be found in the colon-paratyphoid-typhoid-dysentery group.
The absence of exact specificity in agglutination reactions is due to the
SEROLOGICAL METHODS viit4,-9
group agglutinins. In dealing with a bacterial division such as that
cited above, group agglutinogens and agglutinins are encountered
in addition to strain-specific agglutinogens and agglutinins.
AGGLUTININ ABSORPTION
Agglutinin becomes attached to bacteria which are mixed with
an homologous antiserum, and can be removed from the fluid by the
removal of the bacteria. This is known as the absorption of agglu-
tinin. Some inagglutinable organisms retain the capacity to link up
with the antibody (agglutinin) and hence, like agglutinated bacteria,
are capable of absorbing agglutinin. The absorption of agglutinins
with agglutinable and inagglutinable strains of bacteria has become
an extremely important serological procedure for determining
identity of bacterial strains and for establishing group relationshijjs.
The scope of this Manual does not permit consideration of all the
factors involved in this reaction nor the description of the several
technical procedures which have worked well in the hands of differ-
ent investigators. It is to be emphasized that highly significant
results in pure culture studies can be obtained by the application
of this method after the user has become thoroughly conversant
with the technic and is familiar with the conditions which influence it.
Two principles govern the application of the test for the absorption
of agglutinin. According to one principle, the ability of individual
strains to absorb agglutinins from type antisera is tested. A given
organism is considered to be identical with the type strain when it
completely absorbs the agglutinins from the type antiserum and
when the type organism completely removes the agglutinins from
the antiserum for the organism being studied. According to the
second principle, the agglutination of organisms by type sera from
which group agglutinins have been previously removed is tested.
Each method has its special advantages. The first method gives
the more precise results and will be described below, as it includes
the chief procedures which would be used in the application of the
second method.
Procedure for Absorption of Agglutinin: At the start, the agglutinating antisera are
prepared according to the method described. The antigens are prepared in the same
manner as those used in the agghitination test. Dense suspensions are used for the
absorption of agglutinins, while the usual type of suspension (0.5 on McF.arland scale)
is employed in the test? with the absorbed sera.
To prepare the absorbing antigen, wash off the bacteria from agar slants or petri
dishes into a small amount of saline. Filter through absorbent cotton if necessary to
obtain a smooth suspension. Absorption is accomplished by adding the concentrated
antigen to serum diluted 1:20 or l:-iO and removing the bacteria by centrifugation
after a period of incubation at room temperature for one half hour or at 37°C for 1 hour.
The minimal absorbing dose of bacteria for a given volume of serum can be determined
by varying the absorbing dose and selecting the smallest one which completely removes
the agglutinins for the absorbing strain. Successive absorptions with 2 or 3 doses are
more efficient in removing antibodies than a single absorption with the same total
amount of bacterial suspension. In identifying unknown strains, doses 2—1 times the
minimal dose are used. After absorption the serum is tested for its ability to aggluti-
nate the homologous strain, and any other strains of Iiactcria used in the study. These
agglutination tests are. set up with dilutions covering the original serum range and
eviending as low as 2.5% of the original titer of the serum. It is important to cover
the entire range of the titer of the scrum. At times pre-zone phenomena occur which
would lead to a false result if only a single dilution were used in the final test for
agglutination.
viii,;-10 MANUAL OF METHODS FOR PURE CULTURE STUDY
PRECIPITATION
The precipitin reaction may be used in the examination and
identification of bacterial extracts and autolysates. The reaction
involves the mixing of antigen and antiserum, with a resultant
precipitate or ring formation if the two are homologous This is one
of the most delicate serological methods. Sera ina,y be obtained
which detect the specific antigen in dilutions as high as 1:100,000.
A serum which will react in dilutions of. 1:10,000 or 1:20,000 is not
uncommon.
PRODUCTION OF PRECIPITINS
Rabbits are suitable animals for the production of precipitins. It
may be necessary to use several rabbits, since some rabbits are
refractory. Precipitins for bacterial proteins may be produced in
the rabbit by using as antigen bacterial suspensions, filtrates, extracts
or autolysates. However, the antigen employed for the m vilro test
must be in solution, clear, and free from antiseptics. Clarification
TABLE 2
PROTOCOLS FOR THE PRECIPITIN TEST
Vol. of dil'd antigen
Dilution of
Tube
0.85%
Antigen
Vol.
remaining after all
remaining
*
aq. NaCl
•
dilns. are made
antigen
ml.
ml.
ml.
1
0.9
0.1 ml.
1.0
0.9
1:10
2
0.9
0.1 ml. of No.
1
1.0
0.5
1:100
m
3
0.5
0.5 ml. of No.
2
1.0
0.5
1:200
c
1.
0.5
0.5 ml. of No.
3
1.0
0.5
1:400
5
0.5
0.5 ml. of No.
4
1.0
0.5
1 :800
<
G
0.5
0.5 ml. of No.
5
1.0
0.5
1:1,600
7
0.5
0.5 ml. of No.
e
1.0
0.5
1 :3,200
8
0.5
0.5 ml. of No.
7
1.0
0.5
1 :6,400
9
0.5
0.5 ml. of No.
8
1.0
0.5
1:12,800
10
0.5
0.5 ml. of No.
9
1.0
0.5
1 :25,600
Tube
Precipitin
serum
Antigen t
0.85%
aq. NaCl
Result
Dilution
Amount
4^
1
2
3
4
5
(i
7
8
9
10
11
ml.
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
1:100
1:200
1 :400
1 :S00
1:1,600
1 :3,200
1:0,400
1:12,800
1 :25,600
ml.
0.1
0.1
0.1
0.1
0.1
0.1
O.l
0.1
0.1
0.1
.s-
u
m
0.25 ml.)
rol tubes f
Undiluted
*Use ordinary size test tubes for these dilutions. Mix the contents of each tulie
thoroughly before transferring to another tube for further dilution.
fUsing dilutions made in A (above).
jOther controls may be added when deemed advisable.
SEROLOGICAL METHODS viii^,-!!
and sterilization of the antigen may be done by filtration (Berkefeld).
High titered sera have been produced by injecting progressively in-
creasing doses of antigen at 3-day intervals. After 5-6 injections,
a test bleeding is made and if the titer is low additional injections are
given. Bleedings are made a week after the last injection. When a
sufficiently high titer has been reached, the rabbit is bled aseptically
from the heart ;^ the blood is allowed to clot; and the clear serum
removed to sterile ampules which arc sealed and labeled. Preserva-
tives should not be added as they tend to interfere with the preci-
pitin test. The serum should be perfectly clear and free from fat and
hemoglobin. It should be stored at about 4°C. If necessary the
serum may be filtered (Berkefeld). The titer of the precipitating
serum is determined by ascertaining the highest dilution of the anti-
gen with which the serum forms a precipitate or ring test in two
hours at 37°C. (optimum temperature). The precipitate consists
very largely of the globulin and lipids of the precipitating serum.
PROCEDURE
1. Progressively doubled serial dilulions of antigen are prepared in saline beginning
with 1:100. (Tabled).
2. Oue-tcntli ml. of the serum is transferred to the bottom of small tubes (5X50 mm.).
3. An equal volume (0.1 ml.) of each dilution of antigen is layered onto the serum.
4. Incubate at 37° for 2 hours and observe at 30 minute intervals for ring formation
(precipitate at juncture of serum and antigen).
5. Shake tubes and incubate overnight at 4°C. The precipitate will settle out and can
be read by gentle shaking of the tubes.
6. Controls of antigen with saline and serum with saline must be included and should
show no precipitate.
COMPLEMENT FIXATION
The complement fixation test is based upon the observation that
the combination formed between an antigen and its specific antibody
has the property of uniting with complement. On the basis of this
general law, complement can be used to detect the union of an antigen
with its homologous or specific antibody. When a mixture of antigen
and antibody is furnished with an exactly sufficient quantity of com-
plement, all the complement is "fixed", or completely utilized in the
reaction and none is left free in the fluid to take part in any other
reaction between an antigen and its antibody which may be added
subsequently for test purposes.
The test for such fixation is performed by placing together antigen,
antibody and complement in suitable proportions, as determined by
previous titrations, and subsequently testing for the disappearance
of complement. If the complement is not fixed, it indicates that the
antigen and antibody do not have the power to unite, or, in other
words, that the antigen and antibody are not specifically related.
On the other hand, the fixation of complement in the mixture indi-
cates that the antigen and antibody have combined, because of their
specific affinities.
In some cases the union of the complement with the antigen-anti-
body complex produces a solution or lysis of the antigen. In other
'See page VI1147-5-6.
via^,-12 MANUAL OF METHODS FOR PURE CULTURE STUDY
cases no demonstrable lysis occurs, although the three substances,
complement, antigen, an 1 antibody, become united. If no obvious
visible phenomenon accompanies the fixation of complement by a
bacterial antigen an 1 antibody, it becomes necessary to add to the
primary mixture of complement, antigen, and antiboiy, an indi-
cator capable of detecting whether the complement is fixed or is still
free.
The only available indicator is an antigen-antibody mixture which
undergoes visible change in the presence of free complement and
shows no change in the presence of fixed complement. Such an
indicator is a mixture of red blood corpuscles and a specific antibody
for these. For convenience, sheep erythrocytes are most frequently
used for this purpose. An antibody, called hemolysin, or anti-sheep-
cell amboceptor, is prepared by immunizing an animal of a different
species (usually rabbit) by means of injections of sheep's washed
red corpuscles. This hemolytic amboceptor is a thermostabile anti-
body which retains its potency over long periods in suitable storage.
When the amboceptor in the serum of the immunized animal (rabbit)
reaches a suitable potency, the animal is bled, and the amboceptor-
containing serum is preserved for subsequent use. Amboceptor is
freed by heat from the complement in the serum of the immunized
animal which produced it. The combination between red cells and
specific amboceptor plus complement causes hemolysis, or laking of
the cells.
A mixture of this antibody and the red corpuscles for which it is
specific is used as an indicator of the degree of fixation of complement
in any other antigen-antibody combination to which complement had
been originally added in the right proportions. If the complement
has been fixed by the formation of the first antigen-antibody com-
bination, none will be left to bring about hemolysis of the red cor-
puscles: they will not be laked. But if complement is still free, the
red blood cells will be hemolyzed when they are added with ambo-
ceptor to the original mixture. The first type of reaction, shown by
absence of hemolysis, is called a positive reaction, indicating the
specific union of the antigen and antibody being tested. The second
type of reaction, hemolysis or laking of the red cells, is called a
negative reaction, indicating that the original antigen-antibody
mixture did not result in a specific combination.
Innumerable practical apjjlications of the complement fixation
test are made, especially in diagnosing various infectious diseases.
Such applications do not fall within the scope of this Manual; but
there are various ways in which essentially the same technic may be
adapted for use in the pure culture study of bacteria. Antibodies,
in general, are quite specific in their action; in other words, they will
ordinarily unite only with the particular antigen inoculated into the
animal in which the antibodies are produced, or else with some other
related antigen. For this reason, the complement fixation test may
be employed in pure culture study by producing antibodies to
the various strains under investigation (by means of animal inocula-
tion) and then determining the probable relationship of these strains
by noting the action between the known antibodies and the sus-
SEROLOGICAL METHODS vnu^AS
pensions (antigens) of the various strains. If complement fixation
occurs (indicating that such a union has taken place) it is assumed
that the bacterium used as antigen in the test must have antigens in
common with the bacterium used to produce the antibody.
The test requires careful attention to detail and the preparation of
a number of accurately standardized serological reagents (antigen,
antibodies or immune serum, complement, red corpuscles — usually
those of a sheep — and antibodies to red corpuscles, known as hemoly-
sin). A brief discussion of the methods of preparing these reagents is
given below, as well as the methods of making the test. If greater
detail is desired, it may be obtained by consulting standard text
books on Immunology and Serology.
Materials Required: The glassware used for the complement fixation
test, as well as for other serological reactions, should be chosen with
care and kept scrupulously clean. Texts dealing with the Wasser-
mann reaction describe suitable test tubes and pipettes A con-
venient tube is one measuring 100X10 mm. The pipettes should be
serological pipettes: 10 ml. and 5 ml. pipettes graduated in 0.1 ml.;
1 ml. pipettes graduated in 0.1 ml.; and 0.2 ml. pipettes graduated in
0.01 ml. Suitable racks are necessary for holding the tubes.
PREPARATION OF REAGENTS FOR BACTERIAL COMPLEMENT
FIXATION REACTION
(a). Antigen. With 0.85% NaCl solution ("saline") wash off the growth from
a 24i-hour agar slant culture of the organism to be used. The amount of saline neces-
sary to make a satisfactory emulsion varies between 5 and 10 ml. depending upon
the heaviness of growth. Shake well.' Filter through cotton. Heat in a water bath
at 60°C. for 1 hour. Phenol, to make a 0.5% solution, may be added. This is not
advisable, however, as it increases the anticomplementary action. This suspension
may be kept for weeks in the cold without much loss of antigenic power.
For comparative work, the density of the emulsion should be standardized by
nephelometric determinations or by a direct count of the number of organisms con-
tained in 1 ml., as it is important to use approximately similar suspensions. All cell
suspensions, including suspensions of bacteria, have the property of inhibiting the
action of complement. This non-specific property is known as their "anticomple-
mentary action." The titration of the anticomplementary action of the antigen is
given in a subsequent paragraph.
There are a number of other methods of preparing bacterial antigens some of which
are better adapted to certain kinds of bacteria than the one given here. Extracts or
solutions of bacteria and organisms obtained from broth or special culture media may
be used. The Committee realizes the difficulties involved in prej^aring a satisfactory
antigen, but feels that a complete treatise on this important subject is outside the scope
of this Manual. The student must consult with instructors and refer to text bot>ks
for more definite suggestions. A good antigen is the most difficult of all the required
reagents to secure.
(b). Immune Serum (Antibody).^ Immunize an animal against the organism to
be studied by repeated injections of the organism. Rabbits are especially suitable for
this purpose. Injections maj' be made into the marginal veins of the ears, iiitra-
peritoneally, or subcutaneously. For the injections, use light susi)ensions of the
organism in 0.85% saline, made by washing ofi' the culture from a 24-liour agar slant.
As little as possible of the medium should be added to the same with the organism.
Washed broth cultures can be employed in cases where it is desired to use an organism
'A preferable procefhire would be the use of a shaking machine for two days;
centrifuge to give a clear extract.
-See page VI1I47-0-6.
viir47-14 MANUAL OF METHODS FOR PURE CULTURE STUDY
which will not grow well on agar slants. Organisms requiring a carbohydrate for growth
can be grown in sugar broth and then washed free of acid and used as antigen. Before
the suspensions are injected, they should be heated for 1 hour at 60°C. On the first
injection, use 0.5 ml. of this suspension. Increase the dose by increments of 0.5 ml.
at intervals of 5 to 7 days. If the organism is not too virulent and the animal has
not lost weight, the last few injections may be made with unheated suspensions of
living organisms. About one week after the last injection, bleed the rabbit from the
ear vein and obtain sufBcient serum for a preliminary test to determine its potency. If
this test shows that the serum contains antibodies in sufficiently high titer, bleed the
rabbit from the heart, or in some manner which will provide as large an amount of
serum as possible. After the collection of the serum, heat it at 5G° C. for 1 hour to
destroy complement, add 0.3% tricresol as a preservative, and store in sealed ampules
or bottles.
It is not possible to lay down an invariable rule as to the total amount of antigen
which should be injected to bring about a sufficient production of antibodies or to
specify exactly the period of time required for the series of injections. Immune sera
obtained after short periods of immunization are usually more specific than those
obtained after long periods of immunization. By trial the amounts to be used in the
final test can be determined; see p. VIII47-I6-I7.
(c). Complement. Guinea pig serum furnishes an active and easily fixable comple-
ment. It is usually advisable to pool the sera from at least 3 guinea pigs weighing 1 to 2
pounds to obtain a sample of complement having average properties. Bleed the
guinea pigs from the heart, removing 5 to 10 ml. of blood from each animal. Allow the
blood to clot. Pipette ofJ the serum and store in a sterile glass container in the refrig-
erator. The most potent complement can be obtained by allowing the clotted blood
to stand overnight in a refrigerator before separating the serum. Complement rarely
retains its potency longer than 3 days. It is essential to titrate it daily. Very fine
work requires titration twice a day, keeping the complement in the refrigerator as much
as possible when not actually being used. Complement preserved by the lyophile proc-
ess or the cryochem process may be used: see Mudd et al. (1936), Ecker and Pillemer
(1938).
(d). Sheep's Red Blood Corpuscles. With a veterinary needle, or a 19-gauge needle
attached to a 50 ml. syringe, withdraw 10 to 50 ml. of Ijlood from the external jugular
vein of a sheep. Place the blood at once in a sterile flask containing glass beads. Shake
for 15 minutes to defibrinate, and filter through gauze or absorbent cotton to remove
the fibrin. Instead of defibrinating in this manner, the blood may be mixed with an
equal volume of 0.85% saline containing 2% sodium citrate. This prevents coagula-
tion and makes it unnecessary to remove the fibrin. Wash the cells 3 times in 0.85%
saline. This is done by centrifuging the cells at about 1500 r. p. m. for 10 to 15 minutes.
Pipette off the supernatant fluid and add as much fresh saline as the amount removed.
Mix well and repeat the process twice. Final centrifugation should be at 1800 r. p. m.
in order to pack the cells. After the final washing, carefully remove the supernatant
saline without disturbing the packed sediment of cells. With this sediment make a
2.5% suspension of the red cells in saline by adding 2.5 ml. of the packed cells to 97.5
ml. of saline. If it is desirable to keep the cells longer than 3 days, 0.1 ml. of a 1-10
dilution of 40% formaldehyde may be added to 8 ml. of blood. This mixture as well as
any other suspension of blood cells should be kept in the refrigerator until used. Before
use, the cells should be washed 3 times in saline (or until supernatant fluid is clear and
colorless). For accurate work it is best to use fresh cells.
For hemolysin production, red cells which have not been treated with formalin
should be used.
(e). Amboceptor (Anti-sheep-red-cell Hemolysin).^ Very strong hemolysin may
be obtained by the following method: Two healthy rabbits are given intravenous
injections of undiluted and unpreserved washed sheep's corpuscles according to the
following schedule: 1st day, 0.5 ml. packed erythrocytes; 3rd day, 1.0 ml.; 5th day,
1.0 ml.; 7th day, 1.0 ml.; 11th day, 1.5 ml.
Eight days after the last injection a trial bleeding is made from the marginal ear
vein. If the serum is found sufficiently potent the rabbits are bled to death or enough
blood is taken from the ear vein as is desired for stock hemolysin. The latter method
should yield all the serum needed, at least if the bleeding is repeated on two or three
successive days, and if both ears are used.
iSee also Beattie, (1934); von Dardnyi, J., (1928); Stafseth (1932); Ulrich and
McArthur (1942); Sawyer and Bourke (1946).
SEROLOGICAL METHODS
viiij7-15
The serum is allowed to separate from the clot, pipetted off, and treated with 0.4%
phenol, 0.3% tricresol, or an equal amount of 50% neutral glycerol. The potency will
be retained for many months, when stored in the refrigerator. Titrations should be
made at intervals, however, not exceeding three or four months.
The titration of hemolytic amboceptor, using a constant amount of complement, is
discussed below. The hemolytic titer (unit) should be at least 0.25 ml. of a 1-1000
dilution.
If the amboceptor does not have such potency as this, it is advisable to continue
the injection of increasing amounts of the sheep cells. For sharp reactions, in which
a minimal amount of complement can be used, and to have an amboceptor which can
be diluted well beyond its agglutinative effect upon red corpuscles, it is advisable to
prepare an amboceptor with a high titer.
TITRATION OF REAGENTS
Before proceeding with the test, the relative strength of each of
the reagents must be known and the amounts necessary for a suc-
cessful test determined. This process is known as titration. A.11 the
reagents, with the exception of the red corpuscles, and the specific
immune serum (antibody), should be titrated before any test is
conducted. Whenever a freshly prepared reagent is used, it must
be titrated. Daily titrations of complement must be made when
tests are done each day.
Titration of Amboceptor {Hemolysin). In this titration, decreasing amounts of am-
boceptor are mixed with a constant amount of complement and added to sheep's red
corpuscles to determine the smallest amount of amboceptor which will cause hemolysis
of the sheep cells. (To prepare a specimen of complement having good average proper-
ties, mix the blood serum obtained from bleeding at least 3 normal guinea pigs.) Dilute
this complement 1 to 10 with saline. It is advisable to keep the flask containing
complement on ice or in ice water, to prevent the deterioration which takes place
appreciably, even at room temperature. Next make up the following series of dilutions
of the anti-sheep amboceptor: 1-100, 1-200, 1-400, 1-1600, 1-3200, 1-6400. Prepare
a 2.5% suspension of washed red corpuscles (sheep) as described above. Set up the
tubes for this titration according to the following protocol. (Table 3)
TABLE 3
TITRATION OF HEMOLYTIC AMBOCEPTOR WITH RESULTS IN A TYPICAL INSTANCE
Amboceptor
d
Complement
Sheep Cells
?:;=«
Hemolysis
1-10
2.5% susp.
153
Dilution
Amount
ClJ
•^5
1
1-100
0.25 ml.
0.25 ml.
0.25 ml.
Complete
2
1-200
0.25 ml.
0.25 ml.
0.25 ml.
aj -
Complete
3
1-400
0.25 ml.
0.25 ml.
0.25 ml.
^C
Complete
4
1-800
0.25 ml.
0.25 ml.
0.25 ml.
■-E
Complete
5
1-1600
0.25 ml.
0.25 ml.
0.25 ml.
a! -I
Complete
6
1-3200
0.25 ml.
0.25 ml.
0.25 ml.
|s
Partial
7
1-100
0.25 ml.
0.25 ml.
C
None
8
0.5 ml.
0.25 ml.
None
Tubes 7 and 8 are controls used to show whether or not either the amboceptor or com-
plement is hemolytic. If either is hemolytic, that reagent should be discarded. Some
specimens of complement are quite hemolytic.
After the mixtures are made, place the rack containing the tubes in the water bath
at 37° C. and incubate them for 15 min., shaking repeatedly. At the end of the period
of incubation, note hemolysis. The tube containing the highest dilution of the ambocep-
tor which produces complete hemolysis of the cells (tube 5 in instance illustrated in
Table 3) denotes the titer of the amboceptor. In this system, 0.25 ml. of that dilution
of the ambocej)tor is called one iniit of the amboceptor. This unit noiv becomes a fixed
standard, as the amboceptor is a stable substance. In subsequent titrations of comple-
VIII47-16 MANUAL OF METHODS FOR PURE CULTURE STUDY
ment and in the final test, use 3 units of amboceptor (hemolysin). Example: If, in the
above titration,' 0.25 ml. of a 1-1600 dilution of the amboceptor produced complete
hemolysis of 0.25 ml. of the 2.5% suspension of sheep cells, 3 units of amboceptor would
be contained in 0.25 ml. of a 1-533 dilution of the stock amboceptor hemolysin serum,
or a 1-265 dilution of amboceptor serum which has been put up with an equal part of
glycerin.
Titration of Complement. Since the activity of complement in the serum of different
guinea pigs varies, and as the activity of any sample of complement changes on stand-
ing, this reagent must be titrated at least once daily. The activity of the sample to
be used is, therefore, titrated in terms of the arbitrarily established unit of hemolytic
amboceptor. In general, there is a reciprocal relationship between complement and
amboceptor. Within certain limits, hemolysis of a given amount of red corpuscles
can be produced by mixtures containing more of complement and less of amboceptor,
and vice versa. The purpose of the following titration is to determine by dilution the
smallest amount of complement which will cause complete hemolysis of 0.25 ml. of
2.5% sheep red cells in the presence of 3 units of amboceptor. After having obtained
and mixed the serum from at least 3 guinea pigs, dilute the complement 1-10 and
proceed as in Table 4.
TABLE 4
TITRATION OF COMPLEMENT WITH RESULTS IN A TYPICAL INSTANCE
Complement;
Tube
gumea pig
Amboceptor
Sheep Cells
Saline
-o
Hemolysis
serum diluted
3 units
2.5% susp.
1-10
a
U
1
0.15 ml.
0.25 ml.
0.25 ml.
0.60 ml.
Complete
2
0.14 ml.
0.25 ml.
0.25 ml.
0.61 ml.
Complete
3
((.13 ml.
0.25 ml.
0.25 ml.
0.62 ml.
Complete
4
0 12 ml.
0.25 ml.
0.25 ml.
0.63 ml.
Complete
5
0 11 ml.
0.25 ml.
0.25 ml.
0.64 ml.
Complete
6
0 10 ml.
0.25 ml.
0.25 ml.
0.65 ml.
Complete
7
0.09 ml.
0.25 ml.
0.25 ml.
0.66 ml.
<u<^
Partial
8
0.08 ml.
0.25 ml.
0.25 ml.
0.67 ml.
■^c?
None
9
0.25 ml.
0.25 ml.
0.75 ml.
J3"
None
10
0.25 ml.
0.25 ml.
0.75 ml.
c
None
11
0.25 ml.
1.00 ml.
None
Saline is added to the tubes in this series to bring the volume of fluid in each tube
up to 1.25 ml., the amount of fluid used in the final test. Tube 9 is the control for the
hemolytic activity of the complement alone; tube 10 serves a similar purpose as an
amboceptor control, and tube 11 is a control for the isotonicity of the saline solution.
At the end of the 15 min. period of incubation, note the last tube showing complete
hemolysis. This gives the smallest amount of the 1-10 dilution of complement which
will cause the hemolysis of 0.25 ml. of a 2.5% suspension of sheep cells in the presence
of 3 units of amboceptor. In the final test, use 1.5 times as much complement as in
this tube. Example: If, as illustrated in the table, the smallest amount of complement
causing hemolysis were 0.1 ml. of the 1-10 dilution, use 0.15 ml. of a 1-10 dilution of
complement in the final test. With diff'erent specimens of complement, it may be
necessary to use a different series of amounts to arrive at the exact titer of the comple-
ment. This method of titration is devised to permit the use of minimal amounts of
complement in the final test.
Titration of the Antigen. After the bacterial antigen has been prepared by emulsify-
ing the culture in saline, it is necessary to find out by titration three of its properties.
These are: (a) the ability of the antigen alone to inhibit the action of comi)lement,
called the anticomplementary action of the antigen, (b) the hemolytic properties of
the antigen, and (c) the capacity of the antigen to fix complement in the presence of
its specific antiserum, called the binding ])ower of the antigen. These properties can
be determined by the procedure outlined in Table 5.
Interpretation of Results. It will probably be found that most bacterial suspensions
are anticomplementary, and some are slightly hemolytic. In the first series of tubes
SEROLOGICAL METHODS
viii,7-17
in this protocol note the first tube in which complete hemolysis occurs. This denotes
the end of the anticomplementary action of the antigen. In tlie final test do not use
more than one-third of the amount of the antigen which was found to be anticomple-
mentary. Example: If 0.5 ml. of the antigen were found to be anticomplementary, do
not use more than 0.17 nd. of this bacterial suspension in the final test. It is to be cm-
TABLE 5
TITR.A.TION OF ANTIGEN — WITH RESULTS IN A TYPICAL INSTANCE
a
.2
Add after 1st
a
incubation
S)
o
3
3
>
c
-o
4J
a
O
>J
a
g
4)
a
-So
8-^
.2
>5
V
R
0)
3
.60
a
b1
o
_a
i 3
O
Ph
H
<
<;
U
Cfi
<
M
K
1
a
ml.
ml.
ml.
ml.
ml.
ml.
i°
r^ o
1
0.5
0.15
0.1
0.25
0.25
Partial
S!^
2
04
0.15
0.2
0.25
0.25
Complete
Si:-
3
0.3
0.15
0.3
0.25
0.25
Complete
•J3 -^
4
0.2
0.15
0.4
0.25
0.25
Complete
<
5
0.1
0.15
0.5
0.25
0.25
Complete
6
0.05
0.15
0.55
u
0.25
0.25
Complete
_w
>i 3
"o 2
7
0.5
0.5
CI)
0.25
c
None
Slj
8
0.1
0.9
c
0.25
None
s ^
d
a
o
n
GJ
c
u
3
O
Fi
9
0.25
0.25
0.15
0.1
JS
0.25
0.25
to
None
o
10
0.1
0.25
0.15
0.25
a
0.25
0.25
<u
None
60
11
0.075
0.25
0.15
0.275
0.25
0.25
nl
None
3
12
0.05
0.25
0,15
0.3
a
0.25
0.25
3
None
-o
13
0.025
0.25
0.15
0.325
a
0.25
0.25
C
Partial
^
14
0.01
0.25
0.15
0.34
0.25
0.25
Complete
'o
15
0.25
0.15
0.35
0.25
0.25
Complete
3
16
0.25
0.75
0.25
None
U
phasized again that the amounts set down in Table 5 are arbitrarily chosen. A different
series of amounts might be found more suitable for different reagents. There should
be no hemolysis in tubes 7 and 8 showing that the antigen alone does not lake the red
cells. In the third series, tubes 9 to 14, absence of hemolysis denotes fixation or binding
of the complement. The last tube in this series showing complete absence of hemolysis
indicates tlie smallest amount of the antigen which will fix complement in the presence
of the constant amount of its antiserum used in tliis titration. The fixing power of
the antigen should be at least 10 times as great as its anticomplementary action.
The titration of the antigen should be made whenever a new bacterial suspension
is prepared, or at intervals of 3 to 4 weeks if old saspensions are kept on hand.
viii-rlS MANUAL OF METHODS FOR PURE CULTURE STUDY
PROCEDURE FOR COMPLEMENT FIXATION TEST
The amounts of reagents used in the final test for complement
fixation are those which have been found to be appropriate from
the preliminary titrations described above. Stated in the form of
a general protocol, the test should be set up as follows :
Tube 1. Amount 1 of immune serum plus complement plus antigen.
Tube 2. Amount 2 of immune serum plus complement plus antigen.
Tube 3. Amount 3 of immune serum plus complement plus antigen.
These tubes constitute the test for complement fixation. A careful
series of controls is necessary, as follows:
Tube 4. (Anticomplementary serum control) : Double the largest
amount of antiserum plus complement.
Tube 5. (Anticomplementary antigen control) : Twice the amount
of antigen used in test plus complement.
Tube 6. (Hemolytic system control) : Complement alone.
Tube 7. (Saline control) : Saline alone.
Add sufiicient saline so that the total volume of fluid, when all in-
gredients are in the tubes, will be 1.25 ml.
TABLE 6
COMPLETE COMPLEMENT FIXATION TEST — WITH RESULTS IN A TYPICAL INSTANCE
c
;-.
o
u
•3
o
CO
a ^
o p
C
ADD AFTER IST
INCUBATION
5
a
'a
o
CO
1
lO
o
O
t^ .
co^
^1
a V
3 s
a
Tube
Antigen
Im-
mune
Serum
Com-
plement
1-10
dil.
Saline
Ambo-
ceptor
3 units
Sheep
Cells
2.5%
susp.
Hemoly-
sis
1
2
3
4
5
6
7
0.25 ml.
0.25 ml.
0.25 ml.
0.5 ml.
0.1 ml.
0.05 ml.
0.01 ml.
0.2 ml.
0.15 ml.
0.15 ml.
0.15 ml.
0.15 ml.
0.15 ml.
0.15 ml.
0.25 ml.
0.3 ml.
0.34 ml.
0.4 ml.
0.1 ml.
0.6 ml.
1.0 ml.
0.25 ml.
0.25 ml.
0.25 ml.
0.25 ml.
0.25 ml.
0.25 ml.
0.25 ml.
0.25 ml.
0.25 ml.
0.25 ml.
0.25 ml.
0.25 ml.
0.25 ml.
None
None
Partial
Complete
Complete
Complete
None
The optimum temperature of incubation of the mixtures for com-
plement fixation varies under different conditions. The test is in
some cases more sensitive when these mixtures are kept in the refrig-
erator at 5-10°C. for 4 hours. For most purposes incubation in a wa-
ter bath at 37°C. for 1 hour, as given in the protocols, is satisfactory.
After this incubation, add to all tubes except tube 7, 3 units of
amboceptor contained in 0.25 ml. of the diluted amboceptor scrum
and to all tubes add 0.25 ml. of 2.5% sheep cells. Shake well, and
incubate them again, for 15-30 minutes, depending upon the rate
of hemolysis in the control tubes.
At the end of the second period of incubation, note the results.
The partial or complete absence of hemolysis in any of the first
3 tubes denotes fixation of complement, indicating union between the
antigen and antiserum. Hemolysis in these tubes indicates lack of
fixation or a negative reaction.
SEROLOGICAL METHODS viii,v-19
Tubes 4, 5 and 6 should show complete hemolysis, indicating that
the serum and antigen are not anticomplementary and that the
iieraolytic system is working properly.
There should be no hemolysis in tube 7, showing that the salt
solution is isotonic with the sheep cells. When several tests are made
at the same time with the same immune serum, the control tubes
4, 6 and 7 need not be repeated. It is necessary, however, to add an
anticomplementary antigen control whenever a different antigen is
used, and another anticomplementary serum control whenever a
different serum is used.
Example: A specimen protocol, giving amounts of the reagents
presumed to have been decided upon after the preliminary titrations
described above, is given in Table 6. (N. B. The amounts stated here
are arhitrary amounts and are not to he applied to an actual test unless
justified by previous titrations.)
A measurement of the titer of an immune serum can be made by
this test. The specificity of the serum can be judged only by testing
it in this manner against other antigens. In interpreting the results
of this test for the purpose of pure culture studies, it may be assumed
that when an organism causes complement fixation in any of the
tubes 1 to 3 with an antiserum produced by the immunization of an
animal against another organism, the two organisms have common
antigens. The results of complement fixation tests, however, must
not be regarded as a basis for exact determinations of identity, as
certain antigens may show positive reactions with the sera of entire
groups. The test is often more indicative of group relationships
than of identities.
TITRATION OF TOXINS, TOXOIDS AND ANTITOXINS
(Flocculation method)
An unknown toxin or toxoid may be titrated with an antitoxin of
known value or an unknown antitoxin with a toxin or toxoid of known
value in vitro. In serial mixtures of the two, there first occurs a
cloudiness followed by a precipitate in some of the tubes, and finally
a definite flocculation in one tube which is taken as the tube contain-
ing the "indicating mixture". The flocculation in this flrst tube
may be followed by flocculation in other tubes about it within a short
time. The ''indicating mixture" however, is alioays the initial tube
to flocculate and must be watched for rather cautiously. From this
"indicating mixture" is calculated the floccidating unit of the toxin,
which has been designated Lf. The Lf may be defined as the amount
of toxin equivalent to 1 unit of antitoxin as established by flocculation.
There is no complete agreement or relationship between the M.L.D.,
Lo, L, and the Lf values of a toxin. The first three of these units
have been designated as "in vivo units" and the fourth as an "m
vitro unit". Flocculation may occur at any temperature up to 55°C.,
above which the reaction becomes irregular and often completely in-
hibited. A temperature of 40° to 50°C. is the most suitable zone.
The time of incubation and of flocculation vary with different toxins
\ iii„-20 MANUAL OF METHODS FOR PURE CULTURE STUDY
and different antitoxins. The tubes must be observed every 15
minutes at these temperatures in order to observe the tube in which
initial flocculation occurs. The reaction is probably due to a com-
bination of the antigenic portion of the toxin and the antitoxin. The
"floe" formed is composed of both toxin and antitoxin in dissociable
union — dissociated by heat or sodium iodide.
There is no difficulty in obtaining flocculation with the first frac-
tion in the concentration of antitoxins but later fractions may not
flocculate. Concentrated toxoids likewise may not flocculate. The
method of titration has been applied mainly to diphtheria and tetanus
toxins, toxoids, and antitoxins. It has been applied to others and
may be applied still further.
PROCEDURE
To a series of ten test tubes (4" X 3^") add serially amounts of anti-
toxin, differing by 0.005 ml. (or 0.001 ml.) from tube to tube. This
may be done by means of a 0.2 ml. pipette graduated in 0.01 ml. (or
0.001 ml.) or, if greater accuracy is desired, by means of a Trevan
micro-syringe. Add to each tube from a 10 ml. pipette 2 ml. of the
toxin or toxoid to be titrated for its Lf value. The tubes are now
shaken, placed in a ivater bath at 40 to 50° C. and observed every 15
minutes for the first appearance of flocculation.
TABLE 7
TITRATION OF A DIPHTHERIA TOXIN WITH RESULTS IN A TYPICAL INSTANCE
(Flocculation method)
Time in
minutes
Tube
Antotoxin
Toxin
Indicating Mixture
No.
No. 1347
No. 16304
15
30
45
60
1
0.020 ml.
2.0 ml.
2
0.025 ml.
2.0 ml.
3
0.030 ml.
2.0 ml.
P
P
P
4
0.035 ml.
2.0 ml.
P
P
P
5
0.040 ml.
2.0 ml.
C
P
P
P
6
0.045 ml.
2.0 ml.
C
P
P
P
7
0.050 ml.
2.0 ml.
C
P
P
F
8
0.055 ml.
2.0 ml.
C
P
F
F
8th tube in 45 minutes.
9
0.060 ml.
2.0 ml.
C
P
P
F
10
0.005 ml.
2.0 ml.
C
P
P
P
C = Cloudiness; P = Precipitate; F = Flocculation.
Temperature of the water bath 50°C.
Depth of the tubes in water — water % distance to top of the liquid in the tubes.
Size of tubes 4"X3^" (inside dimensions).
Antitoxin used in above titration contained 425 units per ml. (or 1 unit is contained
in 0.00235.)
In Table 7 is given a protocol for the titration of an unknown
diphtheria toxin with the results obtained. For greater accuracy the
toxin would be retitrated using 0.050 to 0.60 ml. of antitoxin with
differences of 0.001 ml. between tubes.
Calculation of the typical instance given in Table 7 is as follows:
SEROLOGICAL METHODS VI1I47-2I
2 ml. of toxin flocculated with 0.055 ml. of antitoxin ("indicating
mixture")
0.00235 of the antitoxin contains 1 unit
Since the Lf = the amount of toxin that will flocculate with 1
unit of antitoxin
Therefore, 2:0.055 = x:0.00235
X = 0.080 which is the Lf of toxin No. 16304
(or 11.6 flocculating units per cc.)
P'or details on the titration of toxins and antitoxins in animals the
reader is especially referred to the recent publication of Gershenfeld
(1939) and to Wadsworth's book (1947).
REFERENCES
Beattie, M.\rgaret. 1934. A new method for the production of antisheep
hemolysis. J. Lab. and CHn. Med., 19, 666-667.
EcKER, Enrique E. and Pillemer, L. 1938. An inexpensive method for the de-
hydration and preservation of complement and other biological material.
Am. J. Pub. Health, 28, 1231-1232.
Gershenfeld, Louis. 1939. Biological Products. Romaine Pierson Publishers,
Inc , New York.
Kolmer, John A. and Boerner, Fred. 1938. Approved Laboratory Technic.
Fourth edition. D. Appleton-Century Co., New York.
Landsteiner, K. 1946. The Specificity of Serological Reactions. Harvard Uni-
versity Press, Cambridge, Massachusetts.
McFarland, John. 1907. The Nephelometer: An instrument for estimating the
numbers of bacteria in suspensions used for calculating the opsonic index
and for vaccines. J. Am. Med. Assoc, 49, 1176-1178.
Marrack, J. R. 1938. Chemistry of Antigens and Antibodies. Medical Research
Council, London.
MuDD, Stuart; Flosdorf, Earl W.; Eagle, Harry; Stokes, Joseph; and McGuin-
NESS, Aims C. 1936. The preservation and concentration of human serums
for clinical use. J. Am. Med. Assn., 107, 956-959.
Ramon, G. 1922. Flocculation dans un melange neutre de toxine antitoxine diph-
therique. Compt. Rend. Soc. Biol., 86, 661-663
Sawyer, H. P. and Bourke, A. R. 1946. Antisheep Amboceptor Production with
Elimination of Rabbit Shock. J. Lab. and Clin. Med., 31, 714-716.
Stafseth, H. J. 1932. On the preparation of hemolytic and precipitating sera.
Science 76, 444.
Trevan, J. S. 1922. An apparatus for the measurement of small quantities of fluid.
Lancet I, 786.
Ulrich, Catherine A. and McArthur, Francis X. 1942. An improved method
for the production of antisheep hemolysin. Am. J. Clin. Path. (Clin. Sect.)
6, 84-85.
von Daranyi, J. 1928. Methods of obtaining and preserving antibodies. J.
Immunol., 15, 521-526.
Wadsworth, Augustus B. 1947. Standard Methods. Williams and Wilkins Co.,
Baltimore.
Zinsser, Hans; Enders, John F.; and Fothergill, Le Roy D. 1939. Immunity.
Principles and Application in Medicine and Public He;,lth. The Macmillan
Company, New York.
LEAFLET IX
Manual of Methods for
Pure Culture Study of Bacteria
THE MEASUREMENT OF pH,
TITRATABLE ACIDITY, AND
OXIDATION-REDUCTION POTENTIALS
loth EDITION
Pure Culture Study of Bacteria, Vol. 16, No. 3-4
September, 1948
Completely Revised by Barnett Cohen
LEAFLET IX
THE MEASUREMENT OF pH, TITRATABLE ACIDITY,
AND OXIDATION-REDUCTION POTENTIALS^
The Measurement of pH
Originally, pH was defined as the logarithm of the reciprocal of
the hydrogen ion concentration. However, certain assumptions
regarding indeterminate factors enter the theoretical treatment of
any method of measuring this quantity. It is now recognized that
the pH scale is standardized on a basis that is arbitrary with respect
to a small and indeterminate uncertainty, although any pH number
closely approximates the logarithm of the reciprocal of the corre-
sponding hydrogen ion activity. The activity of any substance is
virtually the product of that substance's molar concentration and a
factor, called the activity coefficient. This factor expresses the
departure from that behavior which would obtain were there no van
der Waals and Coulomb (attraction and repulsion) forces operating.
The common methods for the measurement of pH are of two types :
(1) potentiometric, and (2) colorimetric. The theoretical and prac-
tical aspects of the subject are treated extensively in the monograph
by Clark (1928).
POTENTIOMETRIC METHODS
The several potentiometric methods to be cited depend upon the
fact that the pH of a solution suitably incorporated in a so-called
half -cell is proportional to the electric potential difference established
between this half-cell and some reference half -cell used as a standard.
The Hydrogen electrode method. This is regarded as the basic
experimental method whereby the various other methods are stand-
ardized. It consists in the measurement of the potential difference
(emf) established under conditions of maximum work between the
"hydrogen half-cell", or "hydrogen electrode", and a calomel or
other half-cell which is employed as a working standard.
The hydrogen half-cell consists of a suitable vessel provided with (a) a platinum
foil electrode, coated with platinum-black, which is immersed or intermittently dipped
in the solution to be measured, and (6) an inlet and outlet for oxygen-free hydrogen to
saturate both solution and electrode at atmospheric pressure.
A convenient reference half-cell is the "saturated calomel electrode" which consists
of a vessel containing a layer of purified mercury covered with a paste of calomel
(HgoCy, mercury, and saturated KCl solution; the calomel paste is layered with crys-
tals of KCl, and the rest of the vessel is filled with saturated KCl solution which has
been saturated with calomel. A platinum wire provides the electrical lead to the
mercury of the calomel cell, and a siphon containing saturated KCl solution provides
liquid junction with the solution to be measured in the hydrogen half-cell.
In the normal hydrogen half-cell, which provides the standard of potential for all
measurements of potential in electrochemistry, the hydrogen partial pressure is one
iThis presentation is confined to the brief description of general procedures that may
be applied in the bacteriological laboratory. For theoretical discussions and the
elaboration of detail, the reader should consult the texts, monographs, and original
references cited.
1X48-2
THE MEASUREMENT OF pH 1x48-3
normal atmosphere and the hydrogen ions are at unit activity. The potential differ-
ence between electrode and solution in the normal hydrogen half-cell is assumed to be
zero at all temperatures.
In standardizing the pH scale by means of measurements with a cell composed of a
hydrogen half-cell and a saturated KCl calomel half-cell, it is customary to ignore
the small and indeterminate liquid junction potential between the saturated solution
of KCl and the solution in the hydrogen half-cell.
The combination of the two half-cells to make an electric cell is indicated schema-
tically as follows:
(Pt)H2; H+ in solution X I Sat. KCl I Sat. KCl; HgaClj; Hg (Pt)
Hydrogen KCl Sat. calomel (reference)
electrode bridge electrode
For a pH determination, purified hydrogen is bubbled through the
test solution to saturate it and the platinized platinum electrode until
equilibrium is attained as indicated by constancy of the emf deter-
mined potentiometrically between the metal terminals of the hydrogen
and the calomel half-cells. The observed emf, in volts^, is converted
to pH by the following equation, where T is the absolute temperature.
Observed emf - Emf of calomel cell Eh
pH = = (1)
0.000,198,322 T 0.000,198,322 T
For this equation to be applicable, the temperature must be constant. For precis-
measurements, a correction must be made for any departure of the hydrogen partial
pressure from one atmosphere. The correction seldom exceeds 0.001 volt (0.017 unit
of pH) for the ordinary ranges of barometric pressure and vapor pressures of solutions.
As indicated by equation 2,
-AEh
= 0.000,198,322 T (2>
ApH
the slope of the straight line relating potential to pH is a constant
dependent on the absolute temperature. For example, at 25°, the
potential of the hydrogen electrode becomes more negative by 0.0591
volt- for each unit increase in pH. Values of this constant at certain
temperatures are shown as constant "A" on p. iX48-4.
Standardization of the saturated calomel half-cell. For ordinary
measurements, the values at different temperatures of the saturated
calomel half-cell, referred to the normal hydrogen half-cell, are as
follows :
°c
Ecal
°c
Ecal.
20
0.250 V.
35
0.238 V.
25
0.246
38
0.236
30
0.242
40
0 234
The potential of this half-cell after continued use may change as a result of dilution
-The electrical units employed in this leaflet are based on the "international" system
in which, according to the National Bureau of Standards, 1 international volt (U. S.)
equals 1.00033 absolute volts. The Bureau has announced that, as of January 1, 1948,
absolute electrical units will supersede international units.
However, the efifect of this new convention for potentiometry is to introduce changes
which may be regarded as negligibly small in ordinary measurements of pH and oxida-
tion-reduction potentials. For example, in equation 2, -AEh 'ApH equals 0.05912
international volt and 0.05914 absolute volt, at 25°C (298.1° absolute).
1X48-4 MANUAL OF METHODS FOR PURE CULTURE STUDY
and contamination, and it is advisable to check its value regularly as a routine pro-
cedure.
The precise standardization of the calomel half-cell is discussed in detail by Clark
(1928). It consists in measuring the potential of this half-cell against the hydrogen
electrode in a solution of known hydrogen ion activity or against other carefully con-
structed half-cells of reproducible, known potential. For measurements of ordinary
precision, the quinhydrone electrode (see below) in 0.1 N HCl can serve for standardi-
zation of the calomel half-cell.
The quinhydrone electrode. Ignoring refinements and minor
details, we may state that the potential of a noble metal electrode in
an acid or neutral solution saturated with quinhydrone varies linearly
with the pH of the solution; and this so-called quinhydrone electrode
may, therefore, be used to measure the pH of such solutions.
The linear relationship of potential to pH holds only for acid and
neutral solutions to about pH 8. In more alkaline solutions two effects
disturb this regularity. One is the ionization of the reductant, and
the other is deterioration of the components of the system.
The quinhydrone electrode within its range of usefulness, may often
be employed in cases where the hydrogen electrode cannot be applied.
It comes to equilibrium rapidly, and its manipulation is simple and
convenient. Consult Clark (1928) for fuller details.
Its utilization may be illustrated in the standardization of the
saturated calomel half-cell. The potential, Ecai, of this half-cell is to
be determined relative to that of a standard solution of fixed pH and
saturated with quinhydrone, e.g., 0.1 M HCl, the pH of which is
1.082 at 38°. This is done with purified quinhydrone and accurately
prepared HCl solution as follows. Place about 5 ml. of the standard
HCl solution in a suitable electrode vessel. Add 50 to 100 mg. of
quinhydrone crystals to saturate the solution; some quinhydrone
in the solid phase must be present. Insert a clean platinum or gold
electrode preferably in contact with the solid phase at the bottom of
the vessel. Then join this half-cell with the calomel half -cell by
means of a siphon containing saturated KCl solution, bring the sys-
tem to constant temperature, and measure the potential which
should reach a constant value in a few minutes.
The observed potential, Eobs, is related to the potential of the
calomel cell, Ecai, as follows:
Ecai = Eq-Eobs-A.pH (3)
Eq and A are constants at any given temperature, and have the following values:
°C Eq A
20 0.7029 0.0581
25 0.6992 0.0591
30 0.6955 0.0601
35 0.6918 0.0611
38 0.6896 0.0617
For example, at 38°, with a quinhydrone electrode in 0.1 M HCl,
Ecal = 0.6896-Eobs-(0.0617X1.082) (4)
from which the value of Ecai- can be calculated after substitution of
the experimentally determined value of Eobs-
THE MEASUREMENT OF pH
1X48-5
To determine the pH of an unknown solution, proceed as above ex-
cept that the unknown solution is substituted for the standard HCl.
The "glass electrode". Under suitable conditions, a properly pre-
pared thin membrane of special glass separating two solutions of
different pH exhibits an electric potential that is proportional to the
difference in pH of the solutions. Based on this property, a device
called the glass electrode is now widely used for the comparative
determination of pH.
The glass probably most generally employed is that known as Corning No. 015;
Beckman type E glass has been advocated for alkaline solutions (pH 9 to 14) because
of its low sodium error as compared with that of glass 015.
One of the common forms of the glass electrode consists of a tube
of the glass terminating in a thin-walled bulb which contains an
electrode of definite potential in a solution of fixed pH. A combina-
tion of electrode and buffer solution frequently employed is a plati-
num wire, silver-plated and then coated with AgCl, in a half-cell
containing 0.1 M HCl. For the construction, operation, and theory
of the glass electrode, consult Dole (19-il).
The carefully rinsed bulb of the electrode, after seasoning in water
or buffer solution, is immersed in the solution to be tested and coupled
through a saturated KCl liquid junction with the saturated calomel
half-cell as indicated schematically below,
Ag; AgCl; HCl (0.1 M) I Glass membrane I Solution X 1 KCl (sat.); HgzCla; Hg
all parts of the cell being maintained at a uniform temperature.
The potential difference between the terminals of this cell can be
related to the pH of solution X if the glass electrode has been stand-
ardized in buffer solutions of known pH.
Standardization of the glass electrode. The potential of a properly
functioning glass electrode should vary linearly with pH, from about
pH 1 to 9, in solutions of low salt content (up to 0.1 M). For this
range, therefore, the electrode requires standardization in buffer
solution at one point of pH, but preferably at two, within this linear
range. Standard buffer solutions convenient for this purpose may
be selected from Tables 1 and 3.
TABLE 1
SOME STANDARD BUFFER SOLUTIOX9
Solution
pH
25"
0.1 MHCl
0.01 M HCl, 0.09 M KCl
0.05 M Acid potassium phthalate
0.025 M KH2PO4. 0.025 M NaaHPOi'gHoO.
0.05 M Na2B4O7-10H..O
1.085
2.075
4.000
6.855
9.180
1.082
2.075
4.015
6.835
9.070
Such standardization should be performed at least daily; preferably,
it should be done immediately before a measurement. As occasion
requires, a series of buffer solutions of known pH should be used to
establish more carefully the linearity of response of the electrode.
VLa-6 MANUAL OF METHODS FOR PURE CULTURE STUDY
In solutions more alkaline than about pH 9, the 015 glass electrode
responds also to cations other than H ions, the potential being in-
fluenced by the activity and kind of such cations. Sodium and
lithium ions produce the most marked effects, potassium and bivalent
cations smaller effects. When working under these conditions, it is
advisable to standardize the electrode with known buffer solutions of
about the same composition and of pH closely above and below the
pH of the sample being tested.
The standardization for linearity of response from pH 1 to 9 is a
necessary check on the operation of the glass electrode, since its re-
sults are comparative, not absolute. The slope, -AEh/ApH,
should be not merely constant at any temperature but also equal or
closely equal to 0.000,198,322 T (the values for this constant are
shown under A on p. 4). Obviously, a "pH-meter" with its pH
scale adjusted to the theoretical slope for a given temperature cannot
give correct readings at all points from pH 1 to 9 if its glass electrode
follows a significantly different slope at the same temperature. For
a brief discussion of the effects of temperature, see Clark (1948).
Cleaning of the glass surface, by immersion in a hot mixture of concentrated nitric
and sulfuric acids followed by soaking in water, may restore a sluggish or erratic
electrode to normal functioning. A somewhat drastic procedure that may be effective
is to dip the glass electrode for a second or two in dilute HF or in a 20% solution of
ammonium bifluoride and then to wash it thoroughly in water. If the electrode still
behaves erratically, it should be discarded. For such an emergency, it is highly
advisable to have available a reserve electrode. This may obviate any mistaken
tendency to carry on with an electrode of doubtful reliability.
The instructions accompanying the various glass-electrode "pH-meters" now on
the market are usually sufficient to aid the user in tracing out sources of trouble and
error in operation. A major source of trouble is electrical leakage due to accumulation
of films of moisture at critical parts of the circuit; and perhaps the most frequent sites
of such accumulation are the electrode support and lead, both of which are apt to be
spattered with water or salt solution during careless manipulation.
The glass electrodes now available are fairly rugged and easily adaptable to use
under a variety of conditions and on difiFerent types of biological material (e.g., liquid
and "solid" culture media). Measurements with an accuracy of 0.05 pH may be
made rapidly in poorly buffered, colored, or turbid solutions, and in blood or serum.
The monograph by Dole (1941) discusses many of its uses.
THE COLORIMETRIC METHOD
The colorimetric method of measuring pH makes use of acid-base
indicators, which, within certain limits, vary in color with the pH of
the solution. Such indicators are compounds capable of existing in
solution as conjugate proton (H-ion) donor and proton acceptor, with
one of the conjugate pair differing in color from the other. The re-
lation of these two forms to pH is defined by the equation
[proton acceptor]
pH=pK'+log (5)
[proton donor]
in which brackets represent concentrations, and pK' (= - log K')
is called the apparent ionization exponent of the indicator's proton
donor-acceptor system. Simple calculations, using, for example,
0.8, 0.5 and 0.3 as values for the ratio [proton acceptor]/ [proton donorl
at each of the pK' values 3, 6, and 9, will show that indicators with
different pK' values cover different ranges of pH. (See Fig. 1). For
THE MEASUREMENT OF pH
1X48-7
PERCENT DISSOCIATION
Fig. 1. — Ionization curves of some sulfonphthalein indicators, illustrating the
general relationships among the acid-base indicators and the applications of equation 5.
Note: In some cases, the positions of the curves on the pH ordinate are approximate.
Table 2 should be consulted for accurate values of pK'.
IX4S-8
MANUAL OF METHODS FOR PURE CULTURE STUDY
a full discussion of the properties and uses of pH indicators, see Clark
(1928), and Kolthoff and Rosenblum (1937).
Within a short range on the pH scale on each side of the pK' value,
every color gradation of the indicator corresponds to a definite pH
number; this zone may be called the sensitive range of the indicator.
Throughout its sensitive range, an indicator can be used to deter-
mine the pH of a solution by comparing its color in the solution with
that produced in standard solutions representing known pH numbers.
The indicators. A selection of indicators is presented in Table
2. All but three of the compounds are sulfonphthaleins which are
particularly useful in bacteriological work because of their high tinc-
torial power, low or moderate salt and protein errors, and relative
resistance to bacterial action. Table 2 gives the pK' values of the
indicators and their sensitive ranges. The last column, and footnote
b of the table give specifications for the preparation of stock solutions
of the mono-sodium salt of each of the sulfonphthaleins.
TABLE 2
ACID-BASE INDICATORS*
Name
pK'
pH-range and Colors
Recom-
mended
Cone. %
(a)
Ml. of
0.01 M NaOH
per 0.1 gm.
(b)
Thymol blue (acid range)
Methyl orange (c)
Bromphenol blue
Bromcresol green
Methyl red
1.7
3.5
4.0
4.7
5.0
6.0
6.2
7.1
7.8
8.3
8.9
9.7
Red 1.2-2.8 yellow
Red 3.1-4.4 yellow
Yellow 3.1-4.7 blue
Yellow 3.8-5.4 blue
Red 4.2-6.3 yellow
Yellow 5.1-6.7 red
Yellow 5.4-7.0 purple
Yellow 6.1-7.7 blue
Yellow 6.9-8.5 red
Yellow 7.4-9.0 red
Yellow 8.0-9.6 blue
Colorless 8.3-10.0 red
0.04
0.05
0.04
0.04
0.02
0.04
0.04
0.04
0.02
0.02
0.04
0.10
21.5
w
14.9
14.3
(e)
Chlorophenol red
Bromcresol purple
Bromthymol blue
Phenol red
23.6
18.5
16.0
28.2
Cresol red
26.2
Thymol blue (alk. range) .
Phenolphthalein
21.5
(f)
*See Clark (1948), and Kolthofif and Rosenblum (1937).
(a) Stock solutions in 95% ethanol for the indicator acids, or in water for the indica-
tor salts, unless otherwise specified.
(b) Grind 100 mg. of the pure indicator acid with the amount of NaOH specified,
and when solution is complete dilute with water to a volume that will yield the con-
centration recommended in column 4.
(c) Do not use with phthalate buffers.
(d) Dissolve 50 mg. in 100 ml. water.
(e) Dissolve 20 mg. in 60 ml. 95% ethanol, and add 40 ml. water.
(f) Dissolve 100 mg. in 65 ml. 95% ethanol, and add 35 ml. water.
It will be noted from footnote a that ethanolic solutions are
ordinarily satisfactory. For precise work, however, aqueous solu-
tions of the indicator salts are preferable to the alcoholic solutions of
the free acids. To obviate the labor of preparing the neutralized
solutions, some makers now offer the soluble salts of the sulfonph-
thaleins. They are ammonium, sodium, or possibly other salts of
these compounds. In ordinary use, the indicator salts contribute
negligibly to the total ions present in a test solution, and the nature
of the cation may be of no consequence. However, in some studies
THE MEASUREMENT OF pH 1x43-9
of bacterial nutrition, the kind of cation and even the small amounts
thus added may be of significance. In such cases, it is advisable to
learn from the maker what cations (Na, NH4, etc.) are present in the
indicator salt in order to make due allowance for their possible effects.
The colorimetric method of pH determination depends on matching
the color of a suitable indicator in the unknown solution with that of
the same indicator in a standard. The standards can be set up in
two different ways: by means of buffer standards or by means of
"drop-ratios". These will be considered in detail presently. In
brief outline, the colorimetric method includes these major steps:
1. Selection of the appropriate indicator.
2. Preparation of color standards.
3. Color comparison for pH determination.
Later paragraphs will outline essential specifications that must
be observed in each of these steps in order to assure reliable results.
Selection of the appropriate indicator. Test successive small por-
tions (1 ml.) of the unknown with a drop of bromthymol blue (BTB).
If the color produced is orange or red then the unknown is probably
in the range of pH covered by thymol blue (acid range). If the BTB
color is yellow, repeat the test with the indicators of successively
lower pK' (see Table 2) until that indicator is found which gives a
color within its sensitive or useful range. If the BTB color is blue,
proceed in like manner with indicators of higher pK' until the ap-
propriate indicator is found. Of course, if the unknown is more acid
than pH 1 or more alkaline than pH 10, none of the indicators listed
in Table 2 will serve.
If the unknown solution is unbuffered (e.g., water or saline) or
very weakly buffered, the buffering effect of the added indicator may
prevail and significantly change the pH of the unknown. In such
cases, special methods are required (see Clark, 1928).
It is plain that a rough idea can be obtained as to the pH value
of a sufficiently buffered solution by simply finding which indicators
give their acid color in it and which give their alkaline color. Indeed,
the intelligent employment of indicators with overlapping pH ranges
can be made to define the upper and lower limits of a relatively
narrow zone of pH within which lies the pH of the solution under
study (Small, 1946). Accuracy, however, can be obtained only by
actual comparison with the colors produced by the indicators in
solutions (buffers) whose pH values are known, or produced by ap-
plication of equation 5 (drop-ratio method, p. 12-14).
Buffer solutions and color standards. A considerable variety of
buffer solutions have been proposed; and many of them are discussed
and described by Clark (1928). Thecompositionsof the series of buffer
standards proposed by Clark and Lubs (1917) are given in Table 3.
Preparation of the stock solutions is described by Clark (1928).
After finding the appropriate indicator, prepare or select a series
of properly graded standard buffer solutions sufficient in number to
bracket the estimated pH of the unknown solution as determined in
the preliminary trials. If, for example, the indicator selected is
bromcresol green and the estimated pH of the unknown is near 6.0,
then not more than five standards, namely buffers of pH 5.6, 5.8,
1X48-10
MANUAL OF METHODS FOR PURE CULTURE STUDY
TABLE 3
COMPOSITION OF MIXTURES GIVING pH VALUES AT 20°C AT INTERVALS OF 0.2
From Clark {1928) p. 200-1.
KCl, HCl mixtures
pH
M/5 KCl
M/5 HCl
Dilute to
1.2
50 ml.
64.5 ml.
200 ml.
1.4
50 ml.
41.5 ml.
200 ml.
1.6
50 ml.
26.3 ml.
200 ml.
1.8
50 ml.
16.6 ml.
200 ml.
2.0
50 ml.
10.6 ml.
200 ml.
2.2
50 ml.
6.7 ml.
200 ml.
Phthalate, HCl mixtures
pH
M/5 KH Phthalate
M/5 HCl
Dilute to
2.2
50 ml.
46.70 ml.
200 ml.
2.4
50 ml.
39.50 ml.
200 ml.
2.6
50 ml.
32.95 ml.
200 ml.
2.8
50 ml.
26.42 ml.
200 ml.
3.0
50 ml.
20.32 ml.
200 ml.
3.2
50 ml.
14.70 ml.
200 ml.
3.4
50 ml.
9.90 ml.
200 ml.
3.6
50 ml.
5.97 ml.
200 ml.
3.8
50 ml.
2.63 ml.
200 ml.
Phthalate, NaOH mixtures
pH
M/5 KH Phthalate
M/5 NaOH
Dilute to
4.0
50 ml.
0.40 ml.
200 ml.
4.2
50 ml.
3.70 ml.
200 ml.
4.4
50 ml.
7.50 ml.
200 ml.
4.6
50 ml.
12.15 ml.
200 ml.
4.8
50 ml.
17.70 ml.
200 ml.
5.0
50 ml.
23.85 ml.
200 ml.
5.2
50 ml.
29.95 ml.
200 ml.
5.4
50 ml.
35.45 ml.
200 ml.
5.6
50 ml.
39.85 ml.
200 ml.
5.8
50 ml.
43.00 ml.
200 ml.
6.0
50 ml.
45.54 ml.
200 ml.
6.2
50 ml.
47.00 ml.
200 ml.
KH,P04,
NaOH mixtures
pH
M/5 KH2PO4
M/5 NaOH
Dilute to
5.8
50 ml.
3.72 ml.
200 ml.
6.0
50 ml.
5.70 ml.
200 ml.
6.2
50 ml.
8.60 ml.
200 ml.
6.4
50 ml.
12.60 ml.
200 ml.
6.6
50 ml.
17.80 ml.
200 ml.
6.8
50 ml.
23.65 ml.
200 ml.
7.0
50 ml.
29.63 ml.
200 ml.
7.2
50 ml.
35.00 ml.
200 ml.
7.4
50 ml.
39.50 ml.
200 ml.
7.6
50 ml.
42.80 ml.
200 ml.
7.8
50 ml.
45.20 ml.
200 ml.
8.0
50 ml.
46.80 ml.
200 ml.
THE MEASUREMENT OF pH
1X48-11
TABLE S—{Conti7iued)
COMPOSITION OF MIXTURES GIVING PH VALUES AT 20°C AT INTERVALS OF 0.2.
From Clark {1928) p. 200-1
Boric acid, KCI, NaOH mixtures
pH
M/5 H3BO3 M/5 KCI
M/5 NaOH
Dilute to
7.8
50 ml.
2.61 ml.
200 ml.
8.0
50 ml.
3.97 ml.
200 ml.
8.2
50 ml.
5.90 ml.
200 ml.
8.4
50 ml.
8.50 ml.
200 ml.
8.6
50 ml.
12.00 ml.
200 ml.
8.8
50 ml.
16.30 ml.
200 ml.
9.0
50 ml.
21.30 ml.
200 ml.
9.2
50 ml.
26.70 ml.
200 ml.
9.4
50 ml.
32.00 ml.
200 ml.
9.6
50 ml.
36.85 ml.
200 ml.
9.8
50 ml.
40.80 ml.
200 ml.
10.0
50 ml.
43.90 ml.
200 ml.
Notes. Overlapping members of the above series should be checked for consis-
tency, i.e., phthalate "5.8" to "6.2" should match phosphates of the same pH numbers
when tested with bromcresol purple; likewise for phosphate and borate "7.8" and
"8.0" when tested with cresol red.
According to more recent assumptions used in standardization, the pH values given
in the above table are too low by about 0.03 to 0.04 unit of pH.
6.0, 6.2 and 6.4, should suffice to safely bracket the actual pH of the
unknown.
In preparing for the actual measurement, the unknown and the color standards
should be contained in clear glass tubes selected for uniform bore, wall thickness, and
inherent color. It is essential that the total concentration of indicator in the unknown
be exactly the same as that in each of the color standards. This is best accomplished
by accurately measuring, with a pipet, equal amounts of indicator {e.g., 0.50 ml.) into
equal amounts {e.g., 10.0 ml.) of each of the selected standard buffer solutions. The
indicator may be satisfactorily measured in drops provided the dropper tip is properly
shaped (not too blunt), and the dropper is held vertically during the measurement.
The use of excessive amounts of indicator may introduce difficulties; the minimum
quantity necessary to produce recognizable coloration is desirable from the theoretical
standpoint. It is essential, of course, that the indicator be uniformly distributed
throughout the solutions to which it is added.
Prepared buffer standards can be obtained from supply houses, either as solutions
or as powders or tablets to be dissolved as needed. They may also be obtained in
sealed glass tubes containing the indicator. Such commercial color standards are
convenient and satisfactory. They presuppose the use of comparable concentrations
of indicator in the solution under test, and they must be used with the understanding
that they are not permanent and may need to be checked or renewed at least once a
year. All such indicator standards should be kept in the dark when not in use.
Color cotyiparison. This procedure, commonly miscalled colorim-
etry, requires intelligent application to yield reliable results. The
subject is adequately discussed by Clark (1928, 1948). Accurate
color comparison of a standard solution with an unknown requires
uniformity of the following conditions: the optical path (i.e., dis-
tance through the solutions traversed by the light), transparency,
wall thickness and color of the containers, concentration of indicator
1X43-12 MANUAL OF METHODS FOR PURE CULTURE STUDY
in each of the solutions, and radiant power incident upon the systems
under comparison. Also, any inherent color in the unknown must
be compensated by an equivalent amount in the optical path through
the standard. These conditions are met by selecting clear, un-
scratched tubes of uniform bore, glass thickness, and color, by having
the same concentrations of indicator in the unknown and the stand-
ard, by dispersing the color uniformly in the solutions, and by employ-
ing proper illumination.
The color comparison is conveniently made in a comparator block of the type
described by Clark (1928, 1948). Various forms of this are obtainable from supply
houses. Two pairs of tubes are arranged in the comparator as follows: 1, a tube
containing buffer standard plus indicator behind which is placed a tube containing
the unknown solution to compensate for inherent color, and 2, a tube containing the
unknown solution plus indicator backed by a tube containing distilled water. The
two pairs of tubes are viewed against a uniform source of white light so placed that
the beams incident upon the two systems are of the same radiant power. The color
standards are successively compared with the unknown until a match is obtained,
thereby establishing the pH of the unknown. If the color of the unknown falls be-
tween those of two adjacent standards an interpolated pH number may be estimated.
Systems of fixed or "permanent" color standards are also available. These
standards consist of colored glasses or other transparent material. Since the spectral
absorptions of such standards would hardly be expected to be exactly the same as
those of the indicators that they are supposed to match, the applicability and accuracy
of these fixed standards must be determined in each case before they are placed in
service. Acceptable sets of such standards can be of great convenience in the bac-
teriological laboratory, especially for approximate determinations.
The drop-ratio standards of Gillespie. If commercial color stand-
ards are not available and there are no facilities for making standard
buffer solutions, color standards may be prepared by the drop-ratio
method as refined by Gillespie (1920). The method of preparing the
standards consists in setting up pairs of tubes, containing stepwise
proportions, of the full alkaline color and the full acid color of an
indicator in such a manner that the resulting color of each pair, when
properly viewed, represents a definite pH within the sensitive range
of that indicator.
A general notion of the arrangement and composition of the drop-
ratio color standards may be obtained from inspection of Table 4.
The preparation of the standards is explained in the next two para-
graphs and in Table 5.
Although the alcoholic solutions of the indicator acids mentioned
in Table 2 may be used, Gillespie recommends for accurate work the
use of aqueous solutions of the indicator salts (the preparation of
which is specified in Table 2), except in the case of methyl red.
Table 5, lower half, gives specifications for the recommended con-
centrations of seven of the indicator stock solutions. The exact
concentration of the indicator solutions is not very significant in
much bacteriological work.
Select 18 test tubes of approximately the same bore (between 12
and 15 mm.). They can be selected by adding 10.0 ml. of water to a
large number of test tubes and choosing a lot in which the columns of
water come to approximately the same height (i.e., ±1.5 mm.).
THE MEASUREMENT OF pH
1x43-13
TABLE 4
DROP-RATIO COLOR STANDARDS FOR pH DETERMINATIONS
Quantit\
of indicator solution to l)e added to each tube later to re-
Tube pairs
ceive
dilute alkali or acid and then brought to final volume of 5 nd.
1
Acid tubes
Alkali tubes
Pair No. 1
9 drops*
1 drop
Pair No. 2
8 drops
2 drops
Pair No. 3
7 drops
3 drops
Pair No. 4
6 drops
4 drops
Pair No. 5
5 drops
5 drops
Pair No. 6
4 drops
6 drops
Pair No. 7
3 drops
7 drops
Pair No. 8
2 drops
8 drops
Pair No. 9
1 drop
9 drops
*If a little more accuracy is desired one may use a 1 ml. pipet graduated in tenths and
use the specified number of tenths of a milliliter instead of drops in preparing these
tubes. In that case each tube should be brought up to a total volume of 10 ml. instead
of 5 ml.
TABLE 5
DATA FOR DETERMINING PH VALUE BY THE DROP-RATIO METHOD
No. of drops
pH value represeu
ted by each pair of tubes
of indicator
Pair
Brom
Brom
Brom
Thv-
Alkali
Acid
phenol
Methyl
cresol
thymol
Phenol
Cresol
mol
tube
tube
blue
red
purple
blue
red
red
blue
1
1
9
3.0
4.05
5.2
6.15
6.85
7.35
7.95
2
2
8
3.4
4.4
5.6
6.5
7.2
7.7
8.3
3
3
7
3.6
4.6
5.8
6.7
7.4
7.9
8.5
4
4
6
3.8
4.8
6.0
6.9
7.6
8.1
8.7
5
5
5
4.0
5.0
6.2
7.1
7.8
8.3
8.9
6
6
4
4.2
5.2
6.4
7.3
8.0
8.5
9.1
7
7
3
4.4
5.4
6.6
7.5
8.2
8.7
9.3
8
8
2
4.6
5.6
6.8
7.7
8.4
8.9
9.5
9
9
1
4.9
5.95
7.0
8.05
8.75
9.25
9.85
Data as to stock solutions
Percent concentra-
0.008
0.008
0.012
0.008
0.004
0.008
0.008
tion of indicator
salt in
acid in
salt in
salt in
salt in
salt in
salt in
salt or acid
water
95%
alcohol
water
water
water
water
water
Quantity N/20
NaOH to produce
alkaline color* . . .
1 drop
1 drop
1 drop
1 drop
1 drop
1 drop
2 drops
Quantity of acidf to
produce acid color
1 ml.
1 drop
1 drop
1 drop
1 drop
1 dropt
1 dropt
*If the standards are prepared by the method suggested in the footnote to Table 4
(i. e., measuring the indicator in tenths of 1 ml. and diluting to 10 ml.) it is well to use
N/10 instead of N 20 NaOH to assure proper strength. The exact concentration or
the exact number of drops used is of no great importance.
fUse approximately N/20 HCl (or N/10 if the method is modified as indicated in
the footnote to Table 4) except in the case of cresol red and thymol blue. In the case of
these two indicators a weaker acid must be used. Gillespie recommends 2 percent
H2KPO41 or in the case of thymol blue no acid need be used, water alone having a suffi-
ciently high pH value to bring out the full acid color.
ix«-U MANUAL OF METHODS FOR PURE CULTURE STUDY
Place these 18 tubes in two rows in a rack, 9 tubes in each row. To
the left hand tube in the front row add 9 drops of the indicator
solution, in the second tube place 8 drops, and so on to the last tube
which should contain 1 drop. In the back row of tubes place 1 drop
in the left hand tube, 2 in the next, etc., up to 9 in the last. Make
up approximately N/20 stock solutions of NaOH and HCl (i.e., 0.2%
NaOH; and 1 ml. concentrated HCl (sp. gr. 1.19) diluted to 240 ml.).
Then, except in the case of those indicators for which different direc-
tions are given in Table 5, add one drop of the stock acid solution
to each tube in the front row and 1 drop of the stock alkali solution
to each tube in the back row; add enough distilled water to each tube
to bring its total contents to 5 ml., thoroughly mix the contents of
each tube and return to its place in the rack. It will be seen from
Table 5 that two of the indicators, namely thymol blue and brom-
phenol blue, require more of the alkali or the acid, respectively, than
the other standards in order to insure the appearance of full alkaline
or acid color. In the case of thymol blue (alkaline range) and cresol
red, the production of the required acid color (yellow) requires not a
strong acid but a weaker one such as mono-potassium phosphate or,
in the case of thymol blue, distilled water alone.
The arrangement of tube-pairs indicated in Table 4 produces pro-
gressively different colors corresponding to steps of 10% in the
transformation of the indicator from its acid to its alkaline color.
That is, each pair of tubes, when aligned between the eye and a
source of white light, will show a color mixture corresponding to a
definite pH. This pH can be computed by means of equation 5
which can be rewritten as
drops of alkalinized indicator
pH = pK'+log (5a)
drops of acidified indicator
The fraction on the right side of the above equation is called the
drop-ratio. The values of the standards for seven of the indicators
are given in Table 5. They may be computed for the other indica-
tors by using the above equation and the pK' values in Table 2.
For approximate work it is often possible to compare the Gillespie
standards with the unknown by merely holding the two tubes of the
standard in the hand between the eye and a source of light. For
accurate work, however, a comparator block must be used, but one
with six holes instead of four, so that a tube of the unknown solution
(without indicator) can stand behind the pair of tubes of the standard.
The tube of the unknown for comparison with the standard should
contain the same amount of indicator as the sum of those in the two
standard tubes, i.e., ten drops per 5 ml.; and, of course, this tube
must be backed by two tubes of water to equalize the optical path
through the standard pair.
Indicator Papers. Passing mention may be made of these laboratory aids for the
approximate measurement of pH. Red and blue litmus papers for the detection of
alkalinity and acidity are well known. Papers impregnated with other indicators,
singly or in various combinations, can be made or obtained on the market. Those
with a single indicator may be of use to detect roughly, (about ±0.3 to 0.4 pH), values
within a relatively narrow zone of pH; those with indicator combinations enable one to
THE MEASUREMENT OF pH ix«-15
detect, more roughly, pH values over wider zones of pH. Such papers are more re-
liable in buffered solutions than in unbuffered ones.
To be emphasized, is the fact that the capillary action of the paper and of the sizing
materials on the paper fibers may interfere, through selective sorption, with the normal
interaction of solution and indicator. Generally speaking, a generous time of soaking
of the paper for the establishment of equilibrium, seems desirable. On the other hand,
a standardization of the procedure may permit a short exposure (30 sec.) to yiehl re-
producible results, which are approximate in any case. See Kolthoff and Rosenblum
(1937). Indicator papers are not recommended, except when the use of indicator
solutions is precluded and a mere approximation is sufficient.
TiTRATABLE AciDITY, BuFFER AcTION, AND pH ADJUSTMENT OF
Culture Media
In the titration of an acid with an alkah, or vice versa, a pH is
reached at which the number of equivalents of acid equals the number
of those of alkali. This pH is the equivalence point ("end-point").
If both the acid and the alkali are completely ionized, e.g., HCl
and NaOH, it is simple to calculate that this pH is about 7, and
that, in the case of 0.1 N reactants, the pH of the HCl solution will
sweep precipitously from about pH 4 to 7 upon the addition of the
last tenth per cent of NaOH; further, the addition of the first tenth
per cent excess of NaOH will cause a shift from pH 7 to about 10.
In other words, the titration curve, constructed by plotting pH as
ordinates and per cent neutralization as abscissas, is very steep at the
equivalence point (pH 7) in this titration.
The ideal indicator for the detection of this equivalence point
would be one capable of giving a distinctive color at pH 7, e.g., brom-
thymol blue. In practice, however, the steepness of the titration
curve of the HCl at the equivalence point in the above example will
permit this indicator to pass sharply from yellow to blue upon the
final addition of a negligibly small excess of NaOH. For this reason,
phenolphthalein (pK' 9.7) is frequently used for this purpose because
the first appearance of its pink color, at about pH 8.5, is a convenient
and usually sufficiently accurate indication of the endpoint of such a
titration.
In fact, except for refinements that may be neglected for ordinary
purposes, pH 8.5, detectable by means of phenolphthalein, is a fairly
satisfactory endpoint for the titration of strong acids and of all weak
acids with pK' values of less than 6.0. In the case of acids with pK'
values greater than 6.0, it is necessary, by application of equation 5,
to calculate the pH of the equivalence point, and to refine the method
of endpoint determination. For a discussion of the elementary
theory of acid-base titration, see Clark (1928).
Titratable acidity of a culture. The titration of an acid (or a base)
to an equivalence point, as discussed above, is a rational application
of simple acid-base theory. On the other hand, in the titration of
complex mixtures such as milk, tissue extract, or culture media, an
equivalence point has no precise meaning. In such a case, the
selection of an endpoint pH is arbitrary, and fixed by custom (e.g.,
pH 8.5 with phenolphthalein) or by some special requirement.
In bacteriology, there is frequent need for determining the so-called
titratable acidity produced during the growth of a culture in a fluid
1X48-16 MANUAL OF METHODS FOR PURE CULTURE STUDY
medium. To do this, it is necessary first to select a baseline — that
is, a pH number which is to be used as an endpoint in the titration
and for the selection of an appropriate acid-base indicator. In the
absence of special criteria, it is reasonable to choose as a baseline the
pH of the uninoculated medium. The selection of pH 7 as a baseline
may be acceptable, because many bacteria grow optimally in this
region, not necessarily because it represents the pH of theoretical
"neutrality". Other baselines may be chosen in accordance with
the special requirements for which the titration is to be made.
The titratable acidity of the culture can be measured by titration
of a known volume of the fluid with 0.1 N NaOH to the predeter-
mined endpoint as shown by a standardized glass electrode or by the
color of a suitable indicator. In the latter case, it is necessary to pre-
pare for comparison an appropriate color standard representing the
pH of the chosen endpoint (see earlier discussion of the essential re-
quirements for adequate color comparison). If the endpoint pH is
other than that of the uninoculated control, a titration is made of the
latter and its titration value is subtracted algebraically as a correction
or "blank", from that of the culture. The result is usually recorded
as ml. of 0.1 normal acid per 100 ml. of the culture fluid. If the
culture produces an alkaline reaction, the titration is performed with
0.1 A^ HCl, and recorded after correction, if any, in the same way but
as a minus quantity of titratable acid. Special precautions are
necessary if the titratable acidity is to include all of the volatile acids,
including COo and bicarbonate, that may be present in the culture
that is being titrated.
It should be emphasized that, in most cases, the titratable acidity
is merely a measure of the buffering capacity (see below) of the
medium within the pH range observed. It does not permit further
interpretation without additional data on the components of the
culture. The titratable acidity is of some importance, along with
final pH, in the comparison of high acid producing organisms. For
such comparisons to be valid, it is necessary that the different organ-
isms be grown in the same medium. Different media which vary
in buffering capacity may yield misleading results.
Buffer action. The titration curve of a weak acid has a sigmoid
shape, each end of the curve having a large (steep) slope, and the
main central portion having a small slope. This small slope ex-
presses the buffer action of the system, that is, the ability of the
system (comprising the weak acid and its salt) to resist large change
in pH on the addition of acid or alkali. The sigmoid shape of the
titration curve expresses, therefore, the fact that the buffer action of
such a system is maximal at the midpoint and decreases on either
side of this point, first gradually and then more extensively as either
end of the curve is approached. The limits of the pH zone of effec-
tive buffer action may be arbitrarily set at 1.5 pH units greater and
less than the pK' of the acid of the buffer system. It is obvious that
increasing the concentration of the buffer system will increase its
buffer action; therefore buffer action also depends upon the concen-
tration of the buffer system.
The buffer action of a culture medium is dependent on its composi-
tion and may vary considerably in different regions of pH. Signifi-
THE MEASUREMENT OF pH ix«-17
cant results as to final pH and titratable acidity in cultures depend to
a large extent on comparisons made in media having buffer action
that is uniform and adjusted in amount to the purpose of the test.
A method for estimating such buffer action is as follows:
Assume, for example, that the initial pH of a culture medium is
6.8 and that it is desired to measure the buffering capacity of the
medium between the pH limits 5.0 and 8.0. This can be done by
titrating an aliquot e.g., 5 ml., of the medium with 0.05 N HCl to
pH 5.0, and another aliquot with 0.05 N NaOH to pH 8.0. The
sum of these titers gives a simple and useful measure of the buffering
capacity of the medium within the pH zone 5.0 to 8.0. Brown (1921)
has described the procedure and some of its practical uses.
The 'pH -adjustment of a culture medium. This is done with the
medium at about 80 to 90% of its final volume. Prepare approxi-
mately normal NaOH and HCl stock solutions, and also, about 100
ml. of each of these solutions diluted with distilled water exactly to
one-tenth concentration. Assume, for example, that the adjustment
of a colorless medium is to be made to pH 7.0 before sterilization.
Test the pH of the medium to establish whether acid or alkali will be
required for adjustment to pH 7. To determine the amount re-
quired, titrate 5 ml. of the medium plus 5 drops of the appropriate
indicator {e.g., bromthymol blue) with the diluted acid or alkali until
the color almost matches that of 10 ml. of standard buffer pH 7.0
plus 5 drops of the same indicator. Next, add water to the tube
with medium to bring the volume to 10 ml., mix w^ell, and make a
proper comparison with the standard. If the color difference is
small, then small additions of either acid or alkali may be made to
bring about a correct match without changing significantly the ne-
cessary volume relations. If the color difference is large, the titra-
tion should be tried again. (In the case of a medium with inherent
color, this should be compensated as previously described.)
From the titration value, a calculation can be made of the amount
of the stronger acid or alkali to be added to bring the bulk of the
medium to the desired pH. The pH of the medium is checked after
the addition and, when correctly adjusted, the medium is diluted
with distilled water to the final volume.
In making a colorimetric pH determination of a well-buffered
medium that is already colored, it is permissible to dilute the test
sample of the medium 1 to 5 or 1 to 10 with distilled water to thin
out the inherent color before proceeding with the test. The change
in pH due to such dilution of a well-buffered solution is usually negli-
gible. On the other hand, caution must be observed in employing
the dilution procedure on poorly buffered solutions, because the
results may be misleading should the distilled water, or even the
indicator solution, be too far from the desired pH.
The Measurement of Oxidation-Reduction Potentials
Introduction. The oxidation-reduction reaction
CIo+21- — ?-2Cl-+l2
represents an exchange of electrons between the chlorine: chloride
system and the iodine: iodide system. These systems may be rep-
1X48-18 MANUAL OF METHODS FOR PURE CULTURE STLTDY
resented by the hypothetical "half-reactions"
Cl2+2e — ^ 2 Cl-
l2+2e 2 I-
-•;
to show the participation of electrons. In the interaction, chlorine
is the electron-acceptor, and iodide the electron-donor.
The chlorine, the iodine and a considerable number of other sys-
tems can be studied by means of electric cells in which such systems
can display their relative oxidation-reduction tendencies in terms of
electrode potentials. The latter permit evaluation of the change in
Gibbs free energy (see later) in the interaction of any two such
oxidation-reduction systems.
Without going into details of derivation or refinements, we may state that the elec-
trode equation for a reversible oxidation-reduction system has the general form:
RT [Reductant] / <• .• r tt j \
p _y 7 \ I a lunction 01 pH and i ,„x
t;, rrk -J ii I dissociation constants I ^ '
nt [OxidantJ \ /
where Eh is the potential, in volts, referred to that of the normal hydrogen electrode;
Eo is a constant characteristic of the system at pH 0; R is the gas constant, 8.315 volt-
coulombs per degree per mole; T is the absolute temperature; n is the number of elec-
trons involved in the oxidation-reduction process; F is the faraday (96500 coulombs);
In is the logarithm to the base e; and brackets represent concentrations of the reduc-
tant and oxidant. At any fixed pH, the first and last terms on the right side of the
above equation may be combined as a constant, E'o, then,
RT [Reductant]
Eh = E'o In (7)
nF [Oxidant]
That is, Eh = E'o at anj- fixed pH when [Reductant] = [Oxidant].
It is apparent from equation 6, that the potential of such a system may be influenced
by the pH of the solution; and the potential of one system may vary relative to that of
another as the pH is varied. In fact, cases are known where system A can oxidize
system B at one pH level, and system B oxidize system A at another. Hence the
importance of comparing such potentials at the same pH, as well as the same tempera-
ture, and the desirability of specifying pH in connection with a statement of the Eh
of a system.
Elaboration of the theory of reversible oxidation-reduction potentials can be found
in Clark (1928, 1948), Clark, Cohen, et al. (1928), and modern texts on electrochemistry,
such as Glasstone (1942).
There are two methods of measuring oxidation-reduction potentials, the potentio-
metric method and the colorimetric. Each has its advantages and disadvantages;
but the potentiometric method is generally preferable for reasons that will appear
below. In either case, it is usually necessary to deaerate the container and the solution
to be measured by evacuation or by displacing gaseous and dissolved oxygen with an
inert gas such as purified nitrogen. Deoxygenation is often accomplished spontaneous-
ly in the depths of an actively growing culture of facultative bacteria.
THE MEASUREMENT OF 0/R POTENTIALS ix^s-l*
THE POTENTIOMETRIC METHOD
Electrode vessel. This may be a test tube with a constriction and
bulb at its lower end or a more elaborate container depending on the
requirements of the experiment. Such vessels are described by
Clark, Cohen, ct al. (1928), Borsook and Schott (1931), Allyn and
Baldwin (1932), and Hewitt (1936).
Electrodes. A "noble" or "unattackable" metal is the electrode of
choice. A coil of bright platinum wire has been frequently employed,
but this is difficult to clean thoroughly and there is danger of entrap-
ment of particulate material during a measurement. Platinum sheet,
about 5 mm. square or larger, is preferable.
Gold-plated platinum electrodes seem to have certain advantages.
They can be readily replated to provide a clean, new surface and
thereby obviate erratic electrode behavior. Secondly, gold, being
relatively impervious to hydrogen, should have less tendency to act
as a hydrogen electrode in a culture producing appreciable quantities
of molecular hydrogen. However, some observers do not consider
this of much practical importance.
Electrodes should be checked for reliability by measuring the potential of a known
oxidation-reduction system (e.g., quinhydrone in 0.1 M HCl, Eh =0.6351 at 25°, see
p. 4.) Where possible, duplicate or multiple electrodes should be employed; and one
that exhibits persistent erratic behavior should be discarded. Unless the solution or
culture under examination is well stirred, the electrode reading may record merely
a local oxidation-reduction potential rather than one representative of the solution as
a whole. In a heavily growing culture, electrodes may become coated with adherent
cell masses, and duplicate electrodes may show widely divergent potentials even when
the culture is well stirred.
The common method of cleaning a platinum electrode involves cautious treatment
with aqua regia, or hot concentrated nitric acid, or hot bichromate cleaning mixture,
followed by thorough washing in water. For careful oxidation-reduction work, this
procedure may not leave the metal surface altogether "inert". A more suitable pro-
cedure is to electrolyze a 1:1 solution of concentrated HCl with the electrode to be
cleaned as the anode (gold-plated platinum may be deplated in the same way). The
well washed electrode may be stored in distilled water. If the metal surface remains
dry for any length of time, the electrode may be sluggish in reaching an equilibrium
potential.
Calomel half-cell. (See also p. 3). The "saturated" type of any
convenient form is generally suitable, preferably one that permits
flushing of the siphon outlet with saturated KCl solution in order to
wash away contaminations from liquid junction contacts. Liquid
junction between the calomel half-cell and culture should be of a
kind which can be made aseptically when desired. For ordinary
purposes, this is conveniently accomplished by preparing a glass
tube partly sealed at one end over a piece of acid-washed asbestos
fiber. This tube is filled by means of a capillary-tipped pipet with
melted KCl-agar (40 g. KCl per 100 ml. of 3% agar in water) and
autoclaved. The partly sealed end of the tube is inserted into the
culture to provide the "liquid" junction, and the open end is placed
in bubble-free contact with saturated KCl solution leading to the
calomel half-cell.
Potentiometer and galvanometer. Generally speaking, cell suspen-
Pt;
Solution X
KClor
Saturated
or
or
KCl-agar
calomel
tAu;
Culture X
bridge
half-cell
1X48-20 MANUAL OF METHODS FOR PURE CULTURE STUDY
sions and bacterial cultures are poorly stabilized with respect to
oxidation-reduction potential. Consequently, disturbing polari-
zation may occur if even the small amount of current necessary to
operate the usual potentiometer and galvanometer circuit is allowed
to pass through the half-cell containing the biological system under
measurement; and the observed potential may be of uncertain
accuracy and reliability. This difficulty can be minimized by the
employment of a vacuum tube potentiometer-electrometer of the
kind now in common use for glass electrode measurements and pro-
vided with a scale graduated in volts.
The oxidation-reduction cell is set up by joining the saturated
calomel half-cell with the half-cell containing the solution or culture
to be measured as indicated in the following scheme :
(Pt)
Ordinarily, the potential of a culture is negative (reducing) to
that of the calomel half-cell, and the metal terminals of the above
oxidation-reduction cell are connected accordingly to the terminals of
the potentiometer. The reading of potential thus obtained will be
that referred to the calomel half -cell; and this observed potential,
Eobs, can be converted to Eh, the potential referred to the standard
normal hydrogen half-cell, by adding Eobs and Ecai algebraically.
That is, Eh = Eobs+Eeai. Thus, if Ecai= +0.250 v. (see p. 3) and
Eobs = -0.150 v., then Eh= +0.100 v.
Significance of E^. measurements. The potentiometric method is
direct and relatively simple. The interpretation of the results is,
however, another matter. Discounting subsidiary, but sometimes
important, instrumental effects such as potentials due to liquid
junctions, and temperature differences within the oxidation-reduction
cell, all of which can be eliminated or minimized (see Clark, 1928), an
observed Eh of a system such as ferric :ferrous iron, under conditions
of equilibrium and maximum work, is a measure of the Gibbs free
energy change, nFEh = -AG, in the reaction between the components
of the two halves of the oxidation-reduction cell. This is the case for
a considerable number of oxidation-reductions which, alone or in the
presence of catalysts and mediators, can take place more or less
rapidly and reversibly as if a transfer of electrons, with or without
accompanying protons, were direct and complete. These are re-
actions between so-called electromotively active systems, the Eh of
which is fixed, at constant pH, by a characteristic constant and by
the relative concentrations (more accurately, activities) of the
components of each such system. For example, a potential of the
ferric rferrous system in acid solution, can be defined by the relation:
RT [Fe++]
Eh = E'o In (8)
F [Fe+++]
which implies the limitation that definite and significant potentials
are possible only in the presence oi finite ratios of oxidant to reduc-
tant. In addition, the total concentration of the reversible system
may be decreased to and beyond a level at which traces of electromo-
THE MEASUREMENT OF 0/R POTENTIALS
ix,8-21
tively-active contaminants attain dominance and an observed po-
tential becomes unstable and difficult to interpret.
In contrast to the above mentioned reversible processes which are
readily amenable to Eh measurement, there are a great many oxida-
tion-reductions that proceed by a variety of mechanisms that do not
permit formulation and precise measurement in terms of equili-
brium states. Electrode potentials in such cases are difficult to
interpret and of uncertain significance.
In cell suspensions and bacterial cultures, especially when de-
prived of free access of oxygen, there develops with time a progressive-
ly more negative potential which traverses the zones characteristic of
reversible oxidation-reduction indicators (see next section). Polari-
zation of the electrode or a small dose of an oxidant may reverse the
trend of reduction potential temporarily, but the trend is resumed
after a while to levels of potential that may sometimes be associated
with the type of cell and the various metabolites in the suspension or
culture. Duplicate electrodes in such systems may not be in good
agreement at the start, but they will reach about the same limiting
value in time. For examples, see Clark, Cohen, et al. (1928), Allyn
and Baldwin (1932), and Hewitt (1936).
THE COLORIMETRIC METHOD
The empirical use of substances such as litmus or methylene blue
as indicators of reduction in bacterial cultures is well known. For
the determination of various degrees of reduction intensity an ap-
propriate series of indicators is necessary. Among those available
TABLE 6
A SELECTION OF OXIDATION-REDUCTION INDICATORS
E'o at pH 7, (30°)
(Values of E'o between pH 5 and 9 will be found in Table 7)
Compound
Phenol-7ri-sulfonate-indo-2,6-dibromophenol . . .
TO-Chlorophenol-indo-2,6-dichIorophenol
o-Chlorophenol-indophenol
2,6-Dichlorophenol-indophenol
2,6-Dichlorophenol-indo-o-cresol
l-Naphthol-2-sulfonate-indo-2,6-dichlorophenol
Lauth's violet (Thionin) ,
Cresyl blue
Methylene blue
Indigo tetra sulfonate
Methyl Capri blue
Indigo trisulfonate
Indigo disulfonate
Gallophenine
Brilliant alizarine blue
Phenosafranine
Tetramethyl-phenosafranine
Saf ranin T
Induline scarlet
Neutral redj
Rosindone sulfonate No. 6
(Hydrogen at 1 atmosphere)
*At 25°. fSe^ footnote 3 in text.
E'
g-
h.
i.
i-
k.
1.
m.
n.
o.
P-
q-
r.
s.
t.
u.
0.273 V.
0.254
0.233
0.217
0.181
0.119
0.062
0.047
+0.011
-0.046
-0.061
-0.081
-0.125
-0.142*
-0.173*
-0.252
-0.273
-0.285
-0.299
-0.324
-0.385
(-0.421)
1X48-22 MANUAL OF METHODS FOR PURE CULTURE STUDY
TABLE
SELECTED OXIDATION-
Relation at
(Letters refer to compounds listed in
pH
a
b
c
d
e
f
g
h
i
J
5.0
+ .390
+ .391
+ .366
+ .335
+ .262
+ .138
+.149
+.101
+ .065
5.5
.360
.359
..332
.300
.230
.109
.117
.072
.035
6.0
.330
.326
+ .301
.295
.261
.196
.094
.189
.047
+ .006
6.5
.301
.290
.269
.255
.220
.158
.077
.066
.028
-.022
7.0
.273
.254
.233
.217
.181
.119
.062
.047
+ .011
-.046
7.5
.246
.220
.195
,182
.145
.080
.047
.030
-.005
-.066
8.0
.218
.188
.155
.150
.112
.046
.030
+ .015
-.020
-.083
8.5
.192
.159
.117
.119
.081
+ .016
+ .017
-.001
-.035
-.099
9.0
.168
.133
.082
.089
.051
-.012
-.001
-.016
-.050
-.114
are reversible oxidation-reduction systems, the oxidants of which are
usually colored and the reductants practically colorless. A number
of such indicator systems have been characterized and may be
employed, with due precautions, in determining an oxidation-reduc-
tion potential colorimetrically.
A selection of such indicators^ is listed in Tables 6 and 7. Similar
tabulations are given by Hewitt (1936). Fuller details can be found
in Clark, Cohen, et al. (1928) and Cohen (1933, 1935). Table 6
gives the names of the indicators, listed in the order of their E'o values
at pH 7.0; and Table 7 gives the corresponding E'o values at suc-
cessive levels between pH 5.0 and 9.0. The magnitude of the salt
and protein errors of these compounds has not been established.
Each indicator system listed in Tables 6 and 7 involves a two-
electron transfer, and the relation of E'o to other factors at fixed pH is
given by equation 9.
RT [Reductant]
Eh = E'o -■ In ■
2F [Oxidant]
(9)
Converted to ordinary logarithms after insertion of numerical values,
this equation becomes, at 30°C,
[Reductant]
Eh = E'o- 0.030 log (10)
[Oxidant]
The relation of percentage reduction to potential as defined by the
last term in equation 10 is given in Table 8. For example, if methy-
lene blue is observed to be 80% reduced at pH 7, Eh = 0.01 1-0.018 =
-0.007 volt.
^A special comment is necessary in regard to neutral red (compound t in Tables 6
and 7). It undergoes reversible reduction in tlie usual manner, and the colorless
solution of reductant formed upon rapid reduction reoxidizes very rapidly when ex-
posed to air. However, the reductant on standing in solution at pH 4 to 6 for a little
time undergoes transformation to a fluorescent substance which is stable for days in the
presence of air, but reoxidizes rapidly upon acidification. As an oxidation-reduction
indicator, therefore, neutral red must be employed with due caution and can be used
only for rough comparisons.
THE MEASUREMENT OF 0/R POTENTIALS
1X48-23
BEDUCTION INDICATORS
E'o to pH (30°)
Table 6; the values listed are E'o in volts)
k
1
m
n*
o*
P
q
r
s
tt
u
+ .038
+ .032
-.010
-.003
-.040
-.158
-.157
-.197
-.235
-.205
+ .006
-.002
-.040
-.042
-.080
-.188
-.194
-.227
-.253
-.236
-.021
-.028
-.069
-.077
-.112
-.215
-.225
-.251
-.268
-.265
-.298
-.043
-.056
-.098
-.110
-.142
-.234
-.252
-.270
-.284
-.294
-.349
-.061
-.081
-.125
-.142
-.173
-.252
-.273
-.285
-.299
-.324
-.385
-.077
-.103
-.148
-.172
-.203
-.269
-.288
-.300
-.314
-.352
-.425
-.093
-.121
-.167
-.202
-.226
-.284
-.303
-.316
-.329
-.382
-.460
-.108
-.137
-.184
-.232
-.251
-.299
-.319
-.331
-.344
-.410
-.491
-.123
-.152
-.199
-.262
-.279
-.314
-.334
-.347
-.359
-.438
-.520
*At 25°
fSee footnote 3 in text.
Color standards. Since the compounds listed in Tables 6 and 7 are
practically one-color oxidation-reduction indicators, color standards
of sufficient approximation can be prepared simply by graded dilu-
tions of the colored component, the oxidant. It should be borne in
mind that some of the compounds are also acid-base indicators,
therefore it may be necessary to set up the color standards in a buffer
at the same pH as the solution or culture under test.
Color imetric measurement. The general principles of color com-
parison, as outlined for the indicator method of pH-determination,
are applicable here. In addition, special precautions are required to
make certain that the measurement is a valid one. An indicator
may fade in a test solution for reasons other than simple reduction.
The compound may precipitate or adsorb on suspended particles, or
it may be decomposed; in such cases judicious treatment with
a suitable oxidizing agent (e.g., ferricyanide, or air) will not
immediately restore the initial color of the oxidant. Moreover,
many reversible oxidation-reduction systems are so sensitive to
oxygen as to require extreme precaution for its exclusion. This ap-
plies to the electrometric method as well as to the colorimetric.
TABLE 8
Reduction
-0.03 log ratio
Reduction
-0.03 log ratio
%
Volts
%
Volts
1
+ 0.060
60
-0.005
10
0.029
70
-0.011
20
0.018
80
-0.018
30
0.011
90
-0.029
40
0.005
99
-0.060
50
0.000
100
(- «)
It is a fact that many biological systems act as if they contain,
at any moment, only minute amounts of electromotively active
oxidation-reduction substances, therefore the addition to such a
system of even a small amount of indicator-oxidant may suffice to
oxidize the system at once without appreciable reduction of the indi-
ix,8-24 MANUAL OF METHODS FOR PURE CULTURE STUDY
cator. This drawback cannot be overcome except by allowing suffi-
cient time for the biological system to overcome the poising* effect
of the added indicator. However, the time required may be very
long (especially in relation to the most active period of a growing
bacterial culture) so that it may be difficult or impossible to deter-
mine successive Eh values colorimetrically at brief intervals.
Furthermore, the indicator may not merely come into simple
oxidation-reduction equilibrium with the components of the system
under test. It may act catalytically to displace the oxidation-reduc-
tion equilibrium that it is supposed to measure; or it may be toxic
toward living cells, or combine chemically with components of the
system under test.
In summary, the indicator method, often applicable where it is
impossible to employ an electrode, may give results that require
considerable caution in interpretation, especially the results obtained
on unstable oxidation-reduction systems or on biological material
containing them.
REFERENCES
Allyn, W. p., and Baldwin, I. L. 1932. Oxidation-reduction potentials in re-
lation to the growth of an aerobic form of bacteria. J. Bact., 23, 369-398.
BoRSOOK, H., and Schott, H. F. 1931. The role of the coenzyme in the succinate-
enzyme-fumarate equilibrium. J. Biol. Chem., 92, 535-557.
Brown, J. H. 1921. Hydrogen ions, titration and the buffer index of bacteriological
media. J. Bact., 6, 555-568.
Clark, W. M. 1928. The Determination of Hydrogen-Ions. 3rd Ed. Williams
and Wilkins, Baltimore.
Clark, W. M. 1948. Topics in Physical Chemistry. Williams and Wilkins, Balti-
more.
Cl.\rk, W. M., Cohen, Barnett, et al. 1928. Studies on Oxidation-Reduction,
I-X. Hygienic Laboratory Bulletin No. 151, U. S. Public Health Service,
Washington.
Cl.\rk, W. M. and Lubs, H. A. 1917. The colorimetric determination of hydro-
gen-ion concentration. J. Bact., 2, 1-34, 109-136, 191-236.
Cohen, Barnett. 1926. Indicator properties of some new sulfonphthaleins.
Public Health Rpts., 41, 3051-3074.
Cohen, Barnett. 1933. Reversible oxidation-reduction potentials in dye systems;
(also) Reactions of oxidation-reduction indicators in biological material, and
their interpretation. Cold Spring Harbor Symposia on Quantitative Biology,
1, 195-204; 214-223.
Cohen, Barnett. 1935. Oxidations and Reductions. Chapt. XIX in: A Text-
book of Biochemistry, by B. Harrow and C. P. Sherwin. W. B. Saunders
Co., Phila.
Dole, M. 1941. The Glass Electrode. John Wiley and Sons, New York.
Gillespie, L. J. 1920. Colorimetric determination of hydrogen-ion concentration
without buffer mixtures, with especial reference to soils. Soil Sci., 9, 115—136.
Glasstone, Samuel. 1942. An Introduction to Electrochemistry. Van Nostrand,
N. Y. See Chapt. VIII.
Hewitt, L. F. 1936. Oxidation-Reduction Potentials in Bacteriology and Bio-
chemistry. Ii-th Ed. London County Council.
Kolthofp, I. M., and Rosenblum, Charles. 1937. Acid-base Indicators. Mac-
millan, New York.
Small, James. 1946. pH and Plants. Van Nostrand, New York.
^Poising action of an oxidation-reduction system is analogous to buffer action of an
acid-base system. (Compare paragraph on buffer action, p. 16.)
LEAFLET X
INOCULATIONS WITH BACTERIA CAUSING
PLANT DISEASE
Prepared by
A. J. RiKER
Committeeman on Plant Pathological Methods
In collaboration with
P. A. Ark, Charlotte Elliott, and E. M. Hildebrand
3rd EDITION
Pure Culture Study of Bacteria, Vol. 13, No. 1-2
February, 1945
Note. — This leaflet is issued in accordance with the j)olicy of including in the M.\ncal
material drawn up by committeemen or sub-committees who assume responsibility
for methods outlined and opinions expressed. The committee will appreciate it if users
of the Manual who have any fault to find with the methods or their presentation will
communicate with the committee chairman or with the committeeman who has written
this leaflet.
LEAFLET X
INOCULATIONS WITH BACTERIA CAUSING
PLANT DISEASE
Introduction
The methods for studying the pathogenicity of bacteria in plants,
and for making a few selected cognate investigations are briefly
treated in this Leaflet. The procedures, in relation to handling certain
organisms and to studying the diseases they induce, vary so widely
that no given directions apply to the group as a whole. The selected
representative methods included are thus to be considered primarily
as guides to the beginner, and are to be modified as circumstances war-
rant.
Difficulty in interpretation is frequently encountered from varia-
tions in results, depending on the methods used. A given bacterial
character may sometimes be positive when measured by one method
and be negative when measured by a slightly different technic. Stu-
dents should employ a known positive and a known negative as
controls when making critical determinations. The method used
should always be given or cited when a character is listed, so that
the validity of the character can be correspondingly estimated by
the reader. Some of the technical pitfalls to be avoided have been
listed by Frobisher (1933).
A number of topics discussed in Leaflet VII regarding bacteria
pathogenic on animals are applicable to bacteria pathogenic on plants.
These include particularly: (1) identification of the active agent as
the bacterial cell or its products; (2) distinction between invasion
and the power to cause disease after entry; (3) variability in virulence
of the pathogen, which requires single-cell cultures, and in suscepti-
bility of the host, which frequently calls for plants with known genetic
constitution, when critical studies are involved; and (4) relations
between reactions induced in the test tube and in the host.
The pathogenicity of a microorganism may be proved by fulfilling
Koch's postulates, which have been stated and modified in various
ways, and which are so important that they are repeated here. One
summarized statement follows: (1) The causal agent must be associ-
ated in every case with the disease, and conversely the disease must
not appear without this agent. (2) The causal agent must be isolated
in pure culture and its specific characters determined. (3) When the
host is inoculated under favorable conditions with suitable controls,
the characteristic symptoms of the disease must develop. (4) The
causal agent must be reisolated, usually by means of the technic
employed for the first isolation, and identified as that first isolated.
Obviously, the demonstration of pathogenicity is made only after
repeated trials, preferably with a number of different isolates which
are of unquestioned purity. When the technic for cultivating causal
X4S-4 MANUAL OF METHODS FOR PURE CULTURE STUDY
agents on artificial media has not yet been worked out, their patho-
genicity is established in other ways (e. g.. Rivers, 1937). When
causal relations are being worked out, one may well differentiate be-
tween predisposing, inciting, and continuing causes. Various
factors that influence the physiology of the plant may also affect
pathogenicity.
The simpler methods for making isolations, for preparing and using
both ordinary and differential media, and for studying the morphol-
ogy and physiology of such bacteria have been adequately described
in Leaflets II, IV, V, VI, and elsewhere (e. g., Rawlins, 1933; Riker
and Riker, 1936; and Smith, 1905-1914, 1920). This Leaflet, there-
fore, is concerned primarily with methods of inoculation.
To insure against erroneous conclusions, the environmental con-
ditions for experimental inoculations should be maintained as nearly
as possible like those occurring in nature at the time of natural infec-
tion. When difiiculty is experienced in artificial inoculation, careful,
continued observation of the host plant at the time of natural infec-
tion may reveal the cause of the trouble.
In advanced research it appears that investigators working on
pathogens, whether with plants, animals, or men, have many common
interests. These include, for example, (1) life cycles, referring to
changes in the morphology of individual cells and the relation of these
different forms to virulence; (2) changes in colony characters and
physiology, including particularly changes in pathogenicity; (3)
factors attending changes, such as the time, frequency, and conditions
of origin, as well as the influence of environment, and relations to
earlier and succeeding generations; (4) statistical analyses to classify
the origin and frequency of the variations observed; and (5) life
histories of the pathogens in relation to entrance into the host, loca-
tion, exit, and transmission to a new host, the well-known essentials
of studies in epidemiology which are vitally influenced by variations
in the pathogens.
Certain characteristics of plants not possessed by animals facilitate
basic research on pathogenicity. Among the advantages in experi-
mental work are the following: (1) Large numbers of hosts are easily
available. The number used, whether 10 or 10,000, is selected on
the basis of experimental needs. (2) The initial cost and expense of
maintaining plants are relatively low. (3) The species of plants
studied frequently contains varieties or selections possessing several
degrees of resistance and susceptibility. (4) Plants are suited to a
wide range of experimental procedures, such as regulation of internal
temperature and moisture, that are not feasible with animals. (5)
Epidemics^ are induced with relative ease and without concern for
the health of the technician or the public. (6) The genetic purity of
the host can be assured. Seed from long fines of successively self-
i"Epidemic," in the original Greek meaning "on the people," was early applied to
plant diseases, together with many other medical terms. It is an old and common
word in plant pathology, although on etymological grounds its use for human disease
alone is preferred by some medical authorities. In this paper, however, the broad
definition from Gould's Medical Dictionary is followed, "Epidemic: of a disease affect-
ing large numbers or spreading over a wide area."
INOCULATIONS WITH BACTERIA CAUSING PLANT DISEASE x.^-S
fertilized parents is often available. When this is not sufficient, one
can commonly find or develop experimental units all genetically
identical through vegetative propagation. With such material any
variations secured can be studied without concern that the host may
have been obscuring pathogenicity. (7) Certain plant materials
can be cultivated in vitro on media containing only nutrients for
which the chemical formulae are known (reviewed by White, 1943).
Simple Representative Inoculation Methods
The actual method of making inoculations varies with circum-
stances. Some simpler methods are considered briefly by way of
illustration. Methods for testing the relative efficiency of several
technics are considered in a later section.
SOIL "inoculation"
The introduction of large numbers of pathogenic bacteria into the
soil depends upon growing sufficient quantities in cultures, either on
agar or in liquid media. Special flasks, bottles, and other containers
having adequate flat surfaces are employed. Most of the plant patho-
gens are aerobic and need to be incubated under pronounced aerobic
conditions if the best growth is to be secured. When agar is used the
surface growth on a suitable medium is washed or scraped off after
sufficient growth has appeared, and a suspension is made. When a
liquid medium is employed, a greater bacterial count per cubic centi-
meter is secured, with an organism like Phytomonas tumefaciens
(Smith and Town.) Bergey et al., by use of a medium less than 2 cm.
deep or well aerated by some other means. Satisfactory aeration may
be secured in deep liquid cultures by bubbling sterile air through a
scintered glass or other aerator placed in the medium. In large
containers aeration can be improved by a few pounds of pressure
which forces more air to dissolve in the liquid. Maintaining such
pressure also reduces contamination from leaky valves. Chemicals
that poise the oxidation-reduction potential may be helpful. The
highest count of active bacterial cells may be reached somewhat
before the maximum turbidity is attained. Considerable turbidity
is caused by bacterial gum. Usually the whole culture is employed
for soil treatment; but one should avoid adding too much extraneous
matter with the inoculum. Such aerated liquid cultures also work
well with some fungi.
Soil may be "inoculated" by pouring liquid suspensions on rela-
tively dry soil, allowing the water to be absorbed long enough to
avoid puddling, and mixing. The quantity of culture used for each
plant varies. One might begin with 1 part of culture to 10 parts of
soil and use a handful of this mixture about the roots of each plant.
Inoculations through the soil are considerably more difficult than
those with various other methods.
SEED inoculation
Perhaps the easiest way to infect a large population is through
treatment of the seed. Legume root nodule bacteria from a fresh,
X4S-6 MANUAL OF METHODS FOR PURE CULTURE STUDY
active culture grown on agar are shaken into a water suspension and
are commonly spread on the seed just before planting. Many com-
mercial "inoculations" are prepared by mixing the culture with some
moisture absorbing powder, such as autoclaved ground peat. Wood
flour is also quite absorbent, and contains almost no bacteria. If the
seed is drill-sown, it is made only moist enough to distribute the bac-
teria well, and then dried sufficiently not to clog the drill. To secure
uniform results it is best to use plenty of bacterial culture, for
example, 5000 bacteria (plate count) per seed. For convenience
in estimating the number of bacteria per seed a brief table is given
by Fred, Baldwin and McCoy (1932), w^ho review this general
subject, showing the average number of seeds per pound of many
legumes.
SPRAY INOCULATION
Spraying is one of the methods most commonly used in plant inoc-
ulation. It is particularly useful in diseases where the bacteria enter
the host plants through natural openings such as stomata, water
pores, and nectaries. For many simple tests, suspensions of bacteria
are merely sprayed on the surfaces of susceptible leaves, stems, flow-
ers, fruits, etc. For more exact tests, however, such as those for com-
parative virulence, it is common to suspend the growth from an agar
culture in water, saline solution (0.9% NaCl), or a selected buffer
(such as suitable mixtures of dilute K2HPO4 and KH2PO4), and to
standardize the concentration according to a selected and measured
turbidity. If the bacteria have been grown in liquid culture, the
entire culture may be used. This procedure, however, is often un-
satisfactory because, after spraying, secondary organisms may grow
in the nutrient medium. It is frequently better to separate the
bacteria from the medium by means of a centrifuge and to resuspend
the cells as with the growth from agar media.
The number of bacteria in a suspension may be determined, for
example, (1) by direct examination in a Petroff-Hausser counting
chamber; or (2) by mixing a known volume of the bacteria with previ-
ously counted suspensions of yeast or red blood cells, making smears,
and determining the relative number of bacteria and cells. Bacterial
suspensions are often duplicated by comparing their turbidity with
that of a graded series of barium sulfate standards (described by Riker
and Riker, 1936). A common density for a bacterial suspension has
the turbidity of a solution obtained by mixing 1 ml. of a 1% solution
of barium chloride with 99 ml. of dilute sulfuric acid. Turbidity can
be measured accurately and rapidly in an Evelyn densiometer.
The prepared bacterial suspension is filtered through cheesecloth,
to remove small pieces of agar or other materials which might clog
the spray nozzle, and is placed in the spraying device. The plants are
sprayed so that good coverage is given especially to the lower sides of
leaves which commonly have more stomata. The plants are placed in
an environment where they will not dry off for a number of hours.
Certain additional precautions are sometimes necessary for best
results, of which several are mentioned briefly. (1) The relative
humidity of the air surrounding the host plant is maintained at satu-
INOCULATIONS WITH BACTERIA CAUSING PLANT DISEASE x^-?
ration before as well as after inoculation. The length of time varies
with the host plant and the parasite. A saturated atmosphere for
6 to 36 hours in both instances favors infection with many leaf para-
sites. Various kinds of moist chambers, e. g., that described by
Keitt et al. (1937), can be used in the greenhouse. Small outdoor
plantings can be covered for a short time with a cloth tent (Keitt,
1918) and water sprayed over the exterior. The amount of moisture
in the air apparently influences the inter-cellular humidity and, corre-
spondingly, the susceptibility of the host. (2) If the plant parts are
diflicult to wet because of a waxy covering, the surface can be gently
rubbed with a moist cloth. For work on a large scale, the suspension
of the organism can be made in a solution of a spreader (e. g., castile
soap, 1:1000) to reduce surface tension. The concentration is arbi-
trary and is varied according to requirements. Some spreaders,
however, are toxic for certain pathogens. (3) A reduced oxygen
supply may be important if the pathogen is a facultative anaerobe.
For example, the protective wound-cork formation in potato tubers
requires abundant oxygen, while certain bacterial pathogens, such as
Erwinia carotovora (Jones) Holland, grow well with little oxygen. (4)
Water pressure, suction, prolonged spraying, and other means can
be used to saturate the intercellular spaces below the stomata and
thus to improve the penetration of bacterial suspensions into these
regions. This is particularly important with a pathogen, like that
causing black fire of tobacco (Johnson, 1937), which is often not
aggressive. With this method it is possible to induce necrotic
areas on plants not ordinarily considered hosts of the microorganism
used. Since bacteria that are usually considered as saprophytes
have caused damage under these circumstances, care is necessary
while interpreting such results. For example, such saprophytes
would hardly fulfill the first of Koch's postulates, as given earlier.
WOUND INOCULATION
Suspensions of bacteria, small portions of culture, or of diseased
tissue can be introduced into healthy plants through wounds when
they do not readily gain entrance through natural openings or when
heavier or more rapid infection is desired. The simplest procedure is
to smear the point of a dissecting needle with the bacterial mass and to
insert the needle into the plant tissue. If large numbers of inocula-
tions are to be made, various instruments are useful. For example,
an inoculator is described in detail by Ivanoff (1934). It consists of
a hypodermic needle (size varied according to needs) with end closed
and smooth-walled opening made on one side, of a suitable chamber
to hold a bacterial suspension, and of a valve to regulate flow. This
needle with a side opening may be used with an ordinary syringe.
The common type of needle clogs too easily to be practical.
Known small numbers of bacteria may be introduced into micro-
wounds by means of a micromanipulator. Such wounds may re-
semble those made by insects (Hildebrand, 1942).
zi^S MANUAL OF METHODS FOR PURE CULTURE STUDY
INSECT INOCULATION
The translocation of microorganisms causing plant disease and
their introduction into susceptible plants by insects is a large and rela-
tively undeveloped field. The simplest technic with active insects
like cucumber beetles or leaf hoppers is merely to place the plant to be
inoculated in the same insect cage with an infested diseased plant.
(Leach, 1940.)
For virus diseases, inoculation with slow-moving insects, like
aphids, is accomplished by placing a paper on a caged plant to be
inoculated, and by laying on this paper a portion of a diseased leaf
which carries aphids. As the new leaf tissue dries, the insects crawl
over the paper to the fresh leaf below. When insects are involved, a
variety of special cages (Leach, 1940) may be used.
All stages in the life cycle of the insect employed must be considered
because inoculation capabilities often vary in this respect. The insect
should be identified by a competent authority, and if significant re-
sults are obtained a specimen should be deposited in a permanent
reference collection.
A detailed discussion of methods for studying insect transmission
has been given by Leach (1940). Some knowledge of the mouth parts
of insects and of their feeding and breeding habits is necessary if
insects are to be used successfully in inoculating bacterial plant
pathogens. Insects are particularly important as carriers of virus
diseases.
Before claims are made about insect transmission of a plant disease,
demonstrations of the following (Leach, 1940) seem a minimum for
proof: (1) close, but not necessarily constant association of the insect
with diseased plants; (2) regular visits by the insect to healthy plants
under conditions suitable for the transmission of disease; (3) presence
of the pathogen or virus in or on the insect in nature or after visiting
a diseased plant; (4) experimental production of the disease by insect
visitation under controlled conditions and with adequate checks.
FUNGUS INOCULATION
In general, inoculations with the spores or mycelia of fungi differ
only in detail from those made with bacteria. For pathogenic fungi,
variations in the mode of entrance and in other important characters
require modified procedures. Some of the more common methods
are discussed by Riker and Riker (1936).
VIEUS INOCULATION
Brief mention is given to inoculations with viruses without impli-
cation that they are microorganisms. Experimental inoculations are
more commonly accomplished by mechanical processes, insects (see
Insect Inoculations), and grafting.
Mechanical inoculation of a virus is frequently made by grinding
diseased tissue in a mortar with a little water and by rubbing the juice
lightly over leaves of the host plant. With some viruses, the following
modifications may be helpful. A favorable reaction between pH 7.0
INOCULATIONS WITH BACTERIA CAUSING PLANT DISEASE x«-9
and 8.5 may be obtained by placing a little M/10 K2HPO4 in the
mortar before the leaves are triturated. Sometimes viruses have to
be protected from rapid oxidation by means of 0.5% anhydrous
Na^.SOs. Just enough friction by a finger, cheesecloth, or similar
agent is employed to injure the leaf hairs. With viruses difficult to
transmit, better infection may be induced if a fine abrasive material
(e. g., carborundom powder, 600 mesh) is lightly dusted on the leaf
before it is rubbed. Some plant viruses are highly infectious. (Usu-
ally washing with soap and water is sufficient to remove infectious
material from the technician's hands.) When the mechanical
methods and insect vectors fail, two possibilities are left.
Budding or another form of grafting may be employed and is
sometimes the only successful means of virus transmission. When
grafts are made, special precautions must be taken to prevent desic-
cation of the grafted parts before union has been accomplished.
This may be achieved by providing high air humidity, by suitable
wrappers, or by spraying the scions with one of the commercial wax
emulsions.
By means of dodder (Bennett, 1940) certain viruses not otherwise
transmitted have been carried even from woody to herbaceous plants.
TREATMENT WITH BACTERIAL PRODUCTS
The metabolic products found in bacterial cultures are prepared
and employed in a variety of ways which are not yet well worked out.
Perhaps the least change occurs in the bacterial cells if they are centri-
fuged from a liquid culture and dried while frozen ("lyophile" appar-
atus described by Flosdorf and Mudd, 1935). The culture filtrate
may be concentrated under reduced pressure at a little above room
temperature and then "lyophilized" if desired.
A fermented culture or an aqueous extract may be sterilized and
placed in a small container. If leaves w4th petioles or growing tops
are removed from the host plant and are placed with the cut sur-
faces in such liquids, they commonly show injury within one day if
much toxic material is present. Care is necessary while interpreting
such injury because many constituents of media may be toxic, e.g.,
ammonia in alkaline material. Likewise, some non-parasitic as
well as parasitic fungi produce toxic substances in culture that are
not necessarily the reason for pathogenicity.
The metabolites are commonly applied either in liquid form or in a
paste made with inert material, like lanolin or flour. The paste has
the advantage of furnishing a continuous supply of material over
a longer period with relatively less desiccation. It is commonly
applied to a wound. The liquid can be introduced into the vascular
system of a potted plant by placing cut roots extending from the
base of the pot, or a cut petiole, into a container of the material.
Likewise, a cup can be made from a rubber stopper and sealed on
a plant stem with vaseline. The cup is filled with liquid, under
which a cut is made into the vascular system, so that the liquid is
taken by the plant directly into the transpiration stream. Or finally,
the stem can be opened to form a small cavity which is kept filled by
X45-10 MANUAL OF METHODS FOR PURE CULTURE STUDY
means of a capillary tube and funnel. If an enzyme like pectinase
is being tested, thin sections of tissue need merely be immersed in a
few drops of the liquid.
So many substances appearing in cultures influence plants in one
way or another that rigid controls are necessary in searching for the
products responsible for pathogenicity. Whenever feasible, an at-
tenuated culture of the same organism or a closely related non-
pathogenic culture is carried in a parallel series of trials.
The methods of testing for plant "hormones" and "vitamines" are
being revised so rapidly that an active investigator should be con-
sulted for the latest procedure.
ANTIBODY PRODUCTION
Questions on the development of antibodies in plants following
inoculation or natural infection are discussed in a considerable litera-
ture reviewed by Chester (1933). A number of controversial points
are involved.
The injection of plant bacteria into an experimental animal (see
Leaflet VIII) commonly results in the production of antibodies useful
for various investigations. Serological work with plant pathogens is
described by Link and his associates (1929, and earlier papers) and
by various other investigators. Methods of applying the precipitin
test to a study of certain viruses are given by Chester (1935).
Cognate Considerations
STRAIN variations
When studies involving strain variations are made it is well to
consider Frobisher's (1933) comment, "Plating and fishing of colonies,
while generally useful, is not a sufficiently reliable method of purify-
ing cultures in work involving bacterial variations. It is sometimes
extremely difficult, if not impossible, to separate bacterial species by
this means. Single-cell methods are much more reliable and, it would
seem, furnish the only satisfactory means of solving our problems,
but even such procedures as are at our disposal require very expert
manipulation and may lead to error." The relative unreliability of
the poured-plate technic for such studies has been discussed by
Riker and Baldwin (1939). The need for cultures with a known
origin from a single cell has stimulated much work on methods for
securing them. Literature on this work has been reviewed by several
writers, e. g., Hildebrand (1938). Unfortunately, several recent
reports on bacterial variations have appeared in which the cultures
were purified merely by several successive dilution plates, and such
purified cultures were called "single-cell cultures." This misleading
use of a well-established phrase provides both the investigator and the
reader with a false sense of security.
INOCULATIONS WITH BACTERIA CAUSING PLANT DISEASE x.^-li
Variations may be induced among plant pathogens by procedures
very similar to those employed on other bacteria. Some of the con-
siderations involved in such studies are discussed by Riker (1940).
When there seems to be a bacteriophage in the complex, the general
discussion by Krueger (1936) and the account by Thomas (1940) of a
precursor may well be consulted.
The pathogenicity of crowngall bacteria can be destroyed (Van
Lanen, Baldwin, and Hiker, 1940) with certain amino acids and
related compounds added to common media. Attenuation was com-
monly secured in 20 to 30 successive transfers. The rate of attenua-
tion was increased if bacterial growth was reduced by the strength of
the compound (e. g., 0.1 to 0.3% glycine) and by an alkaline re-
action (e. g., pH 8.0).
The virulence of partly attenuated cultures was restored by long
cultivation on suitable media and by ultra-violet irradiation (Duggar
and Riker, 1940). Likewise, when a virulent culture was inoculated
into a tomato stem above an attenuated culture, the gall about the
attenuated culture was approximately as large as that about the
virulent culture. A chemical gall served as well as that from a
virulent culture (Riker, 1942).
PATHOGENS ACTING TOGETHER
Combinations of microorganisms sometimes induce symptoms
different from those caused by any one alone. So long as the patho-
gens can be cultivated on artificial media, the principles in Koch's
postulates can be applied with two or more causal agents. For
example, a simple inoculation with one organism may involve a series
of susceptible plants growing in a suitable environment with the liv-
ing causal agent; and a parallel control series. With two causal
agents, however, there should be four series of plants as follows: (1)
with both living pathogens, (2) with only one living pathogen, (3)
with only the other living pathogen, and (4) with neither living patho-
gen. Correspondingly, three causal agents would require eight series
of plants,
CULTURES FROM ANOTHER LOCALITY
The use of a culture of a pathogen not already present on local
plants requires critical consideration. The progress of bacteriology
calls for reasonable freedom in the movement of cultures. This sci-
ence, however, has a duty in the protection of local plant popula-
tions and requires that cultures or strains brought into a new locality
should be handled with proper consideration of all the factors in-
volved. While reasonable freedom in the shipment of such cultures
from one laboratory to another is essential for certain work, it must
be insisted that they be secured and studied only after both the
investigators and their administrators have fully considered and
accepted the responsibility involved. Younger research workers
and particularly graduate students are advised to employ such cul-
tures only after detailed plans have been made in conference with
their advisors.
X4S-12 MANUAL OF METHODS FOR PURE CULTURE STUDY
RELATIVE EFFICIENCY IN TECHNIC
The best methods of procedure for making inoculations and for
recording results have not always been worked out and are not obvi-
ous from inspection. If the question is of sufficient importance, the
answer may be secured statistically. There may be a question, with
a leaf-spot organism, for example, as to whether it is better when
making inoculations to spray or to make needle punctures. Likewise,
when infection is secured, the question may occur whether the results
should be recorded in terms of total number of lesions, of total tissue
involved, the effect of the disease on yield, or of some other criterion.
Such possibilities may be tested by means of the frequently described
"analysis of variance" (e. g., Goulden, 1939). Thus the best
method for making the trials and for recording the results may be
determined. In general, the method that gives the greatest value
for the variance ratio, "F", is the most desirable. This value indi-
cates a greater uniformity in readings from different trials with the
same technic, or a greater differentiation of the varieties used or
treatments employed without a proportional increase in error.
ANTIBIOTICS
A recent survey (Osborn, 1943) has shown that various plants
contain substances adversely affecting certain bacteria. Doubtless
many instances (cf.. Link and Walker, 1933; Ark and Hunt, 1941;
Trussel and Sarles, 1943) occur in w^hich various higher and lower
forms of plant life contain chemicals that inhibit successful plant
inoculations.
Records
Taking notes on plant inoculations presents various problems
depending upon the experiment in hand. To assist with such records
a tentative protocol (Table 1) has been prepared. For some lines of
work it is obviously too complex while for others it is clearly inade-
quate.
A number of the items listed for records may be critical factors for
the success or failure of an experiment. Since each one cannot be
discussed, several examples are mentioned. (1) Infection may fail if
the incubation temperature is either too low or too high. Many plant
pathogens operate best between 18° and 30° C. (2) Plenty of moisture
is important for disease development, a deficiency of water often
being responsible for negative results. (3) The age of the plant or
of the part inoculated may influence the result. The relatively young
leaves are frequently more susceptible than old leaves to bacterial
leaf spots. (4) Some varieties of plants are highly resistant to patho-
gens which readily attack other varieties. Similarly, different strains
of bacteria often vary in pathogenicity.
INOCULATIONS WITH BACTERIA CAUSING PLANT DISEASE x^-lS
Table 1. Tentative Protocol for Plant Inoculations
Host: Manner of inoculation:
Variety Through soil
History Through wounds
Age Ry sprays
]Mon)hological condition Spreader used ....
Physiological condition By insects (name) . .
Susceptibility ^ , Stage in life cycle .
Enyironment Other means
Treatment before .
Treatment after..
Incubation:
Time
Enyironment:
Pathogen : Temperature .
Strain Moisture .
History Light .
Culture on Soil nutrients .
at °C.
for days Symptom.s :
Location
J , J Age of parts affected ,
Inoculum used: c t
Severity .
Diseased tissue Description : '
Entire culture
Early
Bacteria: Medmm ,
Turbidity Final . . .
Number per cc
Filtrate Effect on yield:
Products Quantity ....
Amount used per plant Quality
INTERPRETATION OF RESULTS
The results of research are vaHd only in accord with the reliability
of the methods employed and the accuracy of their interpretation.
After an experiment has been performed it is insisted that a report
of such work must not be published for the use of others until repeated
determinations have been made and the results have been satisfac-
torily analyzed. The simpler experiments are commonly performed
with suitable controls at least in duplicate or triplicate, and carried
through three separate times. A good investigator does not become so
enthusiastic about an experiment that he fails to view it impartially
and to accept sound evidence against it. On the contrary, he makes
every reasonable effort before publishing to find an error in the experi-
ment itself or in the conclusions drawn from it.
X45-14 MANUAL OF METHODS FOR PURE CULTURE STUDY
REFERENCES
Ark, p. a., and Hunt, M. J. 1941. Saprophytes antagonistic to phytopathogenic
and other microorganisms. Science, 93, 35-i.
Bennett, C. W. 1940. Acquisition and transmission of viruses by dodder {Cuscuta
suhinclusa). Phytopath., 30, 2 (Abstract).
Chester, K. S. 1933. The problem of acquired physiological immunity in plants.
Quart. Rev. Biol., 8, 129-54; 275-324.
. 1935. Serological evidence in plant-virus classiBcation. Phytopath., 25,
686-701.
DuGGAR, B. M., and Riker, A. J. 1940. The influence of ultraviolet irradiation
on the pathogenicity of Phytomonas tumejaciens. (Abstract). Photopath., 30, 6.
Flosdorf, E. W., and Mudd, S. 1935. Procedure and apparatus for preservation in
"lyophile" form of serum and other biological substances. J. Immun., 29, 389-
425.
Fred, E. B., Baldwin, L L., and McCoy, E. 1932. Root Nodule Bacteria and
Leguminous Plants. Univ. of Wis. Studies in Science No. 5.
Frobisher, M. 1933. Some pitfalls in bacteriology. J. Bact., 25, 565-71.
G GULDEN, C. H. 1939. Methods of Statistical Analysis. Wiley, New York and
London.
HiLDEBRAND, E. M. 1938. Techniques for the isolation of single microorganism!
Bot. Rev., 4, 627-64.
. 1942. A micrurgical study of crown gall infection in tomato. J. Agr.
Res., 65, 45-59, illus.
IvANOFF, S. S. 1934. A plant inoculator. Phytopath., 24, 74-6.
Johnson, J. 1937. Relation of water-soaked tissues to infection by Bacterium
angulatum and Bact. tahacum and other organisms. J. Agr. Res., 55, 599-618.
Keitt, G. W. 1918. Inoculation experiments with species of Coccomyces from stone
fruits. J. Agr. Res., 13, 539-69.
Keitt, G. W., Blodgett, E. C, Wilson, E. E., and Magie, R. O. 1937. The
epidemiology and control of cherry leaf spot. Wis. Agr. Expt. Sta. Research
Bui. No. 132.
Krueger, a. p. 1936. The nature of bacteriophage and its mode of action. Physiol.
Rev., 16, 129-72.
Leach, J. G. 1940. Insect Transmission of Plant Diseases. McGraw-Hill, New
York and London.
Link, G. K. K., Edgecombe, A. E., and Godkin, J. 1929. Further agglutination
tests with phytopathogenic bacteria. Bot. Gaz., 87, 531-47.
Link, K. P., and Walker, J. C. 1933. The isolation of catechol from pigmented
onion scales and its significance in relation to disease resistance in onions.
J. Biol. Chem., 100, 379-383.
OsBORN, E. M. 1943. On the occurrence of antibacterial substances in green plants.
Brit. J. Exp. Path., 24, 227-231.
Rawlins, T.E. 1933. Phytopathological and Botanical Research Methods. Wiley,
New York and London.
RiKER, A. J. 1940. Bacteria pathogenic on plants. In The Genetics of Pathogenic
Organisms, Pub. No. 12 of the A.A.A.S., Lancaster, Pa.
. 1942. The relation of some chemical and physico-chemical factors
to the initiation of pathological plant growth. Growth, Sup. to v. 6, 4 Sym.
Devlpmt. and Growth, 105-117, illus.
RiKER, A. J., and Baldwin, I. L., 1939. The efficiency of the poured plate technic
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RiKER, A. J., and Riker, R. S. 1936. Introduction to Research on Plant Diseases.
John S. Swift, St. Louis, Chicago, etc. [Planographed]
Rivers, T. M. 1937. Viruses and Koch's postulates. J. Bact., 33, 1-12.
Smith, E. F. 1905-1914. Bacteria in Relation to Plant Disease. Vols. 1, 2, & 3.
Carnegie Inst., Washington, D. C.
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Philadelphia and London.
Thomas, R. C. 1940. Additional facts regarding bacteriophage lytic to Aplanobacter
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Trussel, p. C, and Sarles, W. B. 1943. Effect of antibiotic substances upon
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INOCULATIONS WITH BACTERIA CAUSING PLANT DISEASE x,^l5
Van Lanen, J. M., Baldwin, I. L., and Riker, A. J. 1940. Attenuation of cell-
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White, P. R. 194.3. A Handbook of Plant Tissue Culture. Jaques Cattell Press ,
Lancaster, Pennsylvania.
INDEX
TO
MANUAL
OF METHODS FOR
PURE CULTURE STUDY OF BACTERIA
EDITION OF 1948-49
Issued Mat, 1949
Note. — This index applies lo the following editions of the various leaflets: Leaflet I,
9th, 1944: Leaflet II, 9th, 1944; Leaflet III, 4th, 1943; Leaflet IV, 9th, 1946; Leaflet V,
10th, 1947; Leaflet VI, 9th, 1942; Leaflet VII, 5th, 1948; Leaflet VIH, 6th, 1947;
Leaflet IX, 10th, 1948; Leaflet X, 3rd, 1945.
INDEX^
Acetone, determination of VI42 8
Acetyl-methyl-carbinol, determination of VI42 10-11
Acid dyes IV46 3
Acid-fast staining V47 7
recommended and alternate procedures IV46, 10-11
Acid fuchsin II44 7
Acid production in milk V47'21-22
Acids, fatty, determination of VI42 7-8
Acids in fermentation, nature of VI42 7-8
Acidity, determination of IX4S 15-17
Acidity, titratable VI42 7; IX48 15-16
Aerobes, special media for II44 9-14
Aerobes to absorb oxygen III43 6
Agar, ammonium phosphate II44 14; VI42 6
beef-extract II44 5; V47 15
blood II44 12; VI42 17
Churchman's gentian violet II44 9
meat infusion II44 5
semi-solid II44 5, 1 1
sugar II44 7
yeast extract II44 5
Agglutination VIII47 3, 5-9
as test for obligate anaerobes III43 13
Agglutination test, macroscopic,
preparation of antigen for VIII47 6
procedure for \ III47 7-9
tabulation of VIII47 7
microscopic, procedure for VIII47 6-7
Agglutinin absorption VIII47 9-10
procedure for VIII47 9
Albert's diphtheria stain (including Laybourne modification) IV46 13
Alcohols, cleavage of V47 14-18; VI42 5-12
determination of VI42 9
Alkaline egg medium II44 20
Alkaline gentian violet (Kopeloff & Beerman's) IV46 9
Amboceptor, anti-sheep cell VIII47 14
Amino nitrogen, determination of VI42 13-14
Ammonia, determination of VI42 14
Ammonia tests VI42 15
Ammonium oxalate crystal violet IV46 8
Ammonium phosphate media (liquid and agar) II44 14; VI42 6
Ammonium salts, action of bacteria on VI42 14
Anaerobic bacteria III43 3-23
enrichment of III43 16
fermentation reactions of III43 12
inoculation technics III43 9-20
isolation methods for III43 18-19
media for II44 14-23
determination of physiological reactions II44 18-22
plating for purification II44 17-18
microscopic examination of III43 14-16
morphology of III43 14
motility of III43 14
pathogenicity tests for III43 12
preliminary culture of III43 3-5
presumptive tests for III43 5
putrefactive changes of III43 1 1
separation from aerobes III43 13, 18-19
serological reactions III43 13
^In this index Roman numerals refer to the leaflet number, inferior Arabic numerals
to the year of the edition cited, and large Arabic numerals to the page of the leaflet.
INDEX 3
spore formation of 11143 15
storage of III43 20
toxin production by III43 21
Andrade's indicator II44 7
Anilin gentian violet (Ehrlich) IV46 7
Animals, laboratory, autopsy of VII48 9-10
use of VII48 5
Anthony's capsule stain IV46 19
Antibody, definition of VIII47 4
Antigen(s), definition of VIII47 4
for complement fixation test, preparation of VIII47 14
titration of VIII47 16-17
for macroscopic agglutination test, preparation of V'IIl47 6
Antigenic variations VII48 11
Antitoxins, titration of VIII47 19-20
Ascitic fluid agar II44 10
Ashby's mannitol solution II44 14
Autopsy of laboratory animals VII48 9-10
Bacteremia VII48 8
Bacterial juices, preparation of VI42 3
Bacterial smears, preparation of IV46 3
Bacterial stains, general ; IV46 6-8
Bailey's flagella stain (Fisher and Conn modification) IV46 17
Basal media for fermentation study VI42 6
for special groups of aerobes II44 9-11
Basic dyes I V46 3
Basic fuchsin II43 1 1 ; I V46 5, 6, 10
Beef-extract agar II44 5; V47 15
broth II44 5; V47 15
gelatin II44 5
Beef heart infusion medium II44 16
Beef liver infusion medium II44 17
Biochemical methods V47 9-22; VI42 2-18
in study of pathogenic properties VII48 13
Bismuth-sulphite agar II44 12
Biuret reaction VI49 13
Blood agar II44 12; VI42 17
broth II44 12; VI42 17-18
corpuscles, red, sheep's, preparation of VIII47 14
culture VII48 8-9
Bohme test for indole V47 12
Brain medium II44 21
Brewer anaerobic jar III43 10
Brewer culture dish III43 12
Brilliant green bile medium II44 12
Brom chlor phenol blue II44 7, 8
Brom cresol green II44 7, 8; V47 16; IX48 7, 8
Brom cresol purple II44 7, V47 16; IX48 7, 8, 13
in milk V47 20, 21
Brom phenol blue II44 8; V47 16; IX48 7, 8, 13
Brom thymol blue II41 6, 7, 8; IX48 7, 8, 13, 17
Broth, beef-extract II44 4; V47 15
blood II44 12; VI42 17-18
glucose III37 4
meat infusion II44 5
nitrate II44 8
sugar II44 6
yeast-extract II44 5
Buffered peptone solution for methyl red test II44 12
BufiFer action IX48 16
Buffer standards IX48 9-1 1
Burke's modification of Gram stain IV46 9
2,3-Butylene glycol, determination of 'VI42 11
Calomel electrode IX48 2-4
Calomel half-cell IX4, 19
Capsule stains III43 15; V47 8
recommended procedures IV46 18-20
4 PURE CULTURE STUDY OF BACTERIA
Carhohydrates, cleavage of V47 15-18; VI^: 5-12
Carhohydrate media, synthetic II44 1-1
Carhol-fuchsin (Kinyoun's or Ziehl's) IV46 5, 6, 10
Carl)ol gentian violet (Nicolle) IV46 7
Carhon componnds tor fermentation study VI42 5
Carl)on dioxide, determination of VI42 9-10
Casares-Gil's flagella stain IV46 IC
Chlor phenol red IX4S 7, 8
Chromium and sulfuric acid method III43 9
Chromogenesis V47 11
Churchman's gentian violet agar II44 9
Cleavage of sugars, alcohols and glucosides V47 15-18; VI42 5-12
Color changes of indicators 1X45 8
Color standards IX48 10-11, 12-14
Colorimetric measurement of pH IX4S 6-15
Complement, definition of VIII47 4
preparation of VIII44 14
titration of VIII47 16
Complement fixation VIII47 3, 11-19
preparation of reagents for VIII47 13-15
procedure for VIII47 18-19
tabulation of VIII47 18
titration of reagents for VIII47 15-19
Conklin modification of Wirtz spore stain IV46 12
Cooper method of acid-fast staining IV46 11
Corn liver medium II44 1 7
Cresolphthalein IX48 7
Cresol red II44 7; IX4S 7, 8, 13
Crystal violet II44 9; IV46 8-9
Dehydrogenases, determination of VI42 12
Demonstration of capsules and spores (by anaerobes), medium for II44 22
Descriptive chart, standard, description of V47 3-4, 12
history of I41 5-9
routine tests for V47 3-23
Descriptive chart for instruction, description of V47 4, 12
Desoxycholate agar II44 13
Desoxycholate-citrate agar II44 13
Determination of pathogenicity, factors interfering VILs 10-15
Differential media for special groups of aerobes ." II44 12-14
Diphtheria stain (Loeffler's, Albert's, Neisser's, Ponder's) IV46 13-15
Dissociation V47 6; VIII47 4
Dissociation constants (pk') of indicators IX48 7-8
Dorner's spore stain IV46 H
Douglas' trypsin broth II44 9
Drop-ratio standards of Gillespie IX48 12-14
Dyes, anilin IV46 3-22
Dye solubilities IV46 21-22
Egg medium with glycerol II44 11
Eh measurements IX48 18-20
Ehrlich-Bohme test for indole V47 12
Eldredge fermentation tube VI42 9-10
Electrode, glass IX48 5
hydrogen IX48 2
quinhydrone IX48 4
Electrometric measurement of pH IX48 2-6
Endo medium II44 11
Endospores, determining presence of V47 7
Eosin-methylene-blue agar, Levine's II44 12
Erythrocytes, bacterial action upon VI42 16-18
Fatty acids, determination of VI42 7-8
Fermentation V47 15-18; VI42 5-12
Fermentation tubes V47 17; VI42 9-10
Final H-ion concentration VI42 6
Fisher and Conn's flagella stain IV46 17
FlagelLi stain(s) III43 14; IV46 15-18
Fluorescent method for acid-fast staining IV46 10
INDEX 5
Fontana stain for spirochaetes I V^e 20
Formol titration VI42 13-14
Fuc'hsin, acid 11^4 7
basic ^ 1144 11; IV46 5, (), 10
carbol-, Kinvoun's iy^i\ (>
Zielil's . . ; IV4G 7, 10
Gelatin liquefaction, tests for V47 1-1-15; VL^ l;}-14
Gelatin media II44 5, (i, 20
General bacterial stains, recommended IV46 5
alternate IVje 6
(Jentian, violet agar. Churchman's II44 9
(Jentian violet, alkaline (Kopeloff & Beernian's) IV46 9
anilin (Ehrlich) IV46 7
carbol (NicoUc) IV46 7
Gillespie, drop-ratio standards of IX4S 12-14
Glass electrode IX48 5
Glossar.v I44 13-16
Glucosidcs, cleavage of V47 15-18; VI42 5-12
Gnezda test for indole V'47 12
Gore test for indole V47 12
Gram stain III43 14; IV43 9-10; V47 8
Burke and Kopeloff-Beernian modifications IV46 9
Hucker's modification IV46 8
interpretation of IV46 10
recommended procedures IV46 8-10
Granulose reaction 11143 16
Gray's flagella stain IV46 17
Hansen test for ammonia VI4.2 15
Hemolysin Vni47 14
preparation of Vni47 14
titration of VUL? 15
Hemolysis Vin47 4, 13
H-ion concentration IX48 2-24
final VI42 6
Hiss' capsule stain IV46 19
Hitchens' semi-solid glucose agar n44 5, 11
Hormone heart infusion broth, Huntoon's n44 H
Hucker modification of Gram stain I V46 8
Huntoon's hormone heart infusion broth n44 H
Hydrogen, determination of VI42 10
Hydrogen electrode IX47 2-4
Hydrogen half-cell IX48 2
Hydrogen sulfide, production of n44 21
Hydrogen sulfide production, determination of VI42 14
media for II44 9
tests for V47 13
Hydrolysis of starch V47 18-20
Immune serum, for agglutination, preparation of VIII47 5-6
for complement fixation test, preparation of VIII47 13-14
Incubation V47 5
Indicator(s)._ IL,4 6,7-8; V48 16; 1X48 7-9
Andrade's II44 7
color changes of IXjs 8
dissociation constant (pk') of IXis 7-8
Eh 1X48 21
Indicator media II44 7-8; VI42 16
milk ^ V47 21
Indole production, tests for II44 21 ; V47 11-13
Injection ^ II48 5-8
by cisterna puncture VILs 8
by inhalation ^ II4S 7
by insufl9ation VII48 7
cutaneous VII4S 6
intracardial VII4S 8
intracerebral VII48 7
intracranial VII48 7
6 PURE CULTURE STUDY OF BACTERIA
intracutaneous VII48 6
intramuscular VII48 6
int raperitoneal VII4S 7
int raplcural VII48 7
intraspinal VII48 8
intratracheal VII48 7
intravenous VII48 6
ophthalmic V^ILs 7
per OS VII48 7
subcutaneous VII48 6
Inoculation of animals VII48 5-8
Inoculations with bacteria causing plant disease X45 3-15
Iodine solution, Lugol's, Gram's modification IV46 8, 20
Kopcloff & IJeerman's modification IV46 9
Irregular forms, study of V47 8
Isolation methods for obligate anaerobes III4.3 18-19
Juices, bacterial, preparation of VI42 3
Koch's postulates VII48 4; X45 3
Kopeloff and Bcerman modification of Gram stain IV46 9
Kovacs test for indole \U7 12
Kracke and Teasley's medium II44 10
Laljoratory animals, autopsy of VII48 9-10
use of VII48 5-8
Lead acetate test strips II44 9
Leifson's capsule stain IV46 19
Leifson's flagella stain IV46 17
Levine's eosin-methylene-blue agar II44 12
Liquefaction of gelatin V47 14-15
Litmus II44 7, 8
Litmus milk II44 19; V47 21
Ljubinsky stain IV46 14
LoefHer's alkaline methylene blue IV46 6
LoefBer's blood serum II44 10
Lugol's iodine solution, Gram's modification IV46 8, 20
Macchia Velio's stain for Rickettsiae IV46 20
Macroscopic agglutination test, procedure for VIII47 7-9
Manual, history of I44 5-9
purpose of I44 2-5
use of I44 9-12
Mcintosh and Fildes jar IIL.i 1 1
Meat infusion agar and broth II44 5
Media, adjusting reaction of II44 6
l)asal, for fermentation study VI42 6
cultivation and storage II44 4-6
differential II44 6-9
for anaerobic bacteria II44 14-23
for special groups of aerobes II44 9-14
indicator II44 7-8
natural II44 6
preparation of II44 3-24
semi-solid II44 5, 1 1
sterilization of II44 3
synthetic II44 14
Mercaptans, volatile, determination of VI42 14
Meta-cresol purple IX48 7
Methyl orange IX48 8
Methyl red IX43 7, 8, 13
Methyl red test II44 12; V47 20-21
Methyh'ue blue II44 13
as indicator of anaerobiosis III43 4
Loeff^er's alkaline IV43 6, 13
Microscopic agglutination test, procedure for VIII47 6-7
Milk agar II44 21
Milk, acid production in V47 21-22
Milk as storage medium II44 6
INDEX 7
Milk, litmus V47 21
Milk, I'lricli indicator in V'j? 21
Morphology of obligate anaerobes 11141 4-16
Morphology, study of V47 6-8
Motility, determination of III43 14; V47 6-7
Much's method of acid-fast staining IV46 H
Mutations V47 6
Neisser's diphtheria stain IV48 14
Neutral solvents, determination of VI42 !)
Nigrosin solution, Dorner's IV46 7, 11
Nitrate agar II44 22
Nitrate broth II44 8
Nitrate medium, synthetic II44 14
Nitrate(s), action of bacteria on V47 9-11; VI42 14-16
reduction of V47 9-1 1 ; VI42 15
test for V47 10
Nitrite tests V47 9-10; VI42 15-16
Obligate anaerobes, (see anaerobic bacteria)
Optimum conditions for growth, determining V47 4-5
Optimum oxygen tension VI42 4
Oxidation-reduction indicators IX48 21
Oxidation-reduction potentials IX48 17-24
colorimetric measurement of IX48 21-24
potentiometric measurement of IX48 19-21
Oxygen, free, relation of bacteria to V47 9; VI42 4-5
Oxygen removal by combustion III43 10
Oxygen removal, biological methods for III43 5
Oxygen removal, chemical methods for III43 7
Oxygen tension, optimum VI42 4
Pathogenic aerobes, the study of VII48 3-15
Pathogenicity, determination, factors interfering VII48 10-13
Peptone solution, buffered, for methyl red test Il44 12
Peptone-tryptone-glucose agar II44 18
pH, colorimetric measurement of IX48 6-15
potentiometric measurement of 1X48 2-6
pH adjustment of media IX48 17
Phase mutations V47 6
Phenolphthalein IX48 7, 8, 15
Phenol red II44 6, 7; IX48 7, 8, 13
Phosphorus jar 11143 7
Plant disease, inoculations with bacteria X45 3-15
Plant inoculation methods:
Fungus inoculation X46 8
Insect inoculation X45 7
Seed inoculation X45 5
Soil "inoculation" X45 5
Spray inoculation X45 6-7
Virus inoculation X45 8-
Wound inoculation X45 7
Plant inoculations, protocol for X45 IS
Ponder's diphtheria stain IV46 14
Potentiometric measurement of pH IXis 2-6
Precipitation VIII47 3, 10-11
Precipitin, test, tabulation of VIII47 10
Precipitins, production of VIII47 10
Proteins, cleavage of VI42 13-14
Pyrogallol, alkaline III43 8
Pyruvic acid, determination of VI42 8
Quinhydrone electrode IX47 4
Reaction, determination of IX48 2-17
Reaction of media II44 6
Reducing medium for anaerobes III43 12
Reduction of nitrates V47 9-11
Reed and Orr's basal medium II44 19
8 PURE CULTURE STUDY OF BACTERIA
Rennet production V47 22
Residual sugar of fermentation VL12 7
Respiratory quotient VI42 4-5
Rickettsiae, stain for IV^e 20
SchaefTer and Fulton modification of Wirtz spore stain IV46 13
Semi-solid agar II44 5,11
Serological reactions VIII47 3-21
Sheep's red blood corpuscles, preparation of VIII47 14
Skatole II44 21
Soil as storage medium II44 6
Solubility of dyes IV46 21-22
Spirochaete stains IV46 20
Spore formation of ol)ligate anaerobes III43 15
Spore production (by anaerobes), medium for II44 22
Spore staining, recommended procedures IV46 11
alternate procedures IV46 13
Spray plate cultures III43 8
Spray's basal medium II44 19
Staining acid-fast bacteria IV46 10-11; V47 7
capsules IV46 18-20; V47 8
diphtheria organisms, recommended procedures IV46 13-14
alternate procedures IV46 14
flagella, recommended procedures IV46 15-18
spirochaetes I V46 20
spores IV46 11-13
Staining methods IV46 3-22
Starch, hydrolysis of V47 18-20
Sterilization II44 3
Succinic acid, determination of VI42 8
Sugar agar II44 7
broths II44 6
Sugars, cleavage of V47 15-18; VI42 5-12
determination of VI42 7
residual, of fermentation VI42 7
Sulphonphthalein indicators II44 7-8
Synergism VII48 1 2
Synthetic media II44 14
Tellurite agar II44 13
Thermal death point V47 5
ThioglycoUate agar II44 f5, 17
Thomas' test for ammonia \T42 15
Thymol blue IX48 7, 8, 13
Titratable acidity VI42 7; IX43 15-17
Titration of reagents for complement fixation VIII47 15-19
Titration of toxins, toxoids and antitoxins VHI47 19-20
Toxins, bacterial VII48 4
titration of Vni47 19-20
Toxin production (by anaerobes) III43 21
medium for II44 22
Toxoids, titration of VHI47 19-20
Trypsin Ijroth, Douglas' II44 9
Tunnicliff's stain for Spirochaetes IV46 20
Ulrich indicator in milk V47 21
Variation V47 6; VH48 11-12
Vegetable tissue jar III43 5
Virulence VII48 4
Voges-Proskauer test II44 12; V47 20-21; VI42 10-11
Wirtz spore stain (Schaeffer- Fulton and Conklin modifications) IV46 12, 13
Yeast-extract broth and agar 1144 5
Yeast infusion glucose agar II44 18
Ziehl-Neelsen method of acid-fast staining IV46 10
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