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MANUAL 

OF METHODS FOR 

PURE CULTURE STUDY OF 
BACTERIA 



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^^ ociETY OF American Bacteriologists 

65 

.S6 
1946 



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This Manual is published in loose-leaf form so 


that it can be revised, leaflet by leaflet, and thus kept 


up to date. The revised leaflets are issued in the , 


semi-annual publication, Pure Culture Study of 


Bacteria ; each leaflet thus issued is punched to fit 


the cover of this Manual. The subscription rate of 


Pure Culture Study of Bacteria is $1.25 per year 


($1.00 cash). 


To keep this copy of the Manual up to date, 


detach the card below and mail it, with remittance 


to Biotech Publications, Lock Box 269, Geneva, 


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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. 

REFERENCES 

Barker, H. A. 1936. Studies upon the methane-producing bacteria. Arch. Mikrob., 
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- 

sulfuric acid method for anaerobic cultures. J. Bact., 41, 301-303. 
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. 
Prevot, A.-R. 1925. Les streptocoques anadrobies. Ann. In.st. Pasteur, 39, 417- 

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. 

Ann. Inst. Pasteur, 60, 285-307. 
Pr^vot, A.-R. 1940a. Etudes de syst(5matique hactdrienne. V. Essai de classifi- 
cation des vihrions anadrobies. Ann. In.st. Pasteur, 64, 117-125. 
Prdvot, A.-R. 1940b. Manual de Classification et de Ddtermination des Bacleries 

Anadrobies. Masson et Cie., Paris. 223 pp. 
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, 309-320. 
Reed, G. B., Orr, J. H., and Baker, M. C. 1939. Gas-gangrene-toxin production 

Soc. Expt. Biol, and Med., Proc, 42, 620-621. 
Robertson, M., and O'Brien, R. A. 1929. The organisms associated with gas 

gangrene. Chap. IX in System of Bacteriology, Vol. 3, (Gt. Brit.) Med. 

Research Council. 
Roe, A. F. 1940. Report on viability of 200 cultures of anaerobes desiccated for 

six years. J. Bact., 39, 11-12. 
Rosenthal, L. 1937. "Chromium-sulfuric acid" method for anaerobic cultures 

J. Bact., 34, 317-320. 
Snieszko, S. 1930. The growth of anaerobic bacteria in petri dish cultures. Centbl. 

Bakt., II Abt., 82, 109-110. 
Soule, M. H. 1932. Anaerobic technic. J. Lab. and Clin. Med., 17, 519-529. 
Spray, R. S. 1930. An improved anaerobic culture dish. J. Lab. and Clin. Med., 16, 

203-206. 
Spray, R. S. 1936. Semisolid media for cultivation and identification of the sporulat- 

ing anaerobes. J. Bact., 32, 135-155. 
Weinberg, M., and Ginsbourg, B. 1927. Donndes Rdcentes sur les Microbes Anaero- 

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Weinberg, M., and Sdguin, P. 1918. La Gangrene Gazeuse. Masson et Cie., Paris. 
Weinberg, M., Nativelle, R., and Prdvot, A.-R. 1937. Les Microbes Anaerobies 

Masson et Cie., Paris. 1186 pp. 
Zeissler, J. 1930. Anaerobenzuchtung. In Kolle, W., Krause, R., und Uhlennuth, 

P., Handbuch der pathogenen Mikroorganismen, 3 Aufl., 10, 35-144. 
Zeissler, J., und Rassfeld, L. 1928. Die anaerobe Sporenflora der europaischen 

Kriegsschauplatze 1917. VerofTentl. aus der Kriegs- und Konstitutionspathologie, 

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 


0.74 


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 


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 


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. 
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Bartholomew, J. W., and Umbreit, W. W. lOl'l.. Riljonucleic acid and the Gram 

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Beni.'VNS, T. II. C. 1916. Relief staining for bacteria and spirochaetes. Brit. Med. 

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Blumenthal, J. M. and Lipskerow, M. 1905. Vergleichcnde Bewertung der dif- 

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Burke, Victor. 1921. The Gram stain in the diagnosis of chronic gonorrhea. 

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Cooper, F. B. 1926. A modification of the Ziehl-Neelsen staining method for 

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Dorner, W. C. 1930. The negative staining of bacteria. Stain Techn., 5, 25-7. 
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Fontana, Artur. 1912. Verfahren zur intensiver und raschen Farbung des Tre- 
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micro-organisms. Nature, 151, 671. 
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IV46-24 MANUAL OF METHODS FOR PURE CULTURE STUDY 

Mallory, F. B. and Wright, J. H. 1924. Pathological Technique. 8th Ed. 

Saunders, Philadelphia. 
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Schaeffer, Alice B. and Fulton, McD. 1933. A simplified method of staining 

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Techn., 1, 143. 
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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, echinula te, 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 



3 rt 









S S 



> M E 



E § .2 '^^ 



fe S M S Q 






p w CO < c/a 



d 

d 
B 

►J 
S 

O 

o 

o 
< 


Q 










































































2 

1 


§ 
1 


3 
o 
■d 

1 


3 
o 

C 

c 

(2 


c 
o 

a! 

'S 
O 

a 


o 

•T3 3 



6 

d 
G 

2; 

o 

<J 

Z 
W 

:s 

w 






















asojong 










SSOJDBq 










osooniQ 










Medium: 

Carbohydrate: % 
Indicator: 


>. 


< 


•a 


O 



o o o o g w 



S- S S- 
S" S- s- 

(13 i» [U 

oi CO 3 

p p 3 



o e i 



3 ?3 a 

° r 
- > 



O 
P w 



9 ?. S 



5 9-9- 

z 



3 <2 



S S 



w 

> 
H 

o 
Z 

H 
O 

fO 

w 
w 

o 

X 
►<! 

o 
w 
z 



? s ? 



o- 3 



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.) 

REFERENCES 

American Gas Association. 1929. Gas Chemists' Handbook, JrcZ. ed. 725 pp. New York. 
Association of Official Agricultural Chemists. 1940. Official and Tentative 

Methods of Analysis, oth ed. Washington, D. C. 
AuERBACH, F. and Zeglin, H. 1922. Beitrage zur Kenntnis der Ameisensaure. Zts. 

physik. Chem., 103, 161-77. 
Booth, V. H. and Green, D. E. 1938. A wet crushing mill for microorganisms. Bio- 

chem. J., 32, 855-61. 
Brockmann, M. C. and Werkman, C. H. 1933. Determination of 2, 3-butylene glycol 

in fermentations. Ind. Eng. Chem., Anal. Ed., 5, 206. 
Brown, J. H. 1919. The use of blood agar for the study of streptococci. Monograph 

No. 9, Rockefeller Institute for Medical Research. 
Bronfenbrennhr, J. and Schlesinger, M. J. 1920. A study of nitrate reduction by 

bacteria. Abstr. Bact., 4, 2. 
Brown, J. H. 1923. The formol titration of bacteriological media. J. Bact., 8, 245-67. 
Conn, H. J. 1936. On the detection of nitrate reduction. J. Bact, 31, 225-33. 
Dixon, M. 1934. Manometric Methods. The Macmillan Co., New York. 
FoLTN, O. 1902. Eine neue Methode zur Bestimmung des Ammoniaks im Harne und 

anderen thierischen Fliissigkeiten. Zts. Physiol. Chem., 37, 161-76. 
Friedemann, T. E. and Graeser, J. B. 1933. The determination of lactic acid. J. Biol. 

Chem., 100, 291-308. 
Fromageot, Claude and Desnuelle, Pierre. 1935. Eine neue Methode zur 

Bestimmung der Brenztraubensaure. Biochem. Zts., 279, 174-83. 
Gillespie, L. J. and Walters, E. H. 1917. The possibilities and limitations of the 

Duclaux method for the estimation of volatile acids. J. Am. Chem. Soc, 39, 

2027-55. 
Goodwin, L. F. 1920. The analysis of acetone by Messinger's method. J. Am. Chem. 

Soc, 42, 39-45. 
GozsY, B. 1935. Mikrobernsteinsaurebestimmung und ihre Anwendung. Zts. Physiol. 

Chem., 236, 54-8. 
Green, D. E. 1936. a-Glycerophosphate dehydrogenase. Biochem. J., 30, 629-44. 
Hansen, P. A. 1930. The detection of ammonia production by bacteria in agar slants. 

J. Bact., 19, 223-9. 
Hewitt, L. 1932. Bacterial metabolism. L Lactic acid production by hemolytic 

streptococci. Biochem. J., 26, 208-17. 
Hopkins, F. G. and Dixon, M. 1922. On glutathione. H. A thermostabile oxidation- 
reduction system. J. Biol. Chem., 54, 527-63. 
Hucker, G. J. and Wall, W. A. 1922. The use of agar slants in detecting ammonia 

production by bacteria. J. Bact., 7, 513-6. 



FURTHER BIOCHEMICAL METHODS VI4.-I9 

Johnson, M. J. 1932 Determination of small amounts of etliyl and butyl alcohol. 
Ind. Eng. Chem., Anal. Ed., 4, 20-22. 

Johnson, M. J., Peterson, W. H. and Fred, E. B. 1931. Oxidation and reduction 
relations between substrate and products in the acetone-butyl alcohol 
fermentation. J. Biol. Chem., 91, 569-91. 

Kltjyver et al. 1925. Ueber die Bildung von Acetylmethylcarbinol und 2, 3-Butylen- 
glykol im Stoffweclisel dor Ilefe. Bioch. Zts., 161, 3G1-78. 

Krebs, H. a. 1937. Role of fumarate in respiration of B. coli commune. Biochem. J., 
31, 2095-2124. 

Langlykke, a. F. and Peterson, W. H. 1937. Determination of acetylmethyl- 
carbinol. Ind. Eng. Chem., Anal. Ed., 9, 163-6. 

Leifson, Einar. 1932. Types of bacteria on blood and chocolate agar and the im- 
mediate cause of these types. J. Bact., 24, 473-84. 

Lemoigne, M. 1913. Assimilation du saccharose par les bacteries du gro upe du "B. 
subtilis". Fermentation butyleneglycolique. Ann. Inst. Past., 27,856-85. 

MoYLE, D. M. 1924. A quantitative study of succinic acid in muscle. Biochem. J., 
18, 351-64. 

McCuLLOCH, Andrew. 1938. Gas Analysis, 166 pp. Witherbjs London. 

OsBURN, O. L. and Weukman, C. II. 1931. Determination of organic acids. V. Applica- 
tion of the partition method to quantitative determinations of acetic, proi)io- 
nic and butyric acids in mixtures. Ind. Eng. Chem., Anal. Ed., 3, 264. 

OsBURN, O. L., Wood, H. G. and Werkman, C. H. 1933. Determination of formic, 
acetic and propionic acids in a mixture. Ind. Eng. Chem., Anal. Ed., 5, 
247-50. 

OsBURN, O. L., Wood, H. G. and Wekkman, C. H. 1936. The determination of 
volatile fatty acids by the partition method. Ind. Eng. Chem., Anal. Ed., 8. 
270-5. 

Peters, J. P. and Van Slyke, D. D. Quantitative Clinical Chemistry. Baltimore, Md. 

1931. Vol. 1. Interpretations. 

1932. Vol. 2. Methods. 

Potter, V. R. and Elvehjem, C. A. 1936. A modified method for study of tissue 

oxidations. J. Biol. Chem., 114, 495-504. 
Shaffer, P. A. and Hart.mann, A. F. 1921. The iodometric determination of copper 

and its use in sugar analysis. II. Methods for the determination of reducing 

sugars in blood, urine, milk and other solutions. J. Biol. Chem., 45, 365-90. 
Shwachm.\n, H., Hellerman, L., and Cohen, B. 1934. On the reversible inactivation 

of pneumococcal hemolysin. J. Biol. Chem., 107, 257-65. 
Simon, L. J. and Piaux, L. 1924. Detection and estimation of small amounts of pyruvic 

acid. Bull. Soc. Chem. Biol., 6, 477-87. 
SouLE, M. II. 1928. Gas Metabolism of Bacteria. Chap, xviii in Jordan and Falk's 

Newer Knowledge of Bacteriology and Immunology. Univ. of Chicago Press. 
Stahly, G. L., Osburn, O. L. and Werkman, C. H. 1934. Quantitative determination 

of acetone and ethyl, butyl and iso-propyl alcohols in fermentation liquors. 

The Analyst, 59, 319-25. 
Stahly, G. L. and Werkman, C. H. 1936. Determination acetylmethylcarbinol in 

fermentation liquors. Iowa State Col. J. Sci., 10, 205-11. 
Stiles, H. R., Peterson, W. H. and Fred, E. B. 1926. A rapid method for the deter- 
mination of sugar in bacterial cultures. J. Bact., 12, 427-39. 
Straub, F. B. 1936. Bestimmung der Brenztraubensaure mit Salicylaldehyd. Zts. 

Physiol. Chem., 244, 117-9. 



VI42-20 MANUAL OF METHODS FOR PURE CULTURE STUDY 

Thunberg.T. 1933. Zur Kenntnis der Spezifitat der Dehydrogenasen. Biochem. Zts 
258, 48-64. 

VanSlyke, D D. 1913. The quantitative determination of aliphatic amino groups. 
J. Biol. Chem., 12, 275-84. 

Van Slyke D. D. 1929. Manometric determination of primary amino nitrogen and its 
application to blood analysis. J. Biol. Chem., 83, 425-47. 

Van Slyke, D. D. and Cullen, G. E. 1916. The determination of urea by the urease 
method. J. Biol. Chem., 24, 117-22. 

Weil-Malherbe, H. 1937. Studies on brain metabolism. II. Formation of succinic 
acid. Biochem. J., 31, 299-312. 

Werkman, C. H. and Wood, H. G. 1940. Gewinnung freigeloster Enzyme Spezial- 
methoden fiir Bacterien. From: Bamann and Myrback, Die Methoden der 
Fermentforschung. Geo. Thieme, Leipzig, pp. 1191-1214. 

WiGGERT, W. P., Silverman, M., Utter, M. E. and Werkman, C. H. 1940. Prepara- 
tion of an active juice from bacteria. la. State Col. J. of Sci., 14, 179-86. 

Virtanen, a. I. and Pulkki, L. 1928. The volatility with steam of water soluble 
organic substances. J. Am. Chem. Soc, 50, 3138-51. 

Wendel, W. B. 1932. The determination of pyruvic acid and the preparation of 
lithium pyruvate. J. Biol. Chem., 94, 717-25. 

YuDKiN, John. 1933. The dehydrogenases of Bacterium coli. I. The effect of dilution: 
with a note on the existence of a co-enzyme of glucose dehydrogenase. Bio- 
chem. J., 27, 1849-58. 

ZoBell, C. E. 1932. Factors influencing the reduction of nitrates and nitrites by bac- 
teria in semisolid media. J. Bact., 24, 273-81. 



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 


12 ml. 


0.25 ml. 


0.25 ml. 


0.63 ml. 




Complete 


5 


11 ml. 


0.25 ml. 


0.25 ml. 


0.64 ml. 




Complete 


6 


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 


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 

as applied to bacterial plant pathogens. Phytopath., 29, 852-63. 
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. 
. 1920. An Introduction to Bacterial Disea.ses of Plants. Saunders, 

Philadelphia and London. 
Thomas, R. C. 1940. Additional facts regarding bacteriophage lytic to Aplanobacter 

Stewartii. Phytopath., 30, 602-611. 
Trussel, p. C, and Sarles, W. B. 1943. Effect of antibiotic substances upon 

Rhizobia. J. Bact., 45, 29. 



INOCULATIONS WITH BACTERIA CAUSING PLANT DISEASE x,^l5 

Van Lanen, J. M., Baldwin, I. L., and Riker, A. J. 1940. Attenuation of cell- 
stimulating bacteria by specific amino acids. Science, 92, 512-513. 

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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