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DIFCO MANUAL

DEHYDRATED CULTURE

MEDIA and REAGENTS

for

MICROBIOLOGICAL and CLINICAL

LABORATORY PROCEDURES

NINTH EDITION

DIFCO LABORATORIES

INCORPORATED

DETROIT 1, MICHIGAN

First Edition

1927

Second Edition

1929

Third Edition

1931

Fourth Edition

1933

Fifth Edition

1935

Sixth Edition

1939

Seventh Edition

1943

Eighth Edition

1948

Ninth Edition

1953

Reprinted

1953

INCORPORATED

Detroit 1, Michigan, U. S. A.
Printed in U. S. A.

APPRECIATION

Difco Laboratories desires hereby to acknowl-
edge its indebtedness to the many microbiolo-
gists from whom it has so often sought advice
and whose cooperation and assistance it has so
frequently received in the development, im-
provement and standardization of these prod-
ucts.

Acknowledgment is also made of the use of
published papers and textbooks as a source of
much of the material in this book.

Foreword

This edition of the Difco Manual, the ninth published since 1927, has
been completely revised and rewritten. The Ninth Edition is the first to
include the reagents used in the preparation of media for in vitro propa-
gation and maintenance of tissue cells.

When we first introduced prepared dehydrated culture media to the
scientific world, they were regarded as laboratory curiosities. Today, Difco
products are used in microbiological laboratories in all parts of the world.
Difco products are not only being used by a continually increasing number
of investigators, but are also constantly extending into new fields of micro-
biology. Bacto Dehydrated Culture Media and laboratory reagents are
used in municipal and state health departments, water and sewage works,
in dairies, in medical and technical colleges and the larger universities, in
private diagnostic laboratories, hospitals, and commercial laboratories.
Research laboratories maintained by the Federal Government are also
users of Difco products.

Recommendation and approval have been extended to our products by
the authors of many standard textbooks of bacteriology and by the com-
mittees on methods and procedures of scientific societies such as the
American Public Health Association, the American Dairy Science Associa-
tion and others.

Bacto Dehydrated Culture Media and laboratory reagents are prepared
according to available standards or accepted formulae. Each ingredient is
thoroughly tested and the final product is subjected to rigid examination
and is standardized before being offered for distribution. The new section
entitled Tissue Culture Media Reagents describes the products prepared
in collaboration with the Tissue Culture Association and are tested and
certified for tissue culture procedures.

The dehydrated media are stable and resist deterioration over long
periods of time. They are readily and quickly prepared for use and yield
uniform lots of media which provide identical nutritional conditions at all
times. Aside from these readily apparent advantages, Bacto Dehydrated
Culture Media are truly economical when actual cost comparisons are
made.

Grateful acknowledgment is made of the support we have received
from bacteriologists the world over. It is the desire of our organization to
continue and to extend our services to the advancement of microbiology
and related sciences.

Difco Laboratories

Table of Contents

PAGE

Foreword 7

Introduction 11

Origin of Dehydrated Culture Media 15

General Conditions Pertaining to the Cultivation of Microorganisms . . 16

Preparation of Media from Dehydrated Culture Media, Difco ... 21

Dehydrated Culture Media 23

Guides for the Selection of Culture Media 23

Media for Examination of Water and Sewage 29

"Standard Methods" Media 29

Supplementary Media for Water and Sewage Examination ... 41

Media for Examination of Dairy and Other Food Products .... 57

"Standard Methods" Media 57

Supplementary Media for Dairy and Other Food Products .... 68

Media for Lactobacilli 72

Media for Cultivation of Pathogenic Microorganisms 76

Infusion Media 76

Peptone Media (without infusions) 99

Differential Media 130

Schema for Examination of Stools 130

Primary Plating Media 131

Differential Liquid Enrichments 156

Differential Tube Media 159

Sterility Test Media 195

Media for Microbiological Assays 203

Media for the Assay of Antibiotics 203

Media for the Assay of Vitamins and Amino Acids 212

Media for Mycology 237

Ingredients of Culture Media 255

Peptones, Difco 255

Enzymatic Hydrolysates 255

Hydrolysates, Acid 265

Amino Acids 268

Extracts 269

Enrichments 271

Enzymes 278

Bile Products 286

Dehydrated Meats for Infusions 288

Solidifying Agents 290

Carbohydrates, Polyhydric Alcohols and Glucosides 291

Carbohydrate Solutions in Ampuls 292

9 69966

10 CONTENTS

Dyes and Indicators 293

Biochemicals 296

Miscellaneous Ingredients 299

Tissue Culture Media Reagents, Difco 301

Methods of Tissue Culture 307

Serological Reagents for Diagnosis of Syphilis 309

Reagents for Complement-Fixation Tests 309

Reagents for Precipitation Tests 311

Cardiolipin Antigens 313

Reagents for Preparation of Antigens 315

Diagnostic Reagents 317

Miscellaneous Products 338

Dehydrated Media in Special Packages 338

Prepared Media in Tubes and Bottles 338

Index 343

Introduction

Bacteriology emerged as a definite branch of science as a result of the monu-
mental and immortal researches of Pasteur and Koch. When in 1876 Robert
Koch, for the first time in history, propagated a pathogenic bacterium in pure
culture outside the body, he not only established Bacillus anthracis as the cause
of a specific disease in cattle, but he inaugurated a method of investigation of
disease in general which is still pursued with unabated vigor. The decade immedi-
ately following Koch's epoch-making introduction of solid culture media for the
isolation and growth of bacteria ranks as one of the brightest in the history of
medicine because of the number, variety, and brilliance of the discoveries made
in that period. These discoveries, which, as Koch himself expressed it, came "as
easily as ripe apples fall from a tree," were all dependent upon and resulted from
the evolution of correct methods for the "in vitro" growth of bacteria.

The fundamental principles elaborated at that time, of which the most im-
portant was the introduction of the "poured plate" method for isolation of pure
cultures, still constitute the foundation of bacteriological research. Nevertheless,
it has become more and more apparent that a successful attack upon the problems
still unsolved is closely related to, if not dependent upon, a thorough understand-
ing of the minutiae influencing bacterial nutrition. With a suitable culture
medium, properly used, advances in bacteriology are more readily made than
when either the medium or method of use is inadequate. The bacteriologist of
today is, therefore, largely concerned with the evolution of methods for the
development and maintenance of growth of bacteria, upon which an understand-
ing of the biological and biochemical phenomena resulting from bacterial activity
must largely be dependent.

The chemical analyses of bacteria indicate that they are essentially water plants,
the protoplasm of which contains chemical elements found in other types of plant
protein (Ford). In order to build up the cells of bacteria in their anabolic phase,
the protoplasmic elements must be found in the immediate environment. So vast
is the problem of bacterial metabolism, and so numerous and diverse are the
minutiae it includes, that a comprehensive discussion of its ramifications is pre-
cluded here. It is the purpose of this discussion to present briefly only some of
the outstanding developments in this phase of bacteriology, especially as they con-
cern the evolution of new methods for the cultivation and study of bacteria.

Almost without exception wherever bacteria occur in nature, and this is particu-
larly true of the pathogenic forms, nitrogenous materials and carbohydrates are
present. These are utilized in the maintenance of growth and for the furtherance
of bacterial activities. So complex is the structure of many of these substances,
however, that before they can be utilized by bacteria they must be broken down
into simpler compounds. Such alterations are effected by processes of hydrolysis,
oxidation, reduction, deamination, etc., and are the result of bacterial activities of
primary and essential importance. These changes are ascribed to the activities
of bacterial enzymes which, obviously, are both numerous and varied. The proc-
esses involved, as well as their end-products, are exceedingly complex; those of
fermentation, for example, result in the production of such end products as acids,
alcohols, ketones, and gases including hydrogen, carbon dioxide, methane, etc.

Abstract as such studies of bacterial metabolism may seem, their practical
application is soon apparent. From these studies has come a better understanding
of the nutritional needs of bacteria, and from this in turn has come the develop-
ment of culture media productive of rapid and luxuriant growth — essential req-
uisites for the isolation and study of specific organisms.

12 INTRODUG TION

Studies to determine the forms of carbon, hydrogen, oxygen, and nitrogen
which could most easily be utilized by bacteria for their development were
originally carried on by Naegeli between 1868 and 1880, and were published by
him^ in the latter year. Naegeli's report covered the use of a large variety of sub-
stances including carbohydrates, alcohols, amino acids, organic nitrogen com-
pounds, and inorganic nitrogen salts.

The first reference to the use of peptone for the cultivation of microorganisms
is that made by Naegeli in the report referred to above, when in 1879 he compared
peptone and ammonium tartrate. Because of its content of amino acids and other
nitrogenous compounds which are readily utilized by bacteria, peptone soon be-
came one of the most important constituents of culture media, as it still remains.
In the light of our present knowledge, proteins are believed to be complex com-
pounds of amino acids joined together mainly by means of the peptide linkage.
When subjected to hydrolysis proteins yield metaproteins, proteoses, peptones,
polypeptides, and finally the chemically simpler amino acids and their analogs.
The intermediate products should be considered as classes of compounds, rather
than individual substances, for there exist no sharp lines of demarcation between
the various classes — one group shades by imperceptible degrees into the next. All
bacteriological peptones, thus, are mixtures of various products of protein hy-
drolysis. Not all the products of protein decomposition are equally utilizable by
all bacteria. In their relations to proteins, bacteria may be divided into two
classes; those which decompose naturally occurring proteins, and those which re-
quire simpler nitrogenous compounds such as peptones and amino acids.

The relation of amino acids to bacterial metabolism, and the ability of bacteria
to use these compounds, have been studied by many workers. DuvaP.^, for ex-
ample, reports that cysteine and leucine are essential in the cultivation of Myco-
bacterium leprae. Kendall, Walker, and Day* and Long^ report that the growth
of M. tuberculosis is dependent upon the presence of amino acids. Many other
workers have studied the relation of amino acids to the growth of other organisms,
as for example, Hall, Campbell, and Hiles^ to the meningococcus and strepto-
coccus; Cole and Lloyd''' and Cole and Onslow^ to the gonococcus; and Jacoby
and FrankenthaP to the influenza bacillus. Indispensable as amino acids are to
the growth of many organisms, certain of them in sufficient concentration may
exert an inhibitory effect upon bacterial development.

From the data thus far summarized, it is apparent that the problem of bacterial
metabolism is indeed complicated, and that the phase concerned with bacterial
growth and nutrition is of the utmost practical importance. It is not improbable
that bacteriological discoveries of the greatest etiological value await merely the
evolution of suitable culture media and methods of utilizing them, just as in the
past important discoveries were long delayed because of a lack of similar require-
ments. Bacteriologists are therefore continuing to expend much energy on the
elucidation of the minutiae of bacterial metabolism, and are continuing to seek
methods of applying, in a practical way, the results of their studies.

While the importance of nitrogenous substances for bacterial growth was
recognized early in the development of bacteriological technique, it was also real-
ized, as has been indicated, that bacteria could not always obtain their nitrogen
requirements directly from protein. It is highly desirable, in fact essential, to
supply nitrogen in readily assimilable form, or in other words, to incorporate in
media proteins which have already been partially broken down into their simpler
and more readily utilizable components. Many laboratory methods, such as
hydrolysis with alkali^^, acid^^'^-'^^, enzymatic digestion^^'^'^^'^^'^^-^^, partial di-
gestion of plasma^^, et al., have been described for the preparation of protein
hydrolysates.

The use of protein hydrolysates, particularly gelatin and casein, has stimu-

INTRODUCTION

lated a revival of interest in studies of the development of bacterial toxins and
has opened entirely new fields of research such as the microbiological assay of
vitamins and amino acids. The importance of these new paths of investigation
can, as yet, only be estimated but it is already apparent that the knowledge which
has been acquired is of essential value in an understanding of bacterial metabo-
lism. Among studies in the first of these fields may be mentioned the work of
Mueller and his associates^^'^^'^^'^^'^*-^^ on the production of diphtheria toxin;
that of Tamura et al.^® of toxin of Clostridium welchii; that of Bunney and
Loerber2'''28 on scarlet fever toxin, and of Favorite and Hammon^^ on staphylo-
coccus enterotoxin. The work of Snell and Wright^^ may be mentioned as but one
of many references to investigations on the assay of vitamins by means of
microbiological methods. Closely associated with research of this nature are such
studies as those of Mueller^^'^^ on pimelic acid as a growth factor for Corynebac-
terium diphtheriae, and those of O'Kane^^ on synthesis of riboflavin by staphylo-
cocci.

In this brief discussion of certain phases of bacterial nutrition we have at-
tempted to indicate the complexity of the subject and to emphasize the impor-
tance of continued study of bacterial nutrition. Difco Laboratories has been en-
gaged in research closely allied to this problem in its broader aspects since 1914
when Bacto-Peptone was first introduced. Difco Dehydrated Culture Media, and
ingredients of such media, have won universal acceptance as useful and depend-
able laboratory adjuncts in all fields of microbiology.

'I:

1 Sitz'ber, math.-physik. Klasse Akad. Wiss.
Muenchen, 10:277:1880.

a J, Exp. Med., 12:46:1910.
Exp. Med., 13:365:1911.
Infectious Diseases, 15:455:1914.

6 Am. Rev. Tuberculosis, 3:86:1919.

«Brit. Med. J., 2:398:1918.

■^ J. Path. Bact., 21:267:1917.

8 Lancet, 11:9:1916.

8 Biochem. Zeit., 122:100:1921.
10 Centr. Bakt., 1:29:617:1901.
"Indian J. Med. Research, 5:408:1917-18.
13 Compt. rend. soc. biol., 78:261:1915.
"J. Bact., 25:209:1933.
i*Ann. de I'lnst., Pasteur, 12:26:1898.
15 Indian J. Med. Research, 7:536:1980.
18 Sperimentale, 72:291:1918.

"J. Med. Research, 43:61:1922.
18 Can. J. Pub. Health, 32:468:1941.
lOCentr. Bakt., 1:77:108:1916.
» J. Bact., 29:515:1935.

21 Brit. J. Exp. Path., 27:335:1936.

22 Brit. J. Exp. Path., 27:342:1936.

23 J. Bact., 36:499:1938.

24 J. Immunol., 37:io3:i939-

25 J. Immunol., 40:21:1941.

28 Proc. Soc. Expl. Biol. Med., 47:284:1941.

27 J. Immunol., 40:449:1941.

28 J. Immunol., 40:459:1941.

29 J. Bact., 41:305:1941.

30 J. Biol. Chem., 139:675:1941.

31 J. Biol. Chem., 119:121:1937.

32 J, Bact., 34:163:1940.
83 J. Bact., 41:441:1941.

THE ORIGIN

of Dehydrated Culture Media

It is a pleasure to include, as a part of this book, the abstract given below
which is believed to be the earliest reference to the preparation and use of de-
hydrated culture media, at least in this country. It is to be noted that Dr. Frost's
arguments in favor of these preparations are just as forceful today as they were
in 1909.

DESICCATED CULTURE MEDIA

W. D. Frost

University of Wisconsin

Abstract of Paper at Boston (1909) Meeting of the Society of American Bacteri-
ologists. Science, 31:555: (Apr. 8) 1910.

In order to overcome the generally recognized faults of bacterial culture media,
such as variation in composition of small batches, time consumed in preparation,
rapidity with which it deteriorates, its unavailability in small institutions or pri-
vate practice, the preparation of culture media in large batches in establishments
especially equipped for it and then desiccated is suggested.

The author's work on this problem, covering nearly a decade of time, is con-
sidered and samples are submitted.

There is apparently no reason why the different culture media cannot be put
on the market in the form which requires merely the addition of water and sterili-
zation to make it ready for use. Not only the ordinary, but probably most of the
special media, can be prepared in this way and could be put up where desired,
in the form of tablets, these to be of such size that they could be put directly in
test tubes and when the proper amount of water is added they would be ready
for sterilization and use.

It is interesting to note that Doerr, in Kraus and Uhlenhut: Handbuch der
Mikrobiologischen Technik, states he also prepared powdered culture media by
drying on glass in 1909.

The practical application of the dehydration of culture media was initiated and
pioneered by Difco in 1915, under the direction of Dr. J. W. M. Bunker.

16 DIFCOMANUAL

GENERAL CONDITIONS

Pertaining to the Cultivation of Microorganisms

The development of microorganisms upon culture media is dependent upon a
number of very important factors :

(a) The proper food elements must be available.

(b) Oxygen must be available as required.

(d) The medium must be of the proper reaction.

(e) Proper temperature relations must prevail,
(f ) The medium must be sterile.

(g) Contamination must be prevented.

A satisfactory microbiological culture medium must contain available sources
of carbon, nitrogen, inorganic salts and, in certain cases, vitamins or other growth-
promoting substances. These were originally supplied in the form of the meat in-
fusions which were, and still are in certain cases, widely used in culture media.
Beef extract frequently replaces meat infusions, but the preparation of this sub-
stance subjects it to the loss of its heat labile nutritive factors in much the same
way as infusions are affected. The addition of peptone provides a readily available
source of nitrogen and carbon.

Peptone is used in culture media to supply an available form of nitrogen since
native proteins are not generally attacked by bacteria. Most organisms are capable
of utilizing the amino acids and other simpler nitrogenous compounds present in
peptone. Continued investigations in our laboratories indicate that for the isola-
tion and propagation of many organisms the complicated infusion media can be
replaced by simpler media prepared by using the proper peptones in place of the
meat infusions heretofore employed.

Certain bacteria require additions of other food substances such as serum,
blood, or ascites to the culture medium upon which they are to be propagated.
Carbohydrates may also be desirable at times, and certain salts such as those of
calcium, manganese, magnesium, sodium, and potassium seem to be required.
Dyes may be added to media as indicators of metabolic activity or because of their
selective inhibitory powers. Growth promoting substances of a vitamin-like nature
are essential or assist greatly in the development of certain types of bacteria.

The consistency of a liquid medium may be modified by the addition of agar,
gelatin or albumin in order to change it into a solid or semisolid state. Solid
media, which were originally devised for the isolation of organisms in pure cul-
ture, are now universally used for almost all general cultural work. The semi-
solid media are used chiefly for carrying stock cultures or for propagating the
anaerobes.

One of the principal landmarks in bacteriology was the preparation of a
satisfactory medium by Hesse's introduction of agar as a solidifying agent for
bacteriological culture media, Hueppe: Die Methoden der Bakterienforschung,
250: 1891. Previous to that time infusions of plant and animal tissues, solutions
of organic compounds, and gelatin media only were employed. Until the intro-
duction of gelatin media by Koch in 1881, the only method for obtaining pure
cultures was the very unsatisfactory dilution procedure devised by Lister. The

DIFCOMANUAL 17

solid media used after the introduction of gelatin were only partially effective
since many of the organisms under investigation would not develop satisfactorily
at temperatures below the melting point of the gelatin, while others liquefied the
gelatin. Bacteriology as a science began with the development of methods for the
cultivation of bacteria, and the use of agar was a step of greatest importance.

Most bacteria are capable of growth under ordinary conditions of oxygen
tension. Certain types, however, are capable of deriving their oxygen from their
food substances. The aerobic organisms require the free admission of air, while
the anaerobes grow only in the exclusion of atmospheric oxygen. Between these
two groups are the microaerophiles which develop best under partial anaerobic
conditions and the facultative aerobes and anaerobes which develop over a wide
Eh range.

Anaerobic conditions for growth of microorganisms are obtained in a number
of ways :

(a) Addition of small amounts of agar to liquid media.

(b) Addition of fresh tissue to the medium.

(d) Addition of a reducing substance to the medium.

(e) Displacement of the air by carbon dioxide.
(f ) Absorption of the oxygen by chemicals.

(g) Removal of oxygen by direct oxidation of readily oxidizable substances
such as burning a candle, heating of palladiumized asbestos, copper, hydro-
gen, phosphorus or other readily oxidizable metals.

(h) Incubation in the presence of germinating grain or pieces of potato.

( i ) Inoculation into the deeper layers of solid media, or under a layer of oil

in liquid media.
( j ) Combinations of these methods.

Methods of readily obtaining anaerobic conditions in the laboratory are dis-
cussed in detail on page 118. Swancara^ describes a method of removing oxygen
from individual tubes of culture media and also a method of supplying partial
carbon dioxide tension. For anaerobic conditions a cotton plug is placed just
above the culture medium in tubes and a gelatin capsule containing pyrogallic
acid in a Durham fermentation tube is placed on top of this plug. A solution of
sodium hydroxide is placed in the Durham fermentation tube and the culture
tube then sealed with a rubber stopper or screw cap. The tube is then ready for
incubation, the oxygen being removed by the action of the sodium hydroxide on
the pyrogallic acid. He also describes a method of obtaining partial carbon
dioxide tension in individual tubes by similarly placing a broken household match
on a cotton plug just above the medium. Remove the percussion tip from the
match prior to placing in the tube. Seal the culture tube with a rubber stopper
or screw cap and ignite the match head by applying heat to the outside of the
tube.

Proper moisture conditions must prevail in the culture media employed for the
propagation of microorganisms. A moist medium and a moist atmosphere are
necessary for the continued luxuriant growth of the vegetative cells.

The pH or reaction of the culture medium, expressing its hydrogen ion concen-
tration, is extremely important for the growth of microorganisms. The majority of
the microorganisms prefer culture media which are approximately neutral, while
others may require a medium which is distinctly acid. The pH or reaction of the
culture medium is determined by colorimetric or electrometric measurement of
its hydrogen ion concentration. It should be noted that additions of acid or alkali
which are insufficient to prevent the growth of bacteria in a medium may inhibit

18 DIFCOMANUAL

or prevent them from proceeding with the normal functions of their metabolic
processes.

The usual range of temperature suitable for the growth of microorganisms lies
between 15° and 43 °C. Microorganisms have, however, been known to develop
at 0°G., and others, such as the soil organisms, may grow at 80°C. The patho-
genic organisms in general are limited by a comparatively narrow range of tem-
perature around 37°G. while the saprophytes usually have a much broader
latitude.

All organisms exhibit three cardinal points in their thermic relations :

(a) A minimum below which development ceases.

(b) An optimum at which growth is luxuriant.

In addition to a suitable temperature for growth of microorganisms it is
necessary to provide sufficient moisture in the atmosphere. Some organisms re-
quire a moist surface for growth. For example, media in plates inoculated with
the gonococcus, may fail to show growth of the organism if placed in an
ordinary incubater at 35-37 °G. A duplicate inoculation, in contrast, in a sealed
container in which is placed moist cotton or a wet towel to provide moisture,
will show profuse growth. Incubators should have open containers filled with
water at all times to provide sufficient moisture for growth and prevent drying of
media. Growth of most microorganisms is obtained in the absence of light. Sun-
light is to be avoided.

The media upon which microorganisms are grown must be sterile or free from
all other forms whose development might influence or prevent the normal growth
of the inoculated type. The usual method for immediate sterilization of culture
media is by means of the autoclave in which steam under pressure is the steriliz-
ing agent. The proper operation of the autoclave to insure sterilization of media
requires careful manipulation.

Autoclave sterilization for 15 minutes at 15 pounds pressure (121°G.) is recom-
mended for quantities of liquid media up to one liter. If larger volumes are to
be sterilized in one container, and especially if the medium is not hot when
placed in the autoclave, a longer period should be employed. The medium is
prepared according to formula, distributed in tubes or flasks which are then
plugged with nonabsorbent cotton or loosely capped and placed in the autoclave.
Tubes should be placed in racks or packed loosely in baskets. Flasks should never
be more than two-thirds full.

In the operation of the autoclave, all the enclosed air must be allowed to
escape and must be completely replaced by steam. Pressure-temperature relations
of a properly operated autoclave are shown in the table below.

Pressure-Temperature Relations in Autoclave
(Figures based upon complete replacement of air by steam.)

Pressure in Pounds Temperature

o G op

5 108 226

10 116 240

15 121 250

20 127 260

25 131 267

30 134 274

DIFGO MANUAL

If all the air is not removed from the sterilizing chamber, which condition is
best shown by use of a thermometer in the exhaust line of the autoclave, an en-
tirely different pressure-temperature relationship exists. Through the courtesy of
Dr. F. W. Tanner 2 we are able to reproduce the following chart which plainly
shows the actual temperature in the autoclave when the air is not completely
exhausted.

Effect of Entrapped Air on Temperature of Autoclave

•250i

PER CENT AIR IN SItAM,

When the operator is assured that all the air is replaced by steam, which is best
indicated by a thermometer placed in the exhaust line, the outlet valve of the
autoclave is closed and the steam pressure is raised to 15 pounds. When the ther-
mometer indicates a temperature of 121°C. heating is continued for 15 minutes.
A maximum of 15 minutes is recommended for the sterilization of carbohydrate
media in tubes to be used for fermentation studies. After the sterilization period
has been completed, the source of steam is cut off and the autoclave is allowed
to return to atmospheric pressure. Pressure should not drop too rapidly or the
media will boil over, blowing the plugs from the tubes or flasks. Pressure should,
however, drop rapidly enough to prevent excessive exposure of the media to heat
after the sterilization period. Ordinarily about 8, and not more than 1 2, minutes
should be required for the usual bacteriological laboratory autoclave to reach
atmospheric pressure without danger of prolonged heating. The media should be
removed from the autoclave shortly after sterilization and should not be per-
mitted to remain in the autoclave for any appreciable length of time after the
sterilization period.

For the sterilization of coagulable material such as serum, see the method given
for Bacto-LoeflPier Blood Serum, page 128.

Oversterilization or prolonged heating will change the composition of the
medium. For example, in our laboratories we have shown that Phenol Red Lac-
tose Broth which has been sterilized for 15 minutes at 15 pounds pressure
(121°C.), or sterilized by filtration, produces no demonstrable amount of acid
when inoculated with Salmonella typhosa. The same lot of medium sterilized for
30 or 45 minutes at 121°C. showed appreciable acid production under the same
conditions of testing. This demonstrates that oversterilization resulted in a break-
down of the lactose.

Agar media on prolonged sterilization or heating are apt to show a precipitate.

20 DIFGOMANUAL

Repeated melting of solidified agar, or long holding of melted agar at high tem-
perature, may likewise cause a precipitate to form in the media. Media contain-
ing agar may also form a flocculent precipitate if the liquid medium is held in
the water bath at 43 to 45 °G. for longer than 30 minutes. This flocculent agar
precipitate, however, may be dispersed by reheating the medium.

Excessive heating of media also results in an increase in acidity. The reaction
of the media will become more acid as heating is prolonged. Some media which
are acid, such as Wort Agar (pH 4.8) will, upon prolonged heating, cause de-
struction of the agar. It is possible to destroy completely the jellifying properties
of agar by prolonged heating, and this destruction is hastened as the acidity in-
creases.

Culture media which may be injured by autoclaving are sometimes sterilized
by the discontinuous or intermittent method. This procedure consists of heating
the medium in a chamber of flowing steam for a period of 20 or 30 minutes, or
longer, on several successive days. Body fluids and sera are sometimes sterilized
in the inspissator at 53° to 70° C. for one hour on six successive days. Liquid
media may be sterilized by filtration through unglazed porcelain or earthenware
candles, or through a sterilizing pad.

External contamination of culture media is prevented by plugging the tubes or
flasks with nonabsorbent cotton before sterilization. Plugs should fit neither too
loosely nor too tightly and should protect the lip of the container against the ac-
cumulation of dust. Screw cap tops or metal covers may also be used to close the
tubes or flasks. Marcus and Greaves^ have called attention to the fact that atypi-
cal cultural reactions may be obtained in sealed tubes of media used to test
biochemical activities due to anaerobic conditions. Tubes of Kligler Iron Agar
and Russell Double Sugar Agar, for example, gave aberrant reactions in tubes
sealed with screw caps or rubber stoppers. The same medium with tubes loosely
capped, or caps replaced with cotton plugs, showed typical reactions.

Media should always be stored in a cool moist atmosphere to prevent evapora-
tion. Prolonged storage of sterile media cannot, however, be recommended. If
tubes of media have been kept for any length of time they should be reheated
just before use. Liquid media should be heated in a boiling water bath or in flow-
ing steam for a few minutes, to drive off dissolved gases, and then cooled quickly
in cold water without agitation just prior to inoculation. Agar tubes should be
melted and allowed to solidify in order to secure a moist surface which is desired
by most microorganisms. These precautions for both liquid and solid media are
extremely important for the initiation of growth of highly parasitic organisms
such as those encountered in blood culture work.

Blood or other body fluids to be cultured should always be taken prior to the
administration of the therapeutic agent. If drugs have been administered their
bactericidal effects should be neutralized, if possible. The addition of j!?-amino-
benzoic acid (PAB) in 0.5 mg. per cent to the medium will assure the inactiva-
tion of any sulfa drug carried over with the inoculum. Bacto-Penase, a concen-
trated purified penicillinase, should be added to the sterile cooled medium used
for blood culture if the patient is under penicillin therapy. Bacto-Brain Heart
Infusion with P.A.B. and Agar with added Bacto-Penase is an ideal medium for
blood culture work. The small amount of agar present will give all degrees of
anaerobiosis, permitting the development of aerobes as well as the strictest
anaerobes, the PAB will inactivate any sulfa drug, the added Bacto-Penase will
inactivate any penicillin in the inoculum, while 100 ml. of the medium itself will
inactivate up to 1000 units of streptomycin.

'Am. J. Med. Tech., 14:214:1948.
2 Am. J. Pub. Health, 25:301:1935
•J. Lab. Clin. Med., 36:134:1950.

DIFCOMANUAL 21

THE PREPARATION

of Media from Dehydrated Culture Media, Difco

The advantages of dehydrated media and their efficiency for the cultivation of a
large variety of saprophytic and pathogenic microorganisms have been recognized
by the authors of many bacteriological texts, including "Standard Methods for
the Examination of Water and Sewage," "Standard Methods for the Examination
of Dairy Products" and "Diagnostic Procedures and Reagents" of the American
Public Health Association.

The preparation of media from Bacto Dehydrated Culture Media is a time-
saving and economical process by which large or small quantities of standard and
uniform culture media are made available with the minimal expenditure of effort.

The composition of each medium is stated on the label of the bottle with the
quantities of the ingredients present in one liter of the finished medium. All that
is necessary to rehydrate the medium is to weigh accurately the dehydrated
medium, dissolve the powder in freshly distilled water or boiled distilled water
and sterilize the solution. For ease of preparation and for best results with Bacto
Dehydrated Media, a discussion of these methods is given in detail.

Distilled water should be used in the rehydration of dehydrated culture media.
Distilled water that has been stored at room temperature for any length of
time is apt to absorb sufficient gases to actually alter the final reaction and com-
position of the medium. It is recommended that freshly distilled water or boiled
distilled water at room temperature be used in the preparation of all media.

The quantity of dehydrated culture medium to use per liter is specified on each
package. Amounts for small or large quantities may be easily calculated, and the
desired amount weighed. Best results will be obtained by adding the powder to
a dry flask, and then adding the freshly distilled water a little at a time with con-
stant agitation to prevent the formation of lumps. A stirring rod may be used to
secure an even mixture. The entire amount of distilled water is added when all
of the powder is thoroughly wetted. Broth or liquid media are readily soluble in
v/ater at room temperature. Agar media must be heated to the boiling point for
complete solution. This may be accomplished in several ways. For small quanti-
ties (up to 300 ml.) the best procedure is to heat the flask over a free flame,
keeping the contents of the flask well agitated to prevent burning; for larger
quantities, solution of the agar media may be more easily effected in flowing
steam, stirring from time to time, or in the autoclave. Another satisfactory
method, which reduces the heating period of the medium is to boil three-quarters
of the distilled water over a free flame and suspend the dehydrated medium in the
remaining cold distilled water, taking care that all of the medium is thoroughly
vv^etted and evenly suspended. The suspension is then finally added to the boiling
water and boiling continued until solution is complete. Gelatin media are best
dissolved by heating to 50 °G. in a water bath. Agar or gelatin media must be in
complete solution before being dispensed into the containers in which they are
to be sterilized.

Care should be exercised to avoid contamination of media during the rehydra-
tion process. Only chemically clean glassware should be used in dissolving the
media, and for distribution in the final container. It has been shown that deter-
gents used in washing glassware, if not satisfactorily removed by repeated rinsing,
may be responsible for growth inhibition and changes in reaction. Three highly

22 DIFCOMANUAL

advertised soapless cleansers commonly employed for cleaning laboratory glass-
ware were investigated and found quite bacteriostatic for many bacteria. Glass-
ware on which two of the three cleansers were used required two thorough rinses,
and that on which the third detergent was used required three complete rinses in
cold water to remove the toxic effect of the cleanser for bacteria. Clean glassware
is particularly required in media for microbiological assay procedures where ex-
tremely small amounts of material may give rise to erratic results. For example,
in the assay of Vitamin B12, as small a quantity as 0.01 millimicrogram will give
rise to a definite growth response. The importance of clean glassware in the assay
of this vitamin was stressed by the U.S. Pharmacopeia Vitamin B12 Study Panel,
since they stated that glassware for this purpose required special handling, and
as many as twelve rinses appeared to be necessary for satisfactory results.

Following rehydration, Difco culture media require no filtration. In some
cases the medium contains a slight flocculent precipitate, such as, for example,
Levine E.M.B. Agar and Bacto-Niacin Assay Medium. Removal of these precipi-
tates will result in an inferior growth response. The filtration of any medium
through cotton is especially to be avoided. Wright^ showed that cotton-wool con-
tained a material inhibitory to the growth of the pneumococcus. Drea^ showed
that absorbent cotton stoppers contained inhibitory material for the tubercle
bacilli. Boyd and Gasman^ filtered 100 ml. quantities of Tryptose Agar through
10 gram portions of 15 different commercial brands of absorbent cotton and
failed to obtain growth from a dilute inoculum of Brucella abortus while the un-
filtered medium supported good growth of the test organism. They were able to
extract a fatty acid-like substance from the cotton which completely inhibited
growth when added to the medium in a concentration of 1 mg. per liter, as did
unsaturated fatty acids from cottonseed oil. The addition of starch to the medium
neutralized this toxic effect. The same observation was also made with centri-
fuged and cotton filtered Potato Infusion Agar for B. abortus. The centrifuged
medium gave excellent growth while medium filtered through cotton was in-
ferior.

Adjustment of the reaction of the medium is not required. The final reaction
of the sterilized medium at 25 °C. is shown on the label of each bottle. Tenipera-
ture and method of standardization of the potentiometer in the determination of
pH is important. The potentiometer should be standardized with a standard
buffer solution, near the same reaction of the medium. The temperature of the
standard buffer used in standardizing the instrument should be the same as the
medium when making readings. The ionization constant increases with a rise in
temperature. For example, the pH of distilled water free from carbon dioxide at
25°G. is practically pH 7.0, but at 40°C. is 6.7.

Uniform standardized media are readily prepared in large or small amounts
from Bacto Dehydrated Gulture Media. By the use of these products microbiolo-
gists are able to provide themselves daily with freshly prepared and wholly satis-
jfactory media of known composition and definite reaction.

ij. Path. Bact., 38:499= i943-

2 J. Bact., 44:149:1942.

8 Public Health Reports, 66:44:1951.

Dehydrated Culture Media

GUIDES

for the Selection of Culture Media

The tables on the following pages will assist in the selection of media for vari-
ous purposes. The media listed are discussed in detail in this Manual and they
are recommended as being those most generally employed for specific purposes.
For the reason that media other than those listed may be preferred in some lab-
oratories, we suggest that consideration also be given to similar media which are
discussed in the same section of the manual.

DIFCO MANUAL

Culture Media for the Examination of Water and Sewage
"Standard Methods" Procedures

Tests for Coliform Organisms
Plate Counts Presumptive Confirmed Completed

Nutrient Agar ^
Nutrient Gelatin
Tryptone Glucose Extract
Agar

Lactose Broth
Lauryl Tryptose
Broth

Endo Agar

Levine E.M.B. Agar

Brilliant Green

Bile 2%
Formate Ricinoleate

Broth
Lauryl Tryptose

Broth

Nutrient Agar
Lactose Broth

Control of Water Filtration Plant Operation

Selective Broths

Selective Agars

Differential Test Media

Fuchsin Lactose Broth
Brilliant Green Bile 2%
M.B.-B.C.P. Medium
Formate Ricinoleate Broth
Crystal Violet Broth
Eijkman Lactose Medium
E C Medium
MacConkey Broth

MacConkey Agar
Violet Red Bile Agar
Desoxycholate Lactose

Agar
Brilliant Green Bile Agar
Levine E.M.B. Agar

Bacto-Tryptone
M.R.-V.P. Medium
Koser Citrate Medium

Culture Media for the Examination of Dairy and Other Food Products

Plate Counts

Tryptone Glucose Extract Agar

Proteose Tryptone Agar

Beef Lactose Agar

Nutritive Caseinate Agar

Heart Infusion Agar
Brucella

Tryptose Agar
Lactobacilli

Tomato Juice Agar

Trypsin Digest Agar

Peptonized Milk

Skim Milk

Micro Assay Culture Agar

Micro Inoculum Broth

Snyder Test Agar
Hemolytic Streptococci

Heart Infusion Agar

Coliform Organisms

Brilliant Green Bile 2%

Formate Ricinoleate Broth

Violet Red Bile Agar

Desoxycholate Agar

Desoxycholate Lactose Agar

MacConkey Agar

Lactose Broth

Endo Agar

Levine E.M.B. Agar

Bacto-Tryptone

M.R.-V.P. Medium

Koser Citrate Medium
Thermophiles

Dextrose Tryptone Agar

Thermoacidurans Agar
Molds and Yeasts

Potato Dextrose Agar

Malt Agar

y.-^

v ^

1^2

art Infusio

Phosphate

Starch Ag

Agar

Broth

Agar

ium

^ (D <D O d, V'V

rain H
ryptos
extros
ryptos
extros
extros
G Me

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s <

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CO eg

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ti S C O bo

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Tj ° o o

4^. ^ 4J a;

DEHYDRATED CULTURE MEDIA 29

MEDIA FOR THE EXAMINATION
OF WATER AND SEWAGE

The dehydrated culture media listed in this section conform to the formulae
given in "Standard Methods for the Examination of Water and Sewage," Ninth
Edition, 1946, of the American Public Health Association and the American
Water Works Association. Carefully selected standardized ingredients are used in
the exact proportions specified in the "Standard Methods" formulae. The reac-
tion of each medium is carefully adjusted so that the final reaction will fall
within the range of pH recommended in "Standard Methods."

The American Public Health Association has permitted the use of dehydrated
media of "Standard Methods" composition continuously since 1923 in every
edition of its "Standard Methods for the Examination of Water and Sewage,"
including the current Ninth Edition.

The U. S. Public Health Service Drinking Water Standards^ specify that the
procedures recommended in "Standard Methods of Water Analysis" of the Amer-
ican Public Health Association be followed in all details, including the culture
media. It is, therefore, permissible for Bacto Dehydrated Culture Media to be
used in the testing of the potability of water according to the U. S. Public Health
Service Drinking Water Standards.

A guide for the selection of culture media listed in this section is given on

page 25.

1 Public Health Reports, 61:376:1946
(Reprint No. 2697).

"STANDARD METHODS" MEDIA

The media listed in this section are used for the bacteriological examination of
water in accordance with the procedures of "Standard Methods for the Examina-
tion of Water and Sewage."

BACTO

NUTRIENT BROTH (B3)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Peptone 5 g.

Bacto-Nutrient Broth is a peptone meat extract liquid medium recommended
for general laboratory use for the cultivation of the microorganisms that are not
exacting in food requirements. Bacto-Nutrient Broth is prepared in accordance
v/ith the formula specified by the American Public Health Association and Amer-
ican Water Works Association's "Standard Methods for the Examination of Water
and Sewage"^ and "Standard Methods for the Examination of Dairy Products"^
this medium has been prepared to give to bacteriologists an Extract Broth of
approved and standard formula. It is also recommended as a base to which a

30 DIFGOMANUAL

variety of materials may be added, for the preparation of a large number of dif-
ferential, selective, and enriched media requiring only the addition of dyes, salts,
carbohydrates, tissues, or serous fluids. An enriched medium such as Rosenow*
described for the isolation of streptococci from infected teeth, was prepared by
adding dextrose, pieces of sterile brain tissue, and marble chips to Nutrient Broth.

Bouillon or Beef Broth as suggested by LoefRer was one of the earliest media
used in bacteriology. An infusion of meat and a peptone constituted the nutri-
ments of this medium. Later it was shown that for many routine purposes beef
extract could satisfactorily replace the infusion of fresh meat and had the decided
advantage of ease of preparation, uniformity, and economy. The American Public
Health Association recognized the advantage of beef extract in standard culture
media and in 1917 discontinued the use of infusion of beef in standard media.
Bacto-Nutrient Broth has been prepared to duplicate the formula approved by
the American Public Health Association and since 1927 our label has carried a
statement to this effect.

Bacto-Nutrient Broth is recommended for use in the tube dilution method of
testing the sensitivity of microorganisms to antibiotics. Waisbren, Carr and
Dunnett* showed that Bacto-Nutrient Broth, Bacto-Dextrose Broth or Bacto-
Tryptose Phosphate Broth were suitable media for comparative sensitivity test
studies while a Tryptic soy medium inhibited the action of Neomycin, Aureo
mycin, Terramycin and Polymyxin against the test organism.

To rehydrate the medium, dissolve 8 grams of Bacto-Nutrient Broth in 1000
ml. of distilled water. Distribute in tubes and sterilize in the autoclave for 15
minutes at 15 pounds pressure (121°C.). The final reaction of the medium will
be pH 6.8.

One pound of Bacto-Nutrient Broth will make 56.7 liters of medium.

^Standard Methods for the Examination ^ J. Dental Research, 1:205:1919.

of Water and Sewage, 9th Edition: 185:1946. ^ Am. J. Clin. Path., 21:884:1951.
3 Standard Methods for the Examination

of Dairy Products, 9th Edition: 166:1948.

BACTO

LACTOSE BROTH (B4)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Peptone 5 g.

Bacto-Lactose 5 g.

Bacto-Lactose Broth is recommended for use in the presumptive test for mem-
bers of the coliform {Escherichia- Aerobacter) group from water and other ma-
terials. This medium is prepared according to the formula specified in "Standard
Methods for the Examination of Water and Sewage,"^ and in "Standard Methods
for the Examination of Dairy Products."^

In the determination of the potability of drinking water one of the most im-
portant tests is the detection of possible fecal contamination. The presence of
Escherichia coli is generally considered to be an indication of fecal pollution of
the water.

The demonstration of the presence of coliform bacteria in water has been re-
duced to a relatively simple process as outlined in "Standard Methods." Since
1917 the recommended procedure has been:

1. The determination of gas production in Lactose Broth resulting from the
direct inoculation of water (presumptive test).

2. The inoculation of differential or selective media from tubes of fermented
Lactose Broth (confirmed test),.

DEHYDRATED CULTURE MEDIA 31

3. The identification of Gram negative, non-sporulating, aerobic organisms
capable of producing gas when reinoculated into Lactose Broth (completed
test).

Since 1917 "Standard Methods of Water Analysis," in the interests of greater
uniformity, has recommended the use of a Beef Extract Lactose Broth in place
of the infusion medium formerly employed. Bacto-Lactose Broth is prepared from
the ingredients recommended in "Standard Methods" and requires only solution
in distilled water and sterilization. No adjustment of reaction is required.

Bacto-Lactose Broth prepared in the normal concentration contains 0.5 per
cent each of Bacto-Lactose and Bacto-Peptone together with 0.3 per cent Bacto-
Beef Extract. This concentration of sugar and of peptone yields an optimum
growth of the organisms of the coli-aerogenes group.^ "Standard Methods"^
specify that when using inocula greater than 1 ml., multiple strength Lactose
Broth shall be prepared to maintain the concentration of nutriments as given in
the formula (1 ml. or less of sample may be added to 10 ml. of single strength
medium).

"Standard Methods" procedures apply to the U.S.P.H.S. Drinking Water
Standards* in all details of technique in the bacteriological examination, includ-
ing apparatus and media. These specifications provide for the use of 10 ml. and
100 ml. as standard portions of water for the bacteriological test. They recom-
mend standard dilution water bottles of 160 to 180 ml. capacity as containers for
the presumptive test where 100 ml. water samples are to be tested; and also
authorize the use of either the ordinary inverted vial or a Cowles^ tube for col-
lection of gas produced in the medium. The inverted vial or Cowles tube must
be of sufficient length so that it will be in at least a 45° angle to the base of the
bottle. For convenience it is recommended that the bottles used for the test be
marked at the volume of medium to be used and also at the total volume of the
medium plus the water sample. For instance, if it is desired to use regularly 35
ml. of concentrated medium for each 100 ml. of water sample the bottle can be
marked at the 35 ml. and the 135 ml. points.

In order to maintain a constant final concentration of nutriments in all tests,
it is suggested that the medium be prepared in the concentrations given in the
table below.

To rehydrate the medium, dissolve 13 grams, or other specified quantity, of
Bacto-Lactose Broth in 1000 ml. of distilled water, distribute into test tubes or
bottles with fermentation vials and sterilize in an autoclave for 15 minutes at
15 pounds pressure (121° C). The final reaction of the medium will be pH 6.7.

One pound of Bacto-Lactose Broth will make 34.9 liters of medium.

Concentrations of Dehydrated Medium Required
to Maintain the Proper Concentration of Ingredients

Bacto-Lactose Broth

Amount Medium

Vol. Medium

used per

Inoculum

Tube

and Inoculum

1000 ml.

1 ml.

10 ml.

or more

11 ml. or more

13 g.

10 ml.

30 ml.

40 ml.

17.3 g.

10 ml.

20 ml.

30 ml.

19.5 g.

100 ml.

50 ml.

150 ml.

39 g.

100 ml.

35 ml.

135 ml.

49.4 g.

100 ml.

20 ml.

120 ml.

78 g.

1 Standard Methods for the Examination 3 Public Health Reports, 44:2865:1929.
of Water and Sewage, 9th Edition: 186:1946. * Public Health Reports, 61:376:1946

2 Standard Methods for the Examination (Reprint No. 2697).
of Dairy Products, gth Edition: i33:!048. ^^ J. Bact., 38:677:1939.

32 DIFCOMANUAL

BACTO

NUTRIENT GELATIN (Bll)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Peptone 5 g.

Bacto-Gelatin 120 g.

Bacto-Nutrient Gelatin is recommended for the 20°C. plate count of water
according to "Standard Methods for the Examination of Water and Sewage"^
of the American Public Health Association and the American Water Works Asso-
ciation. It is also recommended for the determination of gelatin liquefaction in
the identification of anaerobes and other microorganisms.

Gelatin was one of the first solidification agents used in the science of bac-
teriology, enabling the direct plate count to be used rather than the dilution
method for determination of bacterial populations, and for the isolation of pure
cultures. However, there are certain limitations to the use of gelatin in plating
and isolation procedures, as incubation must be carried out at approximately
20° C., a temperature lower than the optimum for many organisms. Further,
many organisms have the ability to attack and liquefy the gelatin. Although
Nutrient Gelatin is still used in the plate count of water with incubation at
19-2 1°C., plating procedures now most generally utilize media containing agar
as a solidifying agent. The chief use of Nutrient Gelatin is for the detection of
proteolysis (the elaboration of gelatinolytic enzymes) as evidenced by the lique-
faction of gelatin.

Bacto-Nutrient Gelatin is prepared according to the formula specified in
"Standard Methods" and since 1925 our label has carried a statement to that
effect. Plates are incubated at 19-21 °C. for 48 hours and the colonies counted.
In determining gelatin liquefaction of pure cultures in this medium, tubes are
incubated for at least five days at 19-21°C. If the optimum temperature for the
organism in question is above 20°C., incubate at the desired temperature and
after incubation cool the tubes to 20 °C., to determine if the gelatin has been
liquefied.

To rehydrate the medium, suspend 128 grams of Bacto-Nutrient Gelatin in
1000 ml. of cold distilled water and warm to about 50° C. to dissolve the medium
completely. Distribute in tubes and sterilize in the autoclave for 15 minutes at
15 pounds pressure (121°G.). The final reaction of the medium will be pH 6.8.

One pound of Bacto-Nutrient Gelatin will make 3.5 liters of medium,

1 Standard Methods for the Examination of Water and Sewage, gth Edition: 186:1946.

BACTO

NUTRIENT AGAR ( B 1 )

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Peptone 5 g.

Bacto-Agar 15 g.

Bacto-Nutrient Agar is recommended for the examination of water according
to "Standard Methods for the Examination of Water and Sewage"^ of the Ameri-
can Public Health Association and the American Water Works Association. It is
also recommended as a general culture medium for the cultivation of the majority
of the less fastidious microorganisms, as well as a base to which a variety of
materials are added to give selective, differential, or enriched media.

DEHYDRATED CULTURE MEDIA 33

Infusions of meat were first generally employed together with peptone as
nutriments in culture media. Later it was found that for many routine procedures
beef extract gave fully as good results and had the decided advantages of greater
ease of preparation, greater uniformity, and economy, A simple medium com-
posed of beef extract, peptone, and agar has been one of the most generally used
media in bacteriological procedures. It is used for the ordinary routine examina-
tions of water, sewage, and food products; for the carrying of stock cultures; for
the preliminary cultivation of samples submitted for bacteriological examination;
and for isolating organisms in pure culture.

Bacto-Nutrient Agar was originally prepared to duplicate an extract agar of
approved and standard formula. The American Public Health Association in its
earliest reports on methods for water analysis emphasized the necessity of the
universal use of a standard medium, and since the Third Edition of "Standard
Methods of Water Analysis" in 191 7^ has recommended the use of beef extract
rather than infusion of meat in the preparation of Nutrient Agar. In the Fifth
Edition of "Standard Methods of Water Analysis," 1923,^ and the Fourth Edition
of "Standard Methods of Milk Analysis," 1923,* the use of dehydrated media of
"Standard Methods" composition has been permitted for the bacteriological ex-
amination of water and milk. In the Fifth Edition of "Standard Methods of Milk
Analysis," 1927,^ the use of Bacto dehydrated media was approved as being on
a par with laboratory-made media for the bacteriological plate count of milk,
and in this connection the work of Norton and Seymour^ was cited. Bacto-
Nutrient Agar is prepared in accordance with the formula specified in "Standard
Methods of Water Analysis"'^ and since 1924 our label has stated "Conforms to
Standard Methods Formula." The "Standard Methods for the Examination of
Dairy Products" Ninth Edition^ specifies a medium known as Tryptone Glucose
Extract Milk Agar, as discussed on page 57, to take the place of Nutrient Agar
in the bacteriological examination of dairy products.

To rehydrate the medium, suspend 23 grams of Bacto-Nutrient Agar in 1000
ml. of cold distilled water and heat to boiling to dissolve the medium completely.
In preparing large volumes of the medium the heating period required to effect
complete solution may be reduced by boiling about three-fourths of the distilled
water over a free flame and suspending the dehydrated medium in the remaining
cold distilled water, taking care that all particles of the medium are thoroughly
wetted and evenly suspended. The suspension is then slowly added to the boiling
water and boiling continued for a minute or two to complete solution. The
medium is distributed in tubes or flasks and sterilized in the autoclave for 15
minutes at 15 pounds pressure (121°C.). The final reaction of the medium will
be pH 6.8.

One pound of Bacto-Nutrient Agar will make 19.7 liters of medium.

1 Standard Methods for the Examination ^ Standard Methods of Milk Analysis,

of Water and Sewage, gth Edition: 186:1946. 5th Edition: 7:1927

2 Standard Methods of Water Analysis. « Am. J. Pub. Health, 16:35:1926.
3rd Edition: 93:191 7- 'Standard Methods of Water Analysis,

3 Standard Methods 0' '*' ' ' ' « . .. .
5th Edition: 97:192

* Standard Methods c
4th Edition: 4:1923

itandard Methods of Water Analysis, 8th Edition: 201:1936.

th Edition: 97:1923- s Standard Methods for the Examination

tandard Methods of Milk Analysis, of Dairy Products, gth Edition: 93: 1948.

BACTO

TRYPTONE GLUCOSE EXTRACT AGAR

DEHYDRATED

The option of using Bacto-Tryptone Glucose Extract Agar as discussed on
page 57 for plate counts of water is permissible in following "Standard Methods"
procedures in laboratories where both milk and water counts are made, so that

34 DIFCOMANUAL

two different media need not be carried in stock. In "Standard Methods for the
Examination of Water and Sewage," Ninth Edition, 1946, page 191, it is stated that
experimental data indicate Bacto-Tryptone Glucose Extract Agar and Bacto-
Nutrient Agar give practically the same counts with water samples, and that
colonies are larger and more easily counted when the former medium is used.

BACTO

ENDO AGAR (B6)

DEHYDRATED

Bacto-Peptone 10 g.

Bacto-Lactose 10 g.

Dipotassium Phosphate 3.5 g.

Bacto-Agar 15 g.

Sodium Sulfite 2.5 g.

Bacto-Basic Fuchsin 0.4 g.

Bacto-Endo Agar is recommended for the confirmation of the presumptive
test for members of the coliform group in the bacteriological examination of
water, milk, and other dairy products, according to "Standard Methods for the
Examination of Water and Sewage"^ and "Standard Methods for the Examina-
tion of Dairy Products."^

Endo^ originally described a medium using a fuchsin sulfite indicator to differ-
entiate lactose fermenting and lactose non-fermenting organisms of the intestinal
tract. Upon plates of this medium, in which the fuchsin has been decolorized by
sodium sulfite, typhoid and other lactose non-fermenting organisms appear as
clear, colorless, glistening drops against the faint pink background of the medium.
Coliform organisms fermenting lactose become red and color the surrounding
medium. The typical reactions of this medium are not caused by acid production
but by the intermediate product acetaldehyde, which is fixed by the sodium sul-
fite as was shown by Margolena and Hansen* and Neuberg and Nord.^

Endo's original formula has been subjected to many modifications, due largely
to variations in the available dyes and sulfites and to new uses of the medium
advocated by individual investigators. The result has been a multiplicity of varia-
tions of the formula. Harris^ investigated the problem of Endo Agar, studying
various ingredients, reaction, and available dyes. He reported that by using
Levine's modification'^ several sources of error were eliminated and that Bacto-
Peptone as recommended by Levine gave satisfactory results. In this modification
Dr. Harris found that a basic fuchsin composed of almost equal parts of rosanilin
and pararosanilin gave color reactions which were exceedingly sensitive and con-
sistent. Bacto-Endo Agar was developed in cooperation with Dr. Harris, conform-
ing to the Levine modification with the dye combination proposed by Harris.

Endo Agar was originally developed for the isolation of typhoid bacilli. Since
that time more satisfactory media have been developed for this problem and
Endo Agar has proved of most value in the bacteriological examination of water.
Endo Agar and Levine Eosin Methylene Blue Agar are the two solid media
specified in the Ninth Edition of "Standard Methods for the Examination of
Water and Sewage" for the confirmation of the presence of coliform organisms
in Lactose Broth tubes giving a positive presumptive test. The formula used in
the preparation of Bacto-Endo Agar is identical with Formula II of "Standard
Methods for the Examination of Water and Sewage,"^ and since 1932 our label
has carried a statement to this effect. For the isolation of members of the Sal-
monella and Shigella group see Bacto-Bismuth Sulfite Agar, page 139; Bacto-
S S Agar, page 134; Bacto-MacConkey Agar, page 131; Bacto-Tetrathionate

PEHYDRATED CULTURE MEDIA 35

Broth Base, page 157; Bacto-Selenite Broth, page 158; Bacto-Brilliant Green
Agar, page 144.

Endo Agar inoculated the same day as rehydrated may be used without auto-
clave sterilization. Under these conditions the medium need be heated only to
boiling to dissolve it completely before pouring into plates.

To rehydrate the medium, suspend 41.5 grams of Bacto-Endo Agar in 1000
ml. of cold distilled water and heat to boiling to dissolve the medium completely.
Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). The characteristic flocculant precipitate present in
the medium following autoclaving may be evenly dispersed by twirling or gently
shaking the flask just prior to pouring into sterile petri dishes. The final reaction
of the medium will be pH 7.5.

One pound of Bacto-Endo Agar will make 10.9 liters of medium.

» Standard Methods for the Examination ^ Blochem. Zeit., 96:133:1919.

of Water and Sewage, 9th Edition: 195:1946. ^ MiHtary Surgeon, 57:280:1925.

2 Standard Methods for the Examination ' Abst. Bact., 2:13:1918.

of Dairy Products, gth Edition: 33:1948. 8 Standard Methods for the Examination

8 Centr. Bakt., Abt. I, Orig., 35:109:1904. of Water and Sewage, 9th Edition: 187:1946.
* Stain Tech., 8:131:1933.

BACTO

LEVINE E.M.B. AGAR (B5)

DEHYDRATED

Bacto-Peptone 10 g.

Bacto-Lactose 10 g.

Dipotassium Phosphate 2 g.

Bacto-Agar 15 g.

Bacto-Eosin Y 0.4 g.

Bacto-Methylenc Blue 0.065 g.

Levine Eosin Methylene Blue Agar is recommended for the confirmation of
presumptive tests of members of the coliform group in the bacteriological exami-
nation of water, milk, and other dairy products according to "Standard Methods
for the Examination of Water and Sewage"^ and "Standard Methods for the
Examination of Dairy Products. "^ It may also be used for differential plate count
as mentioned in Appendix I of "Standard Methods for the Examination of Water
and Sewage."^ While pathogens concerned in enteric fevers caused by members
of the Salmonella and Shigella group will develop on this medium, forming
translucent, colorless colonies which are readily differentiated from lactose-fer-
menters, its principal function is to demonstrate the presence of coliform bacteria
from water and milk samples.

Levine* developed an Eosin Methylene Blue Agar which gave excellent differ-
entiation of Escherichia coli and Aerobacter aerogenes. The colon colonies usually
show a dark center and have a greenish metallic sheen, but occasionally variants
have been observed similar to the type described but having no sheen. Another
variant grows effusely in colonies somewhat larger than the typical growth and
has a distinct metallic sheen. Colonies of A. aerogenes are usually much larger
than typical E. coli and tend to run together. The centers are usually brown in
color and not as dark as E. coli. The metallic sheen is only occasionally observed.
A more detailed differentiation is indicated in the accompanying table.

To obtain the most satisfactory reactions, especially in the differentiation of
E. coli and A. aerogenes, it is very important that particular care be taken in the
choice of the dyes and a meticulous determination be made of their proper pro-
portions. The Bacto dyes in this medium are selected to satisfy the extreme
delicacy of the medium. Levine recommends that the reaction should not be

< ea

? bo .

S> «i t>

too rt S

O •— ' o

u CO
^^

coli and
Striated
lonies.

•§1

S-i

^^8

•rj O

•■S-S

rt t;

• s

3
Ih

'S
o

bO
G

^ o o

S*?"^

^•2

c-3

iJ^ c

-^y,

C d cj

(3i.l

•

fl «

rt b

Si2

^ rt fl

u
o

4^ --^ ^

-s«°

•^

."S

«*-!

^ "t^

•-"'

t)ifi

O (U

e^i

.-^

c-S a

§1

^ _

u
bo

^ bo

6§

tJ p3 U
h

M bo

>' w

« o

« z

« 3

(^ ^

e* fe

^ «

pj 3

< H

< S

DEHYDRATED CULTURE MEDIA 37

adjusted and the medium should not be filtered. The medium is relatively stable
and may be stored for short periods of time. It is usually used in petri dishes;
however, some laboratories have found tubes to be convenient. The tubes should
be prepared with long slants and no butts.

Levine E.M.B. Agar is prepared according to the formula specified in "Stand-
ard Methods for the Examination of Water and Sewage"^ and since 1926 our
label has carried a statement to this effect.

The "Standard Methods for the Examination of Water and Sewage" in the
Ninth Edition permits the use of either Endo or Eosin Methylene Blue Agar as
solid media for confirming tubes of Lactose Broth showing positive presumptive
tests.

Taft and Daly® prepared a selective medium for the primary isolation of
pathogenic intestinal bacteria by adding normal sodium hydroxide solution (gen-
erally 1.5 ml. per liter medium) to Bacto-Levine E.M.B. Agar. This selective
medium gave superior results to that obtained with Desoxycholate Citrate Agar,
and gave uniformly good results as did S S Agar in the examination of stools.
The authors suggest that the selective Levine E.M.B. Agar be supplemented by
an enrichment such as Tetrathionate Broth in laboratories doing routine stool
examination.

Levine-E.M.B. Agar inoculated the same day as rehydrated may be used with-
out autoclave sterilization. Under these conditions the medium need be heated
only to boiling to dissolve it completely before pouring into plates. '

To rehydrate the medium, suspend 37.5 grams of Levine E.M.B. Agar in 1000
ml. of cold distilled water and heat to boiling to dissolve the medium completely.
Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes at 15
pounds pressure ( 121°C.). The characteristic flocculant precipitate present in the
medium following autoclaving may be evenly dispersed by gently twirling or
shaking the flask just prior to pouring into sterile petri dishes. The final reaction
of the medium will be pH 7.1.

One pound of Levine E.M.B. Agar will make 1 2 liters of medium.

* Standard Methods for the Examination of Water and Sewage, gth Edition: 328:1946.
of Water and Sewage, gth Edition: 195:1946. * Bull. 62, Iowa Eng. Exp. Sta., 1921.

* Standard Methods for the Examination ^ Standard Methods for the Examination

of Dairy Products, gth Edition: 134:1948. of Water and Sewage, gth Edition: i87:ig46.

"Standard Methods for the Examination ^Am. J. Clin. Path., 17:561:1947.

BACTO

BRILLIANT GREEN BILE 2% (B7)

DEHYDRATED

Bacto-Peptone 10 g.

Bacto-Lactose 10 g.

Bacto-Oxgall 20 g.

Bacto-BrilHant Green 0.0133 g.

Bacto-Brilliant Green Bile 2% is recommended for the confirmation of pre-
sumptive tests for members of the coliform group in the bacteriological examina-
tion of water according to "Standard Methods for the Examination of Water
and Sewage"^ and for the detection of members of the coliform [Escherichia-
Aerobacter) group in milk and dairy products as specified in "Standard Methods
for the Examination of Dairy Products."^ This medium may also be used in the
control of water filtration plant operation as described in Appendix I of "Stand-
ard Methods for the Examination of Water and Sewage"^.

The development of a selective medium which would inhibit organisms other
than members of the coliform group has long been of interest to sanitary bac-
teriologists. The principal interest has been in media containing bile and brilliant

38 DIFCO MANUAL

green. The American Water Works Association made extensive studies of this
problem through its Committee No. 1 on "Standard Methods of Water Analysis."
Dunham and Schoenlein^ have recorded their investigations of the proportions
of bile and brilliant green giving optimum results. They reported that, under
the conditions of their investigations, a reduced bile content and a dilution of dye
higher than that originally suggested by Muer and Harris^, improved conditions
for the development of Escherichia coli. The necessity of maintaining the proper
concentration of ingredients after the water sample is added was emphasized.
Jordan^ indicated in his report that this medium is slightly superior to Lactose
Broth and to the more concentrated bile medium in the detection of the coliform
group in water. McCrady and Langevin'' reported that Bacto-Brilliant Green
Bile 2% is satisfactory for the detection of the coliform group in controlling the
pasteurization of milk. McCrady^ in studies on media for the detection of the
presence of coliform organisms in water, found that while selective media were
not as satisfactory as Lactose Broth for the presumptive test, they could be
recommended for confirmation of the presumptive test and that for this purpose
Brilliant Green Bile 2% was most satisfactory.

Bacto-Brilliant Green Bile 2% is prepared according to the formula specified
in "Standard Methods for the Examination of Water and Sewage"^ and "Stand-
ard Methods for the Examination of Dairy Products"^, and since 1933 our label
has carried a statement to that effect.

When the medium is to be used in water purification plant control where
the inoculum is greater than 1 ml., particular care must be taken to preserve the
correct concentration of dye and bile in the medium after dilution with the
sample. The table given below indicates the quantity of dehydrated medium to
use per 1000 ml. distilled water to maintain the correct concentrations of dye
and bile.

In the presumptive test for members of the coliform group in the examination
of dairy products a series of tubes of Brilliant Green Bile 2% are inoculated with
appropriate dilutions of the sample. Use 5 tubes of each solution. Select dilutions
to provide at least one positive and one negative tube in the series inoculated.
Incubate tubes for 48 hours at 35-3 7 °C. Gas formation constitutes a positive pre-
sumptive test.

To rehydrate the medium, dissolve 40 grams of Bacto-Brilliant Green Bile 2%
in 1000 ml. of distilled water. The medium is distributed in fermentation tubes
and sterilized by autoclaving for 15 minutes at 15 pounds pressure (121°C.). The
final reaction of the medium will be pH 7.2.

One pound of Bacto-Brilliant Green Bile 2% will make 11.3 liters of medium.

Concentrations of Dehydrated Medium Required
to Maintain the Proper Concentration of Ingredients

Bacto-Brilliant

Volume Medium

Green Bile 2%

Inoculum

in Tube

and Inoculum

used per 1000 ml.

1 ml. or less

10 ml.

40 g.

10 ml.

20 ml.

30 ml.

60 g.

10 ml.

30 ml.

40 ml.

53 g.

1 Standard Methods for the Examination ^ Am. J. Pub. Health, 10:874:1920.

of Water and Sewage, gth Edition: 194:1946. « J. Am. Water Works Assoc, i8:337:i927.

2 Standard Methods for the Examination 'J. Dairy Science, 15:321:1932.

of Dairy Products, gth Edition: 152:1948. ^ Am. J. Pub. Health, 27:1243:1937.,

' Standard Methods for the Examination » Standard Methods for the Exammation

of Water and Sewage, 9th Edition: 226:1946. of Water and Sewage, 9th Edition: 188:1946.
* Stain Tech., 1:129:1926.

DEHYDRATED CULTURE MEDIA 39

BACTO

FORMATE RICINOLEATE BROTH

DEHYDRATED

Bacto-Formate Ricinoleate Broth is used in the confirmed and completed tests
for the presence of members of the coliform group in water and sewage examina-
tion and for the detection (presumptive) test of members of the coliform group,
as given in the "Standard Methods for the Examination of Dairy Products,"
Ninth Edition, page 131, 1948. Its use is confined to the study of the possible
presence of spore-forming lactose-fermenting organisms as given in "Standard
Methods for the Examination of Water and Sewage," Ninth Edition, 1946, pages
196 and 197. This selective broth may also be used in parallel planting with
Lactose Broth in the control of water filtration plant operations as described in
Appendix I of the Ninth Edition, page 226. A complete discussion of this me-
dium is given on page 60.

BACTO

LAURYL TRYPTOSE BROTH (B241)

DEHYDRATED

Bacto-Tryptose 20 g.

Bacto-Lactose 5 g.

Dipotassium Phosphate 2.75 g.

Monopotassium Phosphate 2.75 g.

Sodium Chloride 5 g.

Sodium Lauryl Sulfate 0.1 g.

Bacto-Lauryl Tryptose Broth is prepared according to the formula of Mallmann
and Darby^ and is recommended for use in the standard tests for the coliform
group as specified in "Standard Methods for the Examination of Water and
Sewage"-.

The fermentation of lactose with the production of gas has been used as an
indicator of the potability of water for many years. The formation of gas from
Lactose Broth constitutes a presumptive test for the coliform group (this terra
includes all aerobic and facultative anaerobic Gram-negative non-spore-forming
bacilli which are capable of producing gas from lactose)-. Cowls^ showed that
the addition of sodium lauryl sulfate to Lactose Broth gave a medium selective
for the coliform group. Darby and Mallmann* demonstrated the value of Bacto-
Tryptose in the detection of coliform organisms. In a 2 per cent concentration
of Bacto-Tryptose the rate of reproduction during the early logarithmic growth
phase was increased over that obtained with Bacto-Peptone. The addition of
phosphate buffer to the Bacto-Tryptose medium caused a greater growth in the
late logarithmic phase and slightly greater increase during the lag phase than
did the non-buffered medium. When sodium chloride was added to the medium
a marked increase in the rate of reproduction during the lag and early growth
phases was observed. Their final medium permitted the so-called "slow lactose
fermenters" to produce gas in greater quantities in a shorter period of time. The
medium consisted of 2 per cent Bacto-Tryptose, 0.5 per cent lactose, 0.4 per cent
dipotassium phosphate, 0.15 per cent monopotassium phosphate, and 0.5 per cent
sodium chloride, and had a final reaction of pH 6.8.

In an attempt to improve the methods used to demonstrate members of the
coliform group from water, Mallmann and Darby^ investigated a large number
of wetting agents, and showed that sodium lauryl sulfate gave best results as a

40 DIFCOMANUAU

non-inhibitive selective agent for members of the coliform group. Optimum
results were obtained by the addition of 1:10,000 sodium lauryl sulfate to the
buffered Tryptose Lactose Broth. In their comparative study in the checking of
various types of water it was shown that the Lauryl Sulfate Tryptose Broth
(Bacto-Lauryl Tryptose Broth) gave a higher colon index than did the confirm-
atory "Standard Methods" media, and that gas production in the Lauryl Tryp-
tose Broth served not only as a presumptive test, but was also confirmatory of
the presence of the coliform group for routine testing of water.

In a study of the coliform bacteria from chlorinated waters Levine^ compared
Lactose Broth and Lauryl Tryptose Broth. The latter medium gave fewer false
positive presumptive tests than did Lactose Broth and suppressed the spore form-
ing aerogenic bacteria. However, organisms showing a delayed fermentation of
lactose were not eliminated by Lauryl Tryptose Broth.

Bacto-Lauryl Tryptose Broth was studied by 17 collaborating laboratories
situated throughout the United States and Canada. The results of this compara-
tive survey are reported by McCrady^. The study comprised the use of different
types of water and embraced different methods of treatment of samples. The re-
sults showed that the substitution of Lauryl Tryptose Broth for "Standard Meth-
ods" Lactose Broth would result in a reduction in the number of primary gas
positives to be confirmed, and an increase in the number of positive coliforms. It
was recommended that further study be made by different laboratories with par-
ticular reference to use of the medium in the examination of finished waters.

Perry and Hajna'' in a comparative study of E G Medium and Lauryl Tryp-
tose Broth reported both media to be highly sensitive and specific for coliform
bacteria from water, shellfish and sewage. A positive presumptive test with either
medium was more dependable than the usual "confirmed" or "completed" test.

The Ninth Edition of "Standard Methods for the Examination of Water and
Sewage" permits the substitution of Lauryl Tryptose Broth for Lactose Broth in
the standard tests for members of the coliform group in the examination of all
waters except final filtered, treated and filter-treated waters: "It may be sub-
stituted for lactose broth also in the examination of final filtered, treated and
filtered-treated waters provided the laboratory worker has amply demonstrated
by correlation of positive completed tests (isolations of coliform organisms)
secured through the use of lauryl sulfate tryptose broth with those secured
through the use of lactose broth, in the examination of such waters, that the
substitution results in no reduction from the density of coliform organisms indi-
cated by the standard procedure using lactose broth"^.

Bacto-Lauryl Tryptose Broth duplicates the formula described by Mallmann
and Darby^ and specified in "Standard Methods for the Examination of Water
and Sewage," Ninth Edition^. It may be prepared in single strength when ex-
amining 1 ml. or less of water as an inoculum. For inocula of 10 ml. consult the
table given below.

Concentration of Dehydrated Medium Required
to Maintain the Proper Concentration of Ingredients

Bacto-Lauryl
Amt. Medium Vol. Medium Tryptose Broth

Inoculum in Tube and Inoculum used per 1000 ml.

1 ml. or less 10 ml. 10 ml. 35.6 g.

10 ml. 20 ml. 30 ml. 53.4 g.

10 ml. 30 ml. 40 ml. 47.3 g.

DEHYDRATED CULTURE MEDIA 41

To rehydrate the medium, dissolve 35.6 grams of Bacto-Lauryl Tryptose Broth
in 1000 ml. distilled water. Distribute into fermentation tubes and sterilize in the
autoclave for 15 minutes at 15 pounds pressure (121°C.). The final reaction of
the medium will be pH 6.8.

One pound of Bacto-Lauryl Tryptose Broth is suiBcient for 12.7 liters single
strength medium.

1 Am. J. Pub. Health, 31:127:1941. * J. Am. Water Works Assoc, 31:689:1939.

2 Standard Methods for the Examination of ^ Am. J. Pub. Health, 31:351:1941.
Water and Sewage, gth Edition: 193:1946. « Am. J. Pub. Health, 33:ii99: i943-

3 J. Am. Water Works Assoc, 30:979:1938. '^ Am. J. Pub. Health, 34:735:1944.

SUPPLEMENTARY MEDIA

for Water and Sewage Examination

The media in this section are listed in Appendix I of "Standard Methods for
the Examination of Water and Sewage," Ninth Edition, 1946, or have been used
for a long period of time in the examination of water and sewage.

In addition to the media discussed in detail in this section, other media
described elsewhere in the Manual are also used in Appendix I procedures. A
complete discussion of these media is given as indicated in the following listing.

Brilliant Green Bile 2%
Formate Ricinoleate Broth
MacConkey Agar
Violet Red Bile Agar
Desoxycholate Lactose Agar
Levine E.M.B. Agar

BACTO

FUCHSIN LACTOSE BROTH (BIO)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Peptone 5 g.

Bacto-Lactose 5 g.

Bacto-Basic Fuchsin 0.015 g.

Bacto-Fuchsin Lactose Broth is a selective medium which may be used in
parallel planting with Lactose Broth in the control of water filtration plant oper-
ation as described in Appendix I of "Standard Methods for the Examination of
Water and Sewage"^.

As a result of investigations of media to reduce the number of false positives
occurring in examination of waters for Escherichia coli, Ritter^ devised a medium
composed of Lactose Broth with the addition of basic fuchsin. The dye is present
in the medium in a concentration which has been found to inhibit satisfactorily
Gram-positive organisms and other bacteria which may be responsible for false
positive tests for E. coli. McCrady,^ in his study on procedures for the detection
of the presence of coliform organisms in water, found that Fuchsin Lactose Broth,
although satisfactory for some waters as a direct presumptive medium, could not
be used as satisfactorily as Lactose Broth with all waters, and, therefore, recom-

42 DIFCOMANUAL

mended that it be used as a confirmatory medium. Bacto-Fuchsin Lactose Broth
is prepared according to the formula specified in "Standard Methods for the
Examination of Water and Sewage"^ and since 1936 our label has carried a
statement to that effect.

To rehydrate the medium, dissolve 13 grams of Bacto-Fuchsin Lactose Broth in
1000 ml. of distilled water. The medium is distributed in fermentation tubes and
sterilized by autoclaving for 15 minutes at 15 pounds pressure (121°G.). The
final reaction of the medium will be pH 6.8.

For water filtration plant control work, where the inoculum is greater than 1
ml., particular care must be taken to preserve the correct concentration of the
ingredients after dilution with the sample. For example, if 10 ml. of water are
to be added to 10 ml. of medium, the medium should be prepared in double
strength.

One pound of Bacto-Fuchsin Lactose Broth will make 34.8 liters of medium.

1 Standard Methods for the Examination ^ Am. J. Pub. Health, 27:1243:1937.

of Water and Sewaee, gth Edition: 226:1946. * Standard Methods for the Examination

*J. Am. Water Works Assoc, 24:413:1932. of Water and Sewage, 8th Edition: 204:1936.

BACTO

M.B.-B.G.P. MEDIUM (B21)

DEHYDRATED

Bacto-Beef Extract 5 g.

Bacto-Peptone 7.8 g.

Bacto-Lactose 4.7 g.

Dipotassium Phosphate 1.7 g.

Monopotassium Phosphate 0.3 g.

Bacto-Erythrosin (L-D) 0.0064 g.

Bacto-Methylene Blue (L-D) . . . 0.064 g.

Bacto-Brom Cresol Purple 0.01 g.

Bacto-M.B.-B.G.P. Medium has been suggested for the detection of coliform
bacteria in water supplies and is included in Appendix I of "Standard Methods
for the Examination of Water and Sewage"^ for use in parallel planting with
Lactose Broth in the control of water filtration plant operations. It is prepared
according to a modification of the original formula of Dominick and Lauter^
and varies slightly from the formula in "Standard Methods for the Examination
of Water and Sewage."^

Dominick and Lauter^ devised the M.B.-B.C.P. Medium so as to decrease
the length of time required for determining the presence of coliform bacteria in
water. Their medium is essentially a buffered Lactose Broth to which a com-
bination of dyes has been added. The test depends upon reduction of methylene
blue by coliform organisms so that when the dye has been reduced the acid
(yellow) color of the brom cresol purple is brought to view. The dyes employed
inhibit lactose fermenting organisms w^hich are not members of the coliform
group. A positive test is generally assured within 24 hours incubation and a heavy
contamination will yield a positive test within 12 hours. The incubation of tubes
for 48 hours is recommended when they are not definitely positive after 24 hours.
The production of gas without the characteristic color change or, conversely, a
color change without gas production is regarded as a negative test. In the positive
test there must be gas accompanied by a typical color reaction. Bartram and
Black* found Bacto-M.B.-B.G.P. Medium to be the most productive of the
selective media used in their study of strains of Escherichia, Aerobacter, and inter-
mediates which had been recently isolated from raw milk.

To rehydrate the medium, dissolve 19.6 grams of Bacto-M.B.-B.G.P. Medium

DEHYDRATED CULTURE MEDIA 43

in 1000 ml. of distilled water. Dispense into fermentation tubes in 10 ml. quanti-
ties. Sterilize in the autoclave for 15 minutes at 15 pounds pressure (121°C.).
The final reaction of the medium will be pH 6.9.

For water filtration plant control work or other procedures where the inocu-
lum is greater than a loop or 1 ml., particular care must be taken to preserve the
correct concentration of the ingredients after dilution with the sample. For ex-
ample, if 10 ml. of water are to be added to 10 ml. of medium, the medium
should be prepared in double strength.

One pound of Bacto-M.B.-B.C.P. Medium will make 23.1 liters of medium.

1 Standard Methods for the Examination ^ Standard Methods for the Examination

of Water and Sewaee, gth Edition: 226:1946. of Water and Sewage, 8th Edition: 261:1936.

a J. Am. Water Works Assoc, 21:1067:1929. * J. Bact., 37:37' :i939-

BACTO

CRYSTAL VIOLET LACTOSE BROTH (B8)

DEHYDRATED

Bacto-Peptone 5 g.

Bacto-Lactose 5 g.

Dipotassium Phosphate 5 g.

Monopotassium Phosphate 1 g.

Bacto-Crystal Violet 0.00143 g.

Bacto-Crystal Violet Lactose Broth is a selective medium used in parallel plant-
ing with Lactose Broth in the control of water filtration plant operations as de-
scribed in Appendix I of "Standard Methods for the Examination of Water and
Sewage"^ It is prepared according to the formula suggested by Salle.^

Crystal Violet Lactose Broth is a buffered Lactose Broth containing crystal
violet (gentian violet) in a concentration sufficient to inhibit Gram-positive or-
ganisms and other bacteria which may be responsible for false positive tests. The
concentration of this bacteriostatic agent in the medium is not sufficient to affect
the growth of members of the coliform group. Salle^ suggested that such a me-
dium be employed in the bacteriological examination of water, and McCrady^ in
studies on media for the detection of the presence of coliform organisms in water,
found that while selective media were not as satisfactory as Lactose Broth for
the presumptive test, they could be recommended for confirmation of the pre-
sumptive test, and that for this purpose Crystal Violet Lactose Broth could be
recommended.

The Ninth Edition of "Standard Methods"* permits the use of Crystal Violet
Lactose Broth as an alternate medium for the confirmed test for the presence
of members of the coliform group when it has been shown to yield a maximum
number of coliform organisms as indicated by a series of completed tests. Bacto-
Crystal Violet Lactose Broth is prepared according to the formula specified in
"Standard Methods for the Examination of Water and Sewage,"^ and since
1936 our label has carried a statement to that effect.

To rehydrate the medium, dissolve 16 grams of Bacto-Crystal Violet Lactose
Broth in 1000 ml. of distilled water. The medium is distributed in fermentation
tubes and sterilized by autoclaving for 15 minutes at 15 pounds pressure.
(121°C.). The final reaction of the medium will be pH 7.4.

For water filtration plant control work, where the inoculum is greater than 1
ml., particular care must be taken to preserve the correct concentrations of the
ingredients after dilution with the sample. For example, if 10 ml. of water arc
to be added to 10 ml. of medium, the medium should be prepared in double
strength.

44 DIFCOMANUAL

One pound of Bacto-Crystal Violet Lactose Broth will make 28.3 liters of
medium.

1 Standard Methods for the Examination * Standard Methods for the Examination

of Water and Sewage, gth Edition: 226:1946. of Water and Sewage, gth Edition: 195:1946.

*J. Bact., 20:381:1930. 5 Standard Methods for the Examination

* Am. J. rub. Health, 27:1243:1937. of Water and Sewage, 8th Edition: 203:1936.

BACTO

EIJKMAN LACTOSE MEDIUM (B17)

DEHYDRATED

Bacto-Tryptose 15 g.

Bacto-Lactose 3 g.

Dipotassium Phosphate 4 g.

Monopotassium Phosphate 1.5 g.

Sodium Chloride 5 g.

Bacto-Eijkman Lactose Medium is used in the differentiation of Escherichia
coli from other coliform bacteria. This medium may also be used in parallel
planting with Lactose Broth in the control of water filtration plant operations.
Its use is suggested in Appendix I of the Ninth Edition of "Standard Methods for
the Examination of Water and Sewage"^ for this purpose.

In 1904 Eijkman^ described a method of separating the strains of coli originat-
ing from the feces of warm-blooded animals from the strains characteristic of
cold-blooded animals. His method consisted essentially in placing the water under
investigation in fermentation tubes or flasks and adding one-eighth its volume of
a sterile solution containing 10 per cent dextrose, 10 per cent peptone, and 5 per
cent sodium chloride. This mixture was then incubated at 46° C. The presence
of a uniform turbidity and gas production was considered indicative of the pres-
ence of fecal colon strains. Many investigators have studied this method with
water samples and pure cultures with varying results. One of the factors limiting
the value of this method was the inability to obtain growth of transplants from
positive tubes incubated at 46° G. Undoubtedly, the acidity at the relatively high
temperature of incubation was responsible for the death of the culture within 24
to 48 hours.

Perry and Hajna^ modified Eijkman's original method of decreasing the carbo-
hydrate content and adding a phosphate buffer. Their study demonstrated that
with 0.3 per cent dextrose and a potassium phosphate buffer the reaction of the
medium, after inoculation with E. coli and incubation, was pH 5.6, while under
the conditions described by Eijkman the reaction of the medium was pH 4.5.
As a result, they were able to culture E. coli in every instance after incubation
at 46° C. for 96 hours and for longer periods.

Perry* in 1939 reported on the use of a modified Eijkman Medium using 0.3
per cent lactose. This medium had been used successfully and routinely for the
isolation of E. coli for a number of years. In a personal communication, Dr.
Perry° recommended that Bacto-Tryptose replace Bacto-Peptone in this formula.
The specificity of this medium for the detection of E. coli of fecal origin requires
that the composition of the medium be uniform and exact, and that the incuba-
tion temperature be properly controlled at 45.5 to 46° G. at all times. The formula
for Bacto-Eijkman Lactose Medium conforms to the formula for Eijkman Broth
as specified in Appendix I, "Standard Methods for the Examination of Water
and Sewage."^

To rehydrate the medium, dissolve 28.5 grams of Bacto-Eijkman Lactose
Medium in 1000 ml. of distilled water and dispense in fermentation tubes. Steri-

DEHYDRATED CULTURE MEDIA 45

lize in the autoclave for 15 minutes at 15 pounds pressure (121°C.). The final
reaction of the medium will be pH 6.8.

When the medium is to be used in water filtration plant control work where
the inoculum is greater than 1 ml., particular care must be taken to preserve
the correct concentration of the ingredients after dilution with the sample. For
example, if 10 ml. of water are to be added to 10 ml. of medium, the medium
should be prepared in double strength.

One pound of Bacto-Eijkman Lactose Medium will make 15.9 liters of
medium.

1 Standard Methods for the Examination * Food Research, 4:381:1939.

of Water and Sewage, 9th Edition: 226: 1946. ^ Personal Communication, 1939.

2 Centr. Bakt., II. Abt., 37:742:1904. « Standard Methods for the Examination

3 J. Bact., 26:419:1933. of Water and Sewage, gth Edition: 232: 1946.

BACTO

E G MEDIUM (B314)

DEHYDRATED

Bacto-Tryptose 20 g.

Bacto-Lactose 5 g.

Bacto-Bile Salts No. 3 1.5 g.

Dipotassium Phosphate 4 g.

Monopotassium Phosphate 1.5 g.

Sodium Chloride 5 g.

Bacto-E C Medium conforms to the formula given by Hajna and Perry^ for the
medium described for the detection of coliform bacteria at 37°G. and of Escher-
ichia coli at 45.5°C. It can be used either as a primary medium for the growth of
E. coli or as a satisfactory secondary medium for E. coli confirmation. This selec-
tive broth may be used in parallel planting with Lactose Broth in the control of
water filtration plant operation as described in Appendix I of "Standard Methods
for the Examination of Water and Sewage."^

In an effort to improve further the methods for the detection of members of
the coliform group and E. coli Hajna and Perry^ developed E C Medium. This
medium consisted of a buffered Lactose Broth to which was added 0.15 per cent
of Bacto-Bile Salts No. 3. Growth of spore formers and fecal streptococci was
inhibited by the bile salts while growth of coli was enhanced by its presence. The
medium can be used at 37 °C. for the detection of coliform organisms or at
45.5 °C. for the isolation of E. coli. In a further evaluation study of the E G
Medium and Lauryl Tryptose Broth, Perry and Hajna^ reported the results ob-
tained from eleven different laboratories examining a variety of waters, milk,
and shellfish. The results indicated that these media were highly specific for
coliform bacteria. A presumptive test reading with the E G Medium or Lauryl
Tryptose Broth seemed more dependable than the usual "confirmed" or "com-
pleted" test for coliform bacteria.

To rehydrate the medium, dissolve 37 grams Bacto-E G Medium in 1000 ml.
of distilled water. Sterilize in the autoclave for 15 minutes at 15 pounds pressure
(121°G.). Final reaction of the medium will be pH 6.9.

For water filtration plant control work, where the inoculum is greater than
1 ml., particular care must be taken to preserve the correct concentration of the
ingredients after dilution with the sample. For example, if 10 ml. of water are to
be added to 10 ml. of medium, the medium should be prepared in double
strength.

One pound of Bacto-E G Medium will make 12.2 liters of medium.

lAm. J. Pub. Heahh, 33:550: i943- 3 Am. J. Pub. Health, 34:735:i944.

* Standard Methods for the Examination
of Water and Sewage, 9th Edition: 226: 1946.

46 DIFGO MANUAL

BACTO

H D BROTH (B443)

DEHYDRATED

Bacto-Tryptose 20 g.

Bacto-Lactose 5 g.

Sodium Desoxycholate 0.1 g.

Dipotassium Phosphate 4 g.

Monopotassium Phosphate 1.5 g.

Sodium Chloride 5 g.

Bacto-H D Broth (Hajna Desoxycholate Broth) is recommended as a pre-
sumptive and confirmatory medium for coliform bacteria from drinking water,
swimming pools, sewage, urine, shell fish and dairy products. This medium is
prepared according to the formula given by Hajna,^ using sodium desoxycholate
as a selective agent.

The coliform group as defined by the "Standard Methods for the Examination
of Water and Sewage"^ includes all aerobic and facultative anaerobic Gram-
negative non-spore-forming bacilli which ferment lactose with gas formation.
Leifson^ suggested the use of sodium desoxycholate in Lactose Broth for the de-
tection of coliform bacteria. Hajna^ determined that 0.1 g. sodium desoxycholate
per liter of Buffered Tryptose Lactose Broth to be optimum for the development
of coliforms. This medium proved to be equal or superior to standard Lactose
Broth as a presumptive medium and equal or superior to Brilliant Green Bile 2%
as a confirmatory medium.

In comparing the efficiency of the Desoxycholate Broth and standard Lactose
Broth using a variety of specimens including market oysters, crab meat, cottage
cheese, water samples of all types, swimming pool water samples, milk and urine,
2460 tubes showed gas in the H D Broth, all of which confirmed as members of
the coliform group; 2811 showed gas in standard Lactose Broth tubes with 2421
confirming as coliform by the Brilliant Green Lactose Bile 2% method.^ As a
confirmatory medium from 7669 presumptive Lactose Broth tubes, H D Broth
gave 7499 confirmations for coliform against 7263 with Brilliant Green Bile 2%.^

To rehydrate the medium, dissolve 35.6 grams of Bacto-H D Broth in 1000 ml.
distilled water. Distribute in tubes with fermentation vials and sterilize in the
autoclave for 15 minutes at 10 pounds pressure (116°G.). Sterilization at 121 °C.
is not recommended. Final reaction of the medium will be pH 6.9.

When the inoculum is larger than 1 ml. per 10 ml. of medium, particular care
must be taken to preserve the correct concentration of the ingredients after dilu-
tion with the sample. For example if 10 ml. of water are to be added to 10 ml.
of medium, the medium should be prepared in double strength.

One pound of Bacto-H D Broth will make 12.7 liters of medium.

iPub. Health Lab., 9:77:1951. Water and Sewage, gth Edition: 193: 1946.

2 Standard Methods for the Examination of 3 j. Path. Bact., 40:581 :i935-

BACTO

S F MEDIUM (B315)

DEHYDRATED

Bacto-Tryptone 20 g.

Bacto-Dextrose 5 g.

Dipotassium Phosphate 4 g.

Monopotassium Phosphate 1.5 g.

Sodium Azide 0.5 g.

Sodium Chloride 5 g.

Bacto-Brom Gresol Purple 0.032 g.

DEHYDRATED CULTURE MEDIA 47

Bacto-S F Medium is recommended as a selective liquid medium for the detec-
tion of fecal streptococci from milk, water, swimming pools, sewage and feces. In
this medium coliform and other Gram-negative organisms are inhibited. Bacto-S
F Medium is prepared according to the formula of Hajna and Perry i.

Sodium azide has been employed as a selective agent in media for the isolation
of streptococci by a number of investigators. Snyder and Lichstein^ and Lichstein
and Snyder^ reported that 0.01 per cent sodium azide in Blood Agar prevented
swarming of proteus and permitted the isolation of streptococci from stools and
other infected material without overgrowth by Gram-negative organisms. Mall-
mann* used 1:5000 sodium azide in a buffered Tryptose broth for the exami-
nation of sewage and reported that streptococci grew while coliform organisms
were inhibited. This method made possible an easy procedure for routine labora-
tory detection of streptococci. Packer^-^ used sodium azide and crystal violet
in the preparation of a selective Blood Agar for streptococci and Erysipelothrix
rhusiopathiae. Hajna and Perry^ devised a selective medium containing 0.05 per
cent sodium azide, dextrose and the indicator, brom cresol purple, for the detec-
tion of fecal streptococci in swimming pools, water samples and milk. They
specified 45.5 °C. as the incubation temperature, and reported that growth ac-
companied with an acid reaction, as shown by the change in color of the medium,
was almost complete evidence of the presence of fecal streptococci.

To rehydrate the medium dissolve 36 grams Bacto-S F Medium in 1000 ml.
distilled water. Distribute in tubes and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°G.). Final reaction of the medium will be pH 6.9.

When the inoculum is greater than 1 ml., particular care must be taken to
preserve the correct concentration of the ingredients after dilution with the
sample. For example, if 10 ml. of water are to be added to 10 ml. of medium,
the medium should be prepared in double strength.

One pound of Bacto-S F Medium will make 12.6 liters of medium.

lAm. J. Pub. Health, 33:55o:i943- * Sewage Works J., 12:875:1940.

3 J. Infectious Diseases, 67:113:1940. e j. Bact., 42:137:1941.

•J. Bact., 42:653:1941. e J. Bact., 46:343:1943.

BACTO

B A G G BROTH (B442)

DEHYDRATED

Bacto-Tryptose 20 g.

Bacto-Dextrose (Glucose) 5 g.

Dipotassium Phosphate 4 g.

Monopotassium Phosphate 1.5 g.

Sodium Chloride 5 g.

Sodium Azide 0.5 g.

Bacto-Brom Cresol Purple 0.015 g.

Bacto-B A G G Broth (Buffered Azide Glucose Glycerol Broth) is a selective
liquid medium for the detection of fecal streptococci from all types of specimens.
It is prepared according to the formula given by Hajna^ and is a modification of
SF Medium as described by Hajna and Perry. 2 Coliform and other Gram
negative organisms and buccal streptococci are inhibited on this medium as they
are on SF Medium while fecal streptococci develop unrestricted.

In using SF Medium for the detection and isolation of fecal streptococci,
particularly Streptococcus fecalis, Hajna noted that acid production in the
medium was much more rapid when the inoculum consisted of a stool received
in buffered glycerol saline. Investigation of this observation showed that glycerol

48 DIFGOMANUAL

facilitated the fermentation of dextrose by members of Streptococcus Group D
and Hajna^ described the addition of glycerol to SF Medium to give the same
rapid acid production with pure cultures of S. fecalis as with stool specimens.
The concentration of Brom Cresol Purple was decreased to detect more readily
the color change from purple to yellow within 24 hours incubation.

Hajna^ reported that growth with acid production is almost definite evidence
of the presence of fecal streptococci. Streptococcus zymogenes, durans and lique-
faciens develop at 37°C. or 45°G. Streptococcus lactis does not grow at 45°G.
making possible a differentiation of these organisms using this incubation tempera-
ture. An incubation temperature of 37°G. is suggested for the detection of fecal
streptococci from swimming pools, water samples, food products such as oysters
and crab meat and from pathological material such as catheterized urine and
exudates.

To rehydrate the medium, dissolve 36 grams Bacto-B A G G Broth in 1000 ml.
of distilled water containing 5 ml. Bacto-Glycerol. Distribute in tubes in 10 ml.
amounts and sterilize in the autoclave for 15 minutes at 10 pounds pressure
(116°G.). Sterilization at 121 °G. is not recommended. Final reaction of the
medium will be pH 6.9.

When the inoculum is larger than 1 ml., particular care must be taken to pre-
serve the correct concentration of the ingredients after dilution with the sample.
For example, if 10 ml. of water are to be added to 10 ml. of medium, the
medium should be prepared in double strength.

One pound of Bacto-B A G G Broth will make 12.6 liters of medium.

iPub. Health Lab., 9:80:1951. a Am. J. Pub. Health, 33:550:t943.

BACTO

AZIDE DEXTROSE BROTH (B387)

DEHYDRATED

Bacto-Beef Extract 4.5 g.

Bacto-Tryptone 15 g.

Bacto-Dextrose 7.5 g.

Sodium Chloride 7.5 g.

Sodium Azide 0.2 g.

Bacto-Azlde Dextrose Broth is a liquid medium selective for streptococci, and
is recommended for use in the detection of these organisms from milk, water,
swimming pools, sewage, feces and other specimens. This medium is prepared
according to a formula given by Rothe^ as emanating from the laboratory of
the Illinois State Health Department.

The use of sodium azide as an inhibitor of Gram-negative organisms in an
attempt to detect streptococci has been pointed out by a number of investigators.
Edwards^ in 1933 used a liquid medium containing crystal violet and sodium
azide as a selective broth in the isolation of mastitis streptococci. Hartman^
reported the value of sodium azide as a selective agent for the isolation of
streptococci causing mastitis. Bryan, Devereux, Hirschey and Gorbett* reported
that sodium azide in a concentration of 1 :5000 was a better selective preservative
for milk cultures and gave more accurate results for the microscopic and Hotis
tests for Streptococcus mastitis than 1 : 50,000 brilliant green.

Mallmann, Botwright and Ghurchill^ in studying the selective bacteriostatic
effect of slow oxidizing agents reported that sodium azide exerted a bacterio-
static effect on Gram-negative bacteria and permitted the growth of Gram-
positive organisms. Hajna and Perry ^ used this principle in designing a selective

DEHYDRATED CULTURE MEDIA 49

medium for the detection of fecal streptococci. Their medium, Bacto-S F
Medium, is described in detail on page 46 of the Manual. Ritter and Treece^
also used 0.02 per cent sodium azide in a broth containing 2 per cent Proteose
Peptone No. 3, 0.1 per cent dextrose and 0.5 per cent sodium chloride for the
detection of cocci in swimming pools. The selectivity of this medium was later
increased by the addition of crystal violet to this medium by Ritter.^

In a comparative study of media for the detection of streptococci in water
and sewage Mallmann and Seligmann^ used Bacto-Azide Dextrose Broth. They
reported the medium offered a new means of measuring the presence of strepto-
cocci in water, sewage, shellfish and other materials in which sewage pollution
is suspected.

To rehydrate the medium dissolve 34.7 grams Bacto-Azide Dextrose Broth in
1000 ml. distilled water. Distribute in tubes and sterilize in the autoclave for 15
minutes at 15 pounds pressure (121°C.). Inocula of 1 loop or 1 ml. may be
added to 10 ml. or more of medium. The medium should be prepared in mul-
tiple strength for larger inocula to preserve the correct concentration of in-
gredients. For example, if 10 ml. of inoculum is to be added to 10 ml. of me-
dium, the medium should be prepared in double strength. Final reaction of the
medium will be pH 7.2.

One pound of Bacto-Azide Dextrose Broth will make 13.1 liters single
strength medium.

^ Rothe, Personal Communication, 1948. ^ Am. J. Pub. Health, 33:550:1943.

2 J. Comp. Path. Therap., 46:211:1c " ' ' '^

8 Milchw. Forsch., 18:116:1936.

* North Am. Vet., 20:424:1939.

6 J. Infectious Diseases, 69:215:1941.

2 J. Comp. Path. Therap., 46:211:1933. "Am. J. Pub. Health, 38:1532:

8 Milchw. Forsch., 18:116:1936. ^Ritter, Personal Communication, 1949.

* North Am. Vet., 20:424:1939. » Am. J. Pub. Health, 40:286:1950.

BACTO

BORIC ACID BROTH (B439)

DEHYDRATED

Proteose Peptone, Difco 10 g.

Bacto-Lactose 5 g.

Dipotassium Phosphate 12.2 g.

Monopotassium Phosphate 4.1 g.

Boric Acid 3.25 g.

Bacto-Boric Acid Broth is a liquid medium for the enrichment and presump-
tive identification of Escherichia coli from water and food, as indicated by
growth and gas production at 42.5°^3.5°C. This medium is prepared accord-
ing to the formula of Vaughn, Levine and Smith.^

The presence of E. coli is considered by many to be a reliable index of
human or animal fecal contamination, and for some food products the presence
of coliform bacteria may not be a true indication of the sanitary quality
of the food. Thus a method which would permit the selective enrichment and
identification of E. coli would be of decided value in the determination of the
sanitary significance of the various coliform bacteria found in foods. Many
methods have been devised for this purpose. Bacto-E G Medium as described
on page 45 is a modification of the Eijkman test designed to detect E, coli at an
incubation temperature of 45.5 °C. Boric acid has been added to lactose media to
favor the growth of E. coli as described by Levine^.* and Levine, Epstein and
Vaughn.3 Bartram and Black^ and Wolford and Berry^ reported boric acid to
be of value in the detection and differentiation of E. coli from other coliform
bacteria. Vaughn, Levine and Smith^ made a study of the various ingredients,

50 DIFCOMANUAL

reaction, concentration, and incubation temperature and presented data to
show that their medium is useful both for primary enrichment and rapid pre-
sumptive identification of E. coli. They stressed that all conditions of the test
must be adhered to or the medium is of little value. The concentration of
boric acid must be maintained at 3.25 g. per liter. Multiple strength medium
must be prepared when inocula greater than 1 ml. per 20 ml. of medium are
used. The temperature of the medium must be constantly kept between 42.5°-
43.5 °C., and experience has shown that an oil bath with adequate stirring device
to insure even distribution of heat is required. They report that the use of
ordinary or water jacketed air incubators and water baths have re-emphasized
the desirability of an oil bath. Maximum inhibition of the boric acid resistant
members of the Aerobacter and E. freundii groups are obtained only under these
conditions, permitting the growth and development of gas by only E. coli in 48
hours. The productivity of Boric Acid Broth was investigated by the authors.
They were able, in pure culture studies, to demonstrate gas production in every
combination of E. coli and other coliforms, including a boric acid resistant Aero-
bacter aerogenes strain when E. coli was present in the mixture. In the absence of
this organism no gas was formed in 48 hours. The detection of E. coli from water
samples was compared using Lactose Broth at 35 °G. and the Boric Acid Broth at
42.5°-43.5°G. The results of this series of experiments showed that the boric
acid medium could be considered more reliable than Lactose Broth for enrich-
ing and recovering E. coli from contaminated water. Similar results have been
obtained using food samples.

Bacto-Boric Acid Broth duplicates the formula described by Vaughn, Levine
and Smith^. They stated that their medium might be prepared with the addition
of Andrade or other suitable indicator, if desired. Similarly any desired indicator
may be added in preparing the medium from Bacto-Boric Acid Broth.

To rehydrate the medium, dissolve 34.6 grams in 1000 ml. distilled water.
Distribute in tubes in 20 ml. amounts with inverted fermentation vials and
sterilize in the autoclave for 15 minutes at 15 pounds pressure (121°C.). If
inocula greater than 1 ml. is used the medium must be prepared in multiple
strength to maintain the correct concentration of the ingredients. Final reaction
of the medium will be pH 7.0.

One pound of Bacto-Boric Acid Broth will make 13.1 liters single strength
medium.

1 Food Research, 16:10:1951. * J. Bact., 29:24:1935.

3 Am. J. Pub. Health, 11:21:1921. ^ J. Bact., 31 :24:i936.

3 Am. J. Pub. Health, 24:505:1934. « Food Research, 13:172:1948.

BACTO

ENTEROGOGGI PRESUMPTIVE BROTH (B300)

DEHYDRATED

Bacto-Tryptone 5 g.

Bacto- Yeast Extract 5 g.

Bacto-Dextrose 5 g.

Sodium Azide 0.4 g.

Bacto-Brom Thymol Blue 0.032 g.

These Enterococci media are prepared according to the formulae given by
Sandholzer and Winter^ for the detection of Enterococci in water supplies, swim-
ming pools, sewage or other specimens suspected of containing these organisms.
The procedure, as described, consists of a presumptive test in which the produc-

DEHYDRATED CULTURE MEDIA 51

tion of acid and turbidity (growth) in an azide presumptive broth after incuba-
tion at 45 °C. is considered positive presumptive evidence for the presence of
Enterococci. The positive presumptive tests are then confirmed by inoculating a
slant-broth combination prepared with an azide agar medium overlaid with a
salt-azide-penicillin broth. Pinpoint colonies on the slant, growth sediment in the
broth, the presence of Gram-positive ovoid streptococci in the broth, and a nega-
tive catalase test is considered confirmed positive evidence of the presence of
Enterococci.

To rehydrate the medium (single strength) dissolve 15.4 grams Bacto-Entero-
cocci Presumptive Broth in 1000 ml. of distilled water. Tube in 8 ml. amounts
and sterilize in the autoclave for 15 minutes at 15 pounds pressure (121°G.). The
concentrated or 5 strength presumptive enrichment broth is prepared by dis-
solving 77 grams of Bacto-Enterococci Presumptive Broth in 1000 ml. distilled
water. Inocula of 1 loop to 2 ml. are used for 8 ml. tube of the single strength
medium. The 5 times strength medium is distributed in 2 ml. amounts and fol-
lowing sterilization as above, is inoculated with 10 ml. of sample per tube. The
final reaction of the medium is pH 8.4.

One pound of Bacto-Enterococci Presumptive Broth will make 29.4 liters of
medium.

1 Commercial Fisheries Leaflet TLa, November, 1946.

BACTO

ENTEROCOCCI CONFIRMATORY AGAR (B301)

DEHYDRATED

Bacto-Tryptone 5 g.

Bacto-Yeast Extract 5 g.

Bacto-Dextrose 5 g.

Sodium Azide 0.4 g.

Bacto-Agar 15 g.

Bacto-Methylene Blue 0.01 g.

A discussion of the use of this medium is given above under Bacto-Enterococci
Presumptive Broth.

To rehydrate the medium, suspend 30.4 grams Bacto-Enterococci Confirma-
tory Agar in 1000 ml. of cold distilled water. Heat to boiling to dissolve the me-
dium completely and distribute in tubes. Sterilize in the autoclave for 15 minutes at
15 pounds pressure (121°C.). The medium is allowed to cool in a slanted posi-
tion. The final reaction of the medium is pH 8.0.

One pound of Bacto-Enterococci Confirmatory Agar will make 14.9 liters of
medium.

52 DIFCOMANUAL

BACTO

ENTEROGOGGI GONFIRMATORY BROTH (B302)

DEHYDRATED

Bacto-Tryptone 5 g.

Bacto- Yeast Extract 5 g.

Bacto-Dextrose 5 g.

Sodium Azide 0.4 g.

Sodium Chloride 65 g.

Bacto-Methylene Blue 0.01 g.

A discussion of the use of this medium is given above under Bacto-Enterococci
Presumptive Broth.

To rehydrate the medium, dissolve 80.4 grams Bacto-Enterococci Confirma-
tory Broth in 1000 ml. of distilled water. Distribute in flasks in 100 ml. quanti-
ties and sterilize in the autoclave for 15 minues at 15 pounds pressure (121°G.).
Final reaction of the medium will be pH 8.0. When cooled to room temperature
and just prior to use, add 65 units of penicillin to each 100 ml. of medium.
Enough Enterococci Confirmatory Broth containing penicillin is added to each
Enterococci Confirmatory Agar slant to cover approximately one-half the surface
of the slant.

One pound of Bacto-Enterococci Confirmatory Broth will make 5.6 liters of
medium.

BACTO

BRILLIANT GREEN BILE AGAR (B14)

DEHYDRATED

Bacto-Peptone 8.25 g

Bacto-Lactose 1.9 g

Bacto-Oxgall 0.00295 g

Sodium Sulfite 0.205 g

Ferric Chloride 0.0295 g

Monopotassium Phosphate 0.0153 g

Special Agar (Noble) 10.15 g

Erioglaucine 0.0649 g,

Bacto-Basic Fuchsin 0.0776 g

Bacto-Brilliant Green 0.0000295 g

Bacto-Brilliant Green Bile Agar duplicates the medium described by Noble
and Tonneyi for determining the relative density of coliform bacteria in water
and sewage. This medium is not recommended for the determination of the abso-
lute density of coliform organisms in water samples, but rather as an indication
of the degree of contamination of the sample. It is suggested as a selective agar
medium for this purpose in Appendix I of "Standard Methods for the Examina-
tion of Water and Sewage."^

In the enumeration of coliform bacteria using Bacto-Brilliant Green Bile Agar
plates should be poured in dilutions which will show not less than ten colonies of
coliform organisms per plate and it is, therefore, suggested that several dilutions
be plated from each sample. When 10 ml. quantities of water are to be plated
an equal quantity of double strength medium should be employed. Inoculated
plates are incubated at 37°C. for 17 hours and for not longer than 19 hours.
Colonies of coliform bacteria are deep red at the center with a pink halo sharply

DEHYDRATED CULTURE MEDIA 53

outlined against a uniformly blue background. The colonies vary from 0.4 to 0.8
mm. in diameter.

The medium is rather sensitive to light, particularly direct sunlight, which
produces a decrease in the productivity of the medium and change in color from
deep blue to purple or red. It is recommended that the medium be prepared just
prior to use and when necessary to store the medium, it should be kept in the dark.

To rehydrate the medium, suspend 20.6 grams of Bacto-Brilliant Green Bile
Agar in 1000 ml. of cold distilled water and heat to boiling to dissolve the
medium completely. Distribute in tubes or flasks and sterilize in the autoclave for
15 minutes at 15 pounds pressure (121°C.).The final reaction of the medium
will be pH 6.9.

One pound of Bacto-Brilliant Green Bile Agar will make 22 liters of medium.

* J. Am. Water Works Assoc, 27:108:1935. 2 Standard Methods for the Examination

of Water and Sewage, 9th Edition: 228: 1946.

BACTO

TRYPTONE (B123)

DEHYDRATED

Bacto-Tryptone in a 1 per cent concentration is specified in "Standard Methods
for the Examination of Water and Sewage"^ for the performance of the indole
test, and is also specified in "Standard Methods for the Examination of Dairy
Products,"^ for use in preparing media for the standard plate count.

A complete discussion of Bacto-Tryptone, and other uses of this peptone, is
given on page 260.

The test for indole recommended in "Standard Methods" is that of Kovacs^
which is also recommended in "Diagnostic Procedures and Reagents"* of the
American Public Health Association. This test is performed by adding 0.2 to 0.3
ml. of Kovacs' reagent to 5 ml. of a 24-hour culture of the organism under in-
vestigation. A dark red color in the surface layer constitutes a positive test; the
original yellow color of the solution is a negative test when this reagent is used
for indole detection. Kovacs' reagent is made by dissolving 5 grams of p-dimethyl-
aminobenzaldehyde in 75 ml. of amyl alcohol and adding 25 ml. of concentrated
hydrochloric acid.

Among the many other tests for the detection of indole, particular mention may
be made here of the Ehrlich-Boehme^ procedure, the Gore^ modification of this
reaction, and the Gnezda'^ technique.

For the Ehrlich-Boehme test two solutions are necessary. Solution I consists
of 1 gram p-dimethylaminobenzaldehyde in 95 ml. of ethyl alcohol (95 per cent)
and 20 ml. of concentrated hydrochloric acid. Solution II is a saturated aqueous
solution of potassium persulfate. To about 10 ml. of the culture showing good
growth of the organism 5 ml. of Solution I are added, then 5 ml. of Solution II
are added, and the mixture is shaken well. A red color developing in five minutes
indicates a positive test for the presence of indole. Pure Culture Study of Bacteria^
states that this test may be performed by first shaking up the culture with ethyl
ether and then adding Solution I, dropping it down the side of the tube so that it
spreads out as a layer between the ether and the culture fluid. The formation of
a purplish red color at the interface of the two liquids within 5 minute§ indicates
indole production.

For the Gor^ test the same solutions used for the Ehrlich-Boehme test are
employed. The cotton plug, which must be of white absorbent cotton, used for
plugging the culture tube is moistened with four to six drops of Solution II and

54 DIFCOMANUAL

then with the same amount of Solution I. The plug is replaced in the tube and
is pushed down until it is about one inch above the surface of the medium. The
tube is heated in a boiling water bath for 15 minutes without allowing the
medium to touch the plug. The appearance of a red color on the plug indicates
the presence of indole.

The Gnezda test is carried out as follows : A strip of filter paper is dipped into
a warm saturated solution of oxalic acid and is then dried thoroughly. The strip
is bent at an angle and is inserted into the culture tube in such a manner that it
presses against the sides and remains immediately below the cotton plug. Although
it is not necessary to sterilize the paper, aseptic precautions should be observed
when it is placed in the tube. The cotton plug is reinserted into the tube which
is then incubated at 37 °C. Development of a pink color on the paper indicates
the formation of indole.

1 Standard Methods for the Examination ^ Centr. Bakt. I Abt. Orig., 40:129:1905.
of Water and Sewage, gth Edition: 230: 1946. ^ Indian J. Med. Research, 8:505:1921.

2 Standard Methods for the Examination ' Compt. rend. acad. sci., 128:1584:1899.

of Dairy Products, gth Edition: 92: 1948. ^ Manual of Methods of Pure Culture Study

sZeit. Immunitaetsf. Exper. Therap., of Bacteria, 7th Edition: Leaflet V,

55:311:1928. page 9, 1939.

4 Diagnostic Procedures and Reagents,

2nd Edition: 53: 1945.

BACTO

M.R.-V.P. MEDIUM (B16)

DEHYDRATED

Buffered Peptone 7 g.

Bacto-Dextrose 5 g.

Dipotassium Phosphate 5 g.

Bacto-M.R.-V.P. Medium is recommended for the performance of the Methyl
Red and Voges-Proskauer Tests in differentiation of the coli-aerogenes group.
The use of this medium is recommended in Appendix I of "Standard Methods
for the Examination of Water and Sewage"^ for this purpose.

Clark and Lubs^ first pointed out that in a suitable medium coli organisms
produced a high acidity which was constant, while the aerogenes group produced
a much less acid reaction, and on continued incubation became more alkaline.
This difference in the acidity produced in the cultivation of coli or aerogenes
could be recognized by the addition of the indicator methyl red. This Clark and
Lubs test has become known as the Methyl Red Reaction.

Closely associated with the Methyl Red Reaction is the test described by Voges
and Proskauer,^ who noted that a color reaction took place if certain cultures in
a suitable medium were treated with potassium hydroxide and allowed to stand
for some time. This color reaction develops particularly in that part of the
medium exposed to the air, and is very similar to that of a dilute alcoholic solu-
tion of eosin. The development of the color reaction upon treatment of cultures
with potassium hydroxide was found to be due to the presence of acetyl-methyl-
carbinol, (3-hydroxy-2-butanone). Levine* recommended that the term "Voges-
Proskauer Reaction" be restricted to designate the formation of acetyl-methyl-
carbinol from dextrose.

Bacto-M.R.-V.P. Medium was developed as a simple and reliable medium for
use in the performance of the Methyl Red and Voges-Proskauer Tests. Ruch-
hoft, Kallas, Chinn, and Coulter^ reported that Bacto-M.R.-V.P. Medium is
uniform and superior to laboratory-made media for these tests.

For performance of the Methyl Red and Voges-Proskauer Tests, each tube
should be inoculated with a pure culture.

DEHYDRATED CULTURE MEDIA 55

The Methyl Red Test is performed after 5 days incubation at 30° G. as recom-
mended by Vaughn, Mitchell and Levine^ and "Standard Methods."^ For the
Voges-Proskauer Test the culture should be incubated for 24 to 48 hours at

Methyl Red Test

To 5 ml. of culture add 5 drops of methyl red solution. A positive reaction is
indicated by a distinct red color, showing the presence of acid. A negative reac-
tion is indicated by a yellow color. The indicator solution is prepared by dissolv-
ing 0.1 gram Bacto-Methyl Red in 300 ml. of 95 per cent alcohol and diluting
to 500 ml. with distilled water.

Voges-Proskauer Test

To 5 ml. of culture add 5 ml. of a 10 per cent solution of potassium hydrox-
ide, mix well, allow to stand exposed to the air, and observe at intervals of 2,
12, and 24 hours. A positive test is indicated by the development of an eosin
pink color. This is the test as originally described. ^

Various other tests have been suggested as being excellently adapted to the
demonstration of the development of acetyl-methyl-carbinol, chief among which
are those of Werkman,'^ O'Meara,^ Levine, Epstein and Vaughn^ and Vaughn,
Mitchell and Levine.^

Werkman's tesf^ consists of the addition of 2 drops of a 2 per cent solution
of ferric chloride to 5 ml. of culture, followed by 5 ml. of 10 per cent sodium
hydroxide, and shaking the tube well to mix. A stable copper color appearing in
a few minutes is indicative of a positive test.

O'Meara^ recommends the addition of approximately 25 mg. of solid creatine
to 5 ml. of culture and then adding 5 ml. concentrated (at least 40 per cent)
sodium hydroxide. The development of a red color in a few minutes, after
thorough agitation of the tube, is a positive test. Levine, Epstein and Vaughn^
modified the O'Meara technique by dissolving the creatine in a concentrated
solution of potassium hydroxide.

"Standard Methods"^ specifies the use of the Barritt^^ method as recom-
mended by Vaughn, Mitchell and Levine^ for the performance of this test. The
test is made by adding 0.6 ml. of 5 per cent alpha-naphthol in absolute ethyl
alcohol and 0.2 ml. of 40 per cent potassium hydroxide to 1 ml. of culture.

To rehydrate the medium, dissolve 17 grams of Bacto-M.R.-V.P. Medium in
1000 ml. of distilled water. Distribute in 10 ml. quantities in test tubes. Sterilize
in the autoclave for 15 minutes at 15 pounds pressure (121°C.). The final reac-
tion of the medium will be pH 6.9.

One pound of Bacto-M.R.-V.P. Medium will make 26.7 liters of medium.

1 Standard Methods for the Examination ^ J. Am. Water Works Assoc, 3i:993:i939'
of Water and Sewage, gth Edition 1230: 1946. ' j, Bact., 20:121:1930.

2 J. Infectious Diseases, 17:160:1915. ^ J. Path. Bact., 34:401:1931.
»Zeit. Hyg., 28:20:1898. » Am. J. Pub, Health, 24:505:1934.
*J. Bact., 1:153:1916. 10 J. Path. Bact., 42:441:1936.

6 J. Bact., 22:125:1931.

BACTO

KOSER CITRATE MEDIUM (B15)

DEHYDRATED

Sodium Ammonium Phosphate 1-5 g.

Monopotassium Phosphate 1 g.

Magnesium Sulfate 0.2 g.

Sodium Citrate 3 g.

56 DIFCOMANUAL

Bacto-Koser Citrate Medium is a liquid medium recommended for the differ-
entiation of Escherichia coli and Aerohacter aero genes. It conforms to the
formula specified in Appendix I of "Standard Methods for the Examination of
Water and Sewage"^ for this purpose. In this medium an ammonium salt serves
as the sole source of nitrogen, and sodium citrate as the only source of carbon.

The desirability for a means of differentiating coli-aerogenes organisms in
sanitary studies has long been recognized and a wide variety of media have been
devised to accomplish the same. Koser^ showed that either citric acid or its
sodium salt, when employed in a synthetic medium as the only source of carbon,
is readily utilized by A. aero genes, while E. coli fail to develop. This property
was quite constant and not quickly acquired or lost.

In using this medium, coli-like colonies from Endo or Eosin Methylene Blue
Agar plates are inoculated into tubes of Koser Citrate Medium and, after 24 to
48 hours incubation, tubes showing marked turbidity may be assumed to contain
organisms of the aerogenes group. Inasmuch as coli-type organisms fail to grow
in this medium all tubes inoculated from coli-like colonies on Endo or Eosin
Methylene Blue Agar plates and remaining clear after 36 hours incubation may
be considered as coli.

Bacto-Koser Citrate Medium is prepared according to Koser's^ original for-
mula. Chemically pure salts are used in the preparation of the medium and it is
carefully tested to be sure that no sources of carbon, other than the citrate
radical, or nitrogen other than ammonium salts, are present.

To rehydrate the medium, dissolve 5.7 grams of Bacto-Koser Citrate Medium
in 1000 ml. of distilled water. The medium is distributed in tubes and sterilized
by autoclaving for 15 minutes at 15 pounds pressure (121°C.). The final reac-
tion of the medium will be pH 6.7.

One pound of Bacto-Koser Citrate Medium will make 79.6 liters of medium,

1 Standard Methods for the Examination ^ j, Bact., 8:493:1923.

of Water and Sewage, 9th Edition : 231 : 1946.

BACTO

MacGONKEY BROTH (B20)

DEHYDRATED

Bacto-Peptone 20 g.

Bacto-Lactose 10 g.

Bacto-Oxgall 5 g.

Bacto-Brom Cresol Purple 0.01 g.

Bacto-MacConkey Broth is prepared according to a modification of the bile
salt broth recommended by MacConkey^ which contained 0.5 per cent sodium
taurocholate and litmus as an indicator. In later publications^'^ MacConkey
suggested other variants of this medium and employed neutral red instead of
litmus as an indicator.

In recent investigations of the original formula, taking into consideration its
probable use as a presumptive medium in the detection of the coliform group
in water and milk examination, it was demonstrated that Bacto-Oxgall was well
adapted for use in this medium instead of sodium taurocholate. Medium pre-
pared from Bacto-MacConkey Broth is clear and does not require filtration or
adjustment of the reaction. Bacto-MacConkey Broth is now prepared with the
sulfonephthalein indicator Bacto-Brom Cresol Purple. The color of the uninocu-
lated medium is the same as when the indicator litmus was used, but after
incubation, acid is demonstrated by the color of the medium changing to yellow.

To rehydrate the medium, dissolve 35 grams of Bacto-MacConkey Broth in

DEHYDRATED CULTURE MEDIA 57

1000 ml. of distilled water and distribute in 10 ml. quantities in fermentation
tubes. Sterilize by autoclaving for 15 minutes at 15 pounds pressure (121°C.).
The final reaction of the medium will be pH 7.3.

When the medium is to be used with inocula greater than 1 ml., particular
care must be taken to preserve the correct concentration of the ingredients
after dilution with the sample. For example, if 10 ml. of water are to be added
to 10 ml. of medium, the medium should be prepared in double strength.

One pound of Bacto-MacConkey Broth will make 12.9 liters of medium.

1 Centr. Bakt., 29:740:1901. 2 J. Hyg., 8:322:1908.

2 J- Hyg., 5:333:1905.

MEDIA FOR THE EXAMINATION OF
DAIRY AND OTHER FOOD PRODUCTS

The dehydrated culture media listed in this section are recommended for the
examination of milk and other dairy products. In many cases the formulae con-
form to those in "Standard Methods of the Examination of Dairy Products" of
the American Public Health Association and the Association of Official Agricul-
tural Chemists, Ninth Edition 1948, and "Methods and Standards for the Pro-
duction of Certified Milk," 1950, of the American Association of Medical Milk
Commissions, or other official procedures.

Carefully selected standardized ingredients are used in the exact proportions
specified in the "Standard Methods" formulae. The reaction of each medium is
adjusted so that the final reaction of each preparation made from the dehydrated
product will fall within the range of pH recommended in "Standard Methods."

The American Public Health Association has permitted the use of dehydrated
media of "Standard Methods" composition continuously since 1923 in every
edition of its "Standard Methods of Milk Analysis," including the current Ninth
Edition of "Standard Methods for the Examination of Dairy Products."

A guide for the selection of culture media listed in this section is given on
page 25.

"STANDARD METHODS" MEDIA

The media listed in this section conform to the specifications for the examina-
tion of milk and other dairy products as outlined in the "Standard Methods for
the Examination of Dairy Products," Ninth Edition.

BACTO

TRYPTONE GLUCOSE EXTRACT AGAR (B2)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Tryptone 5 g.

Bacto-Dextrose (<i-glucose) 1 g.

Bacto-Agar 15 g.

Bacto-Tryptone Glucose Extract Agar is recommended for use in determining
the standard plate count of milk and other dairy products according to the

58 DIFGOMANUAL

methods specified in "Standard Methods for the Examination of Dairy Prod-
ucts."^ In determining the total bacterial counts of certified milk the use of
Tryptone Glucose Extract Milk Agar is approved by the Committee on Methods
and Standards for the Production of Certified Milk. 2 The Ninth Edition of
"Standard Methods for the Examination of Water and Sewage"^ permits the
use of Tryptone Glucose Extract Agar in place of Nutrient Agar for standard
plate counts of water.

Bacto-Tryptone Glucose Extract Agar is a modification of the Tryptone Glu-
cose Skim Milk Agar of Bowers and Hucker.* Extensive investigations in many
widely separated laboratories established the superiority of their medium over
Nutrient Agar for estimations of bacteria in milk and other dairy products.
This work has been ably summarized by Yale^ in his report on the use of the
medium. Robertson^ has employed this medium in a study of the bacterial count
of ice cream, and Dennis and Weiser''' employed it in their study of the influence
of the incubation temperature on bacterial counts of milk. Prickett^ used a Glu-
cose Agar containing Bacto-Tryptone in his study of the thermophilic bacteria
in milk which was described in the Sixth Edition of "Standard Methods of Milk
Analysis"^ and was prepared in the dehydrated form as Bacto- Yeast Dextrose
Agar. "Standard Methods for the Examination of Dairy Products"^^ presently
recommends the use of Tryptone Glucose Extract Agar for the detection of
thermophilic bacteria. Media similar to that used by Prickett were recommended
by Downs, Hammer, Cordes, and Macy^^ in their report on the bacteriological
methods for the analysis of dairy products.

A committee on Standard Methods for the Examination of Dairy Products
evaluated, comparatively, the modified medium of Bowers and Kucker to deter-
mine whether it was superior to the then standard Nutrient Agar for the plate
count of milk. As a result of these studies the committee^^.ia recommended the
adoption of Tryptone Glucose Extract Milk Agar as the standard medium for
the bacteriological plate count of milk. Details of the "Standard Methods" com-
mittee's studies are reported by Abele.^*

Dilutions of the sample are plated according to Standard Methods procedures
on this medium and the plates incubated for 48 hours ± 3 hours, at 32°C. or
35 °C. Comparison of total counts with Tryptone Glucose Extract Milk Agar
and the former standard Nutrient Agar have indicated greatest differences in
low grade milk and milk products. Certified milk and other high grade milks
have shown little difference in final counts.

The dehydrated medium does not contain skim milk since the "Standard
Methods" committee has recommended that skim milk be added to the medium
only when the dilution of the specimen is greater than 1 to 10. Bacto-Skim Milk,
as discussed on page 74, is recommended for the preparation of a standardized
skim milk solution to be used with Bacto-Tryptone Glucose Extract Agar in the
preparation of the skim milk medium.

To rehydrate the medium, suspend 24 grams of Bacto-Tryptone Glucose
Extract Agar in 1000 ml. of cold distilled water. Tap water, or unsatisfactory
distilled water, may give a precipitate in the final medium. Heat to boiling to
dissolve the medium completely. Distribute in tubes or flasks and sterilize in the
autoclave for 15 minutes at 15 pounds pressure (121°C.). The final reaction of
the medium will be pH 7.0. When the medium is to be prepared with skim milk,
10 ml. of skim milk are added to one liter of medium just before sterilization.

If directions for the preparation of the medium are not followed carefully, or
if the medium is subjected to excessive heat before, during or after the steriliza-
tion period, a precipitation may occur. The following recommendations to over-
come difficulties in the preparation of the medium are suggested.

It is necessary to stir the suspended medium during the heating period to

DEHYDRATED CULTURE MEDIA

prevent the medium from settling out and burning on the bottom of the flask.
In the preparation of large quantities of medium (500 ml. or more) about three-
fourths of the water may be heated to boiling. The dehydrated medium is evenly
suspended in the remaining unheated distilled water, and this suspension is
promptly added to the boiling distilled water. The medium should be boiled for
several minutes to effect complete solution.

Not all samples of fresh skim milk are satisfactory for the preparation of the
medium. For that reason we recommend the addition of 10 ml. of a 10 per cent
solution of Bacto-Skim Milk per liter of Tryptone Glucose Extract Agar after
the medium is in complete solution.

The use of Pyrex or other hard glass containers is recommended for steriliza-
tion.

Do not overheat medium in the autoclave. Care should be taken that the
autoclave reaches 15 pounds pressure (121°C.) promptly and be maintained
there for 15 minutes after which the steam supply is cut off and the autoclave
allowed to come to atmospheric pressure without unnecessary delay. Not more
than thirty to forty minutes for the complete operation should be required. The
medium should not be allowed to cool or solidify in the autoclave, but should be
removed promptly after sterilization.

Do not hold melted agar at 45 °G. for over 30 minutes. Tryptone Glucose
Extract Milk Agar will flocculate if held at 43 to 45 °G. for longer than 30 min-
utes. This flocculation can be dispersed by re-heating the medium. The fioccula-
tion will not develop if the medium is held at 48 to 50°G.

If it is not practical to follow the above directions in detail, and a troublesome
precipitate persists, the complete medium may be prepared by the addition of
sterile skim milk to sterile liquid Tryptone Glucose Extract Agar under aseptic
conditions, just prior to pouring plates.

One pound of Bacto-Tryptone Glucose Extract Agar will make 18.9 liters of
medium.

1 Standau-d Methods for the Examination

of Dairy Products, gth Edition: 93: 1948.
3 Methods and Standards for the Production

of Certified Milk. 21:1950.
8 Standard Methods for the Examination

of Water and Sewage, gth Edition: 191 : 1946.
* Tech. BuU. 228. N. Y. State Agr.

Exp. Sta., 1935.
6 Am. J. Pub. Healtk, 28:148:1938.
« Proc. 36th Cong. Intern. Assoc. Ice

Cream Manufacturers, 2:132:1936.
'J, Dairy Science, 20:445:1937.

8 Tech. BuU. 147, N. Y. State Agr.

Exp. Sta., 1928.
8 Standard Methods of Milk Analysis,

6th Edition: 60: 1934.
^ Standard Methods for the Examination

of Dairy Products, gth Edition: 343: 1948.
^ J. Dairy Science, 18:647:1935.
"Am. J. Pub. Health, 28:1447:1938.
^ Ninth Annual Year Book (ig38-39) p. 79,

Suppl., Am. J. Pub. Health, 29:No. 2:1939.
i*Am. J. Pub. Health, 29:821:1939.

BACTO

BRILLIANT GREEN BILE 2%

DEHYDRATED

A complete discussion of this medium as used in the presumptive test for coli-
form organisms in milk and other dairy products is given under Bacto-Brilliant
Green Bile 2%, page 37. This medium has also been referred to as Brilliant
Green Lactose Peptone Bile, and similar variations.

60 DIFCOMANUAL

BACTO

FORMATE RIGINOLEATE BROTH (B9)

DEHYDRATED

Bacto-Peptone 5 g.

Bacto-Lactose 5 g.

Sodium Formate 5 g.

Sodium Ricinoleate 1 g.

Bacto-Formate Ricinoleate Broth is prepared according to the formula speci-
fied in "Standard Methods for the Examination of Dairy Products"^ and
"Standard Methods for the Examination of Water and Sewage."^ It is used for
the presumptive test for the members of the coliform group in the bacteriological
examination of milk according to "Standard Methods."^ The procedure for use
of this medium in water examination is discussed on page 39.

In research on the development of a medium which would be sufficiently-
selective, yet at the same time not inhibitive to any member of the coliform
group which might be present in the water or milk sample, Stark and England*
devised Formate Ricinoleate Broth. The sodium formate present in the medium
accelerates growth and gas production of coli and related organisms, while
sodium ricinoleate inhibits the development of Gram-positive bacteria and other
organisms which give positive presumptive tests that do not confirm. It is inter-
esting to note that gas production appears earlier in this medium than in other
media under the same conditions, and that it is present in larger proportions at
the completion of the test. The buffer action of the medium, due largely to the
destruction of the sodium formate, maintains a more constant reaction, as shown
by the fact that, even after 48 hours incubation with coli, the reaction of the
medium is rarely more acid than pH 6.0. Noble and White,*^ in their report on
the relative productivity of various fermentation media, state that media pre-
pared from Bacto-Formate Ricinoleate Broth had a higher productivity rating
than other media with which they were compared.

Tiedeman and Smith,^ in a comparison of several methods for the detection
of coliform organisms in pasteurized milk stated that in a survey of 896 samples
of pasteurized milk, showing no fermentation in Formate Ricinoleate Broth, 82 1
or 91.6 per cent gave no colonies on Desoxycholate Agar. An additional 56 or
6.2 per cent gave a count of between 1 and 3 colonies per ml. A total of 97.8
per cent negative fermentation tests gave less than 3 colonies on the plating
medium, which was considered good correlation. They further state that the
Formate Ricinoleate Broth fermentation test is a critical one but does not give
information as to the number of coliforms present as does the use of Violet Red
Bile Agar or Desoxycholate Agar.

In the presumptive test for members of the coliform group in the examination
of dairy products a series of tubes of Formate Ricinoleate Broth are inoculated
with appropriate dilutions of the sample. Use 5 tubes of each dilution. Select
dilutions to provide at least one positive and one negative tube in the series
inoculated. Incubate tubes for 48 hours at 35-3 7 °C. Gas formation constitutes
a positive presumptive test.

When the medium is to be used in water filtration plant control work'' where
the inoculum is greater than 1 ml., particular care must be taken to preserve the
correct concentration of the ingredients after dilution with the sample. The table
given below indicates the quantity of dehydrated medium to use per 1000 ml.

To rehydrate the medium, dissolve 16 grams of Bacto-Formate Ricinoleate
Broth in 1000 ml. of distilled water. The medium is distributed in fermentation
tubes and sterilized by autoclaving for 15 minutes at 15 pounds pressure
(121°C.). The final reaction of the medium will be pH 7.4.

DEHYDRATED CULTURE MEDIA 61

One pound of Bacto-Formate Ricinoleate Broth will make 28.3 liters of

medium.

Concentrations of Dehydrated Medium Required
to Maintain the Proper Concentration of Ingredients

Amt. Medium

Vol. Medium and

Bacto-Formate Ricinoleate

Inoculum

in Tube

Inoculum

Broth used per looo ml.

Loop or 0.1

ml.

10 ml. or more

10 ml.

more 16 g.

ml.

5 ml.

6 ml.

19.2 g.

ml.

10 ml.

20 ml.

32 g.

ml.

15 ml.

25 ml.

26.6 g.

ml.

20 ml.

30 ml.

24 I.

ml.

30 ml.

40 ml.

21.3 g.

1 Standard Methods for the Examination * J. Bact., 29:26:1935.
of Dairy Products, 9th Edition: 131 : 1948. ^ j. Bact., 29:23:1935.

2 Standard Methods for the Examination *• J. Milk Tech., 8:323:1945.

of Water and Sewage, 9th Edition: 188: 1946. 'Standard Methods for the Examination
2 Standard Methods for the Examination of Water and Sewage, 9th Edition: 226: 1946.

of Dairy Products, 9th Edition: 133: 1948.

BACTO

VIOLET RED BILE AGAR (B12)

DEHYDRATED

Bacto- Yeast Extract 3 g.

Bacto-Peptone 7 g.

Bacto-Bile Salts No. 3 1.5 g.

Bacto-Lactose 10 g.

Sodium Chloride 5 g.

Bacto- Agar 15 g.

Bacto-Neutral Red 0.03 g.

Bacto-Grystal Violet 0.002 g.

Bacto-Violet Red Bile Agar is recommended for the direct plate count of
coliform bacteria in water, milk, dairy and other food products. "Standard
Methods for the Examination of Dairy Products"^ recommends the use of Violet
Red Bile Agar for the ml. coliform count in milk and other dairy products. In
Appendix I of "Standard Methods for the Examination of Water and Sewage"^
this medium is suggested as a selective agar medium for direct plating of water
for members of the coliform group.

The original work by Difco Laboratories on the development of a solid selec-
tive medium for the quantitative estimation of the number of viable coliform
organisms in water, milk, dairy and other food products was undertaken in 1932
in cooperation with M. H. McCrady, at that time sub-referee of the committee on
Standard Methods of Milk Analysis of the American Public Health Association.
The formula adopted had exhaustive trials in our laboratories and was also used
extensively by others in the determination of the presence and numbers of coli-
form organisms in milk and dairy products. The use of Bacto-Bile Salts No. 3 in
place of Bacto-Bile Salts originally employed resulted in an improved medium.

Bacto-Violet Red Bile Agar is especially applicable to the control of pasteuri-
zation of milk and cream. The use of this medium makes it possible to determine
quantitatively the coliform count of the milk at any stage of the process. There-
fore, it is possible to determine the cleanliness of apparatus and the efficiency of
the process as well as to detect sources of contamination at various stages.

62 DIFCOMANUAL

Bartram and Black^ in an investigation of media for the isolation of the coli-
form group from raw, pasteurized, and certified milk, found Bacto-Violet Red
Bile Agar to be the most satisfactory solid medium for this work. Babel and
Parfitt^ studying media for the detection of Escherichia-Aerobacter in ice cream,
used Bacto-Violet Red Bile Agar and reported its superiority over other solid
media recommended for such work. Yale^-^ reported the use of Bacto-Violet Red
Bile Agar in making coli counts of ice cream and pasteurized milk. Fabian and
Hook^ used Bacto-Violet Red Bile Agar in establishing the count of Escherichia-
Aerobacter in a study of sanitary conditions of ice cream as served at the foun-
tain. Miller and Prickett^ published a note on the use of this medium in a prac-
tical case concerning recontamination of milk. The Violet Red Bile Agar counts
were completed within 24 hours after plating. This is a considerable saving in
time in comparison with the confirmed Brilliant Green Bile procedure which
requires a minimum period of 48 hours. Quinn and Garnatz^ used Bacto-Violet
Red Bile Agar for the coli-aerogenes count of frozen eggs.

Bacto-Violet Red Bile Agar is a poured plate medium. It is recommended
that 15-20 ml. of medium and not more than 1 ml. of milk be used per petri
dish of 100 mm. diameter. After pouring the plates, the medium is allowed to
solidify. Many investigators prefer to cover the solidified agar with 3-4 ml. of
melted medium as recommended by "Standard Methods for the Examination
of Dairy Products."^*^ Plates are then incubated at 37°C. for 18-24 hours and
at the end of this time are examined by transmitted light. Organisms of the coli-
form group, due to their ability to ferment lactose, form purplish red subsurface
colonies, 1 to 2 mm. in diameter and are generally surrounded by a reddish zone
of precipitated bile. The plates should not be incubated longer than 24 hours,
inasmuch as the organisms whose growth has been suppressed may develop and
confuse the count. Best results are obtained if plates are not too heavily seeded
— the inoculum being diluted so that not more than 150 colonies will develop
per plate.

In using this selective medium best results will be obtained if it is not sub-
jected to autoclave sterilization since organisms, not killed by the boiling re-
quired to dissolve the medium, will not form colonies during the 24-hour
incubation period. Following boiling to dissolve the medium completely, it is
ready for use.

Saccharose in 1 per cent concentration may be added to isolation media, such
as Bacto-Violet Red Bile Agar to permit the detection of certain members of the
coliform group which ferment saccharose more readily than lactose. This prin-
ciple was described by Holt-Harris and Teague^^ and has been employed by
many other bacteriologists.

To rehydrate the medium, suspend 41.5 grams of Bacto-Violet Red Bile Agar
in 1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. Cool to 40-44° C. and pour into plates containing the inoculum.
After solidification of the medium a cover layer may be added, if desired. The
final reaction of the medium will be pH 7.4.

One pound of Bacto-Violet Red Bile Agar will make 11 liters of medium.

^ Standard Methods for the Examination ^ Proc. Intern. Assoc. Ice Cream Manu-
of Dairy Products, 9th Edition: 132: 1948. facturers, 2:30:1936.

2 Standard Methods for the Examination '''Am. J. Pub. Health, 27:564:1937.
of Water and Sewage, 9th Edition: 228: 1946. ^ J. Dairy Science, 21:559:1936.

3 Food Research, 1:551:1936. ^ J. Bact., 45:49:1943.

* J. Dairy Science, 19:497:1936. 10 Standard Methods for the Examination

^ Proc. Intern. Assoc. Ice Cream Manu- of Dairy Products, 9th Edition: 135:1948.

facturers, 2:17:1936. ^ J. Infectious Diseases, 18:596:1916.

DEHYDRATED CULTURE MEDIA 63

BACTO

DESOXYGHOLATE AGAR (B273)

DEHYDRATED

Bacto-Peptone 10 g.

Bacto-Lactose 10 g.

Sodium Desoxycholate 1 g.

Sodium Chloride 5 g.

Dipotassium Phosphate 2 g.

Ferric Citrate 1 g.

Sodium Citrate 1 g.

Bacto-Agar 15 g.

Bacto-Neutral Red 0.03 g.

Bacto-Desoxycholate Agar is used for the direct enumeration of coliform bac-
teria in dairy products as specified in "Standard Methods for the Examination
of Dairy Products."^ It may also be employed as a non-selective primary plating
medium for the isolation of enteric pathogens. The formula of Bacto-Desoxycho-
late Agar is essentially that described by Leifson^ and is recommended for all
procedures specifying the use of a Desoxycholate Agar.

For presumptive determination of coliforms in dairy products a poured plate
of Desoxycholate Agar is prepared. After the medium has solidified, many in-
vestigators prefer to add a thin cover layer of uninoculated medium. Plates are
incubated at 37 °C. for 18-24 hours. Organisms other than those of the enteric
group are inhibited. Coliform colonies are red in contrast to light colorless col-
onies produced by enteric organisms not capable of attacking lactose.

For the isolation of enteric pathogens streak or smear plates are prepared.
Plates should be dry before inoculation for best results. On this medium coli-
form organisms are not inhibited and develop red opaque colonies. Colonies of
Salmonella are colorless and raised with margins varying from smooth to irregu-
lar. Shigella colonies are colorless or opaque presenting somewhat of a ground
glass appearance.

Saccharose in 1 per cent concentration may be added to isolation media, such
as Bacto-Desoxycholate Agar to permit the detection of certain members of the
coliform group which ferment saccharose more readily than lactose. This prin-
ciple was described by Holt-Harris and Teague^ and has been employed by
many other bacteriologists. In some laboratories pathogenic significance is as-
signed to these organisms, and under such conditions, saccharose should not be
added to the medium.

To rehydrate the medium, suspend 45 grams of Bacto-Desoxycholate Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. Do not sterilize in the autoclave. Cool the dissolved liquid medium to
45-50 °C. before using it in making poured plates for the coliform count. After
the inoculated medium in the petri dish has solidified, cover the surface with a
thin layer of the medium cooled to 45-50° C. The final reaction of the medium
will be pH 7.3.

One pound of Bacto-Desoxycholate Agar will make 10 liters of medium.

-^Standard Methods for the Examination 2 j. Path. Bact., 40:581:1935.

of Dairy Products, 9th Edition: 132: 1948. 3 J. Infectious Diseases, 18:596:1916.

64 DIFCOMANUAL

BACTO

DESOXYGHOLATE LACTOSE AGAR (B420)

DEHYDRATED

Bacto-Peptone 10 g

Bacto-Lactose 10 g

Sodium Desoxycholate 0.5 g.

Sodium Chloride 5.0 g

Sodium Citrate 2.0 g

Bacto-Agar 15 g

Bacto-Neutral Red 0.03 g

Bacto-Desoxycholate Lactose Agar is used for the direct enumeration of coli-
form organisms in milk, water and other materials. It is a modification of the
medium described by Leifson,^ and is prepared according to the formula speci-
fied for Desoxycholate Lactose Agar in "Standard Methods for the Examination
of Water and Sewage"^ and "Methods and Standards for the Production of
Certified Milk."^

Counts of coliform organisms may be made directly using Desoxycholate
Lactose Agar. Gram-positive cocci and rods including the spore-forming bacilli
are generally inhibited. Lactose fermenting coliform organisms grow unrestricted
and form typical red subsurface colonies, surrounded by a zone of precipitated
bile and indicator. Incubation periods should be 24 hours or less since other
organisms may develop, on longer incubation causing confusion.

A thin layer of sterile medium may be used as a cover, thus having only sub-
surface colonies. Following solidification of the inoculated plate a small amount
of sterile medium (about 4—5 ml. per 95 mm. plate) is added and allowed to
solidify forming a thin cover layer.

Bacto-Desoxycholate Lactose Agar differs from Bacto-Desoxycholate Agar
essentially in that it contains less sodium desoxycholate, and is accordingly
slightly less selective against Gram-positive organisms. It is suggested that the
medium be sterilized in the autoclave if not used immediately following heating
to boiling to effect complete solution.

To rehydrate the medium suspend 42.5 grams Bacto-Desoxycholate Lactose
Agar in 1000 ml. of cold distilled water and heat to boiling to dissolve the
medium completely. The medium requires no further sterilization if used at
once. If it is to be stored, distribute in tubes or flasks and sterilize in the auto-
clave for 15 minutes at 15 pounds pressure (121°C.). Avoid overheating of the
medium. Final reaction of the medium will be pH 7.1.

One pound of Bacto-Desoxycholate Lactose Agar will make 10.7 liters of
medium.

1 J. Path. Bact., 40:581:1935. a Methods and Standards for the Production

2 Standard Methods for the Examination of Certified Milk, 26:1953-1954.
of Water and Sewage, 9th Edition: 228: 1946.

BACTO

POTATO DEXTROSE AGAR (B13)

DEHYDRATED

Potatoes, Infusion from 200 g.

Bacto-Dextrose 20 g.

Bacto-Agar 15 g.

Bacto-Potato Dextrose Agar is recommended for the determination of yeasts
and molds in butter and in frozen dessert ingredients according to the procedure

DEHYDRATED CULTURE MEDIA 65

of "Standard Methods for the Examination of Dairy Products." ^-^ This medium
is also used for the isolation and cultivation of yeasts and molds in other dairy
and food products. "Standard Methods for the Examination of Dairy Products"^
specifies a reaction of pH 4.5 for Potato Dextrose Agar in determining the mold
content of dried milk.

In a study of comparative methods and media used in the microbiological
examination of creamery butter, Shadwick^ investigated a number of media and
found that Bacto-Potato Dextrose Agar gave the most consistent and highest
count of yeasts and molds in salted and unsalted butter.

To rehydrate the medium, suspend 39 grams of Bacto-Potato Dextrose Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in tubes or flasks and sterilize by autoclaving for 15 minutes
at 15 pounds pressure (121°C.). The medium will have a final reaction of
pH 5.6.

It is frequently desirable in making yeast and mold counts to inhibit bacterial
growth by acidifying the medium, and "Standard Methods" recommends that
the reaction of the medium be reduced to pH 3.5±:0.1 subsequent to sterilization.
The label of each package of Bacto-Potato Dextrose Agar specifies the quantity
of sterile tartaric acid (10 per cent solution) that should be added to each 100
ml. of the sterile melted medium to adjust the reaction to pH 3.5. After the
tartaric acid has been added to the medium, it is mixed well and poured plates
are prepared as usual. The medium should never be heated after the acid is
added inasmuch as heating in the acid state will hydrolyze the agar, destroying
its solidifying properties.

One pound of Bacto-Potato Dextrose Agar will make 11.3 liters of medium.

1 Standard Methods for the Examination 2 Standard Methods for the Examination

of Dairy Products, gth Edition: 157: 1948. of Dairy Products, gth Edition: 181: 1948.

3 Food Research, 3:287:1938.

BACTO

TRYPTOSE AGAR

DEHYDRATED

"Standard Methods for the Examination of Dairy Products," Ninth Edition
recommends the use of Bacto-Tryptose Agar for the detection of Brucella
organisms in milk. A complete discussion of Bacto-Tryptose Agar is given on
page 111.

BACTO

MALT AGAR (B24)

DEHYDRATED

Malt Extract, Difco 30 g.

Bacto-Agar 15 g.

Bacto-Malt Agar is recommended for the detection and isolation of yeasts and
molds from dairy products, food, and other materials. For the mold count of dry
milk "Standard Methods for the Examination of Dairy Products"^ specifies the
use of dehydrated Bacto-Malt Agar or Bacto-Potato Dextrose Agar. This medium
is also recommended for carrying stock cultures of yeast and molds used in
microbiological assay procedures.

Malt media for yeasts and molds have been in use for many years. Reddish^ in

66 DIFGOMANUAL

1919 prepared a satisfactory substitute for beer wort from malt extract. His
medium was used by Thom and Church^ in their studies of the Aspergilli. A
Malt Agar was also employed by Fullmer and Grimes* for their studies of the
growth of yeasts on synthetic media.

The increasing importance of dairy sanitation has already brought milk under
strict microbiological control, and this control has now been extended to include
other dairy products, such as butter, cheese, concentrated and dried milk. Malt
media are readily adapted to the cultivation of fungi encountered in dairy sani-
tation. Butter and cheese manufacturers have recognized the yeast and mold
content of their products as an index of their sanitary classification. The damag-
ing effects of certain molds, which are of tremendous importance, particularly
in butter, may be greatly reduced through eradicating the sources of contamina-
tion by means of strict sanitary control and frequent checking by routine micro-
biological examination. Test samples may be taken at each step of the manufac-
turing process to indicate the sources of contamination.

Hood and White^ have reported their extensive studies of media commonly
employed for counting molds and yeasts in butter, and evolved a tentative
procedure for making these determinations, recommending Bacto-Malt Agar
acidified with lactic acid U.S. P. to a reaction of pH 3.5 just prior to pouring
of plates. This more acid reaction inhibits the growth of bacteria which would
otherwise form colonies that would be confused with colonies of molds and
yeasts.

The committee of the American Dairy Science Association^''^ recommended
the use of Bacto-Malt Agar in its 1930 and 1933 reports on the microbiological
analysis of butter. Parfitt^ has also recommended Bacto-Malt Agar in order to
secure comparative mold and yeast counts of butter. As mentioned above,
"Standard Methods"^ suggest the use of dehydrated Bacto-Malt Agar for mold
count of dry milk. For this purpose the reaction of the medium should be ad-
justed to pH 4.5.

Bacto-Malt Agar is recommended for carrying stock cultures of yeast and
molds used in microbiological assays. The American Association of Cereal
Chemists^ specifies the use of Bacto-Malt Agar in carrying cultures of Sac-
charomyces carlsbergensis in stock and also for the preparation of the inoculum
in the assay of pantothenic acid and Vitamin Bg complex. Miller and Golding^^
used Bacto-Malt Agar in their study of the oxygen requirements of molds.

Heating processes during the rehydration and sterilization should be completed
in as short a period as possible. Excessive exposure to heat causes partial hydroly-
sis of the agar with resultant inability to solidify properly when cooled. Normally,
a medium prepared from Bacto-Malt Agar is slightly soft and is ideal for plating
purposes. However, if the medium is desired for streaking, use 54 grams of Bacto-
Malt Agar per 1000 ml. distilled water, or include 5 grams Bacto-Agar with the
45 grams of Bacto-Malt Agar per liter.

To rehydrate the medium, suspend 45 grams of Bacto-Malt Agar in 1000 ml.
of cold distilled water and heat to boiling to dissolve the medium completely.
Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). The final reaction of the medium will be pH 5.5.

Frequently, in establishing the yeast and mold count, it is desired to avoid
confusion from bacterial growth by acidifying the medium to pH 4.5 or 3.5. On
the label of each package of Bacto-Malt Agar there is specified the amount of
lactic acid U.S. P. (85 per cent) which should be added to 100 ml. of sterile
melted Bacto-Malt Agar to adjust the reaction to pH 4.5 or 3.5. After the acid
has been well mixed with the Malt Agar, plates are poured as usual, allowed to
harden, and incubated for 5 days at 25 °C. before counts of yeast and mold
colonies are made. The medium should never be heated after the acid is added,

DEHYDRATED CULTURE MEDIA 67

as heating in the acid state will hydrolyze the agar, reducing its solidifying
properties so that the resulting medium will be soft or mushy.
One pound of Bacto-Malt Agar will make 10 liters of medium.

1 Standard Methods for the Examination ^ J. Dairy Science, 13:394:1930.
of Dairy Products, 9th Edition: 181 : 1948. '^ J. Dairy Science, 16:289:1933.

2 Abs. Bact., 3:6:1919. ^ J. Dairy Science, 16:141:1933.

2 Thom and Church: The Aspergilli, 1926. ^ Cereal Laboratory Methods, 5th Edition:

* J. Bact., 8:586:1923. 132,135:1947.

s Can. Dept. Agr. Pamphlet, 92-N.S. 1° J. Dairy Science, 32:101,1949.

BACTO

DEXTROSE TRYPTONE AGAR (B80)

DEHYDRATED

Bacto-Tryptone 10 g.

Bacto-Dextrose 5 g.

Bacto-Agar 15 g.

Bacto-Brom Cresol Purple 0.04 g.

Bacto-Dextrose Tryptone Agar is recommended for the determination of
thermophilic "flat sour" organisms associated with the spoilage of food products.
It was developed in collaboration with the research laboratories of the National
Canners Association and is recommended for that purpose in their "Bacterial
Standards for Sugar."^ For plate count of mesophilic or thermophilic aerobes in
sweetening agents used in frozen desserts, "Standard Methods for the Examina-
tion of Dairy Products"- recommend the use of Bacto-Dextrose Tryptone Agar.

Bacto-Dextrose Tryptone Agar is primarily used as a plating medium anc'
when employed for determining thermophiles, it should be incubated at 55 °C.
for 36-48 hours in an incubator sufficiently humid to prevent drying of the
medium.

To rehydrate the medium, suspend 30 grams of Bacto-Dextrose Tryptone
Agar in 1000 ml. of cold distilled water and heat to boiling to dissolve the
medium completely. Distribute in tubes or flasks and sterilize in the autoclave
for 15 minutes at 15 pounds pressure (121°C.). The final reaction of the medium
will be pH 6.7.

One pound of Bacto-Dextrose Tryptone Agar will make 15.1 liters of medium.

^ Pub, of National Canners Association, 1933. 2 Standard Methods for the Examination

of Dairy Products, 9th Edition: 185: 1948.

ADDITIONAL MEDIA

Media as used in "Standard Methods for the Examination of Dairy Products"
procedures are listed below. Complete discussions of these media will be found
on the pages indicated.

Heart Infusion Broth page 80

Cooked Meat Medium page 85

Lactose Broth page 30

Nutrient Broth page 29

Tetrathionate Broth page 157

Tryptose Phosphate Broth page 100

Azide Blood Agar Base page 155

Bismuth Sulfite Agar page 139

Endo Agar page 34

Levine E.M.B. Agar page 35

MacConkey Agar page 131

S S Agar page 134

68 DIFCO MANUAL

SUPPLEMENTARY MEDIA

for Dairy and Other Food Products

The routine bacteriological examination of dairy and other food products may
usually be performed upon the standard media listed in the previous section.
Special examinations including visual sanitation tests, however, require media
for specific purposes. Propagation of pure cultures, and the maintenance of stock
cultures also require special media, and the dehydrated culture media listed in
this section are prepared for such uses.

BACTO

PROTEOSE TRYPTONE AGAR (B23)

DEHYDRATED

Bacto-Beef Extract 3 g.

Proteose Peptone, Difco 5 g.

Bacto-Tryptone 5 g.

Sodium Chloride 5 g.

Bacto-Dextrose 1 g.

Bacto-Agar 15 g.

Bacto-Proteose Tryptone Agar is recommended for determining the bacterial
plate count of certified milk. This medium is prepared according to the formula
specified in "Methods and Standards for the Production of Certified Milk"^ of
the American Association of Medical Milk Commissions, Incorporated.

The combination of Proteose Peptone, Difco, and Bacto-Tryptone in place of
a single peptone is excellently suited for the cultivation of bacteria ordinarily en-
countered in milk. The larger size of the colonies which develop on Proteose
Tryptone Agar during the usual incubation period facilitates counting of the
plate. Comparative study of Bacto-Proteose Tryptone Agar by the Committee on
Methods and Standards has indicated that this dehydrated medium is satisfactory
for determining total counts of bacteria in certified milk. Plate counts are made
according to the usual procedure with 0.1 ml. and 0.01 ml. (1 ml. of 1 to 10 and
1 to 100 dilution) of certified milk and the plates incubated for 48 hours at 35-
37°C. For the bacterial count of milk according to "Standard Methods for the
Examination of Dairy Products," Bacto-Tryptone Glucose Extract Agar, as dis-
cussed on page 57, should be employed.

Proteose Tryptone Agar has also been used as a plating medium to determine
total counts of food products. Quinn and Garnatz^ used Bacto-Proteose Tryptone
Agar for total bacterial count of whole eggs in their study of methods of thaw-
ing frozen eggs.

To rehydrate the medium, suspend 34 grams of Bacto-Proteose Tryptone Agar
in 1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in tubes or flasks and sterilize in the autoclave for 15
minutes at 15 pounds pressure (121°C.). The final reaction of the medium will
be pH 7.0.

One pound of Bacto-Proteose Tryptone Agar will make 13.3 liters of medium.

1 Methods and Standards for the Production ^ J. Bact., 45:49: i943.

of Certified Milk, 26:1953-1954.

DEHYDRATED CULTURE MEDIA 69

BACTO

HEART INFUSION AGAR

DEHYDRATED

This medium is recommended in "Methods and Standards for the Production
of Certified Milk" for the preparation of Blood Agar plates for the recognition
and differentiation of hemolytic streptococci. Such Blood Agar plates may also
be used for the total bacterial count of certified milk. A complete discussion of
Bacto-Heart Infusion Agar is given on page 87.

BACTO

BEEF LACTOSE AGAR (B25)

DEHYDRATED

Beef Heart, Infusion from 450 g.

Proteose Peptone, Difco 5 g.

Bacto-Lactose 10 g.

Bacto-Agar 15 g.

Bacto-Beef Lactose Agar is prepared according to the formula for Beef Infu-
sion Lactose Agar recommended by the subcommittee on Microbiological
Methods for Examining Butter^ of the American Dairy Science Association for
determining the total bacterial count in the microbiological analysis of butter.
This medium, with 0.5 ml. of sterile skim milk added to each plate at the time
of pouring, may be used for the detection of proteolytic colonies.

While much of the spoilage in butter is due to the presence of molds and yeasts,
bacteria are also capable of causing extensive losses due to the production of
undesirable flavors. The subcommittee on Microbiological Methods for Examin-
ing Butter of the American Dairy Science Association in its report has included
methods for the bacterial count and the detection of proteolytic and lipolytic
bacteria as well as for determination of yeasts and molds. In this report the
medium recommended for the total bacterial count is Beef Infusion Lactose
Agar.

To rehydrate the medium, suspend 45 grams of Bacto-Beef Lactose Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). The final reaction of the medium will be pH
6.8.

One pound of Bacto-Beef Lactose Agar will make 10 liters of medium.

* J. Dairy Science, 16:289:1933.

BACTO

NUTRITIVE GASEINATE AGAR (B27)

DEHYDRATED

Bacto-Peptonized Milk 7 g.

Bacto-Isoelectric Casein 3 g.

Special Agar 12 g.

Bacto-Nutritive Caseinate Agar is recommended for the differential plate
count of milk. On poured plates of this medium inoculated with dilutions
of milk and other dairy products, strong and weak acid forming colonies and
peptonizing colonies may be readily differentiated from one another.

70 DIFGO MANUAL

Ayers and Mudge^ devised a differential medium for milk control which was
essentially Nutrient Extract Agar, containing skim milk powder extractives. The
medium gave high counts when compared with Nutrient Agar because it was
well adapted for the growth of lactic acid bacteria. Black, Prouty, and Graham,^
and Prouty^ gave a detailed method for the practical use of Bacto-Nutritive
Caseinate Agar and modified the medium by adding the brom cresol purple
indicator to the medium prior to sterilization, using 8 ml. of the indicator
solution per 1000 ml. of Nutritive Caseinate Agar.

Bacto-Nutritive Caseinate Agar was developed in our laboratories to duplicate
the medium of Ayers and Mudge. Modifications of the original formula were
necessary, but the dehydrated culture medium yields results comparable with
the original. Peptonizers and acid formers are readily distinguished, which to-
gether with the total count, give a more complete sanitary knowledge of the
milk sample under investigation than is possible with plain agar media. In
determining the differential count, the plates are incubated for 48 hours, and
then a total count of all colonies is made. Next the strong acid forming colonies
are counted, these being distinguished by a white zone of precipitated casein
surrounding the colonies. The plate is then flooded with brom cresol purple indi-
cator solution (0.4 gram Bacto-Brom Cresol Purple, 7.4 ml. of N/10 sodium
hydroxide and 92.6 ml. distilled water) for five minutes, and a count made of all
acid forming colonies which are yellow against the purple background of the
medium. To determine the number of weak acid formers, the number of strong
acid formers is subtracted from the number of total acid forming colonies. The
plate is then flooded with dilute acetic acid (5 per cent solution), and the pep-
tonizing colonies, which are surrounded by a clear zone are counted.

To rehydrate the medium, suspend 22 grams of Bacto-Nutritive Caseinate
Agar in 1000 ml. cold distilled water and keat to boiling to dissolve the medium
completely. Distribute in tubes or flasks and sterilize in the autoclave for 15
minutes at 15 pounds pressure (121°C.). The reaction of the medium will be
pH 6.5.

One pound of Bacto-Nutritive Caseinate Agar will make 20.6 liters of medium.

1 J. Bact., 5:565:1920. 'J. Dairy Science, 17:115:1934.

s J. Dairy Science, 15:99:1932.

BACTO

THERMO AGIDURANS AGAR (B303)

DEHYDRATED

Bacto- Yeast Extract 5 g.

Proteose Peptone, Difco 5 g.

Bacto-Dextrose 5 g.

Dipotassium Phosphate 4 g.

Bacto-Agar 20 g.

Bacto-Thermoacidurans Agar is recommended for the cultivation of Bacillus
thermoacidurans {Bacillus coagulans) , the organism causing "flat sour" spoilage
of tomato juice. It is prepared according to the formula described by Stern,
Hegarty and Williams^ for the isolation of this organism, and for its cultivation
in pure culture.

For the detection of B. thermoacidurans Stern, Hegarty and Williams^ recom-
mend the plating of 1 ml. of tomato juice per 20 ml. of agar medium. They ob-
served that larger quantities of tomato juice exhibited an inhibitory effect on
the growth of the organism. Plates are poured with the sterile melted agar at
45-55 °C. and, following solidification, incubated at 55 °C. for 48 hours.

DEHYDRATED CULTURE MEDIA 71

To rehydrate the medium, suspend 39 grams of Bacto-Thermoacidurans Agar
in 1000 ml. of cold distilled water. Heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). Since this is an acid medium, overheating dur-
ing the sterilization period or holding in the melted state should be avoided or
a soft medium will result. The final reaction of the medium will be pH 5.0.

One pound of Bacto-Thermoacidurans Agar will make 11.6 liters of medium.

^ Food Research, 7 : 1 86 : 1 94a.

BACTO

NEUTRALIZING BUFFER (B362)

DEHYDRATED

Monopotassium Phosphate 0.0425 g.

Sodium Thiosulfate 0.16 g.

Tamol-N 5.0 g.

Sodium Hydroxide 0.008 g.

Bacto-Neutralizing Buffer is designed for use in the Sterility Tests for Dairy
Equipment as specified in Standard Methods for the Examination of Dairy
Products^ by the swab contact method, and for the bacteriological examination
of food utensils according to the method presented by the Subcommittee on
Food Utensil Sanitation^ of the American Public Health Association. It is a
modification of the "Standard Methods" buffered distilled water. Bacto-Neu-
tralizing Buffer has the ability to inactivate the bactericidal and bacteriostatic
eiTect of chlorine as well as quaternary ammonium compounds, as shown by
laboratory and field tests using mixed flora as well as a large variety of pure
cultures of organisms, including a Sarcina particularly sensitive to quaternary
ammonium compounds. ^ Bacto-Neutralizing Buffer is used in conjunction with
Bacto-Tryptone Glucose Extract Agar for the performance of such viability,
sanitation or sterility tests.

Bacto-Neutralizing Buffer is not toxic for microorganisms. Plate counts made
at intervals up to 5 hours on a suspension of organisms in the Neutralizing
Buffer solution showed no reduction in numbers. This permits transfer of rinse
water or swabs to the laboratory without danger of loss of viable organisms.
Bacto-Neutralizing Buffer may be safely used in a concentration of 10 times the
single strength in procedures requiring multiple strength solutions without
danger of toxicity.

In using Bacto-Neutralizing Buffer in Sterility Tests for dairy Equipment, it is
suggested that the procedure as given in "Standard Methods for the Examination
of Dairy Products"^ be closely followed.

Bacto-Neutralizing Buffer is also recommended in Swab Test Procedures or
Visual Sanitation Tests. Under these conditions it is often desired to examine
more than one individual utensil as a unit. When so employed, the Neutralizing
Buffer is distributed in screw cap tubes or screw cap swab bottles (cotton stop-
pers are not suitable) so that after autoclaving there will be 1 ml. of Neutralizing
Buffer for each surface or utensil to be examined. If four similar utensils are to
be examined as a unit, 4 ml. should be present in the tube. For the examination
of food utensils the following procedure may be employed:

1. Dip a sterile swab into the tube of sterile Neutralizing Buffer.

2. Remove from the solution and squeeze the swab against the inside of the
tube so as to remove the excess solution leaving the swab moist but not wet.
One swab may be used for each group of four or more similar utensils.

72 DIFCO MANUAL

3. Rub the swab slowly and firmly three times over the significant surfaces of
the utensil to be examined, reversing the direction of the swab each time.

The significant surfaces of utensils are generally considered as the upper half
inch of the inner and outer rims of glasses and cups and the entire inner and
outer surfaces of the bowls of the spoons. In the examination of forks and knives
the inner and outer tines of the fork and both sides of the blade of the knife
should be swabbed. Plates and bowls should be swabbed on the inner and outer
surfaces.

After swabbing each utensil return the swab to the Neutralizing Buffer solu-
tion, rotate well and press free from excess solution, before swabbing the next
utensil in the group with the moist swab.

4. When the last utensil, generally four or more, has been swabbed, replace
the swab in the Neutralizing Buffer, and shake vigorously. If separate swabs were
used, break ofT each swab in the container under aseptic conditions.

5. Keep Neutralizing Buffer containing swabs at 0-6°C. until plated in the
laboratory.

6. For procedures requiring a plate count, break the stick of the swab just
above the cotton with sterile forceps, if this has not already been done as indi-
cated above. Shake the swab in the Neutralizing Buffer thoroughly to disintegrate
the cotton swabs. Plate 1 ml. or desired quantity using Bacto-Tryptone Glucose
Extract Agar as the plating medium. Incubate at 32 or 35 °C. for 48 hours before
making the count.

If desired, a Visual Sanitation Test may be run by following the above direc-
tions through Step 4, except that in Step 4 the swab is not broken but following
the swabbing of the last utensil, it is immersed in the Neutralizing Buffer, shaken
vigorously, squeezed to remove excess moisture and then smeared directly on
the surface of a slant of Bacto-Tryptone Glucose Extract Agar. Inoculation may
be made by moving the moist swab across the surface of the slant from top to
bottom horizontally and then vertically. The swab is rotated while being streaked
across the surface of the medium. The inoculated medium is then incubated as
desired.

To rehydrate Bacto-Neutralizing Buffer, dissolve 5.2 grams in 1000 ml. of cold
distilled water. Distribute in screw cap containers and sterilize in the autoclave
for 15 minutes at 15 pounds pressure (121°C.). Final reaction will be pH 7.2.

One hundred grams of Bacto-Neutralizing Buffer will make 19.2 liters.

1 Standard Methods for the Examination ^ Ann. Year Book 1947-48. Suppl.

of Dairy Products, 9th Edition: 216: 1948. Am. J. Pub. Health, 5:68:1948.

3 J. Milk Food Tech., 12:224:1949.

MEDIA FOR LACTOBACILLI

The media listed in this section are useful for the cultivation, enumeration and
study of Lactobacilli, particularly those concerned with the manufacture of dairy
products.

DEHYDRATED CULTURE MEDIA 73

BACTO

TOMATO JUICE AGAR (B31)

DEHYDRATED

Tomato Juice (400 ml.) 20 g.

Bacto-Peptone 10 g.

Bacto-Peptonized Milk 10 g.

Bacto-Agar 11 g.

Bacto-Tomato Juice Agar is recommended for the direct plate count and cul-
tivation of Lactobacilli. It is an excellent plate medium for determining the
relative numbers of organisms in acidophilus products and for estimating the
degree of intestinal implantation.

The Lactobacilli grow poorly on ordinary culture media and require special
nutrients. Mickle and Breed^ reported the use of tomato juice in culture media
for Lactobacilli, and Kulp,^ while investigating the use of tomato juice on bac-
terial development, found that the growth of L. acidophilus was enhanced in
media containing this material. Colonies on plates of this new medium were
large and more characteristic than on other media. Later Kulp and White^
described a modification of the original medium which gave them relatively
high quantitative counts.

Bacto-Tomato Juice Agar is prepared according to Kulp and White's modi-
fication and contains Bacto-Peptone and Bacto-Peptonized Milk. The dehydrated
medium is excellently suited for the cultivation of members of the Lactobacillus
group.

To rehydrate the medium, suspend 51 grams of Bacto-Tomato Juice Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). The final reaction of the medium will be
pH 6.1.

One pound of Bacto-Tomato Juice Agar will make 8.9 liters of medium.

iTech. Bull, no, N. Y. State Agr. 2 Science, 66:512:1927.

Exp. Sta., 1925. 3 Science, 76:17:1932.

BACTO

TOMATO JUICE AGAR SPECIAL (B389)

DEHYDRATED

Tomato Juice (400 ml.) 20 g.

Bacto-Peptone 10 g.

Bacto-Peptonized Milk 10 g.

Bacto-Agar 20 g.

Bacto-Tomato Juice Agar Special is recommended for the direct plate count
of the Lactobacilli from saliva and for the cultivation of other acidophilic
microorganisms. It is prepared according to the formula suggested by Jay.^-^ The
number of Lactobacilli in saliva is an index of predisposition to dental caries as
described by Jay.^-^ Many dentists prefer to use the direct count of Lactobacilli
for the diagnosis of caries rather than the colorimetric method using Bacto-
Snyder Test Agar, as described on page 190.

The reaction of Bacto-Tomato Juice Agar Special is adjusted to pH 5.0 so as
to encourage the growth of Lactobacilli and at the same time inhibit the growth
of many commensal bacteria that may be encountered in saliva. This is a
slightly more selective medium for Lactobacilli than is Bacto-Tomato Juice Agar
or Bacto-Trypsin Digest Agar described on page 74.

74 DIFGOMANUAL

To rehydrate the medium, suspend 60 grams Bacto-Tomato Juice Agar
Special in 1000 ml. cold distilled water. Heat to boiling to dissolve the medium
completely. Distribute in tubes or flasks and sterilize in the autoclave for 15
minutes at 15 pounds pressure (121°C.). Overheating of this acid medium is to
be avoided to prevent hydrolysis of the agar giving a soft medium. Final reaction
will be pH 5.0.

One pound of Bacto-Tomato Juice Agar Special will make 7.5 liters of
medium.

1 Bacteriology and Immunology of Dental 2 Dentistry in Public Health, 1949.

Caries and Dental Science and Dental
Art, 1938.

BACTO

TRYPSIN DIGEST AGAR (B30)

DEHYDRATED

Trypsinized Milk (800 ml.) 20 g.

Tomato Juice (200 ml.) 10 g.

Bacto-Peptone 8 g,

Bacto-Dextrose 4 g.

Dextrin, Difco 4 g.

Bacto-Agar 13 g.

Bacto-Trypsin Digest Agar is recommended for the plate count and cultiva-
tion of Lactobacillus acidophilus. By using a tryptic digest of milk, to which
tomato juice and other nutriments were added. Dr. H. A. Cheplin was able to
secure excellent growth of L. acidophilus.

Bacto-Trypsin Digest Agar readily lends itself to use in isolating strains of
L. acidophilus as well as for carrying them in stock. It is an excellent plate
medium for determining the relative numbers of organisms in acidophilus prod-
ucts and for estimating the degree of intestinal implantation.

Bacto-Trypsin Digest Agar is prepared according to the formula specified by
Dr. Cheplin. Results obtained with this dehydrated medium when it is made up
for use are comparable to those secured when the medium is prepared from
ingredients.

To rehydrate the medium, suspend 59 grams of Bacto-Trypsin Digest Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). The final reaction of the medium will be pH
6.1.

One pound of Bacto-Trypsin Digest Agar will make 7.6 liters of medium.

BACTO

SKIM MILK (B32)

DEHYDRATED

Bacto-Skim Milk is high grade skim milk reduced to powder by the spray
process. It is readily soluble and is easily prepared for use. Bacto-Skim Milk is
recommended for use with Bacto-Tryptone Glucose Extract Agar in the prepara-
tion of the Tryptone Glucose Extract Milk Agar according to "Standard
Methods for the Examination of Dairy Products."^ It is also recommended for
the preparation of skim milk solutions used in the propagation of organisms
occurring in milk products, and to demonstrate coagulation and proteolysis of

DEHYDRATED CULTURE MEDIA 75

Bacto-Skim Milk may be used in media for the detection of proteolyzers, and
as an ingredient in the preparation of media for the cultivation of such organisms
as Mycobacterium tuberculosis and Corynebacterium diphtheria. A solution of
Bacto-Skim Milk containing litmus is a widely used medium for determining
acid production in milk and also the ability to peptonize or coagulate milk in
the identification of microorganisms. Skim Milk is used to detect the so-called
"stormy fermentation" of Clostridium perfringens.

Nungester and Ellingson^ have suggested the addition of 0.1 per cent agar and
iron, either in the form of filings or freshly pickled strips, to adjust the oxygen
tension of the medium and permit more rapid development of organisms in the
"stormy fermentation" test.

To rehydrate Bacto-Skim Milk to obtain the equivalent of fresh skim milk,
dissolve 100 grams in 1000 ml. of cold distilled water. Distribute in tubes and
sterilize in the autoclave for 15 minutes at 15 pounds pressure (121°G.). Over-
heating during sterilization results in the carmelization of the milk sugar. This is
to be avoided since the resulting discoloration may give an atypical appearance
of the sterile medium and may be further reflected in the appearance of growing
cultures. The final reaction will be pH 6.4.

One pound of Bacto-Skim Milk will make 4.5 liters of skim milk.

^ Standard Methods for the Examination ' Personal Communication,

of Dairy Products, 9th Edition: 93: 1948.

BACTO

PEPTONIZED MILK (B35)

DEHYDRATED

Bacto-Peptonized Milk is used alone or as an ingredient in media for isolatioa
and cultivation of the Lactobacilli and organisms encountered in the bacterio-
logical examination of milk and dairy products.

As early as 1894 Kayser^ used Peptonized Milk in his studies on lactic fermen-
tation. Orla-Jensen^ found Peptonized Milk to be exceptionally satisfactory in
the cultivation of lactic acid forming organisms. Bacto-peptonized Milk was
developed in our laboratories to provide a simple medium with readily available
nitrogen for the cultivation of organisms of the Lactobacillus group. It contains
the degradation products of the proteins, albumins, and globulins of milk, and
therefore its nitrogen is more readily available for bacterial assimilation than
the native proteins of milk. Rapid and luxuriant growth of Lactobacilli is readily
obtained in solutions of Bacto-Peptonized Milk. For carrying stock cultures of
lactic organisms, it is recommended that 0.1 per cent of Bacto-Agar be added
to the medium. Kulp and White^ used Bacto-Peptonized Milk with excellent
results in their Tomato Juice Agar for Lactobacillus acidophilus. Devereux*
employed Bacto-Peptonized Milk in his modification of the Cooledge Medium
for testing the quality of milk. Weiss and Rettger^ used Bacto-Peptonized Milk
in their tomato juice media for L. bifidus.

To rehydrate the medium, dissolve 15 grams of Bacto-Peptonized Milk in
1000 ml. of cold distilled water. Distribute in tubes and sterilize in the autoclave
for 15 minutes at 15 pounds pressure (121°C.). The final reaction of the
medium will be pH 6.5.

One pound of Bacto-Peptonized Milk will make 30.2 liters of medium.

^Ann. inst. Pasteur, 8:737:1894. * Am. J. Pub. Health, 22:1291:1932.

aCentr. Bakt., H Abt., 4:96:1898. b j. fiact., 28:501:1934.

8 Science, 76:17:1932.

76 DIFGO MANUAL

BACTO BACTO

MICRO ASSAY CULTURE AGAR MICRO INOCULUM BROTH

DEHYDRATED DEHYDRATED

Bacto-Micro Assay Culture Agar and Bacto-Micro Inoculum Broth have been
developed for the cultivation of Lactobacilli in microbiological assays. These
media are also recommended for the cultivation of other Lactobacilli. A complete
discussion of Bacto-Micro Assay Culture Agar and Bacto-Micro Inoculum Broth
is given on pages 212 and 213.

ADDITIONAL MEDIA

The media listed below have been rather extensively employed in the past for
^'Standard Methods" and supplementary procedures in the examination of water
and milk. Other media have been developed which are considered superior and
serve the purpose more adequately. For the present we will continue to carry
these media in stock for those laboratories where they have been in routine use,
or where it is desired to continue them for comparative purposes.

PRESENT RECOMMENDATIONS

Bacto- Yeast Dextrose Agar Bacto-Tryptone Glucose Extract Agar,

page 57

Bacto-Brilliant Green Bile 5% Bacto-Brilliant Green Bile 2%, page 37

Bacto-Lactose Peptone Bile

Bacto-Purple Bile Salt Agar Bacto- Violet Red Bile Agar, page 61

Bacto-Gentian Violet Lactose Bile Bacto-Brilliant Green Bile 2%, page 37

Bacto-Formate Ricinoleate Broth, page
60

Bacto-Galactose Whey Agar Bacto-Tomato Juice Agar, page 73

Bacto- Whey Agar Bacto-Trypsin Digest Agar, page 74

Bacto-Peptonized Milk Agar

Bacto-Galactose Peptonized Milk Bacto-Peptonized Milk, page 75

Bacto- Whey Broth

Bacto-Purple Milk Bacto-Litmus Milk, page 192

Bacto-Ulrich Milk, page 193
Bacto-Neutral Red Medium
Bacto-Cooledge Broth

MEDIA FOR CULTIVATION
OF PATHOGENIC MICROORGANISMS

INFUSION MEDIA

Dehydrated infusion media, Difco, are prepared under controlled conditions
from large quantities of fresh tissue. Variations due to differences in meat are
thereby minimized, thus assuring greater uniformity of the final medium than
can be obtained when the medium is prepared in small lots from market meat.
Infusions of meat have long been considered an essential part of media for
the cultivation of many pathogenic organisms. Through years of practical use,
Difco infusion media have demonstrated their suitability for the cultivation of

DEHYDRATED CULTURE MEDIA 77

fastidious streptococci, pneumococci, meningococci, and other pathogenic
microorganisms.

Peptones now have been developed which in a 2 per cent concentration satis-
factorily replace the infusion and peptone portion of infusion media for many of
the fastidious pathogens. These peptone media will be discussed in the section,
Peptone Media, Without Infusions, on pages 99-130.

A guide for the selection of culture media listed in this section is given on
pages 26, 27 and 28.

BACTO

BRAIN HEART INFUSION (B37)

DEHYDRATED

Calf Brains, Infusion from 200 g.

Beef Heart, Infusion from 250 g.

Proteose Peptone, Difco 10 g.

Bacto-Dextrose 2 g.

Sodium Chloride 5 g.

Disodium Phosphate 2.5 g.

Bacto-Brain Heart Infusion is a liquid infusion medium recommended for the
cultivation of streptococci, pneumococci, meningococci, and other organisms
generally considered difficult to cultivate. Virulence, antigenicity, and other
serological characteristics of organisms are quite uniformly maintained when
grown on Bacto-Brain Heart Infusion. This medium is especially adapted for
blood culture work. Bacto-Brain Heart Infusion, solidified with agar is recom-
mended for the isolation of pathogenic fungi, and this medium is discussed in
detail on page 90.

Rosenow^ devised an excellent culture medium for the streptococci by adding
pieces of brain tissue to Dextrose Broth. In this medium he was able to secure
excellent results in culturing organisms from focal infections in the teeth or
other tissues. Hayden,^ using the same procedure as Rosenow, but adding
crushed marble to the medium, reported that this medium was favorable for
the growth of organisms from infections of the teeth, especially those showing
a close relationship with eye infections.

Bacto-Brain Heart Infusion is prepared to duplicate the results obtained by
Rosenow and Hayden. It contains the essential nutriments of their medium and
possesses the advantage of yielding an easily prepared clear medium. An infusion
of brains has replaced the nutritive value of the brain tissue, disodium phosphate
replaced the buffer calcium carbonate, and if desired 0.1 per cent agar may
be added to the medium giving conditions of oxygen tension similar to those
produced by the tissue. The addition of a small amount of agar (0.1-0.2 per
cent) to Brain Heart Infusion is particularly recommended for the growth and
isolation of pathogenic microorganisms especially their primary isolation from
blood and other specimen material.

78 DIFCOMANUAt

bacillus and staphylococci, ordinarily considered easy to cultivate, was aided by
the presence of a small quantity of agar. They also showed that growth is
initiated in a much shorter incubation period in such media. They used 0.1 per
cent agar in their routine tests. Bacto-Brain Heart Infusion with PAB and
Agar, as discussed below, contains 0.1 per cent agar.

Dance and Murray^ used Bacto-Brain Heart Infusion with 0.1 per cent
added agar for the cultivation of hemolytic streptococci in their study of hemo-
lytic properties on various Blood Agars. Berens, Nilson and Chapman^ added
3 grams calf brain to 10 ml. of Brain Heart Infusion for the growth of bacteria
from patients with certain inflammatory eye diseases. Chapman, Stiles, and
Berens^ and Chapman^ used Bacto-Brain Heart Infusion in their test for
pathogenicity of streptococci. Reitzel and KohP added 30-50 per cent sterile
ascitic fluid to Bacto-Brain Heart Infusion and stated that some of the gono-
cocci grew more readily in this medium than in a Hormone Brain Broth con-
taining sterile pieces of brain. They also used Bacto-Brain Heart Infusion in
combination with Bacto-Brain Veal Agar and Bacto-Hemoglobin for the isola-
tion of the gonococcus. A detailed discussion of these products is given on
pages 94 and 271.

The coagulation of plasma is one of the characteristics of pathogenic staphy-
lococci. Brain Heart Infusion is especially well suited for the growth of staphy-
lococci or for the preparation of the suspension of the organism used in the
performance of the Coagulase Test, as described by Chapman.^o A complete
discussion of the performance of this test is given under Bacto-Coagulase Plasma,
page 330. Newman,ii in a study of the detection of food poisoning attributable to
dairy products, used staphylococci grown in Brain Heart Infusion for the per-
formance of the coagulase test.

Neter ^^.is added Bacto-Supplement B to Brain Heart Infusion and demon-
strated that Hemophilus influenzae could be cultivated on this medium through
serial transfers. He also used this enriched Brain Heart Infusion in tests designed
to determine the streptomycin sensitivity of strains of H. influenzae,

Bacto-Brain Heart Infusion has been used in the preparation of media for the
isolation of pathogenic fungi. Roseburg, Epps and Clark^* found that Bacto-
Brain Heart Infusion, containing 2 per cent agar, was more satisfactory than
Dextrose Infusion Agar, for the isolation and cultivation of Actinomyces israeli.
Incubation in a 5 per cent carbon dioxide atmosphere was required for best
results. The addition of sheep blood to the medium was not an advantage.
Howell^^ used Bacto-Brain Heart Infusion to which was added 2 per cent Bacto-
Agar and 10 per cent sterile defibrinated horse blood for the cultivation of
Histoplasma capsulatum. A selective medium for this microorganism was pre-
pared by adding 40 units of streptomycin and 20 units penicillin per ml. of
medium. In comparison with a Blood Agar similarly prepared from Potato
Dextrose Agar, the Brain Heart Infusion medium gave a greater number of
positive isolations. Incubation at room temperature was more efficient than at
37 °C. Colonies isolated on the Brain Heart Infusion Blood Agar must be trans-
ferred to a medium such as Potato Dextrose Agar to obtain the characteristic
tuberculate chlamydospores typical of H. capsulatum. Conant^^ recommended
that a plate of Bacto-Brain Heart Infusion Agar be streaked and incubated at
37 °C. under anaerobic conditions with the addition of 5 per cent carbon dioxide
to obtain growth of the microaerophilic Actinomyces hovis in culturing this
organism from infected mucous membranes, skin and subcutaneous tissues.

For blood culture work a procedure frequently used is to add as much as
10 ml. of blood specimen to 150 ml. of sterile Brain Heart Infusion contained
in a 300 ml. Erlenmeyer flask or bottle. This is incubated at 37 °C. and trans-
fers are made to Blood Agar or other media for the isolation and identifica-

DEHYDRATED CULTURE MEDIA 79

tion of the organisms. Blood for blood culturing should always be taken prior
to the administration of the therapeutic agent. If drugs have been administered
their bacteriostatic effects should be neutralized. For example the addition
of p-aminobenzoic acid (PAB) in 5 mg. per cent to the medium will assure
the inactivation of any sulfa drug carried over with the inoculum. Bacto-Penase,
a concentrated purified penicillinase, should be added to the sterile cooled
medium used for blood culture if the patient is under penicillin therapy. Bacto-
Brain Heart Infusion with PAB and Agar with added Bacto-Penase is an ideal
medium for blood culture work. The small amount of agar present will give all
degrees of anaerobiosis, permitting the development of the obligate anaerobes
and microaerophiles as well as the aerobes. The PAB will inactivate any sulfa
drug, the added Bacto-Penase wdll inactivate any penicillin in the inoculum,
while 100 ml. of the medium itself will inactivate up to 1000 units of strepto-
mycin.

For the detection of organisms from the blood of patients known to be under
sulfonamide therapy, Bacto-Brain Heart Infusion with PAB or Bacto-Brain
Heart Infusion with PAB and Agar are recommended. These media contain
5 mg. per cent of PAB which neutralizes the bacteriostatic effect of the sul-
fonamide present in the blood specimen.

To rehydrate the medium, dissolve 37 grams of Bacto-Brain Heart Infusion
in 1000 ml. distilled water. Distribute in tubes or flasks and sterilize in the auto-
clave for 15 minutes at 15 pounds pressure (121°C.). The final reaction of the
medium will be pH 7.4.

If the medium is not used the same day as prepared and sterilized, heat at
100°C. for several minutes to remove absorbed oxygen, and cool quickly without
agitation, just prior to inoculation.

One pound of Bacto-Brain Heart Infusion will make 12.2 liters of medium.

IJ. Dental Research, 1:205:1919. » J. Am. Med. Assoc, 110:1095:1938.

3 Arch. Internal Med., 32:828:1923. ^^° Trans. N.Y. Academy Sciences, 9:52:1946.

3 J. Infectious Diseases, 29:390:1921. ^ J. Milk and Food Tech., 13:226:1950.

*J. Bact., 37:121:1939. 12 Science, 106:350:1947.

6 J. Infectious Diseases, 63:122:1938. ^ ]. Bact., 54:70:1947.

^ Am. J. Ophthalmol., 19:1060:1936. 1* J. Infectious Diseases, 74:131:1944.

■^Am. J. Clin. Path., 9:1939: Tech. Suppl., ^^ Public Health Reports, 63:173:1948.

3:20:1939. "Diagnostic Procedures and Reagents, 3rd
8 Am. J. Digestive Diseases, 13:105:1946. Edition: 452:1950.

BACTO BACTO

BRAIN HEART INFUSION BRAIN HEART INFUSION

with PAB (B37A) with PAB and Agar (B37B)

DEHYDRATED DEHYDRATED

Two modifications of Bacto-Brain Heart Infusion widely used in blood culture
procedures are now available in dehydrated form. These media are especially
recommended for the detection of organisms in the blood of patients under sul-
fonamide therapy there has been included in the formula sufficient /?-amino-
benzoic acid (PAB) to give a final concentration of 5 mg. per cent. This
will neutralize the maximum amount of sulfonamide that might be carried
over into the medium with the blood inoculum, and thereby exert an inhibitory
effect upon pathogens inoculated into the medium.

The formula for Bacto-Brain Heart Infusion with PAB is identical with that
of Bacto-Brain Heart Infusion except for the addition of 0.05 gram of p-amino-
benzoic acid per liter. To rehydrate the medium dissolve 37 grams of Bacto-
Brain Heart Infusion with PAB in 1000 ml. of distilled water.

80 DIFGOMANUAL

The formula for Bacto-Brain Heart Infusion with PAB and Agar is identical
with Bacto-Brain Heart Infusion except for the addition of 0.05 gram of p-amino-
benzoic acid and 1 gram of Bacto-Agar per liter. The addition of this small
amount of agar to the medium provides optimum conditions for the rapid luxu-
riant development of obligate anaerobes, micro-aerophiles and aerobes. To rehy-
drate the medium suspend 38 grams of Bacto-Brain Heart Infusion with PAB
and Agar in 1000 ml. distilled water and heat to boiling to dissolve the medium
completely.

Both of these media are distributed in tubes or flasks and sterilized in the
autoclave for 15 minutes at 15 pounds pressure (121°C.) Final reaction of the
media is pH 7.4.

BACTO

HEART INFUSION BROTH (B38)

DEHYDRATED

Beef Heart, Infusion from 500 g.

Bacto-Tryptose 10 g.

Sodium Chloride 5 g.

Bacto-Heart Infusion Broth is a liquid medium containing the extractives
from fresh beef heart. It is recommended as a general laboratory medium for
pathogenic as well as non-pathogenic bacteria. A liquid medium, prepared from
an infusion of beef heart, is recommended in "Diagnostic Procedures and Re-
agents"^ of the American Public Health Association for blood culturing the
meningococcus and for other purposes. The "Standard Methods for the Exami-
nation of Dairy Products"^ specify the use of a Heart Infusion Broth for their
study of pathogenic streptococci.

Bouillon, or a liquid medium containing an infusion of meat, was one of the
first media used for cultivation of bacteria. Many modifications of this medium
have been used from time to time for a wide variety of purposes. Huntoon^ using
fresh beef heart and Bacto-Peptone, prepared a "hormone" broth in a special
manner, to retain the growth-promoting substances. In order to have such a
liquid infusion medium readily available, Bacto-Heart Infusion Broth was devel-
oped. Bacto-Tryptose, as employed in this medium, is better suited for the nutri-
tional requirements of pathogenic bacteria than is Bacto-Peptone which was
used by Huntoon in the preparation of his "hormone" media.

The cultural value of Bacto-Heart Infusion Broth is greatly increased by the
addition of 0.1-0.2 per cent Bacto-Agar as discussed on page 77 under Bacto-
Brain Heart Infusion. Many other modifications of the medium may also be
made, such as the addition of dextrose, blood, or other ingredients to give an
unlimited number of media for a variety of purposes.

To rehydrate the medium, dissolve 25 grams of Bacto-Heart Infusion Broth
in 1000 ml. distilled water, distribute in tubes and sterilize in the autoclave for
15 minutes at 15 pounds pressure (121°G.). The final reaction of the medium is
pH 7.4.

For best results, the medium should be freshly prepared. If it is not used the
same day as sterilized, heat in boiling water or flowing steam for several minutes
to remove absorbed oxygen and cool quickly without agitation, just prior to
inoculation.

One pound of Bacto-Heart Infusion Broth will make 18.1 liters of medium.

1 Dia^ostic Procedures and Reagents, 2 Standard Methods for the Examination

3rd Edition: 13: 1 950. of Dairy Products, 9th Edition: 141: 1948.

8 J, Infectious Diseases, 23:169:1918.

DEHYDRATED CULTURE MEDIA 81

BACTO

VEAL INFUSION MEDIUM (B40)

DEHYDRATED

Veal, Infusion from 500 g.

Proteose Peptone, Difco 10 g.

Bacto-Agar 1 g.

Bacto-Veal Infusion Medium is prepared from selected lean veal, and is
recommended as a liquid medium for the growth of anaerobes as well as many
other bacteria generally considered difficult to cultivate. The medium contains
0.1 per cent agar which provides, in freshly heated media, various degrees of
oxygen tension making possible the growth of anaerobes without special condi-
tions.

The advantages of a medium with a low agar concentration, and Its influence
on the development of bacteria, particularly the anaerobes, has been described
by Kitchens^. In a broth, to which 0. 1 per cent agar has been added, there is a
clear upper zone well suited for aerobic growth; below this the flocculent agar
develops variable degrees of oxygen tension. This condition makes the medium
suitable for the growth of either aerobic or anaerobic bacteria. Falk, Bucca, and
Simmons^ point out the advantages of the use of small quantities of agar
(0.06-0.25 per cent) in the detection of contaminants in the sterility testing of
biologicals. They show that the growth of even common forms, such as the hay
bacillus and staphylococci, ordinarily considered easy to cultivate, was aided by
the presence of a small quantity of agar, and that growth is initiated in a much
shorter incubation period in such media. Since their experiments showed little
difference between 0.1 and 0.25 per cent agar, they used 0.1 per cent agar in
their routine tests, which is the quantity employed in Bacto-Veal Infusion
Medium. In the report on culture media for non-acid products, Cameron^, as
associate referee, recommended Bacto-Veal Infusion Medium for use in the
examination of canned meat. He reported this medium to be excellent as an en-
richment for the propagation of anaerobes.

To rehydrate the medium, suspend 23 grams of Bacto-Veal Infusion Medium
in 1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in tubes and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). The final reaction of the medium will be pH 7,3.

For best results, Bacto-Veal Infusion Medium should be freshly prepared. If
the medium is not used the same day as sterilized, heat in boiling water or
flowing steam for a few minutes to remove absorbed oxygen and then cool
quickly without agitation, just prior to inoculation.

One pound of Bacto-Veal Infusion Medium will make 19.7 liters of medium.

1 J. Infectious Diseases, 29:390:1921. 3 Assoc. Official Agr. Chem., 20:429:1937.

2 J. Bact., 37:121:1939.

BACTO

VEAL INFUSION BROTH (B344)

DEHYDRATED

Bacto-Veal Infusion Broth is identical to Bacto-Veal Infusion Medium except
the small concentration of agar has been omitted. This infusion medium is suited
for the cultivation of fastidious pathogenic microorganisms. "Standard Methods
for the Examination of Dairy Products"^ describe a Veal Infusion Broth, pre-
pared from finely ground veal and 0.5 per cent Bacto-Tryptone, for the cultiva-
tion of pathogenic streptococci.

82 DIFGOMANUAL

To rehydrate the medium dissolve 22 grams of Bacto-Veal Infusion Broth in
1000 ml. distilled water. Distribute in tubes and sterilize in the autoclave for
15 minutes at 15 pounds pressure (121°C.). The final reaction of the medium
will be pH 7.3.

One pound of Bacto-Veal Infusion Broth will make 20.6 liters of medium.
1 Standard Methods for the Examination of Dairy Products, gth Edition: 141: 1948.

BACTO

PPLO ENRICHMENT BROTH (B410)

DEHYDRATED

Beef Heart, Infusion from 50 g.

Bacto-Peptone 10 g.

Sodium Chloride 5 g.

Bacto-Crystal Violet 0.01 g.

Bacto-PPLO Enrichment Broth is prepared according to the formula sug-
gested by Morton, Smith, Williams, and Eickenberg,^ for the selective enrich-
ment of pleuropneumonia-like organisms (PPLO). This medium is designed to
facilitate the isolation of PPLO from clinical specimens. It is also useful as an
aid in the purification of PPLO colonies contaminated with other microorgan-
isms. The growth of both Gram-positive and Gram-negative organisms is sup-
pressed, while pleuropneumonia-like organisms develop. Following inoculation
with clinical specimens or with bacterial contaminated agar block colonies of
PPLO, incubate at 37°C. for 36-72 hours and then plate on PPLO Agar, con-
taining 1 per cent Bacto-PPLO Serum Fraction, or 25 per cent Bacto-Ascitic
Fluid. Inoculation of the plates is accomplished by transferring a drop of the
enriched culture to the plate and spreading it uniformly over the surface of the
medium.

To rehydrate the medium, suspend 21 grams of Bacto-PPLO Enrichment
Broth in 1000 ml. of distilled water. Sterilize in the autoclave 15 minutes at 15
pounds pressure (121°C.). To each liter add 2.85 ml. of Bacto-Chapman Tellu-
rite Solution to give a concentration of 1-35,000 of potassium tellurite. Add 25
per cent Bacto-Ascitic Fluid and tube in sterile tubes under aseptic conditions.
The final reaction of the unenriched medium will be pH 7.8.

One pound of Bacto-PPLO Enrichment Broth will make 21.6 liters of unen-
riched medium.
ij. Dental Researchj 30:415:1951.

BACTO

THIOL MEDIUM (B307)

DEHYDRATED

Proteose Peptone No. 3, Difco 10 g.

Bacto- Yeast Extract 5 g.

Bacto-Dextrose 1 g.

Sodium Chloride 5 g.

Thiol Complex 8 g.

Bacto-Agar 1 g.

/7-Aminobenzoic Acid 0.05 g.

Bacto-Thiol Medium is recommended for culturing organisms from body
fluids and other materials containing penicillin, streptomycin and sulfa drugs.

DEHYDRATED CULTURE MEDIA 83

This medium does not contain thermolabile factors and accordingly may be
sterilized in the autoclave. Bacto-Thiol Medium has the ability to inactivate
penicillin, streptomycin and sulfa drugs. Luxuriant growth of staphylococci and
other test organisms will be obtained from dilute inocula in 24 hours in 10 ml.
of Bacto-Thiol Medium to which has been added 100 to 1000 units of penicillin,
1000 to 10,000 units of streptomycin, or sulfonamides. Bacto-Thiol Medium con-
tains 0. 1 per cent Bacto-Agar to maintain an" OR potential conducive to growth
of the strictest anaerobes, microaerophiles, and aerobic microorganisms, without
special seal.

The medium should be distributed in tubes or containers to give a depth
of at least 60 mm. for neutralization of penicillin. For the neutralization
of streptomycin, the medium may be employed in tubes or in shallow layers in
flasks. Neutralization of antibiotics and cultural response with Bacto-Thiol
Medium Is best when freshly prepared, or less than 4 days old.

Bacto-Thiol Medium enriched with 1 per cent Bacto-Supplement B has proven
an especially valuable medium for blood culture. Broom^ reported excellent re-
sults with the enriched Thiol Medium in the isolation of Hemophilus influenzae
and meningococci. Huddleson- reported this medium satisfactory for the isola-
tion, cultivation and maintenance of stock cultures of Vibrio fetus. Cultures re-
mained viable in Thiol Medium for at least 150 days without transfer.

To rehydrate the medium, suspend 30 grams of Bacto-Thiol Medium In 1000
ml. freshly distilled water. Heat to boiling to dissolve the medium completely.
Distribute in tubes or flasks as indicated above and sterilize In the autoclave for
15 minutes at 15 pounds pressure (121°C.). The final reaction of the medium
will be pH 7.2.

*^ Personal Communication, 1948. ^ J. Bact., 56:508:1948.

BACTO

THIOL BROTH (B434)

DEHYDRATED

Bacto-Thiol Broth has the same composition as Bacto-Thiol Medium except
it contains no agar. It is prepared for laboratories requiring a medium having
the properties of Thiol Medium except without agar. In rehydrating Bacto-
Thiol Broth 29 grams are used per liter.

The final reaction of the medium will be pH 7.2.

BACTO

KRAGKE BLOOD CULTURE MEDIUM (B41)

DEHYDRATED

Beef Heart 165 g,

Beef Brain 55 g

Proteose Peptone, Difco 10 g

Bacto-Dextrose 10 g,

Sodium Chloride 4 g

Sodium Citrate 1 g

Disodium Phosphate 2 g

Bacto-Kracke Blood Culture Medium is recommended for the culturing of
organisms from the blood in bacteriemias. The medium may also be used for
maintaining cultures isolated from the blood and for carrying stock cultures.

84 DIFCOMANUAL

Kracke and Teasley^ made an exhaustive study of the methods of blood cul-
turing and described a new medium which yielded relatively higher percentages
of positive cultures than other methods commonly employed. In their medium
the finely divided particles of brain and heart tissue aid in fixing the comple-
ment and in removing immune bodies from the blood specimen. The sodium
citrate prevents the blood from clotting and also aids in fixing the complement.
They used their medium in 50 ml. quantities in flasks and added 10 to 15 ml.
of blood specimen. Cultures were incubated for 18 hours at 37°C., and if nega-
tive at that time, reincubated for 3 weeks, with frequent observations for growth,
before being considered negative.

Since the publication of their paper, Dr. Kracke has made a revision of the
formula (personal communication) resulting in a medium which would give
more satisfactory results. Bacto-Kracke Blood Culture Medium is prepared ac-
cording to this revision.

Feder^ gave in detail a new and simplified technique for blood culturing. He
recommended the use of Bacto-Kracke Blood Culture Medium.

Bacto-Kracke Blood Culture Medium when properly prepared, contains about
50 per cent solid meat particles. The supernatant liquid is frequently quite
turbid due to the presence of some of this very finely divided tissue. In blood
culture w^ork it may be necessary to prepare smears or make a transfer to an-
other medium in order to determine the presence of organisms. Bacto-Brain
Heart Infusion with PAB and Agar as discussed on page 79, can be recom-
mended as a clear liquid medium for blood culture work.

To rehydrate the medium, suspend 3.75 grams of Bacto-Kracke Blood Culture
Medium in 50 ml. of distilled water. The suspension, with frequent agitation, is
allowed to stand for at least 15 minutes or until all the particles are thoroughy
wetted. It is essential that this step be adhered to closely, since the final medium
will not be sterile unless all the meat particles are thoroughly wetted before
heating. Maintain an even suspension of the meat particles if distributed in
tubes. The medium is then sterilized in the autoclave for 15 minutes at 15 pounds
pressure (121°C.).

Special precautions must be taken to prevent too quick a decrease in pressure
at the end of the sterilization period, since a too rapid cooling of the autoclave
produces a vigorous boiling of the medium which results in excessive turbidity
of the supernatant liquid and may even occasionally blow the medium and plugs
from the flasks. The final reaction of the medium will be pH 7.4.

For best results, this medium should be freshly prepared, or if not used the
same day as sterilized, heat in boiling water or flowing steam for a few min-
utes and cool quickly without agitation, just prior to inoculation.

One pound of Bacto-Kracke Blood Culture Medium will make 6 liters of
medium.

ij. Lab. Clin. Med., 16:169:1930. 2 J. Lab. Clin, Med., 22:846:1937.

BACTO

EGG MEAT MEDIUM (B42)

DEHYDRATED

Beef Muscle 454 g.

Egg White from 6 eggs

Calcium Carbonate 5 g.

Bacto-Egg Meat Medium is a liquid medium containing particles of meat, egg
white and calcium carbonate in suspension. It is recommended for the deter-

DEHYDRATED CULTURE MEDIA 85

mination of proteolytic activity of organisms, and for carrying stock cultures of
anaerobes.

The use of a combination of meat and egg white as a culture medium was
reported by Rettger^ in his studies on Escherichia coli and Aerobacter aerogenes.
Later he^ described the use of this medium in studies of intestinal putrefaction.
In 1923, Reddish and Rettger^ used it in their detailed study of Clostridium
putrificurn, and in the following year they* employed it in a study of other spore-
forming anaerobes.

The medium is prepared from fresh meat and egg white, retaining the coagu-
lated portion of the meat and egg white as a part of the medium. The experi-
ments of Reddish and Rettger, using twelve different spore-forming anaerobes,
indicate that a final reaction of pH 7.0 is satisfactory for the organisms, and that
the range of pH 7.0-7.4 favors abundant growth and consistent development of
typical morphological forms.

Bacto-Egg Meat Medium was developed with the cooperation of Dr. Reddish.
The use of this dehydrated medium eliminates a tedious and disagreeable task
that confronted the bacteriologist interested in the study of anaerobes. This
medium is of particular value in studies of morphology and the proteolytic
properties, as demonstrated by the digestion of meat, of these organisms. Bacto-
Egg Meat Medium is recommended for carrying stock cultures of anaerobes,
since even when the medium has evaporated to a practically water-free consist-
ency the organisms still retain their original pathogenicity, morphological, cul-
tural and biochemical characteristics to a remarkable degree.

To rehydrate the medium, suspend 15 grams of Bacto-Egg Meat Medium in
100 ml. distilled water. The suspension is allowed to stand, with frequent agita-
tion, for at least 15 minutes or until all the particles are thoroughy wetted. It is
essential that this step be adhered to closely, since the final medium will not
be sterile unless all the meat particles are thoroughly wetted before heating.
An even suspension of the particles should be maintained while dispensing into
tubes. The medium is then sterilized in the autoclave for 15 minutes at 15
pounds pressure (121°C.). Special precautions must be taken to prevent too
quick a decrease in pressure at the end of the sterilization period, since a too
rapid cooling of the autoclave produces a vigorous boiling of the medium which
results in excessive turbidity of the supernatant liquid and may even occasionally
blow the medium and plugs from the tubes. The final reaction of the medium
will be pH 7.2.

If not used immediately after sterilization, the tubes should be heated in a
boiling water bath or flowing steam for a few minutes and cooled without agita-
tion, just prior to inoculation.

One pound of Bacto-Egg Meat Medium will make 3 liters of medium.

»Am. J. Physiol., 8:284:1903. 3 J. Bact., 8:375:1923.

2 J. Biol. Chem., 2:71:1906. * J. Bact., 9:13:1924.

BACTO

COOKED MEAT MEDIUM (B267)

DEHYDRATED

Beef Heart 454 g.

Proteose Peptone, Difco 20 g.

Bacto-Dextrose 2 g.

Sodium Chloride 5 g.

Bacto-Cooked Meat Medium was developed for studies of contaminated
wounds carried on by investigators working under contract with the Office of

86 DIFGOMANUAL

Scientific Research and Development. It is recommended for use as a general
culture medium for both aerobic and anaerobic bacteria. It has proved especially
valuable as a primary medium for culturing pathological specimens and as a
stock culture medium on which to carry most laboratory cultures. This medium,
containing solid meat particles, has the unusual capacity for initiating growth
of bacteria from minute inocula and for maintaining the viability of cultures
over long periods of time.

Theobald Smith^ first made use of fresh unheated animal tissue for cultivating
anaerobic organisms. Tarozzi^ confirmed Smith's findings on the value of un-
heated tissue in broths for anaerobic culture, and discovered further that he
could heat the meat-broth to 104-1 05 °C. for 15 minutes without destroying its
capacity to support anaerobic growth, A steam sterilized emulsion of brain tissue
in water was employed by von Hibler^'* for cultivating anaerobic bacilli, and
found to be particularly valuable in culturing and classifying these organisms.
It was further noted by von Hibler that organisms growing in the cooked brain
mash were less susceptible to the harmful effects of toxic metabolic products
than were those cultured in milk or carbohydrate serum media. Robertson^ care-
fully analyzed von Hibler's results and substituted beef heart for the brain tissue.
She found the Cooked Meat Medium to be equally as satisfactory as the Cooked
Brain Medium. Henry^ employed the Cooked Meat Medium successfully for
culturing the anaerobes and recommended it for differentiating between various
putrefactive and saccharolytic species. Holman'^ used Cooked Meat Medium for
general culture purposes and commented: "Perhaps the most favorable charac-
teristic of the medium, after its general growth stimulating influence, is that the
products of growth do not rapidly destroy the various forms, and of all media in
common use it is to the meat medium that one can constantly return to reisolate
bacteria which have died out or have become hopelessly overgrown in other
media. The meat is the best single medium we have for studying the anaerobes
from war wounds, the reactions are useful for rapid differentiation of groups, as
well as for individual identification, and no other medium we have can so readily
indicate the presence of anaerobes in mixed cultures where they are often not
expected. The cooked meat medium is the most useful medium we have at pres-
ent for obtaining growth of both anaerobic and aerobic bacteria, for storing
mixed cultures for later isolation, as well as pure cultures for further investi-
gation. "Standard Methods for the Examination of Dairy Products"^ specifies the
use of Holman's Alkaline Cooked Meat Medium for use in the examination of
frozen dessert ingredients and "Diagnostic Procedures and Reagents"^ describes
a Ground Meat Medium for the cultivation of anaerobes.

The capacity of Cooked Meat Medium to initiate growth of minute inocula of
both aerobic and anaerobic microorganisms makes it especially valuable for use
in the primary culture of clinical specimens wherein the causative agent may be
present in such small numbers that enrichment is necessary. In such primary
cultures, even though more than one organism be present, the slower growing
organisms continue to proliferate without great danger of overgrowth by the
faster growing organisms, as is true in case of many other culture media.

Bacto-Cooked Meat Medium is also an excellent stock culture medium in
which to carry laboratory cultures of most microorganisms. Experience indicates
that in this medium strains of the Clostridia, such as tetani, novyi, septicum,
botulinum, sporogenes, putrificum, bijermentans, perfringens, and histolyticum
have remained viable as long as ten years. The Gram-negative enteric bacteria,
including Shigella, Salmonella, Proteus, and the coliforms, all remained viable
for more than five years; staphylococci, Corynebacteria and Lactobacilli have
persisted for more than six months.

To rehydrate the medium, suspend 12.5 grams of Bacto-Cooked Meat

DEHYDRATED CULTURE MEDIA 87

Medium in 100 ml. cold distilled water. Mix thoroughly and allow to stand for
15 minutes until all the particles are thoroughly wetted, and while maintaining an
even suspension distribute into tubes. The medium may also be rehydrated by dis-
tributing 1.25 grams into test tubes, adding 10 ml. cold distilled water, and mix-
ing thoroughly, allowing to stand to insure thorough wetting of all particles.
Sterilize in the autoclave for 15 minutes at 15 pounds pressure (121°C.). To
prevent blowing of medium, avoid rapid release of pressure in the autoclave
after sterilization. Properly prepared tubes of Bacto-Cooked Meat Medium
should have a well defined layer of meat particles overlaid by a clear, amber-
colored supernatant liquid. As soon as the sterile medium has cooled to 37 °G.
it is inoculated by loop or pipette, introducing the inoculum well into the meat
layer. The final reaction of the medium will be pH 7.2.

Tubes of Cooked Meat Medium not used the day they are prepared should be
placed in a boiling water bath or flowing steam for a few minutes to drive out
dissolved oxygen, then cooled to 37°C. and inoculated.

One pound of Bacto-Cooked Meat Medium will make 3.6 liters of medium.

iCentr. Bakt., 7:509:1890. ^ J. Path. Bact., 21:344:1917.

sCentr. Bakt., 38:619:1905. 'J. Bact., 4:149:1919-

3 Centr. Bakt., 25:513:1899. 8 standard Methods for the Examination

*Von Hibler: Untersuchungen ueber die of Dairy Products, 9th Edition: 192: 1948.

Pathogen Anaeroben, 1908. s Diagnostic Procedures and Reagents, 3rd

6 J. Path, Bact., 20:327:1916. Edition: 17:1950.

BACTO

HEART INFUSION AGAR (B44)

DEHYDRATED

Beef Heart, Infusion from 500 g.

Bacto-Tryptose 10 g.

Sodium Chloride 5 g.

Bacto-Agar 15 g.

Bacto-Heart Infusion Agar is a solid infusion medium recommended for gen-
eral laboratory use for the cultivation of many pathogenic bacteria. It can be
used as a base to which a large variety of materials, such as blood or carbo-
hydrates, may be added, giving media for many special purposes. It is a satis-
factory medium for mass culture of organisms, making it valuable in the prep-
aration of vaccines. Bacto-Heart Infusion Agar is recommended by the American
Association of Medical Milk Commissions^ for preparation of Blood Agar for
the recognition and differentiation of streptococci in the examination of certified
milk.

Like infusion broth, a solid medium prepared with the extractives of fresh
meat and containing agar was used almost from the beginning of bacteriology.
Huntoon^ was one among the many to show that highly pathogenic organisms,
such as the meningococcus and pneumococcus, could be grown on an infusion
medium without enrichment. Bacto-Tryptose, as employed in this formula, is
better suited to the nutritional requirements of pathogenic bacteria than is Bacto-
Peptone which was used by Huntoon in the preparation of his "hormone" agar.
The blood culture method of Castaneda, as described in detail on page 113
under Tryptose Agar may be used with this medium.

To rehydrate the medium, suspend 40 grams of Bacto-Heart Infusion Agar
in 1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in tubes or flasks and sterilize in the autoclave for 15
minutes at 15 pounds pressure (121°C.). The final reaction of the medium will
be pH 7.4.

88 DIFCOMANUAL

If Blood Agar is to be prepared immediately, the sterile medium is cooled at
once to 45-50° C. and while still liquid 5 per cent sterile defibrinated blood is
added aseptically with thorough mixing, avoiding incorporation of air bubbles,
and distributing in sterile tubes or plates as desired. Blood Agar for study of
cultural characteristics of colonies should be incubated to insure sterility before
use.

Since most microorganisms prefer a fresh medium with a moist surface, it is
recommended that the Heart Infusion Agar be prepared as required, or melted
and re-solidified just prior to use. Blood Agar from this medium should be pre-
pared as required.

One pound of Bacto-Heart Infusion Agar will make 11.3 liters of medium.

1 Methods and Standards for the Production ^ J. Infectiom Diseases, 23:169:1918.

of Certified Alilk, 28:1933-1954.

BACTO

BLOOD AGAR BASE (B45)

DEHYDRATED

Beef Heart, Infusion from, 500 g.

Bacto-Tryptose 10 g.

Sodium Chloride 5 g.

Bacto-Agar 15 g.

Bacto-Blood Agar Base is recommended as a base to which blood may be
added for use in the isolation and cultivation of many fastidious pathogenic
microorganisms. The slightly acid reaction of this medium is conducive to the
preservation of red blood cells. Without the addition of blood, this medium can
be recommended as a slightly acid agar medium for general laboratory work.
Many microorganisms produce earlier and more abundant growth on Blood
Agar with a slightly alkaline reaction, and therefore the use of this same medium
at pH 7.4, Bacto-Heart Infusion Agar as discussed immediately above or Bacto-
Tryptose Blood Agar Base discussed on page 115, is preferred.

Colonies of bacteria upon a Meat Infusion Blood Agar usually grow luxuri-
antly, and the hemolytic types exhibit clear distinct degrees of hemolysis. Nor-
ton^ has recommended the use of such a medium with a reaction of pH 6.8 as
being distinctly advantageous in culturing the pneumococcus and streptococcus
groups. This slightly acid reaction seems to permit the development of clearer
zones of hemolysis than does an alkaline reaction. Bacto-Blood Agar Base is a
medium containing the extractives from fresh beef heart with Bacto-Tryptose.
This medium, which contains no added carbohydrate, is especially recom-
mended for use in the preparation of Blood Agar to study the hemolytic char-
acteristics of colonies. It is also recommended for isolation of organisms directly
from the blood. Bacto-Blood Agar base is mentioned in "Diagnostic Procedures
and Reagents"^ as an excellent basal medium for plate cultures for the pneu-
mococci. The use of 1 ml. of venipuncture blood in a sterile petri dish to which
is added the sterile cooled medium is recommended. The blood culture method
of Castaneda, as described in detail on page 113 under Tryptose Agar may be
used with this medium.

Tarshis and Frisch^ investigated the addition of bank blood to various media
for the cultivation of tubercle bacilli in pure culture and directly from sputa
under routine diagnostic conditions. Three standard tuberculosis media were
used in the comparative study. They recommended the addition of 25 per cent
bank blood to Bacto-Blood Agar Base or Bacto-Bordet Gengou Agar Base with 1
per cent glycerol added since media of this type grew tubercle bacilli from small

DEHYDRATED CULTURE MEDIA 89

inocula producing colonies that were readily recognized. These media were easily
prepared and in addition were economical. They were also satisfactorily em-
ployed in streptomycin sensitivity tests.

To rehydrate the medium, suspend 40 grams of Bacto-Blood Agar Base in
1000 ml. of col J distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). The final reaction of the medium, before add-
ing blood, will be pH 6.8.

If Blood Agar is to be prepared immediately, the sterile medium is cooled at
once to 45-50° C, and, while still liquid, 5 per cent sterile defibrinated blood is
added aseptically with thorough mixing, avoiding incorporation of air bubbles,
and distributing in sterile tubes or plates as desired. Blood Agar should be
incubated before use to insure sterility.

Since most microorganisms prefer a fresh medium wdth a moist surface it is
recommended that Blood Agar Base be prepared as required, or melted and re-
solidified just prior to use. Blood Agar from this medium should be prepared as
required.

One pound of Bacto-Blood Agar Base will make 11.3 liters of the finished
medium.

IJ. Lab. Clin. Med., 17:558:1932. Edition: 70: 1950.

?Di«s>iostic Procedures and Reagents, 3rd "Am. J, Clin. Path., 21:101:1951.

BACTO

VEAL INFUSION AGAR (B343)

DEHYDRATED

Veal Heart, Infusion from 500 g.

Proteose Peptone, No. 3, Difco .... 10 g.

Sodium Chloride 5 g.

Bacto-Agar 15 g.

Bacto-Veal Infusion Agar is prepared from select lean veal and is recom-
mended as a solid medium for the cultivation of fastidious pathogenic bacteria.
It is also a suitable basal medium for enrichment by the addition of blood,
ascitic fluid, serum or other enrichments.

To rehydrate the medium suspend 40 grams Bacto-Veal Infusion Agar in
1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°G.).

Since most organisms prefer a fresh medium with a moist surface, it is sug-
gested that the medium be prepared as required or melted and re-solidified just
prior to use. The final reaction of the medium will be pH 7.4.

One pound of Bacto-Veal Infusion Agar will make 11.3 liters of medium.

BACTO

PPLO AGAR (B412)

DEHYDRATED

Bacto-Beef Heart for Infusions,

Infusion from 50 g.

Bacto- Peptone 10 g.

Sodium Chloride 5 g.

Bacto-Agar 14 g.

90 DIFCOMANUAL

Bacto-PPLO Agar is a base used in preparing a solid medium for the isolation
and cultivation of pleuropneumonia-like organisms (PPLO), as described by
Morton, Smith and Leberman.^ These authors found that Bacto-Peptone, Bacto-
Tryptose and Bacto-Yeast Extract were satisfactory peptones in a medium for
the cultivation of these organisms. Bacto-Peptone in the presence of an infusion
of Bacto-Beef Heart for Infusions gave best results. Bacto-PPLO Agar duplicates
their recommended formula.

Bacto-PPLO Agar, enriched with Bacto-Ascitic Fluid or Bacto-PPLO Serum
Fraction will permit the development of colonies of PPLO visible microscopi-
cally, after 48 hours or longer aerobic incubation at 37°C. After 48 hours incuba-
tion inverted plates are examined under the low power of the microscope,
focusing through the medium to its surface for the presence of surface colonies
on the medium. Cultures should be examined daily after 48 hours for a period of
one week before the plate is considered negative. PPLO colonies are round with
a dense center and a less dense periphery, giving the appearance of a fried egg.
They vary from 10 to 500 microns in diameter (0.01-0.5 mm.) and grow into the
medium. Individual organisms are not resolved, so there is little evidence at this
magnification of any cellular organization. Vacuoles are seen in the periphery of
some of the colonies. They are the large bodies characteristic of the pleuropneu-
monia group.

To rehydrate the medium, suspend 34 grams of Bacto-PPLO Agar in 1000 ml.
of cold distilled water and heat to boiling to dissolve the medium completely.
Distribute in flasks and sterilize in the autoclave for 15 minutes at 15 pounds pres-
sure (121°C.). After the medium has been cooled to 45-60° C, add 1 per cent
Bacto-PPLO Serum Fraction or 25 per cent Bacto-Ascitic Fluid. Mix thoroughly
and pour into sterile petri dishes. The reaction of the unenriched medium will
be pH 7.8.

One pound of Bacto-PPLO Agar will make 13.3 liters unenriched medium.

1 Am. J. Syphilis Gonorrh. Venereal Diseases, 35:361 :i95i-

BACTO

BRAIN HEART INFUSION AGAR (B418)

DEHYDRATED

Calf Brains, Infusion from 200 g.

Beef Heart, Infusion from 250 g.

Proteose Peptone, Difco 10 g.

Bacto-Dextrose 2 g.

Sodium Chloride 5 g.

Disodium Phosphate 2.5 g.

Bacto-Agar 15 g.

Bacto-Brain Heart Infusion Agar is recommended as a solid medium for the
cultivation of fastidious pathogenic bacteria and fungi. A selective medium for
fungi may be prepared by adding penicillin and streptomycin to the sterile
medium.

The liquid medium, Bacto-Brain Heart Infusion, has for many years been
the medium of choice for the cultivation of streptococci, pneumococci, meningo-
cocci and many other organisms considered difficult to cultivate, and has been
especially valuable in blood culture work. This medium, solidified with agar to
give Bacto-Brain Heart Infusion Agar, is a satisfactory solid medium for the
cultivation of these organisms. In addition, the solid medium has been especially
valuable as a neutral or slightly alkaline medium for the cultivation of patho-
genic fungi. Selective media for fungi may be prepared by adding antibiotics or

DEHYDRATED CULTURE MEDIA 91

other selective agents. Roseburg, Epps and Clark^ reported that Bacto-Brain
Heart Infusion with 2 per cent agar was more satisfactory than 1 per cent Dex-
trose Infusion Agar for the isolation and cultivation of Actinomyces israeli.
Incubation in an atmosphere of 5 per cent carbon dioxide was required for best
results. The addition of sheep blood to the medium offered no growth advantage.
HowelP used Bacto-Brain Heart Infusion to which was added 2 per cent Bacto-
Agar and 10 per cent sterile defibrinated horse blood for the cultivation of
Histoplasma capsulatum. A selective medium for the isolation of this organism
was prepared by adding 40 micrograms streptomycin and 20 units penicillin
per ml. of medium. In comparison with a Blood Agar similarly prepared from
Potato Dextrose Agar, the Brain Heart Infusion Agar gave a greater number of
positive isolations. Incubation at room temperature was more efficient than at
37° C. Colonies of H. capsulatum isolated on Brain Heart Infusion Agar must be
transferred to a medium such as Potato Dextrose Agar to obtain the character-
istic tuberculate chlamydospores typical of this fungus. Conant^ recommended
that a plate of Bacto-Brain Heart Infusion Agar be streaked and incubated at
37° G. under anaerobic conditions with the addition of 5 per cent carbon dioxide
to obtain growth of the microaerophilic A. bovis in culturing this organism from
infected mucous membranes and subcutaneous tissues.

Kotcher, Robinson and Miller* compared various media for the isolation of
H. capsulatum from tissues of experimentally infected mice. Their results showed
that Brain Heart Infusion Blood Agar gave the highest percentage recovery of
H. capsulatum from the tissues of the infected mice.

Bacto-Brain Heart Infusion Agar contains 0.2 per cent Bacto-Dextrose, mak-
ing it unsatisfactory for the determination of typical hemolytic reactions when
enriched with blood. Bacto-Heart Infusion Agar, Bacto-Tryptose Blood Agar
Base or Bacto-Blood Agar Base, as discussed on pages 87, 115 and 88, should
be used in preparing Blood Agar for the determination of typical hemolytic
reactions.

To rehydrate the medium, suspend 52 grams Bacto-Brain Heart Infusion Agar
in 1000 ml. cold distilled water. Heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°G.).

A selective medium for fungi is prepared by adding 20 units penicillin and
40 micrograms streptomycin per ml. sterile liquid medium, at 50-55 °C. Final
reaction of the medium will be pH 7.4.

One pound of Bacto-Brain Heart Infusion Agar will make 8.7 liters of
medium.

1 J. Infectious Diseases, 74:131:1944. Edition :452: 1950.

a Public Health Reports, 63:173:1948. * J. Bact., 62:613:1951,

• Diagnostic Procedures and Reagents, 3rd

BACTO

CYSTINE HEART AGAR (B47)

DEHYDRATED

Beef Heart, Infusion from 500 g.

Proteose Peptone, Difco 10 g.

Bacto-Dextrose 10 g.

Sodium Chloride 5 g.

/-Cystine, Difco 1 g.

Bacto-Agar 15 g.

Bacto-Cystine Heart Agar, enriched with Bacto-Hemoglobin, is recommended
for the cultivation of Pasteurella tularensis. The use of this medium is suggested

92 DIFCO MANUAL

for this purpose by "Diagnostic Procedures and Reagents"^ of the American
Public Health Association. Bacto-Cystine Heart Agar without enrichment sup-
ports excellent growth of Gram-negative cocci and other pathogenic micro-
organisms.

Since P. tularensis was first isolated by McCoy and Chapin,^ many media
have been described for its cultivation. A large number of the media first em-
ployed were difficult to prepare and contained egg or serum. Francis^ reported
Blood Dextrose Cystine Agar in his later investigation as being a satisfactory
medium for cultivating this fastidious organism. Shaw* added 0.05 per cent
cystine and 1 per cent dextrose to Bacto-Heart Infusion Agar for the cultivation
of P. tularensis, Shaw^ also showed that the amount of destruction of cystine in
the autoclave at 15 pounds pressure for 15 minutes would be small or negligible
as far as the bacteriological culture medium was concerned.

Rhamy® found Francis' Blood Dextrose Cystine Agar to be excellent but often
it became contaminated due to the difficulties attendant to its preparation. In
his experience an autoclaved solution of Bacto-Hemoglobin added to Bacto-
Cystine Heart Agar proved to be entirely satisfactory for the cultivation of
P. tularensis. In three or four days the growth is sufficient for the preparation of
bacterial antigens. Because of its nutritional value, this medium may also be
used for cultivating many other organisms ordinarily difficult to grow.

Bacto-Cystine Heart Agar was originally developed in collaboration with
Rhamy. As mentioned in the paper by Rhamy, referred to above, W. M. Simp-
son found this formula, with a reaction of pH 6.8, a most satisfactory medium
for the cultivation of this organism. Also cooperating in these preliminary trial
studies of the medium, Francis found a culture medium made with Bacto-
Cystine Heart Agar and Bacto-Hemoglobin entirely satisfactory for growing
P. tularensis.

When used with Bacto-Hemoglobin, the medium is prepared for use as
follows:

A. Suspend 10.2 grams Bacto-Cystine Heart Agar in 100 ml. cold distilled
water and heat to boiling to dissolve the medium completely. Sterilize in the
autoclave for 15 minutes at 15 pounds pressure (121°C.).

B. Place 2 grams Bacto-Hemoglobin in a dry flask and add 100 ml. cold
distilled water, while the flask is being agitated vigorously. The hemoglobin
suspension is shaken intermittently for 10-15 minutes to break up all aggregates
and effect complete solution and sterilized in the autoclave for 15 minutes at 15
pounds pressure (121°C.).

C. Both solutions are cooled to 50-60° C, mixed and poured into sterile petri
dishes or tubes.

When a plain Cystine Dextrose Agar, without hemoglobin, is desired, the
medium is rehydrated by suspending 51 grams of Bacto-Cystine Heart Agar in
1000 ml. cold distilled water and heating to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). The final medium will have a reaction of
pH 6.8.

One pound of Bacto-Cystine Heart Agar will make 8.9 liters of the enriched,
or plain, medium.

1 Diagnostic Procedures and Reagents, * Zentr. Bakt. I Abt. Orig., 118:216:1930.
3rd Edition: 259: 1950. ^ J. Lab. Clin. Med., 16:294:1930.

2 J. Infectious Diseases, 10:61:1912. ^ Am. J. Clin. Path., 3:121:1933.

3 J. Am. Med. Assoc, 91:1155:1928.

DEHYDRATED CULTURE MEDIA 93

BACTO

MUELLER HINTON MEDIUM (B252)

DEHYDRATED

Beef, Infusion from 300 g.

Bacto-Gasamino Acids, Technical . . 17.5 g.

Starch 1.5 g.

Bacto- Agar 1 7 g.

Bacto-Mueller Hinton Medium duplicates the formula recommended by
Mueller and Hinton^ for the primary isolation of the gonococcus and menin-
gococcus. Sulfonamide resistance of gonococci and other microorganisms may
also be determined on this medium.

In an attempt to develop a simple transparent medium containing no heat-
labile materials and capable of withstanding autoclaving, Mueller and Hinton
selected what they considered the most suitable complete medium available, and
attempted to break it into its essential components. The medium chosen for study
was the complex Gordon and Hine^ Pea Meal Extract Agar, which, in the
opinion of the senior author, had proved to be satisfactory for the primary iso-
lation of the meningococcus and the gonococcus. As a result of the fractionation
of the pea extract, it was found that the active portion was starch and not a
protein. Additional work showed that starch could replace the growth-promoting
properties of the pea extract and that the starch probably acts as a "protective
colloid" against toxic materials present in the medium. In addition, they found
that the tryptic digest of meat could be replaced by Bacto-Gasamino Acids,
Technical. Growth of the gonococcus and meningococcus on the developed
medium was highly satisfactory and colonies were usually large and easily recog-
nizable, especially with the aid of the oxidase reagent.

Goodale et al.^ used the Mueller Hinton Medium to identify sulfonamide
resistant strains of the gonococcus. Nelson* used Bacto-Mueller Hinton Medium
in correlating sulfonamide resistance with the clinical picture in gonorrhoea. In
"Diagnostic Procedures and Reagents"^ of the American Public Health Associa-
tion, the Mueller and Hinton Medium is suggested for the isolation and trans-
portation of the meningococcus in the field or whenever blood is unobtainable.

For the cultivation of the gonococcus, it is imperative to have the incubation
atmosphere saturated with moisture. Satisfactory conditions can be obained if
the plates of Mueller Hinton Medium are incubated in a closed container, which
contains cotton, a towel or a sponge saturated with water. A can with a suitable
cover, Novy jar, desiccator or any other convenient sized container capable of
retaining the moisture is entirely satisfactory. About 200 ml. of water added in
this manner is ample for a container of one or two gallons capacity. Plates incu-
bated under these conditions will give a luxuriant growth of many gonococci
when identically inoculated plates incubated in the ordinary manner in the in-
cubator show no growth. If the culture requires carbon dioxide for growth this
may be supplied as indicated under Bacto-Proteose No. 3 Agar, page 116. Car-
bon dioxide is recommended for isolation, but is not generally necessary, in the
presence of abundant moisture, for growth of isolated strains.

To rehydrate the medium, suspend 38 grams of Bacto-Mueller Hinton
Medium in 1000 ml. of cold distilled water and heat to boiling to dissolve the
medium completely. Distribute in tubes or flasks and sterilize for 10 minutes at
10 pounds pressure (116°G.). The final reaction of the medium will be pH 7.4.

It is recommended that the dissolved medium be distributed into test tubes
in 5 ml. quantities for slants or into 120-200 ml. flasks in 20 ml. quantities for
pouring plates. This medium must not be heated more than the sterilization

94 DIFCOMANUAL

period specified. The flasks can be used to pour plates at once and tubes may be
melted in boiling water and used as needed.

One pound of Bacto-Mueller Hinton Medium is sufficient to prepare 11.9
liters of medium.

1 Proc. Soc. Exp. Biol. Med., 48:330:1941. * Personal Communication.

2 Brit. Med. J., 2:678:1916. ^ Diagnostic Procedures and Reagents,

3 J. Am. Med. Assoc, 123:547:1943. 3rd Edition: 192: 1950.

BACTO

BRAIN VEAL AGAR (B49)

DEHYDRATED

Calf Brain, Infusion from 250 g.

Veal, Infusion from 375 g.

Proteose Peptone, Difco 10 g,

Monosodium Phosphate 1.25 g.

Sodium Chloride 3.75 g.

Bacto-Agar 15 g.

Bacto-Brain Veal Agar is a medium containing extractives of fresh calf brain
and veal. It was developed primarily for the cultivation of the gonococcus. This
medium also supports good growth of streptococci, pneumococci, meningococci,
and other microorganisms generally considered difficult to cultivate.

Pelouze and Viteri,^ following a study of the cultural requirements of the
gonococcus, devised a simple medium, Brain Veal Agar, upon which this diplo-
coccus grew luxuriantly. Bacto-Brain Veal Agar is prepared according to their
formula and gives results comparable to the medium prepared by them. In
addition, it is an excellent medium for other highly fastidious microorganisms.
Bacto-Brain Veal Agar is a satisfactory medium for the preparation of bacterial
antigens and vaccines.

For the cultivation of the gonococcus for complement-fixation work Garcia^
reported that Bacto-Brain Veal Agar gave good growth. Saccone^ used Bacto-
Brain Veal Agar for the isolation of the gonococcus. Reitzel and Kohl,* in their
modification of the McLeod method for the isolation of the gonocccus, employed
a medium more adaptable for use in small laboratories. Their medium was pre-
pared by using a combination of Bacto-Brain Heart Infusion as discussed on
page 77 and Bacto-Brain Veal Agar which, when mixed with an equal quantity
of Bacto-Hemoglobin as discussed on page 271, would give a final concentration
of 1.2 per cent agar. Shaw^ added 0.5 per cent cystine and 1 per cent dextrose
to Bacto-Brain Veal Agar for the cultivation of Pasteurella tularensis with ex-
cellent results. Foshay^ also used Bacto-Brain Veal Agar with Nutrose (sodium
caseinate), dextrose, cystine, inorganic salts and serous enrichments for the
propagation of P. tularensis.

Although Bacto-Brain Veal Agar will support good growth of the gonococcus,
media prepared without infusions from meat have been developed, giving much
better growth of these discriminating organisms. For isolation of the gonococcus
we recommend a Chocolate Agar prepared from Bacto-Proteose No. 3 Agar and
Bacto-Hemoglobin, enriched with Bacto-Supplement A or Bacto-Supplement B,
or Bacto-G. C. Medium Base similarly enriched. The procedure is given in de-
tail on pages 116 and 122. For cultivation of the gonococcus in pure culture we
recommend Bacto-Dextrose Starch Agar as discussed on page 124.

To rehydrate the medium, suspend 53 grams of Bacto-Brain Veal Agar in
1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and autoclave for 15 minutes at 15 pounds
pressure (121°C.). The final reaction of the medium will be pH 7.6.

DEHYDRATED CULTURE MEDIA 95

Since best results are obtained with solid media having a moist surface, Brain
Veal Agar should be used the same day it is prepared or, if not used at once, the
medium should be melted and allowed to re-solidify. A moist surface is particu-
larly important for the cultivation of the gonococcus, and for that reason it is
recommended that sterile rubber stoppers be substituted for the cotton plugs as
soon as the slants have cooled.

One pound of Bacto-Brain Veal Agar will make 8.5 liters of medium.

ij. Am. Med. Assoc, 86:684:1926. * J. Am. Med. Assoc, 110:1095:1938.

2 Rev. Med. Malaga, Nov. 6, 1930. ^ Zentr. Bakt., I Abt., Orig., 118:216:1930.

3Rinascenza Medica, i4:No. 17:1936, XIII. « Am. J. Clin. Path., 3:379:1933.

BACTO

POTATO INFUSION AGAR (B51)

DEHYDRATED

Potatoes, Infusion from 250 g.

Bacto-Beef Extract 5 g.

Proteose Peptone, Difco 10 g.

Sodium Chloride 5 g.

Bacto-Dextrose 10 g.

Bacto-Agar 15 g.

Bacto-Potato Infusion Agar is prepared according to the formula used by
Stockman and MacFadyean for the isolation of Brucella abortus. This medium
permits a luxuriant growth of characteristic colonies of B. abortus from infected
materials and may be used with excellent results in mass cultivation of the
organism in the preparation of vaccines and antigens. Bacto-Tryptose Agar,
which does not contain an infusion, as discussed on page 111, is recommended as
being far more satisfactory than Bacto-Potato Infusion Agar for the isolation and
cultivation of the Brucella.

To rehydrate the medium suspend 49 grams of Bacto-Potato Infusion Agar
in 1000 ml. of a 2 per cent solution of glycerol in distilled water and heat to
boiling to dissolve the medium completely. Distribute in tubes or flasks and
sterilize in the autoclave for 15 minutes at 15 pounds pressure (121°G.). The
final medium will have a reaction of pH 6.8 and will contain a slight precipitate
which settles rapidly. The presence of this precipitate in no way interferes with
the use of the medium.

Best results are obtained on freshly prepared media with a moist surface. It
is suggested if the medium is not used the day it is prepared that the agar be
melted and allowed to re-solidify in order to provide most satisfactory condi-
tions for growth.

One pound of Bacto-Potato Infusion Agar will make 6 liters of medium.

BACTO

BORDET GENGOU AGAR BASE (B48)

DEHYDRATED

Potatoes, Infusion from 125 g.

Sodium Chloride 5.5 g.

Bacto-Agar 20 g.

Bacto-Bordet Gengou Agar Base, enriched with 15 to 20 per cent blood, is rec-
ommended for use in the "cough plate" method for the detection and isolation of

96 DIFGOMANUAL

Hemophilus pertussis in the diagnosis of whooping cough. This is a modification
of the medium originally described by Bordet and Gengou^ in 1906 for the culti-
vation of H. pertussis and is prepared according to the formula recommended
in Diagnostic Procedures and Reagents^ of the American Public Health Associa-
tion for the isolation of this organism. The addition of 1 per cent Proteose
Peptone to the medium is suggested if employed for mass culture of H. pertussis
as in vaccine production.

The "cough plate" method for the diagnosis of whooping cough was originally
reported by Chievitz and Meyer^ in 1916. Lawson and Mueller^ in 1927 and
Sauer and Hambrecht^ in 1930 used modifications of the Bordet Gengou Medium
to demonstrate the value of the cultural diagnosis of this disease. This method
has been applied routinely as a diagnostic procedure for public health labora-
tories as a result of the thorough and painstaking investigations of Kendrick and
her associates. Kendrick and Eldering^ first used a modified Bordet Gengou
Medium for the isolation and propagation of H. pertussis. Eldering and Ken-
drick'^ reported that the addition of 1 per cent of Proteose Peptone or Neopep-
tone increased the growth of H. pertussis thereby increasing the yield for vaccine.

With this modification of the Bordet Gengou Medium, enriched with 15 to 20
per cent blood, the appearance of colonies of H. pertussis is typical, being
smooth, raised, glistening and not over 1 mm. in diameter. They are of a pearly,
almost transparent appearance, and are surrounded by a characteristic zone of
hemolysis which is not sharply defined, but which merges diffusely into the
medium. The zone of hemolysis usually is absent if 30 per cent or more blood is
added to the medium. Sterile sheep, rabbit or human blood may be used in pre-
paring the medium. Horse blood should not be used in preparing vaccine.

Kendrick, Miller and Lawson'^ and Kendrick, Lawson and Miller^ recom-
mended that, after exposure, cough plates prepared from the modified Bordet
Gengou Blood Agar should be incubated at 37°C. During the first 48 hours
incubation they are examined for contamination by molds and spreaders, which
are cut aseptically from the medium. The plates are then examined twice daily,
using a hand lens, until typical colonies of H. pertussis are found or until dis-
carded after 6 days of incubation.

Maclean^ used Bacto-Bordet Gengou Agar Base and reported it to be efficient
in the isolation of H. pertussis. He further reported that this medium was a
valuable standard for the comparison of various lots of media prepared from
ingredients.

Tarshis and Frisch^o investigated the addition of bank blood to various media
for the cultivation of tubercle bacilli in pure culture and directly from sputa
under routine diagnostic conditions. Three standard tuberculosis media were
used in the comparative study. They recommended the addition of 25 per cent
bank blood to Bacto-Bordet Gengou Agar Base or Bacto-Blood Agar Base with
1 per cent glycerol added since media of this type grew tubercle bacilli from
small inocula producing colonies that were readily recognized. These media
were easily prepared and in addition were economical. They were also satis-
factorily employed in streptomycin sensitivity tests.

To rehydrate the medium, suspend 3 grams of Bacto-Bordet Gengou Agar
Base in 100 ml. of a 1 per cent solution of glycerol in distilled water and heat
to boiling to dissolve the medium completely. Distribute in flasks and sterilize in
the autoclave for 15 minutes at 15 pounds pressure (121°C.). It is cooled to 45
to 50°G. and aseptically enriched with 15 to 20 per cent sterile sheep, rabbit,
or human blood^ and is poured into sterile petri dishes. The blood should be
used when fresh, never more than 72 hours after it has been obtained. Satisfac-
tory plates should be bright cherry red in color and free from bubbles and lumps
of agar. Plates may be used as long as they remain moist and red, usually two

DEHYDRATED CULTURE MEDIA

or three weeks if kept in the cold room. For mass cultivation of H. pertussis as
in vaccine production dissolve 1 gram of Proteose Peptone in 100 ml. of glycerol
solution used to rehydrate the medium.

One pound of Bacto-Bordet Gengou Agar Base is sufficient for 15 liters of
medium.

1 Am. inst. Pasteur, 20:731:1906. « Am. J. Pub. Health, 24:309:1934.

2 Diagnostic Procedures and Reagents, 'Am. J. Pub. Health, 26:506:1936.

3rd Edition: 141 : 1950. s Sixth Annual Year Book (1935-36), p. 200,

3 Ann. inst. Pasteur, 30:503:1916. Suppl., Am. J. Pub. Health, 26:No. 3:1936.
* J. Am. Med. Assoc, 89:275:1927. » J. Path. Bact., 45:472: i937-

5 J. Am. Med. Assoc, 95:263:1930. "Am. J. Clin. Path., 21:101:1951.

BACTO

END AMOEBA MEDIUM (B53)

DEHYDRATED

Beef Liver, Infusion from 272 g.

Proteose Peptone, Difco 5.5 g.

Disodium Phosphate 3 g.

Sodium Chloride 2.7 g.

Bacto-Agar 11 g.

Bacto-Endamoeba Medium is a Liver Infusion Agar prepared especially for
the cultivation of Endamoeba histolytica from specimens. The formula cor-
responds to that recommended by Cleveland and Sanders^ and Cleveland and
Collier.2

Cleveland and his associates made a comprehensive study of the cultivation of
E. histolytica. They used egg, serum and various other materials for cultivating
the amoeba and state^: "We feel however, that we really did not cultivate Enta-
moeba histolytica until we grew it on slants of liver infusion agar covered with
fresh horse serum-saline 1-6 and containing rice flour." They used about half
strength Bacto-Liver Infusion Agar in this study. Bacto-Endamoeba Medium
has been prepared to duplicate the modified Liver Infusion Agar described by
Cleveland and his co-workers.

This medium, furthermore, is reported by them to be almost specific for
E. histolytica, as far as the intestinal amoebae of man are concerned. They
attempted to cultivate Endolimax nana, Dientamoeba fragilis, and E. coli and
failed, while E. histolytica grew abundantly. In a committee report Spector,^
referee, states that the method of Cleveland and Collier using Bacto-Liver In-
fusion Agar overlaid with sterile serum-saline 1-6 was one of the best for practi-
cal diagnostic purposes and that Wassermann-negative human inactivated serum,
horse, or rabbit serum may be used. This report also mentions that other in-
testinal amoebae do not grow as readily in this culture medium as does E.
histolytica.

To rehydrate the medium, suspend 33 grams of Bacto-Endamoeba Medium in
1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). The final reaction of the medium will be pH 7.0. The
tubes are allowed to solidify in a slanted position. For the cultivation of E. histo-
lytica the slants are covered with Bacto-Horse Serum Saline 1-6 and a loopful
of sterilized Bacto-Rice Powder is added. Bacto-Rice Powder is sterilized in a
dry heat oven at 160°F. for one hour. Scorching must be prevented. These en-
richments are discussed in detail on pages 273 and 200.

One pound of Bacto-Endamoeba Medium will make 13.7 liters of medium.

1 Arch. Protiskenkunde, 70:223:1930. 3 Sixth Annual Year Book (1935-36), p. 130,

* Am. J. Hyg., 12:606:1930. Suppl., Am. J. Pub. Health, 26:No. 3:1936,

98 DIFGO MANUAL

BACTO

STOCK CULTURE AGAR (B54)

DEHYDRATED

Beef Heart, Infusion from 500 g.

Proteose Peptone, Difco 10 g.

Bacto-Gelatin 10 g.

Bacto-Dextrose 0.5 g.

Bacto-Isoelectric Casein 5 g.

Disodium Phosphate 4 g.

Sodium Citrate 3 g.

Bacto-Agar 7.5 g.

Bacto-Stock Culture Agar is recommended for the maintenance of cultures of
streptococci and other organisms. It is a soft, almost semisolid medium.

Ayers and Johnson^ described a medium that gave luxuriant growth and long
life of streptococci. Their medium was developed from a formula originally
given them by Supplee. The success of their medium probably lies in the fact
that the medium has a semisolid consistency, contains casein, is well buffered and
contains a small quantity of dextrose which serves as a readily available source
of energy. They reported that pathogenic streptococci remained viable for at least
four months at room temperature (24°G.) in their medium. Organisms other
than streptococci, such as pneumococci, human tubercle bacilli and others grew
well on their Stock Culture Agar.

Bacto-Stock Culture Agar is prepared to duplicate the medium described by
Ayers and Johnson. This medium, likewise, will support luxuriant growth of
many pathogenic bacteria and preserve their viability over a long period of time.

Bacto-Cooked Meat Medium, as discussed on page 85, is also recommended
as a medium for carrying cultures in stock. For carrying cultures of gonococcus,
meningococcus, and other organisms not capable of splitting starch, the infusion
free Bacto-Dextrose Starch Agar, in half concentration, is recommended as dis-
cussed on page 125.

To rehydrate the medium, suspend 50 grams of Bacto-Stock Culture Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°G.). The final reaction of the medium will be pH 7.5.

One pound of Bacto-Stock Culture Agar will make 9 liters of medium.

■ij. Bact., 9:111:1924,

BACTO

LIVER VEAL AGAR (B59)

DEHYDRATED

Bacto-Liver, Infusion from 50 g.

Veal, Infusion from 500 g.

Proteose Peptone, Difco 20 g.

Neopeptone, Difco 1.3 g.

Bacto-Tryptone 1.3 g.

Bacto-Dextrose 5 g.

Soluble Starch, Difco _ 10 g.

Bacto-Isoelectric Casein 2 g.

Sodium Chloride 5 g.

Sodium Nitrate 2 g.

Bacto-Gelatin 20 g.

Bacto-Agar 15 g.

DEHYDRATED CULTURE MEDIA 99

Bacto-Liver Veal Agar is especially well suited for the isolation and cultiva-
tion of anaerobes. The medium may be used in anaerobic culture dishes or in
deep tube culture.

Numerous methods for the cultiv^atlon of anaerobes have been devised and
many media have been proposed for this purpose. The use of the anaerobic
culture dish as described by Spray^'- is one of the procedures suggested for the
propagation of these organisms. Medium prepared from Bacto-Liver Veal Agar
is identical with the medium described by Spray^-* for use in his anaerobic
culture dishes for cultivation of anaerobes.

Bacto-Liver Veal Agar gives excellent growth of the sporulating anaerobes. In
a personal communication Spray reported the usefulness of this medium for
isolation purposes since Clostridium perfringens colonies were fished within 6
hours from time of inoculation and C. tetani within 8 hours. Gas production is
inhibited when the medium is inoculated sparingly. With proper dilution giv-
ing 10-15 colonies per plate, primary isolations of pure cultures are readily ob-
tained. The medium may also be used in deep tube cultures of the sporulating
anaerobes. Bacto-Liver Veal Agar is also suitable for the routine isolation and
cultivation of many aerobes.

To rehydrate the medium, suspend 97 grams of Bacto-Liver Veal Agar in
1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure ( 121°C.). The final reaction of the medium will be pH 7.3.

When it is used in the anaerobic dish, Spray^ recommends that unless taken
directly from the sterilizer, the medium should be boiled for 10 minutes and then
cooled to 50° C. without agitation. Serial inoculations are then made and the
medium is poured into the dishes. After solidification, 5 ml. sterile Liver Veal
Agar is poured over the medium as a cover layer to prevent the spreading of
surface colonies.

One pound of Bacto-Liver Veal Agar will make 4.6 liters of medium.

^J:

Lab. Clin. Med., 16:203:1930. 'Personal Communication.

Bact., 21:23:1931. *J. Bact., 32:135:1936.

PEPTONE MEDIA

{Without Infusions)

Infusions of meat with added peptone have been employed consistently as
culture media for bacteria. Extractives from fresh meat have been considered
essential in media for all but the most common organisms. In many cases, due
to ease of preparation and demand for greater uniformity in media, beef extract
has replaced the infusion of fresh meat. Media containing peptone without meat
extractives have been used for the routine cultivation of some of the more hardy
laboratory strains, or for routine tests such as the production of indole, methyl
red test, etc. Peptones have been used in semi-synthetic broths for the production
of diphtheria and scarlet fever toxin.

The opportunities for variation in media prepared from fresh meat are ob-
vious. The freshness of the meat used in the infusion influences the amount of
muscle sugar present in the final medium. The age of the animal, the cut of the
meat, amount of trimming, time and conditions of infusion are further factors
contributing to variability of the final media. Likewise, the infusion of meat is
laborious, time consuming and costly. These conditions were recognized by the

100 DIFCO MANUAL

Standard Methods Committee of the American Public Health Association, when
in 1917^ they specified the use of beef extract rather than infusion of fresh meat
for use in the preparation of media for the examination of water. In 1921
"Standard Methods for Milk Analysis,"^ in the interests of greater uniformity,
specified the use of beef extract rather than infusions of beef for media used in
plate counts of milk. A standard medium must be uniform in composition and
easily prepared. Any procedure that simplifies the preparation and maintains
greater uniformity of composition of media is a definite advance in technical
bacteriology.

For isolation or cultivation of strains of many highly pathogenic organisms
such as the Brucella, streptococci, pneumococci, gonococci, meningococci and
others, it has generally been considered necessary to have infusions of fresh meat
in the medium. Bateriological peptones have been pioneered and developed by
Difco Laboratories that make the addition of infusions of fresh meat unnecessary
for the cultivation of many organisms, including even the discriminating gono-
cocci, meningococci, pneumococci, streptococci. Brucella and others. For example,
Bacto-Tryptose Agar, developed and prepared in 1938, proved more satisfactory
for the isolation and cultivation of Brucella, streptococci and pneumococci than
previously employed infusion media. Bacto-Proteose No. 3 Agar and Bacto-
Dextrose Starch Agar also developed in 1938 for the isolation and cultivation of
the gonococci, simplified the cultural diagnosis of gonorrhoea so as to make it a
routine test even for the smallest laboratory. These media contain specially
prepared bacteriological peptones without infusions and have proven in many
years of practical use to be superior to previously recommended complicated
infusion and enriched media used for the cultivation of these organisms. A 2 per
cent solution of Bacto-Tryptose or Proteose Peptone No. 3 will satisfactorily
replace the peptone-infusion portion of media previously employed for the
isolation and propagation of streptococci, pneumococci, meningococci, gonococci
and other fastidious microorganisms. In the discussion of Meat Infusion Broths
in "Diagnostic Procedures and Reagents"^ of the American Public Health As-
sociation, the referee states that in his laboratory a broth with 2 per cent pan-
creatic digest of casein and 0.5 per cent yeast extract has been substituted for
infusion broth and the growth of the more fastidious organisms has been heavier
than that obtained in infusion media.

The simplicity of preparation and uniformity of composition, combined with
the ability to grow the organisms, make the Peptone Media listed in this secton
of practical value and interest.

It is a well recognized fact that infusion media contain varying amounts of
muscle sugar. In preparing media from peptones this variability can be controlled
by the addition of a known and definite quantity of dextrose. A small quantity
of dextrose (0.025-0.05 per cent) in peptone media assists materially in the
initiation of growth of many bacteria.

1 Standard Methods of Water Analysis, ^ Diagnostic Procedures and Reagents,

3rd Edition: 93: 191 7. 3rd Edition: 13: 1950.

" Standard Methods of Milk Analysis,
3rd Edition: 7: 192 1.

BACTO

TRYPTOSE PHOSPHATE BROTH (B60)

DEHYDRATED

Bacto-Tryptose 20 g.

Bacto-Dextrose 2 g.

Sodium Chloride 5 g.

Disodium Phosphate 2.5 g.

DEHYDRATED CULTURE MEDIA 101

Bacto-Tryptose Phosphate Broth is a liquid peptone medium prepared without
an infusion of meat, recommended for the cultivation of streptococci, pneumo-
cocci, meningococci and other organisms generally considered difficult to culti-
vate. This medium is especially adapted for blood culture work. A procedure
frequently used for this purpose is to add as much as 10 ml. blood specimen to
150 ml. of sterile Tryptose Phosphate Broth contained in a 300 ml. Erlenmeyer
flask or bottle. Inoculated flasks are incubated at 37° G. and are observed at in-
tervals for bacterial growth. When growth occurs in the flasks, transfers are
made to Blood Agar or other suitable media for isolation and identification,
according to usual practice.

The addition of 0.1-0.2 per cent Bacto-Agar to Tryptose Phosphate Broth
improves the productivity of the medium for most purposes. The advantages of
a medium with a low agar concentration is discussed fully under Bacto-Brain
Heart Infusion, page 77. In the "Standard Methods for the Examination of
Dairy Products,"^ 0.1 per cent agar is included in the formula for Tryptose Phos-
phate Agar Broth as used in the isolation of pathogenic bacteria from cheese.
"Diagnostic Procedures and Reagents"- of the American Public Health Associa-
tion include the addition of 0.1-0.2 per cent agar in the formula of Tryptose
Phosphate Broth.

Newman^ in a study of the detection of food poisoning attributable to dairy
products, used Tryptose Phosphate Broth to which was added 0.1 per cent agar
and 1 :2500 sodium azide for the cultivation of streptococci. An incubation tem-
perature of 37 °C. was used with this medium. Bacto-Tryptose Phosphate Broth
is recommended for use in the tube dilution method of testing the sensitivity of
microorganisms to antibiotics. Waisbren, Carr and Dunnett* showed that Bacto-
Tryptose Phosphate Broth, Bacto-Dextrose Broth or Bacto-Nutrient Broth were
suitable media for comparative sensitivity test studies while a medium contain-
ing a soy bean peptone inhibited the action of Neomycin, Aureomycin, Terra-
mycin and Polymyxin against the test organism.

To rehydrate the medium, dissolve 29.5 grams of Bacto-Tryptose Phosphate
Broth in 1000 ml. distilled water. Distribute in tubes or flasks. For blood culture
work dispense in 150 ml. quantities in 300 ml. Erlenmeyer flasks or bottles. The
medium is sterilized in the autoclave for 15 minutes at 15 pounds pressure
(121°C.). The final reaction of the medium will be pH 7.3.

If the medium is not used immediately after preparation, it should be heated
in the autoclave or in a boiling water bath in order to drive off dissolved oxygen,
and should then be cooled to 37°G. without agitation, just prior to inoculation.

One pound of Bacto-Tryptose Phosphate Broth will make 15.3 liters of
medium.

1 Standard Methods for the Examination * J. Milk and Food Tech., 13:226:1950.
of Dairy Products, 9th Edition: 165:1948. * Am. J. Clin. Path., 21:884:1951.

2 Diagnostic Procedures and Reagents,
3rd Edition: 16: 1950.

BACTO

DEXTROSE BROTH (B63)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Tryptose 10 g.

Bacto-Dextrose 5 g.

Sodium Chloride 5 g.

Bacto-Dextrose Broth is recommended as a liquid enrichment medium for the
isolation of many bacteria. It is a superior medium for the cultivation of organ-

102 DIFGO MANUAL

isms such as streptococci, pneumococci, meningococci and other microorganisms.

The fact that dextrose (<i-glucose) is a readily available source of energy,
utilized by a large number of organisms, makes Dextrose Broth an important
medium in any laboratory. Dextrose Broth can be recommended to give rapid
growth and hasten the early development of attenuated forms. A liquid medium
containing dextrose is especially adapted to primary culture in the isolation of
pathogenic bacteria. Bacto-Dextrose Broth contains 0.5 per cent dextrose along
with Bacto-Tryptose. This medium is superior to most Infusion-Peptone Dextrose
Broths for the cultivation of a large variety of bacteria, including the pathogenic
cocci, and has the further advantage of greater uniformity. Bacto-Dextrose Broth
is especially suited for use in the preparation of Rosenow's Dextrose Brain Broth^
as described for the isolation of streptococci. The value of Bacto-Dextrose Broth
is greatly increased for the cultivation of organisms like streptococci, pneumo-
cocci, meningococci, gonococci, etc., if 0.1 per cent agar be incorporated in the
medium as discussed on page 77 under Bacto-Brain Heart Infusion.

Bacto-Dextrose Broth is recommended for use in the tube dilution method of
testing the sensitivity of microorganisms to antibiotics. Waisbren, Carr and Dun-
nett^ showed that Bacto-Dextrose Broth, Bacto-Tryptose Phosphate Broth or
Bacto-Nutrient Broth were suitable media for comparative sensitivity test studies
while a medium containing a soy bean peptone inhibited the action of Neomycin,
Aureomycin, Terramycin and Polymyxin against the test organism.

To rehydrate the medium, dissolve 23 grams of Bacto-Dextrose Broth in 1000
ml. of distilled water. Distribute in tubes and sterilize in the autoclave for 15
minutes at 15 pounds pressure (121°C.). Final reaction of the medium will be
pH 7.2.

For best results Bacto-Dextrose Broth should be freshly prepared. If the
medium is not used the same day as sterilized, heat in boiling water or flowing
steam for a few minutes to remove absorbed oxygen, and cool quickly without
agitation, just prior to inoculation.

One pound of Bacto-Dextrose Broth will make 19.7 liters of medium.

1 J. Dental Research, 1:205:1919. 2 Am. J. Clin. Path., 21:884:1951.

BACTO

TRYPTOSE BROTH (B62)

DEHYDRATED

Bacto-Tryptose 20 g.

Sodium Chloride 5 g.

Bacto-Dextrose 1 g.

Thiamine Hydrochloride 0.005 g.

Bacto-Tryptose Broth is prepared without extract or infusion of meat and is
recommended as a general laboratory medium for the cultivation of discriminat-
ing pathogenic as well as saprophytic bacteria. Huddleson^ used a broth contain-
ing 2 per cent Bacto-Tryptose as an enrichment medium in the isolation of
Brucella from man. McCullough, Mills, Herbst, Roessler and Brewer^ reported
that the addition of thiamine, dextrose and iron salts increased the growth of
B. suis. Sanders and Huddleson^ showed that the addition of dextrose and
thiamine hydrochloride to the medium resulted in the stimulation of the growth
of all species of Brucella. Bacto-Tryptose Broth is prepared according to the
formula of Tryptose Dextrose Vitamin B Broth as given in the "Diagnostic
Procedures and Reagents"^ of the American Public Health Association and is
particularly recommended as a blood enrichment medium, with the addition of
sodium citrate, for isolation of Brucella from febrile and afebrile patients accord-

DEHYDRATED CULTURE MEDIA 103

ing to the method of Huddleson^ and as given in "Diagnostic Procedures and
Reagents."^

In the past, it has been considered necessary to have meat extract or meat
infusions in culture media for the cultivation of bacteria, except possibly for
some of the more easily cultivated strains. It has been shown that many fastidious
pathogenic organisms can be isolated and cultivated in media prepared without
meat extract, infusions of meat or other enrichment if a suitable peptone is em-
ployed. Bacto-Tryptose, in 2 per cent concentration, satisfactorily replaces the
usual infusion-peptone portion of many media. Huddleson^ pointed out that the
probability of isolating Brucella from human blood is hastened and more certain
if the blood be incubated in Bacto-Tryptose Broth. Sodium Citrate in 1 per
cent concentration added to the medium serves as an anticoagulant and assists in
fixing the complement of the blood specimen.

The procedure in detail for the isolation of Brucella from human blood is
given in the discussion of Bacto-Tryptose Agar on page 111. In a personal com-
munication Huddleson recommended that the Tryptose Broth blood culture be
incubated in 10 per cent carbon dioxide, rather than 25 per cent as originally
specified.

The addition of 0.1 per cent of agar to Tryptose Broth is highly recommended,
if the use of this small amount of agar is not objectionable. Diagnostic Pro-
cedures and Reagents^ prefers the use of the Tryptose Broth with the addition
of 0.05-0.1 per cent agar for culturing Brucella from whole blood. Growth of
aerobes and anaerobes in liquid media is greatly increased by the addition
of 0.1 per cent of agar, as was demonstrated by Kitchens^ and by Falk,
Bucca, and Simmons.'^ Borman and West^ stated that the addition of 0.05-0.1 per
cent of agar to the Tryptose Broth was preferable in primary blood culture for
Brucella.

Schuhardt, Rode, Foster and Oglesby,^ by special techniques, demonstrated
that a few of the numerous samples of Bacto-Tryptose which had been in his
laboratory exhibited some toxicity for certain Brucella abortus strains used in his
laboratory. The particular samples of Bacto-Tryptose possessing this character-
istic had absorbed moisture and had undergone chemical change. Schuhardt^ in
a discussion of this observation stated that "the ease of neutralization of this toxic
factor by blood, serum, agar and other substances tends to make the practical
significance of the toxicity relatively minor. We probably would not have en-
countered it had we not been doing extensive tests on the in vitro effect of sul-
fonamides on Brucella using decimal dilution inocula". The high productivity of
Bacto-Tryptose Agar, and Bacto-Tryptose used clinically for the isolation and
cultivation of Brucella attests to its value for the primary cultivation of Brucella
as well as other fastidious organisms.

To rehydrate the medium, dissolve 25 grams of Bacto-Tryptose Broth in 1000
ml. of distilled water. Distribute in tubes, bottles or flasks and sterilize in the
autoclave for 15 minutes at 15 pounds pressure (121°C.). The final reaction of
the medium will be pH 7.2.

For best results Bacto-Tryptose Broth should be freshly prepared. If not used
the same day as sterilized, heat in boiling water or flowing steam to remove
absorbed oxygen and cool quickly without agitation, just prior to inoculation.

One pound of Bacto-Tryptose Broth will make 18.1 liters of medium.

1 Huddleson: Brucellosis in Man and Animals, ^ Diagnostic Procedures and Reagents,
14:1939. 3rd Edition: 246: 1 951.

2 J. Bact., 53:5:1947. e J. Infectious Diseases, 29:390:1921.
^ J. Vet. Research, 11:70:1950. '^ J. Bact., 37:121:1939.

* Diagnostic Procedures and Reagents, ^ J. Bact., 57:1:1949.

3rd Edition: 17: 195 1. ^Personal Communication, 1949.

104 DIFGO MANUAL

BACTO

PEPTONE COLLOID MEDIUM (B61)

DEHYDRATED

Bacto-Tryptose 20 g.

Sodium Chloride 5 g.

Bacto-Agar 1 g.

Bacto-Peptone Colloid Medium, containing 0.1 per cent agar, is a liquid
medium recommended for the cultivation of anaerobes, and also for many
aerobes generally considered difficult to cultivate. This medium was developed
as a basal medium to which carbohydrate and other test materials could be
added for studying the physiological properties of anaerobes and other micro-
organisms.

The preparation of the usual peptone-infusion medium employed for patho-
genic bacteria, is a costly, laborious, time-consuming task, resulting in media
that vary from time to time depending on the age of the meat, condition of
infusion and a number of other factors. In Bacto-Peptone Colloid Medium, a 2
per cent concentration of Bacto-Tryptose satisfactorily replaces the peptone-
infusion portion of infusion media. This medium will support excellent growth
of the anaerobes and is also equally well suited for the propagation of men-
ingococci, streptococci, pneumococci and other microorganisms that are gener-
ally considered difficult to cultivate. For many organisms, especially the an-
aerobes, Bacto-Peptone Colloid Medium will be improved by the addition of a
small quantity of dextrose as a source of readily available energy. An added
0.025 to 0.05 per cent dextrose is sufficient to initiate growth and still not enough
to produce gas or to give rise to appreciable acid production. The above men-
tioned quantities of dextrose are well within the limits of muscle sugar normally
obtained in infusion media. It is not practical to include the indicator in the
medium for fermentation studies with anaerobes, since these organisms reduce
the indicators to their leuco bases. Solutions of indicator should be added to the
culture after incubation to detect changes in hydrogen ion concentration.

The advantages of a medium with a low agar concentration and its influence
on the development of bacteria, particularly the anaerobes, has been described
by Kitchens.^ In a broth, to which 0.1 per cent agar has been added, there is a
clear upper zone well suited for aerobic growth; below this the flocculent agar
develops all degrees of anaerobiosis. This condition makes the medium suitable
for the growth of either aerobic or anaerobic bacteria. Falk, Bucca, and Sim-
mons^ point out the advantages of the use of small quantities of agar (0.06 to
0.25 per cent) in the detection of contaminants in testing the sterility of biologi-
cals. They show that the growth of even common forms, such as the hay bacillus
and staphylococci, ordinarily considered easy to cultivate, were aided by the
addition of a small quantity of agar. They also showed that growth is initiated
in a much shorter incubation period in such media. In their routine tests they
used 0.1 per cent agar which is the amount present in Bacto-Peptone Colloid
Medium.

To rehydrate the medium, suspend 26 grams of Bacto-Peptone Colloid
Medium in 1000 ml. of cold distilled water and heat to boiling to dissolve the
medium completely. Distribute in tubes and sterilize in the autoclave for 15
minutes at 15 pounds pressure (121°C.). The final reaction of the medium will
be pH 7.3.

For best results Bacto-Peptone Colloid Medium should be freshly prepared.
If the medium is not used the same day as sterilized, heat in boiling water or
flowing steam for a few minutes to remove absorbed oxygen, cool quickly with-
out agitation, just prior to inoculation.

DEHYDRATED CULTURE MEDIA 105

One pound of Bacto-Peptone Colloid Medium will make 17.4 liters of medium.

ij. Infectious Diseases, 29:390:1921. ^ J. Bact., 37:121:1939.

BACTO

DUBOS BROTH BASE (B385)

DEHYDRATED

Bacto-Asparagine 2 g.

Bacto-Casitone 0.5 g.

Disodium Phosphate 2.5 g,

Monopotassium Phosphate 1 g.

Ferric Ammonium Citrate 50 mg.

Magnesium Sulfate 10 mg.

Calcium Chloride 0.5 mg.

Zinc Sulfate 0.1 mg.

Copper Sulfate 0.1 mg.

Tween 80 0.2 g.

Bacto-Dubos Broth Base is recommended for the preparation of a liquid
medium for the rapid cultivation of pure cultures of Mycobacterium tuberculosis.
It is prepared in accordance with the formula given by Dubos, Fenner and
Pierce.^ This is a modification of the original medium described by Dubos and
Davis^ and Dubos and Middlebrook.^

Liquid media have been used for the cultivation of tubercle bacilli for many
years. Generally the growth on these media appeared as a surface pellicle. Dubos*
reported rapid and submerged growth of M. tuberculosis in Long's Synthetic
Medium to which was added soya bean phosphatide and a polyoxyalkylene de-
rivative of sorbitan monostearate (Tween 60). Dubos and Davis,^ in a study of
factors influencing the growth of M. tuberculosis described, among other media,
a liquid medium containing Tween 80, an oleic acid ester, and albumin (serum
fraction V). In this liquid medium growth of the tubercle bacillus was obtained
in 3-5 days from dilute inocula. The addition of the albumin fraction V to the
medium facilitated the growth of tubercle bacilli from small inocula but did
not increase markedly the total amount of growth produced. Less pure prepara-
tions of the protein increased the amount of growth as well as initiated growth
from minute inocula. '^-^ The character of the growth obtained on this medium
was different from that obtained on liquid media previously described. Subsur-
face, readily dispersible growth was obtained, and pellicle formation occurred
only in old cultures. This type of growth was particularly valuable in obtaining
an even suspension of tubercle bacilli for use in mouse and guinea pig infection
tests and for other techniques requiring an even suspension of organisms.

Dubos and Davis^ obtained typical diffuse cultures of acid-fast bacilli from
sodium hydroxide treated human sputa, and avian bacilli from animals experi-
mentally infected. Even though the medium was capable of supporting growth of
tubercle bacilli from pathological material, they cautioned against its indiscrim-
inate use in diagnostic work. Foley^-''' reported that a liquid medium similar to
that described by Dubos could be used successfully for the isolation of tubercle
bacilli from various pathologic materials, and that a combination of rapid culture
in this medium and guinea pig confirmation should result in a shorter period of
time required for diagnosis of tuberculosis. Goldie^ used the liquid medium for
the cultivation of tubercle bacilli from sputa treated with ammonium carbonate
and penicillin. Other laboratories,^ after thorough comparative study, reported
that the liquid medium as described by Dubos could not be recommended for
the clinical diagnosis of tuberculosis, confirming the statement of Dubos.

106 DIFGO MANUAL

Media similar to the one described by Dubos have been used by numerous
investigators for various pure culture studies of the tubercle bacillus, as, for
example, the work of Middlebrook^° and others. Such a medium is excellent for
carrying stock cultures of M. tuberculosis, as organisms maintain their viability
without change in phase of virulence for long periods of time. It also offers a
suitable means of running chemotherapeutic screening tests and the testing
of the sensitivity of the tubercle bacillus to antibiotics and other materials.

Wolinsky and Steenken^^ and Bernstein, D'Esapo and Steenken^^ ^sed Dubos
Medium and modifications of this liquid medium in testing the resistance of
tubercle bacilli to streptomycin. Wong, Hambly and Anderson^^ xj^sed a modifi-
cation of the Dubos and Davis medium to demonstrate sensitivity of M. tubercu-
losis to subtilin. Beattie^* compared hydrochloric acid and trisodium phosphate
treatment of sputa, followed by culturing on the fluid medium of Dubos and on
Petragnani medium. Her results indicated no superiority of either method of
treatment of sputa, however, the evidence suggested that the use of trisodium
phosphate may adversely affect the growth of the tubercle bacilli. Both the liquid
and solid medium were equally effective in the cultivation of M. tuberculosis in
this study.

Mollov, Hill and Oshinsky used Dubos^^ medium for the routine isolation and
cultivation of M. tuberculosis. They added 10 units of penicillin per ml. of the
Dubos liquid medium as a selective agent. Types of specimens examined were
sputum, gastric contents, pleural fluid, spinal fluid and urine. The necessity of
making smears to determine the presence of the tubercle bacillus was stressed
and they streaked on Petrick's medium to detect tubercle bacilli and show
typical colony characteristics. These authors also found the Dubos liquid medium
to be helpful in the determination of sensitivity and resistance of the tubercle
bacilli to streptomycin.

Bacto-Dubos Broth Base enriched with Bacto-Dubos Medium Albumin or
Bacto-Dubos Medium Serum will give growth in from 3-5 days from a 10~^ mg
inoculation of M. tuberculosis and in 10-15 days using 10"^ mg inoculum. The
medium prepared with Bacto-Dubos Medium Serum will generally give a more
luxuriant growth and initiate growth from a smaller inoculum than that en-
riched with Bacto-Dubos Medium Albumin. Growth is generally more diffuse
in media prepared with the Albumin enrichment, while with the Serum enrich-
ment the growth is granular. Bacto-Dubos Medium Albumin is a 5.0 per cent
solution of albumin fraction V from bovine plasma in normal saline and contains
7.5 per cent Bacto-Dextrose. Bacto-Dubos Medium Serum is beef serum with 7.5
per cent Bacto-Dextrose. Both solutions are filter sterilized.

The early and luxuriant growth of tubercle bacilli in media prepared with
Dubos Broth Base and Dubos Medium Serum make it ideal for the cultivation
and study of pure cultures of these organisms. It is also recommended for
culturing spinal, pleural and peritoneal fluids likely to harbor the tubercle
bacillus in pure culture. Growth is readily visible. In sensitivity tests where the
presence or absence of growth is the determining factor, the medium prepared
with Bacto-Dubos Medium Serum may be recommended. For tests requiring
turbidimetric determinations for quantitative growth, mouse or guinea pig
inoculation work or other techniques requiring a readily dispersible growth, the
medium should be prepared with Bacto-Dubos Medium Albumin.

The microscopic appearance of tubercle bacilli, when viewed with a 16 mm.
objective, is quite typical on this medium. The organisms appear in irregular
clumps or tangled filaments rather than as individual cells. Edges of these
clumps, examined with the oil immersion lens, show typical acid-fast bacilli. If
it is desired to grow tubercle bacilli from concentrated sputa or other pathologi-
cal material it is difficult to distinguish macroscopically between tubercular

DEHYDRATED CULTURE MEDIA 107

granules and debris, or possible growth from extraneous organisms. It is neces-
sary to determine the presence of tubercle bacilli microscopically. Even though
the medium, inoculated with treated specimens, shows obvious contaminants,
a microscopic examination w^U often show that the tubercle bacilli have multi-
plied even in the presence of the contaminants.

The medium may be prepared with or without glycerol. The addition of
glycerol enhances the growth of human strains of tubercle bacilli, and is not
required by bovine or avian strains. Glycerol may be added to the distilled water
at the time of rehydrating the medium. The use of Bacto-Glycerol is recom-
mended since it has been tested bacteriologically to insure freedom from toxic
principles and for suitability for use in this medium.

To rehydrate the medium dissolve 1.3 grams Bacto-Dubos Broth Base in
180 ml. distilled water or 170 ml. distilled water containing 10 ml. Bacto-
Glycerol. Sterilize in the autoclave for 15 minutes at 15 pounds pressure
(121°C.). Cool to below 50°C. and add the contents of one tube Bacto-Dubos
Medium Albumin (20 ml.) or Bacto-Dubos Medium Serum (20 ml.) under
aseptic conditions. Mix thoroughly and distribute in 16-20 mm. diameter sterile
test tubes in 5-7 ml. amounts. Incubate for 24 hours to test sterility. The medium
is then ready for inoculation.

One pound of Bacto-Dubos Broth Base will make 69.8 liters of medium.

1 Am. Rev. Tuberculosis, 61:66:1950. » Personal Communications. 1947-48.

2 J. Expl. Med., 83:409:1946. loProc. N.Y. State Assoc. Pub. Health Lab.,
8 Am. Rev. Tuberculosis, 56:334:1947. 27:28:1947.

* Proc. Soc. Exp. Biol. Med., 58:361:1945. 11 Am. Rev. Tuberculosis, 55:281:1947.

sj. Exp. Med., 85:9:1947. "Am. Rev. Tuberculosis, 58:344:1948.

•Proc. Soc. Exp. Biol. Med., 62:298:1946. ^^ j_ l^Jj^ Clin. Med., 32:837:1947.

'J. Lab. Clin. Med., 32:842:1947. "J. Lab. Clin.. Med., 34:733:1949.

8 Proc. Soc. Exp. Biol. Med., 65:210:1947. ^^ Am. J. Clin. Path., 20:1085:1950.

BACTO

DUBOS BROTH BASE (B435)
without Tween 80

DEHYDRATED

Bacto-Dubos Broth Base without Tween 80 has the same formula as Bacto-
Dubos Broth Base except Tween 80 has been omitted. In certain tests to deter-
mine the sensitivity of tubercle bacilli to various antibiotics or other bactericidal
agents it may be desirable to omit the Tween 80. There is a possibility that this
material present in the medium may influence the sensitivity of the tubercle
bacilli to the test substance.

BACTO

DUBOS OLEIC AGAR BASE (B373)

DEHYDRATED

Bacto-Gasitone 0.5 g,

Bacto-Asparagine 1 g.

Disodium Phosphate 2.5 g.

Monopotassium Phosphate 1 g.

Ferric Ammonium Citrate 50 mg.

Magnesium Sulfate 10 mg.

Calcium Chloride 0.5 mg.

Zinc Sulfate 0.1 mg.

Copper Sulfate 0.1 mg.

Bacto-Agar 15 g.

108 DIFCO MANUAL

Bacto-Dubos Oleic Agar Base is recommended for the preparation of a solid
medium for the isolation of Mycobacterium tuberculosis. It is prepared accord-
ing to the formula described by Dubos and Middlebrook.^ Bacto-Dubos Oleic
Agar Base enriched with Bacto-Dubos Oleic Albumin Complex may also be
used as a basal medium on which to test the sensitivity of M. tuberculosis to
chemotherapeutic agents.

Dubos and Middlebrook in a discussion of media for the cultivation of tubercle
bacilli described an agar medium suitable for diagnostic primary isolations and
also for studies of colony morphology. This medium is prepared without glycerol
or dextrose in order to discourage the growth of commensal organisms that
might not have been killed during the concentration process. Asparagine like-
wise is added in minimal quantities to provide maximum selectivity. The basal
agar is enriched with Bacto-Dubos Oleic Albumin Complex, (page 275). The
sterile base is cooled to 50-55 °C., and oleic acid albumin complex and penicillin
as selective agent are added. The medium is mixed and distributed in sterile
tubes or plates as desired. About 15 ml. of medium is used per 95 mm. dish
and following solidification two drops of an adequately diluted suspension of the
tubercle bacilli or of pathological material are used as an inoculum. Roberts,
Wallace and Erlich^ in a study of methods of isolation of the tubercle bacilli re-
ported that Dubos Oleic Albumin Agar Medium gave better results than other
media, even superior to guinea pig inoculations. Byham,^ comparing different
media for primary isolation work, reported best results with Middlebrook's
medium.* Commensal organism contamination was reduced by the penicillin and
early growth of the tubercle bacilli was obtained. Foley,^ in a limited survey,
too small to permit analysis reported that four strains grew on the solid Dubos
medium, but failed to develop in the liquid Dubos medium. He pointed out that
colonial morphology is strikingly different on the Dubos Agar than on coagu-
lated egg media.

To rehydrate the medium suspend 4.2 grams Bacto-Dubos Oleic Agar Base
in 180 ml. of cold distilled water. Heat to boiling to dissolve the medium com-
pletely. Sterilize in the autoclave for 15 minutes at 15 pounds pressure (121°C.).
Cool to 50-55 °C. and add the contents of one tube of Bacto-Dubos Oleic
Albumin Complex (20 ml.) and 5,000 to 10,000 units of penicillin (25 to 50 units
per ml.), under aseptic conditions. Mix thoroughly and distribute in sterile tubes
or plates as desired. Keep uninoculated medium in the refrigerator.

One pound of Bacto-Dubos Oleic Agar Base will make 21.6 liters of medium.

2 Am. Rev. Tuberculosis, 61:563:1950. 27:28:1947.

3 Am. J. Clin. Path., 20:678:1950. ^ J. Lab. Clin. Med., 32:842:1947.

^ Am. Rev. Tuberculosis, 56:334:1942. * Proc. N.Y, State Assoc, of Pub. Health Lab.,

61:563:1950.
1950.

BACTO

TB BROTH BASE (B291)

DEHYDRATED

Bacto-Yeast Extract 2 g.

Proteose Peptone No. 3, Difco 2 g.

Bacto-Casitone 2 g.

Disodium Phosphate 2.5 g.

Monopotassium Phosphate 1 g.

Sodium Citrate 1.5 g.

Magnesium Sulfate 0.6 g.

Tween 80 0.5 g.

Bacto-TB Broth Base is a modification of the medium described by Dubos
and Davis^ and is recommended for the preparation of a liquid medium for

DEHYDRATED CULTURE MEDIA 109

the rapid cultivation of Mycobacterium tuberculosis. This modification was pre-
pared since we considered it to give earlier and more rapid growth than the
original Dubos formula, as well as the initiation of growth from inocula contain-
ing fewer organisms. This base, when enriched with Bacto-Dubos Medium
Serum, will support luxuriant growth of tubercle bacilli from dilute inocula
after a short incubation period (within 7-10 days). M. tuberculosis produces
a granular growth, settling to the bottom of the tube in this medium. When the
base is enriched with Bacto-Dubos Medium Albumin a readily dispersible or
diffuse growth is obtained.

A discussion of liquid media for the cultivation of the tubercle bacilli is given
in the description of the medium, Bacto-Dubos Broth Base, (page 105). Media
prepared from Bacto-TB Broth Base are recommended for the cultivation and
propagation of pure cultures of AI. tuberculosis, and for culturing pathological
specimens such as spinal, pleural and peritoneal fluids likely to contain the
tubercle bacillus in pure culture. It is also employed for determining the sensi-
tivity of M. tuberculosis to antibiotics and other chemotherapeutic agents. Gen-
erally, solid media have proven more satisfactory for initial propagation of the
organism from specimens.

The medium may be prepared with or without glycerol. The addition of
glycerol enhances the growth of human strains of tubercle bacilli and is not re-
quired by bovine or avian strains. Glycerol may be added to the distilled water
at the time of rehydrating the medium. The use of Bacto-Glycerol is recom-
mended since it has been tested bacteriologically to insure freedom from toxic
principles and for suitability for use in this medium.

To rehydrate the medium, dissolve 2.4 grams Bacto-TB Broth Base in 180 ml.
of distilled water or 170 ml. of distilled water containing 10 ml. Bacto-Glycerol.
Sterilize in the autoclave for 15 minutes at 15 pounds pressure (121°C.). Cool to
below 50° C. and add the contents of one tube Bacto-Dubos Medium Serum
(20 ml.) or one tube Bacto-Dubos Medium Albumin (20 ml.) under aseptic
conditions. The medium is mixed thoroughly and distributed under aseptic
conditions in 16-20 mm. diameter test tubes in 5-7 ml. amounts. The medium is
then ready for inoculation. Final reaction of the base and medium prepared as
above will be pH 7.0.

One pound of Bacto-TB Broth Base will make 37.8 liters of medium.

ij. Exp. Med., 83:409:1946.

BACTO

TB BROTH BASE (B374)
without Tween 80

DEHYDRATED

Bacto-TB Broth Base without Tween 80 has the same formula as Bacto-
TB Broth Base except Tween 80 has been omitted. In certain tests to deter-
mine the sensitivity of tubercle bacilli to various antibiotics or other bacterio-
cidal agents, it may be desirable to omit the Tween 80. There is a possibility that
this material present in the medium may influence the sensitivity of the tubercle
bacilli to the test substance.

110 DIFCO MANUAL

BACTO

PEIZER TB MEDIUM BASE (B400)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Gasamino Acids 10 g.

Bacto-Asparagine 3 g.

Potato Starch 15 g.

Ferric Ammonium Citrate 0.1 g.

Magnesium Sulfate 0.015 g.

Dipotassium Phosphate 3.5 g.

Citric Acid 0.1 g.

Bacto-Agar 15 g.

Bacto-Peizer TB Medium Base with Bacto-Peizer TB Medium Enrichment is
recommended for the isolation and cultivation of Mycobacterium tuberculosis,
and for the determination of sensitivity of this organism to therapeutic agents.
This base and enrichment are prepared according to the formulae described by
Peizer and Schecter.^

Media prepared with coagulated egg or egg yolk have generally been con-
sidered most satisfactory for the isolation and cultivation of the tubercle bacilli.
Peizer and Schecter^ described an agar medium enriched with egg yolk, which
gave comparable results with coagulated egg yolk media for initial cultivation of
the tubercle bacilli from specimens. The medium also gave an early and luxuri-
ant growth with satisfactory inhibition of commensal organisms. The medium
consists of an agar base to which is added an enrichment composed of egg yolk,
glycerol, and dextrose and with malachite green as a selective agent. The basal
medium has been prepared in dehydrated form and the enrichment supplied
ready for addition to the sterile rehydrated agar base.

The ease of preparation of the complete medium from Bacto-Peizer TB
Medium Base and Bacto-Peizer TB Medium Enrichment in amounts as re-
quired, its high nutritional value and its selectivity make it ideally suited for
the isolation of the tubercle bacilli from clinical specimens. In addition, the
medium may be used for determining the sensitivity of the tubercle bacillus to
therapeutic agents. When used for sensitivity testing, the complete medium is
poured either into sterile petri dishes or wide-mouth bottles and allowed to
solidify. The medium is heavily inoculated by smearing a suspension of the organ-
ism over the surface to produce a mass growth. Treated specimens showing a
Gaffky of III or more may also be smeared in a similar manner for testing the
sensitivity of the organisms from primary culture. Bacto-Sensitivity Disks contain-
ing 1, 10 and 100 meg. dihydrostreptomycin are then placed on the inoculated
medium. The plates or bottles are sealed and incubated. Observations are made
for growth of the organism and size of the zone of inhibition of growth around
the disks. See Bacto-Sensitivity Disks (p. 335) for complete discussion of this
technique.

Peizer, Widelock and Schecter^ used this basal medium in testing the sensi-
tivity of the tubercle bacillus to streptomycin. In their method the streptomycin
was included in the enrichment.

To rehydrate the medium suspend 49.8 grams Bacto-Peizer TB Medium Base
in 1000 ml. cold distilled water. Heat to boiling to dissolve the medium com-
pletely. Distribute in 100 ml. amounts and sterilize in the autoclave for 15 min-
utes at 15 pounds pressure (121°G.). Cool to 45-50° C. and add the contents of
one bottle of Bacto-Peizer TB Medium Enrichment to 100 ml. of the sterile
medium. Mix well, avoiding the formation of bubbles. Distribute in sterile tubes

DEHYDRATED CULTURE MEDIA 111

or plates as desired, under aseptic conditions. Tubes are slanted in the desired
position and medium allowed to solidify.

One pound of Bacto-Peizer TB Medium Base will make 11.8 liters of medium.

^Am. J. Clin. Path., 20:682:1950. 2 Am. J. Clin. Path., 21:982:1951.

BACTO

TRYPTOSE AGAR (B64)

DEHYDRATED

Bacto-Tryptose 20 g.

Bacto-Dextrose 1 g.

Sodium Chloride 5 g.

Bacto-Agar 15 g.

Thiamine Hydrochloride 0.005 g.

Bacto-Tryptose Agar is recommended for the isolation, cultivation and differ-
entiation of the Brucella. "Standard Methods for the Examination of Dairy
Products"^ and "Diagnostic Procedures and Reagents"^ of the American Public
Health Association, recommend this medium for this purpose. Bacto-Tryptose
Agar is also recommended as a general medium for the cultivation of a large
variety of pathogenic organisms, especially the streptococci.

Isolation or cultivation of organisms of the Brucella group was originally ac-
complished by means of Liver Infusion Agar as recommended by Stafseth,^ and
Huddleson, Hasley and Torrey.* Completely satisfactory results were not con-
sistently obtained on this medium due to variations of the infusions from liver
tissue. Because of these irregular results on Liver Infusion Agar, an extensive
investigation of media for the propagation of Brucella was undertaken in our
laboratories in cooperation with Dr. Huddleson. This investigation led to the
development of a new medium^ which is excellently adapted to the nutritional
requirements of the Brucella, and which is satisfactory for both primary isolation
and routine propagation of these organisms.^ This medium is prepared with
Bacto-Tryptose as the sole source of nitrogen. The suitability of this peptone for
cultivation of the Brucella is demonstrated by the fact that, in a 2 per cent
solution of Bacto-Tryptose containing 0.5 per cent sodium chloride and 0.1 per
cent Bacto-Dextrose, growth is obtained from small inocula in 24 hours, w^hile
a much larger inoculum and an incubation period of 7-10 days are required to
obtain similar growth in Liver Broth.

For isolation of Brucella strains from infected milk, where Gram-positive con-
taminants are present, crystal violet (gentian violet) is used in a final concen-
tration of 1 to 700,000 in Tryptose Agar. This amount of dye is sufficient to
suppress practically all Gram-positive organisms which might otherwise inter-
fere. To secure the proper concentration, 1.4 ml. of a one-tenth per cent solution
of Bacto-Crystal Violet should be added to each liter of Tryptose Agar before
sterilization of the medium. The sterile medium is poured into sterile petri
dishes, solidified and then inoculated. Each plate receives 0.1-0.2 ml. gravity
cream spread upon the surface of the medium by means of a sterile glass rod
with a right angle bend. The spreading of the inoculum is readily accomplished
by rotating the plate in a horizontal plane.

The inoculated plates are incubated for 5 days at 37 °C. in an atmosphere of
10 per cent carbon dioxide. At the end of this period Brucella generally appear
as purplish, smooth, hemispherical colonies about 1-5 mm. in diameter, and are
transparent to transmitted light. Occasionally the colonies may be flat instead of

112 DIFGO MANUAL

convex. Streptococci, which are the usual interfering contaminants, form large,
opaque, spreading, rough colonies slightly purple in color and not at all to be
confused with those of the Brucella.

If the original plate contains colonies of other bacteria or molds, typical
Brucella colonies should be purified by transfer to Tryptose Agar slants or to
another plate of the crystal violet medium. The organisms should be properly
identified and the species determined. If aerobic types of Brucella such as suis
or melitensis, are present in the milk, their growth will not be inhibited by in-
cubating the inoculated plates in an atmosphere of 10 per cent carbon dioxide.

Bacto-Tryptose and Bacto-Tryptose Agar are also particularly well suited to
the isolation of Brucella from the blood. During 1937, Huddleson^ had an oppor-
tunity to study the use of Bacto-Tryptose in an enrichment medium, and Bacto-
Tryptose Agar in the isolation of Brucella melitensis from 55 cases of undulant
fever on the Island of Malta. Of the total number of cases, 38 were febrile and
17 were afebrile at the time the blood was cultured. Positive cultures were ob-
tained in 32 cases of the former group and 5 of the latter. Growth appeared
within 4 days in 23 of the cultures. One culture required 18 days of incubation
before a positive subculture was obtained.

Briefly, the procedure recommended by Huddleson^ is as follows: An enrich-
ment broth, composed of 2 per cent Bacto-Tryptose, 0.5 per cent sodium chlo-
ride and 1 per cent sodium citrate, or Bacto-Tryptose Broth, page 102, with 1
per cent sodium citrate added, is prepared and distributed in 20 ml. amounts
in 50 ml. serum vials closed with rubber diaphragm stoppers. The vials are then
autoclaved for 15 minutes at 15 pounds pressure (121°C.). The air in the vial
is replaced with carbon dioxide by puncturing the diaphragm with a 23-gauge
needle, removing the air and replacing it with carbon dioxide before introducing
the blood. In a personal communication Huddleson recommended the use of
10 per cent carbon dioxide rather than the 25 per cent originally specified.
Immediately after collection from the patient, the medium is inoculated with
2 to 5 ml. of blood by puncturing the stopper wdth the same needle used in col-
lecting the blood sample. The vial is shaken vigorously to prevent clotting.

The vials are then incubated at 37 °G. At the end of every fourth day the
culture is mixed by shaking, and 0.5 ml. of the contents is removed by means
of a sterile 1 ml. syringe and needle and inoculated on a petri plate of Tryptose
Agar. The plate is incubated under 10 per cent carbon dioxide for 4 days. If no
growth is obtained from the blood culture within 20 days, it may be discarded.

A convenient method for establishing an atmosphere of 10 per cent carbon
dioxide is that described by Thompson'^ in which a solution of sodium bicar-
bonate is mixed with sulfuric acid directly in the container. When a molar solu-
tion (84 grams per liter) of sodium bicarbonate is mixed in equal parts with
dilute sulfuric acid (1 ml. concentrated acid in 29 ml. distilled water), 22.4 ml.
carbon dioxide are liberated for each milliliter of bicarbonate solution. Calcula-
tion of the cubic contents of the container in which the cultures are incubated
will indicate the quantity of each solution required to create a carbon dioxide
concentration of approximately 10 per cent. For example, for a container having
a net volume of 1000 ml., one would use 4.5 ml. of each reagent. The solutions
are preferably introduced separately into the container and are mixed after it
has been sealed. A similar procedure is also described in detail by Shaughnessy.^

A satisfactory carbon dioxide tension can also be supplied by one of the fol-
lowing procedures:

1. Replace about 10 per cent of the air in the container with the gas from a
tank of liquid carbon dioxide.

2. Place a lighted smokeless candle near the top of the container with the
plates, and seal the container.

DEHYDRATED CULTURE MEDIA 113

Another method for isolation of Brucella by blood culture is the double
medium method as described by Castaneda.^ This method may also be used in
culturing other organisms from the blood. His method consisted of preparing a
medium containing 2 per cent Bacto-Tryptose, 0.5 per cent sodium chloride,
0.5 per cent sodium citrate and 3.0 per cent agar. This medium is sterilized in
15 ml. amounts in 100 ml. flat-sided rectangular bottles. The bottles are placed
on their side so that the agar medium solidifies on one of the narrow side walls
forming an even, transparent layer. To each bottle are then added under aseptic
conditions 10 ml. of sterile broth containing 2 per cent Bacto-Tryptose and 2
per cent sodium citrate. The air in the bottles is then replaced by the desired
mixtures of carbon dioxide and air by a suitable mechanical device. The double
medium is incubated at 35-3 7 °G. for 3 or 4 days to test sterility, during which
time the surface of the agar is wetted with the broth by tilting the bottle at 24
hour intervals. The sterile double medium is then inoculated with 10 ml. of the
patient's blood and the mixture of broth and blood washed over the surface of
the agar. Incubation is at 36°G. with the bottle in an upright position. The
medium is examined at daily intervals, and every other day the blood-broth
mixture is allowed to flow over the agar layer. Castaneda reported that if col-
onies developed in the agar layer in 24 to 48 hours it was likely that the culture
had been contaminated. When colonies appear 24 to 48 hours after the second
inoculation the cultivated organism was usually found to be a Salmonella, less
frequently a staphylococcus or a streptococcus. It may be a Brucella; however,
colonies of these organisms are more generally encountered after the sixth day
of incubation, that is after the third inoculation of the Tryptose Agar with the
blood-broth mixture. The culture is discarded as negative after 20 days incuba-
tion. There are many modifications of this method in routine practical use. The
quantity of media, size of bottle, amount of inoculum varying in the various
laboratories. Marvin^^ also described a blood culture bottle utilizing a Tryptose
Agar and a Tryptose Phosphate Broth combination as being a practical method
for blood culture work.

Huddleson^^'^2 h^g established the differentiation of Brucella types by their
behavior in the presence of certain bacteriostatic dyes. Bacto-Tryptose Agar is
employed effectively as a base for the thionin and basic fuchsin media used by
Huddleson, but the dye content of these media must be less than that employed
for Liver Infusion Agar. Bacto-Thionin is employed in 1:100,000 dilution (1 ml.
1 per cent solution of Bacto-Thionin per liter), and basic fuchsin in 1:100,000
dilution (1 ml. 1 per cent solution of Bacto-Basic Fuchsin). The plates should
be inoculated within 24 hours after pouring, as the dyes become reduced in the
medium on standing. The bacteriostatic action of the dyes in these concentra-
tions in Tryptose Agar is in every way comparable with that previously de-
scribed by Huddleson. B. melitensis and B. suis will grow on Tryptose Agar
containing thionin, while B. abortus is inhibited; B. melitensis and B. abortus
develop on Tryptose Agar containing basic fuchsin and B. suis is inhibited.

For the differentiation of the Brucella types on the basis of hydrogen sulfide
production it is recommended that Bacto-Tryptose Agar be dissolved in an
infusion prepared from Bacto-Liver (page 289). Differentiation of the three
Brucella species by means of their hydrogen sulfide production is not clearly de-
fined when distilled water alone is used in preparing the medium.

When voluminous growth of organisms is desired, as in the preparation of
Brucella antigens, it is recommended that a seed culture be prepared first by
propagating the organisms in Bacto-Tryptose Broth. An incubation period of
24 hours at 35-3 7 °G. is generally sufficient to produce a heavy growth. The
seed culture is then spread upon the surface of the medium. A medium pre-
pared by dissolving Bacto-Tryptose Agar in an infusion of Bacto-Liver (page

114 DIFGO MANUAL

289) will yield a somewhat heavier growth on prolonged incubation than will
a medium prepared with distilled water.

Schubert^^ reported that the rapidity and amount of growth of some recently
isolated fastidious strains of B. abortus was markedly improved on Bacto-
Tryptose Agar in the presence of a specially prepared liver extract demonstrat-
ing strong catalase activity.

Bacto-Tryptose Agar is also recommended as a general solid medium for the
cultivation of pathogenic organisms, being an excellent medium, without enrich-
ment, for streptococci, pneumococci, meningococci and others. Blood Agar may
be prepared by adding 5 per cent sterile defibrinated blood to melted sterile
Tryptose Agar at 50° C. Bacto-Tryptose Agar contains 0.1 per cent dextrose,
probably slightly more than is present in the average meat infusion medium in
the form of muscle sugar. Ruediger,^* Brown^^ and Fuller and Maxted^^ have
all demonstrated that the presence of dextrose, or a reducing sugar, inhibits
hemolysin production by streptococci, giving rise to false reactions. For that
reason hemolytic reactions may be atypical on Bacto-Tryptose Agar and should
be confirmed on Blood Agar prepared from Bacto-Tryptose Blood Agar Base,
Bacto-Blood Agar Base, Bacto-Heart Infusion Agar or on a medium prepared
with 2.0 per cent Bacto-Tryptose, 0.5 per cent sodium chloride and 1.5 per cent
Bacto-Agar (Tryptose Agar without dextrose).

Chapman, Stiles, and Berens^^ in their study of the isolation and "in vitro"
testing of pathogenic types of non-exotoxic streptococci, used Bacto-Tryptose
Agar as a base for Blood Agar because it gave more luxuriant growth of strep-
tococci than other base media. Cultures of Pasteurella multocida were cultivated
on Tryptose Agar by Carter.^^ He reported that it was not necessary to freeze
dry cultures for storage when grown on Tryptose Agar. By adding 5 per cent
saccharose to the medium he was able to identify readily blue, intermediate and
fluorescent colonies of the organism. Silverman and Elberg^^ in their study of
Brucella antigens used Tryptose Agar for the cultivation of their strains of
B. abortus, B. suis and B. melitensis. Boyd and Casman^o reported that Tryptose
Agar filtered through absorbent cotton became toxic for a fastidious strain of
B. abortus. The toxic factors extracted from the cotton were characterized as
fatty acids. This toxicity could be nullified by the simple addition of 0.03-0.1
per cent corn starch to the medium, bearing a similarity to the report of Ley
and Mueller^i demonstrating the ability of starch to neutralize toxic factors
found in samples of some agar using the gonococcus as a test organism.

Gray, Stafseth, Thorp, Sholl and Riley ^^ described a new technique for the
isolation of Listeria monocytogenes from infected brain by grinding the medulla
in a mortar with about 10 ml. of Tryptose Broth and then emulsifying with
glass beads in a shaking machine for about 20 minutes. About 0.3 ml. of the
suspension is then plated on Bacto-Tryptose Agar and incubated at 37 °G. for
24 hours. The colonies of Listeria are light green with a finely textured surface
when viewed with a dissecting microscope and sufficiently characteristic that
they can be identified even in cases of extreme contamination. Storage of the
brain suspension in the refrigerator for 24 hours seemed to increase the number
of Listeria developing on the medium. Gray, Stafseth and Thorp-^ added po-
tassium tellurite in 0.1 to 0.05 per cent concentration to Bacto-Tryptose Agar
as a selective medium for the isolation of Listeria. The Listeria colonies were
black, as are other organisms developing on the medium but showed the char-
acteristic green color at the periphery of the colony when viewed by reflected
light with a dissecting microscope. Gray, Stafseth and Thorp^* showed the value
of this method for isolation of L. monocytogenes from 36 sheep and 31 cattle
over a four year period.

Schuhardt, Rode, Foster and Oglesby,-^ by special techniques, demonstrated

DEHYDRATED CULTURE MEDIA

115

that a few of the numerous samples of Bacto-Tryptose which had been in his
laboratory exhibited some toxicity for certain Brucella abortus strains used in his
laboratory. The particular samples of Bacto-Tryptose possessing this character-
istic had absorbed moisture and had undergone chemical change. Schuhardt^^
in a discussion of this observation stated that "the ease of neutralization of this
toxic factor by blood, serum, agar and other substances tends to make the prac-
tical significance of the toxicity relatively minor. We probably would not have
encountered it had we not been doing extensive tests on the in vitro effect of
sulfonamides on Brucella using decimal dilution inocula". The high productivity
of Bacto-Tryptose Agar and Bacto-Tryptose used clinically for the isolation and
cultivation of Brucella attests to its value for the primary cultivation of Brucella
as well as other fastidious organisms.

To rehydrate the medium, suspend 41 grams of Bacto-Tryptose Agar in 1000
ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°G.). The final reaction of the medium will be
pH7.2.

Since most microorganisms prefer a fresh medium with a moist surface, it is
recommended that Bacto-l'ryptose Agar be prepared as required, or melted and
re-solidified just prior to use.

One pound of Bacto-Tryptose Agar will make 1 1 liters of medium.

1 Standard Methods for the Examination
of Dairy Products, 9th Edition: 149: 1948.

2 Diagnostic Procedures and Reagents,
3rd Edition: 246: 1950.

3 Tech. Bull., 49 Mich. Exp. Sta., 1920.
* J. Infectious Diseases, 40:353:1937.

s Huddleson : Brucellosis in Man and Animals,

«J. Am. Med. Assoc, 109:1971:1937.

'Am. J. Clin. Path., 5:313:1935-

«J. Bact., 37:153:1939. , ^

eProc. Soc. Expl. Biol. Med., 64:114:1947-
"^Am. J. Clin. Path., 19:697:1949-
11 Tech. Bull. 100, Mich. Exp. Sta., 1929.
"Am. J. Pub. Health, 21:491:1931-
"Am. J. Clin. Path., 81:894:1951.

J. Infectious Diseases,
Monograph No.

3:633:1906.
Rockefeller Inst. Med.
Research, 1919.
"J. Path. Bact., 49:83:1939-
1'' Am. J. Clin. Path., 9:1939, Tech., Suppl.

3:20:1939.
18 Am. J. Vet. Res., 11:252:1950.
^ J. Immunol., 65:163:1950.

20 Public Health Reports, 66:44:1951.

21 J. Bact., 52:453:1946.

22 J. Bact.. 55:471:1948-

23 J. Bact., 59:443:1950.

2* J. Am. Vet. Med. Assoc, 118:242:1951.

25 J. Bact., 57:1:1949-

28 Personal Communication, 1949-

BACTO

TRYPTOSE BLOOD AGAR BASE (B232)

DEHYDRATED

Bacto-Tryptose 10 g.

Bacto-Beef Extract 3 g.

Sodium Chloride 5 g.

Bacto-Agar 15 g.

Bacto-Tryptose Blood Agar Base is a nutritious, infusion-free peptone medium
designed especially for use as a base in the preparation of Blood Agar. Blood
Agar prepared with this base supports excellent growth of many fastidious
organisms and also gives typical clear-cut hemolytic reactions. Bacto-Tryptose
Blood Agar Base, without added blood, may be recommended as a Nutrient
Agar for general routine culture purposes.

Investigations of the nutritive properties of Bacto-Tryptose demonstrated
that culture media prepared with this peptone were superior to the meat in-
fusion peptone media previously used for the cultivation of Brucella, strepto-
cocci, pneumococci, meningococci and other fastidious pathogenic bacteria. The
use of Bacto-Tryptose in place of infusion in Blood Agar Base yields a medium

116 DIFCO MANUAL

of uniform composition and provides a substrate which maintains the blood cells
in an excellent state of preservation, thus insuring typical hemolytic reactions.
Casmani'2 reported that a medium consisting of 2 per cent Bacto-Tryptose, 0.3
per cent Bacto-Beef Extract, 0.5 per cent sodium chloride, 1.5 per cent Bacto-
Agar and 0.03 per cent dextrose equalled fresh beef infusion base with respect
to growth of organisms. The small amount of carbohydrate, however, interfered
with hemolytic reactions unless the medium was incubated in an atmosphere of
carbon dioxide.

To rehydrate the medium, suspend 33 grams of Bacto-Tryptose Blood Agar
Base in 1000 ml. cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in tubes or flasks and sterilize in the autoclave for 15
minutes at 15 pounds pressure (121°G.). The final reaction of the sterilized
medium, before adding blood, will be pH 7.2.

If Blood Agar is to be prepared immediately, the sterile agar base is cooled
at once to 45-50° C. and while still liquid, 5 per cent sterile defibrinated blood
is added aseptically. Mix thoroughly, avoiding incorporation of air bubbles, and
dispense into sterile petri dishes or sterile tubes as desired. Between 12 and 15
ml. of Blood Agar per 100 mm. petri dish is satisfactory. Blood Agar should be
incubated to insure sterility before use.

One pound of Bacto-Tryptose Blood Agar Base will make 13,8 liters of fin-
ished basal medium or 14.5 liters of Blood Agar.

ij. Bact., 43:33:1942. ''Am. J. Clin. Path., 17:281:1947.

BACTO

PROTEOSE NO. 3 AGAR (B65)

DEHYDRATED

Proteose Peptone No. 3, Difco 20 g.

Bacto-Dextrose 0.5 g.

Sodium Chloride 5 g.

Disodium Phosphate 5 g.

Bacto-Agar 15 g.

Bacto-Proteose No. 3 Agar, enriched with Bacto-Hemoglobin and Bacto-
Supplement A or Bacto-Supplement B, is recommended for the cultural isola-
tion of Neisseria gonorrhoeae from chronic and acute cases of gonorrhoea in the
male and female and in other gonococcal infections. The medium permits excel-
lent growth of the gonococcus without overgrowth by contaminating organisms.
In a survey,^ under carefully controlled conditions, twelve media recommended
for the isolation of the gonococcus were compared. Bacto-Proteose No. 3 Agar
enriched with Bacto-Hemoglobin and Bacto-Supplement A compared very
favorably with other decidedly more complex media. A discussion of a 24 hour
medium for the cultural diagnosis of gonorrhoea is given under Bacto-G G
Medium Base on page 122. In the survey^ this 24 hour incubation medium gave
but slightly better results than Bacto-Proteose No. 3 Agar.

The diagnosis and control of gonorrhoea have been greatly facilitated by im-
proved laboratory methods for detecting, isolating and studying A^. gonorrhoeae.
Foremost among these developments is the cultural method for detecting the
presence of the gonococcus in exudates and body fluids. The greater efficiency
of this procedure over the microscopic technique has established its indispensa-
bility in the routine diagnosis of gonococcal infections. The plating procedure
is not only more sensitive in indicating the presence of the gonococcus, but also
permits isolation of the organism for further study. Improved media developed
for identification of the isolated organisms simplify the procedure making it

DEHYDRATED CULTURE MEDIA 117

practical for use in all laboratories. Furthermore, cultural methods have been
designed to check the response of the gonococcus to chemotherapeutic agents,
thereby enabling the clinician to select more effectively the materials used in
treating gonococcal infections.

Interest in the cultural procedure for the diagnosis of gonococcal infections
was stimulated by Ruys and Jens,^ McLeod and co-workers,^ Thompson,* Leahy
and Carpenter,^ Carpenter, Leahy and Wilson^ and Carpenter,''^ who clearly
demonstrated the superiority of this method over the microscopic technique. The
procedure introduced by McLeod and associates with slight modifications offered
itself most advantageously to practical usage for the detection and isolation of
A^. gonorrhoeae.

Lack of a simple, readily available and easily prepared culture medium re-
tarded immediate and widespread acceptance of this new technique. In coopera-
tion with Dr. Carpenter, and availing ourselves of suggestions received from Drs.
McLeod and Herrold, we devoted considerable effort to the development of a
culture medium suitable for isolation of the gonococcus from urethral and
cervical exudates. The development of this medium was premised upon the
stipulations that it must possess a high degree of efficiency, readily lend itself to
practical use, be of relatively simple composition and that it be duplicable with
ease.

During our experimental work several media were developed which supported
luxuriant growth of the gonococcus in pure culture, but which were unsatisfac-
tory with mixed cultures, since other organisms outgrew the gonococcus. In
diagnostic work the medium must support the growth of the gonococcus not
only when in pure culture, but also permit its development from the mixed
flora encountered in chronic gonococcal infections.

A Chocolate Agar, prepared with Bacto-Proteose No. 3 Agar and Bacto-
Hemoglobin, was developed and proved to be satisfactory for the isolation of
the organism from all types of gonococcal infections. Nearly 3.5 times as many
positives were secured with the cultural method as were obtained by the usual
smear technique. In a number of instances the gonococcus was isolated from
treated female cases which had shown five to seven consecutive negative smears.

Since its introduction in 1938 Bacto-Proteose No. 3 Agar, enriched with
Bacto-Hemoglobin, has become the most generally accepted medium employed
in the laboratory diagnosis of gonorrhoeal infections by the culture method. Car-
penter^ reported that this Difco dehydrated medium was as good as or superior
to the Douglas Digest Agar described by McLeod. Pelouze^ suggested the use
of Bacto-Proteose No. 3 Agar and Bacto-Hemoglobin for the cultural detection
of the gonococcus. Sulkin and Gottlieb^^^ used Bacto-Proteose No. 3 Agar and
Bacto-Hemoglobin with success, and emphasized the simplicity of the medium
and the ease with which it could be prepared. Sewell, Clarke and Nelson^^ re-
ported their findings on 4500 cultures plated upon Bacto-Proteose No. 3 Agar
and Bacto-Hemoglobin and claimed excellent results. Sewell, Salchow, and
Nelson^2 obtained excellent results with urine sediment and urethral secretions
plated upon Bacto-Proteose No. 3 Agar enriched with Bacto-Hemoglobin. Car-
penter^^ suggested the use of the Bacto-Proteose No. 3 Agar and Bacto-Hemo-
globin for isolation of the gonococcus.

In an extended effort to increase the growth rate of the gonococcus by enrich-
ing the Chocolate Agar with plant and animal fluids and thereby shortening the
incubation period, some gonococcal strains were encountered that grew well on
such enriched Chocolate Agar and only sparsely or not at all on the unenriched
medium. Concurrently with this finding Lankford^* reported similar results and
suggested enriching the Chocolate Agar with fresh liver extract. Lankford, Scott,
Cox and Cooke^^ extended their studies on the nutritional requirements of the

118 DIFGO MANUAL

gonococcus and obtained 12 per cent more positive isolations on the Chocolate
Agar enriched with liver extract or yeast extract, than upon the unenriched
medium. Following this work, Lankford and Snell^^ identified the required
growth factor as glutamine. Lankford^^ called our attention to the fact that a
second growth factor, cocarboxylase, was required by a small percentage of
gonococcal strains and that cystine helped under certain conditions. With this
information, an extensive study to provide a satisfactory enrichment for supple-
menting the Chocolate Agar was undertaken, and resulted in the development
of Bacto-Supplement A and Bacto-Supplement B, as discussed on page 276.

Nelson,i8 using the Bacto-Proteose No. 3 Agar and Bacto-Hemoglobin en-
riched with Bacto-Supplement A, increased his positive isolations 5 per cent
over the unenriched medium. In addition to increasing the number of positive
isolations, the efficiency of the medium was improved by reduction of extraneous
growth and an increase in size and number of gonococcal colonies. Rosenblatt,
Meyer, and Robbins^^ found the Chocolate Agar enriched with Bacto-Supple-
ment A superior to the unenriched medium. Morton and Leberman^'^ recom-
mended the use of Bacto-Supplement A in the Chocolate Agar as it restricted
growth of extraneous forms, gave rise to larger gonococcal colonies and in-
creased the positive isolations over that obtained on the unenriched medium. A
practical method for the isolation of the gonococcus using Chocolate Agar pre-
pared from Bacto-Proteose No. 3 Agar, Bacto-Hemoglobin and Bacto-Supple-
ment A, is given in detail by Morton^i.

A recommended procedure for the cultural detection of the gonococcus is
described in detail below.

Collection of Specimen and Inoculation

Methods for preparing the patient, obtaining adequate specimens and cultur-
ing exudates suspected of harboring N. gonorrhoeae are described in detail by
Carpenter^'22 and Morton.^^ Specimens are usually collected on sterile cotton
swabs. These may be used to inoculate the Chocolate Agar directly or preferably
placed in 1-2 ml. of sterile broth. A broth consisting of 2 per cent Proteose
Peptone No. 3, 0.5 per cent sodium chloride and 1 per cent soluble starch and
adjusted to pH 7.2 is recommended as a suspending fluid. Specimens should be
inoculated onto Chocolate Agar as soon as possible after collection and in no
case should plating be delayed longer than 8 hours. Specimens not immediately
inoculated onto plates should be kept in the ice box.

The exudate is suspended in the broth by rotating the swab and pressing
against the inside of the tube to remove as much material as possible, after
which the swab is discarded. About 0.05-0.1 ml. (1-2 drops) of the suspension
is then transferred onto sterile plates and smeared over the surface of the
medium with a bent glass rod.

Incubation

The plates after inoculation should be incubated in an inverted position at
35-37°C. for 36-48 hours.

Best results are obtained in an atmosphere containing carbon dioxide. Cans
with a tight fitting cover or Novy jars are satisfactory containers for the plates
during incubation. Carbon dioxide can be supplied in one of the following pro-
cedures outlined by Christensen and Schoenlein.^s

1. Place a lighted, smokeless candle near the top of the container, with the
plates and put the cover on the container.

2. Replace about 10 per cent of the air in the container with carbon dioxide.

3. Place sodium bicarbonate in a beaker in the container with the inverted
plates. Cover the sodium bicarbonate with cotton to reduce foaming. Add dilute

DEHYDRATED CULTURE MEDIA 119

sulfuric acid to the beaker and place cover on the can at once. One gram sodium
bicarbonate with 3 ml. of sulfuric acid diluted to 100 ml. with water will give
about a 10 per cent carbon dioxide atmosphere to a container of about 2.5 liters
capacity.

Laboratory workers wishing more detailed information on optimum carbon
dioxide tensions should consult Ferguson^* and Morton.-^

Observation

Remove plates from the containers after the incubation period, keeping them
in the inverted position until the time of examination to prevent flooding of the
plate with water of condensation.

The following procedure as given by Carpenter^'-^^ {^ recommended: Make a
direct examination of the plates for colonies of the gonococcus. Typically such
colonies are convex, transparent, from 1 to 3 mm. in diameter, with undulate
margins. By their transparency and character of their margins they can usually
be differentiated from young colonies of streptococci and diphtheroids which
they simulate. Films are prepared from the selected colonies, stained and exam-
ined. Cultural confirmation of typical Gram-negative diplococci is made by
subculturing typical colonies on enriched Chocolate Agar for purification and
inoculation into the appropriate carbohydrate media.

When no typical gonococcus colonies can be detected by direct inspection, the
culture is subjected to the oxidase test which is of especial value in detecting
colonies of A^. gonorrhoeae in mixed cultures. The test is based upon the pro-
duction of an enzyme, oxidase, by organisms belonging to the genus Neisseria.
From 1 to 2 ml. of a one per cent solution of para-aminodimethylaniline mono-
hydrochloride (dimethyl-p-phenylenediamine hydrochloride) or the oxalate
salt, are dropped on each primary plate culture and the plate tilted to spread
the reagent over the entire surface. The plate is observed for a period of 6-10
minutes for evidence of color change of the colonies. The series of color reac-
tions, i.e., pink, maroon, and black, identifies the colonies of Neisseria. Films are
made from the "oxidase-positive" colonies, stained and examined microscop-
ically. If subcultures are to be made for further identification, the colonies
should be picked as soon as they become pink, because the dye component is
toxic for the organisms.

Confirmation

In the routine diagnosis of gonococcal infections and in the release of patients
under treatment, the oxidase test followed by a confirming Gram stain, in the
hands of an experienced operator, is generally sufficient for the identification of
the gonococcus. The identity of the organism may be further confirmed by
studying the reactions of purified cultures on carbohydrate media. The follow-
ing table shows the characteristic reactions of A^. gonorrhoeae and other Neis-
seria which are occasionally encountered.

Microorganism Dextrose Maltose Saccharose Lactose

Neisseria catarrhalis — — — —

Neisseria gonorrhoeae + — — —

Neisseria meningitidis -f- + — —

Neisseria sicca + + + —

In the study of carbohydrate fermentation we recommend the use of Bacto-
Phenol Red Carbohydrate Broths, or of Bacto-Phenol Red Broth Base contain-
ing 0.5 per cent of the desired carbohydrate, and to which has been added 0.15

120 DIFCO MANUAL

per cent Bacto-Agar. The carbohydrate media should be freshly prepared or
reheated and cooled without agitation before inoculation. The inoculum should
be fairly heavy and should be placed on the surface layer of the medium not to
exceed a depth of 0.5 cm. Bacto-Phenol Red Broth Base with 0.5 per cent added
carbohydrate and 0.15 per cent agar is suggested as a satisfactory medium for
fermentation determinations as given in Diagnostic Procedures and Reagents--.
Some laboratories prefer a more solid fermentation medium containing en-
richment substances. A satisfactory medium of such character can be prepared
by adding 0.8 per cent Bacto-Agar to Bacto-Phenol Red Broth Base, sterilizing
it in the autoclave for 15 minutes at 15 pounds pressure (121°G.), cooling below
60°G., and adding 0.5 per cent of the carbohydrate previously sterilized in 10 or
20 per cent solution, and 5 per cent sterile fresh rabbit serum. Sera from other
animals have not been found satisfactory. A similar medium for determination
of fermentation by N. gonorrhoeae has been described by Faber, Gonzales and
Pelczar.26

Other Primary Isolation Media

Some laboratories prefer to use a clear medium rather than a Ghocolate Agar
for culturing A^. gonorrhoeae. This may be accomplished by omitting the Bacto-
Hemoglobin and enriching the Proteose No. 3 Agar with other substances capa-
ble of supporting growth of the gonococcus. Peizer and Steffen-^ reported the
use of a horse plasma hemoglobin dextrose nile blue A enrichment which, when
added to Proteose No. 3 Agar, gave a clear medium and yielded excellent results
in the culturing of the gonococcus. Steinberg and Mollov-^ enriched Proteose
No. 3 Agar with starch and blood and produced a satisfactory, clear medium.

Mueller and Hinton-^ described a casein hydrolysate infusion medium which
they reported to give good results in the primary culturing of the gonococcus.
This medium is available in the dehydrated form as Bacto-Mueller Hinton
Medium as discussed on page 93.

Sulfonamide Resistance of N. Gonorrhoeae

Goodale, Gould, Schwab and Winter^^ developed a culture technique for test-
ing the resistance of N. gonorrhoeae to sulfonamides. The method consisted of
inoculating plates of Mueller Hinton Medium containing 0.10, 0.25 and 0.50
mg. per cent of sulfathiazole, respectively. Susceptible strains fail to grow in the
presence of the sulfonamides, while resistant strains do grow.

Nelson,^^ using the Bacto-Mueller Hinton Medium as Goodale, et al.^° had
described for checking sulfonamide resistant gonococci, obtained very close cor-
relation with the clinical picture on thousands of cases. Nelson also employed
the Proteose No. 3 Hemoglobin Agar in the same manner for sulfonamide re-
sistance tests with equally good results. Frisch, Edwards, and Edwards^^ found
that the Proteose No. 3 Hemoglobin Agar worked well as a basal medium for
testing the sulfonamide resistance of A^. gonorrhoeae.

Mass Culture and Stock Strains

Mass cultivation of newly isolated strains for vaccine production is readily
accomplished on Bacto-Dextrose Starch Agar as discussed on page 124. This
medium supports luxuriant growth of the organism, and colonies of the gono-
coccus frequently exceed 3-5 mm. in diameter. Bacto-Dextrose Starch Agar
cannot be recommended for cultural detection of the gonococcus due to over-
growth by extraneous organisms. Bacto-Dextrose Starch Agar, prepared in half
strength, is an ideal medium for maintaining stock cultures of the gonococcus.
In tubes of this medium the gonococci generally are viable after 6-8 weeks in-
cubation at 37°C.

DEHYDRATED CULTURE MEDIA

121

Preparation of Media

Prepare a double strength Proteose No. 3 Agar by suspending 9.0 grams in
100 ml. of cold distilled water, and steaming or boiling to dissolve the medium
completely. At the same time prepare a double strength hemoglobin solution by
placing 2 grams Bacto-Hemoglobin in a dry flask and adding 100 ml. cold dis-
tilled water, while the flask is being agitated vigorously. The Bacto-Hemoglobin
suspension is shaken intermittently for 10-15 minutes to break up all aggregates
and effect complete solution. Both double strength solutions of Proteose No. 3
Agar and Bacto-Hemoglobin are sterilized in the autoclave for 15 minutes at
15 pounds pressure (121°C.).

The sterile solutions are allowed to cool to 50-60° C. Add 1 per cent of Bacto-
Supplement A or Bacto-Supplement B, based on the final volume of medium, to
the double strength Proteose No. 3 Agar (2 ml. per 100 ml. double strength
sterile agar base). Mix well and add an equal volume of double strength sterile
Bacto-Hemoglobin solution at 50-60° G. Mix well and distribute into sterile petri
dishes. Caution: Do not add Bacto-Supplement A or Bacto-Supplement B to the
hemoglobin solution before mixing with the agar base. Best results will be ob-
tained if the medium is prepared in advance so that the surface of the medium
is dry before inoculation.

When the single strength medium, without enrichment, is desired, the medium
is rehydrated by suspending 45 grams of Bacto-Proteose No. 3 Agar in 1000 ml.
of cold distilled water and heating to boiling to dissolve the medium completely.
Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). The final reaction of the medium without enrich-
ment will be pH 7.3.

One pound of Bacto-Proteose No. 3 Agar will make 10 liters of medium.

Acknowledgment

We are indebted to the personnel of the Division of Social Hygiene of the
Detroit Department of Health for their cooperation in supplying the clinical
material for these studies.

1 Am. J. Syphilis Gonorrh. Venereal Diseases,
33:164:1949.

2 Muench. Wochschr., 80:846:1933.
8 J. Path. Bact., 39:221:1934.

* J. Infectious Diseases, 61:129:1937.
^ Am. J. Syphilis, 20:347:1936.
«Am. J. Syphilis, 22:55:1938.
'Seventh Annual Yearbook (1936-37),

p. 133, Suppl., Am. J. Pub. Health,
27:No. 3:1937.
^ Bull. Genitoinfectious Diseases, Mass. State
Health Dept., 2:1:1938.

* Pelouze : Gonorrhea in the Male and
Female, 3rd Edition: 55: 1939.

^0 Am. J. Syphilis Gonorrh. Venereal Diseases,

and
3rd

25:22:1941.
^ Am. J. Public Health, 31:457:1941,

Venereal Disease Inform.,
Venereal Disease Inform.,
Bact., 44:139:1942.
Bact., 45:321:1943.
Bact., 45:410:1943.

24:218:1943.
24:133:1943.

" Personal Communication 1943.
^ Personal Communication 1944.
"^ Am. J. Syphilis Gonorrh. Venereal Diseases,
28:634:1944.

20 U.S. Naval Med. Bull., 43:409:1944.

21 Encyclopedia of Medicine, Surgery
Specialties, 3rd Edition. Now in press.

22 Diagnostic Procedures and Reagents,
Edition: 107: 1950.

23 J. Bact., 40:162:1940.
2* Am. J. Syphilis Gonorrh. Venereal Diseases,

29:19:1945-

25 J. Bact., 50:589:1945.

26 Am. J. Clin. Path., 18:256:1948.
2^ J. Venereal Disease Inform., 23:224:1942.
2S J. Lab. Clin. Med., 27:656:1942.

29 Proc. Soc. Exp. Biol. Med., 48:330:1941.

30 J. Am. Med. Assoc, 123:547:1943.

31 Personal Communication 1945.

^ Am. J. Syphilis Gonorrh. Venereal Diseases,
28:397:1944.

122 DIFGO MANUAL

BACTO

G G MEDIUM BASE (B289)

DEHYDRATED

Proteose Peptone No. 3, Difco 15 g.

Corn Starch 1 g.

Dipotassium Phosphate 4 g.

Monopotassium Phosphate 1 g.

Sodium Chloride 5 g.

Bacto-Agar 10 g.

Bacto-G C Medium Base, enriched with Bacto-Hemoglobin and Bacto-Supple-
ment A or Bacto-Supplement B, is recommended as a 24 hour culture medium
for the cultural diagnosis of gonorrhoea from acute and chronic cases in the
male and female. This medium has the advantage of requiring a shorter incu-
bation period than the medium prepared with Bacto-Proteose No. 3 Agar.

In a survey^ under carefully controlled conditions twelve media recommended
for the isolation of the gonococcus were compared. Bacto-G C Medium Base
enriched with Bacto-Hemoglobin and Bacto-Supplement A or Bacto-Supplement
B was as effective in the number of gonococci cultures isolated as was any of the
media. In Diagnostic Procedures and Reagents- a medium prepared with Bacto-G
C Medium Base enriched with Bacto-Hemoglobin and Bacto-Supplement B pos-
sessed several advantages over the other media described for the isolation of the
gonococcus. It was pointed out that this medium is always immediately avail-
able and can be prepared in either large or small quantities, insuring a supply
of fresh moist medium essential for dependable results. This medium needs only
an incubation period of 24 hours instead of 48 hours usually required by other
media.

Johnston^ described a medium for culturally detecting Neisseria gonorrhoeae
in exudates within 24 hours. The medium employed was a Chocolate Agar pre-
pared from Bacto-Proteose No. 3 Agar and Bacto-Hemoglobin to which was
added 30 per cent ascitic fluid and 1:50,000 tyrothricin. Inoculated plates of
this medium incubated in a partial carbon dioxide atmosphere in closed con-
tainers at 37 °C. for 24 hours yielded more positives than did the same medium
incubated for 48 hours.

Attempts to elucidate the cause for the accelerated early growth response on
Johnston's modified medium over that of the regular enriched Chocolate Agar
indicated that the variation in growth rates on the two media was due prin-
cipally to the difference in solidity of the media. As a result of this observation
a new medium, Bacto-G C Medium Base, was designed for use as a 24 hour
medium in the laboratory diagnosis of gonococcal infections.

In the examination of over 500 clinical specimens Christensen and Schoen-
lein* found the Bacto-G G Medium Base enriched with Bacto-Hemoglobin and
Bacto-Supplement A or Bacto-Supplement B to give equally as good or better
results after 24 hour incubation as did the Chocolate Agar prepared from Bacto-
Proteose No. 3 Agar, Bacto-Hemoglobin and Bacto-Supplement A or Bacto-Sup-
plement B incubated for similar or longer periods. Johnston^ compared Bacto-G
G Medium Base enriched with Bacto-Hemoglobin and Bacto-Supplement A or
Bacto-Supplement B with the medium containing ascitic fluid and tyrothricin
which she described and found their effectiveness to be comparable. Johnston
preferred the use of Supplement A as an enrichment since the crystal violet
helped in suppressing extraneous contaminating organisms. McRoy and Sala-
chow® of the Detroit Venereal Disease Clinics also reported excellent results
with Bacto-G G Medium Base enriched with Bacto-Hemoglobin and Bacto-
Supplement A and incubating for only 24 hours. Garpenteri'"^ in his report on a

DEHYDRATED CULTURE MEDIA 123

study evaluating the media employed for the cultural diagnosis of gonorrhoea
declared that the G C Medium Base enriched with Bacto-Hemoglobin and
Bacto-Supplement B yielded results in 24 hours comparable with those obtained
on the best plating media requiring a similar or longer incubation period.

Wax^ used Bacto-G C Medium Base enriched with Bacto-Hemoglobin and
Bacto-Supplement B, with tyrothricin as a selective agent in the isolation of
Neisseria other than N. gonorrhoeae from the genito-urinary tract.

For a complete discussion of the development of the cultural diagnosis of
gonorrhoea, including detailed recommendations for the collection of specimen,
inoculation of plates and observation and confirmation of results, reference
should be made to the discussion given under Bacto-Proteose No. 3 Agar, page
116-123.

Since Bacto-G G Medium Base contains only 1 per cent Bacto-Agar, care
must be exercised not to penetrate through the surface during inoculation.
Following inoculation, plates should be incubated at 35-37 °C. for 24 hours.
Best results are obtained in an atmosphere of carbon dioxide obtained by one of
the procedures outlined under Bacto-Proteose No. 3 Agar, page 118.

The enriched Chocolate Agar, using Bacto-G C Medium Base and Bacto-
Hemoglobin, is prepared in the following manner:

Prepare a double strength G G Medium Base by suspending 7.2 grams in 100
ml. cold distilled water and steaming or boiling to dissolve the medium com-
pletely. At the same time prepare a double strength hemoglobin solution by
placing 2 grams Bacto-Hemoglobin in a dry flask and adding 100 ml. cold dis-
tilled water, while the flask is being agitated vigorously. The Bacto-Hemoglobin
suspension is shaken intermittently for 10-15 minutes to break up all aggregates
and effect complete solution. Both double strength solutions of G C Medium
Base and Bacto-Hemoglobin are sterilized in the autoclave for 15 minutes at
15 pounds pressure (121°G.).

The sterile solutions are allowed to cool to 50-60° G. Add 1 per cent of Bacto-
Supplement A or Bacto-Supplement B, based on the final volume of medium, to
the double strength G G Medium Base (2 ml. per 100 ml. double strength
sterile agar base). Mix well and add an equal volume of double strength sterile
Bacto-Hemoglobin solution at 50-60° G. Mix well and distribute into sterile petri
dishes. Gaution: Do not add Bacto-Supplement A or Bacto-Supplement B to the
hemoglobin solution before mixing with the agar base.

Best results will be obtained if the medium is prepared in advance so that
the surface of the medium is dry before inoculation.

When the single strength medium, without enrichment, is desired, the medium
is rehydrated by suspending 36 grams Bacto-G G Medium Base in 1000 ml. cold
distilled water and heating to boiling to dissolve the medium completely. Dis-
tribute in tubes or flasks and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°G.). The final reaction of the medium without enrich-
ment will be pH 7.2.

One pound of Bacto-G G Medium Base will make 12.6 liters of medium.

^ Am. J. Syphilis Gonorrh. Venereal Diseases, ^Personal Communication 1947.

33:164:1949. ^Personal Communication 1947.

8 Diagnostic Procedures and Reagents, 3rd ^ Paper read at the annual meeting of the

Edition: 30-1 07: 1 950. Am. Pub. Health Assoc, 1947.

8 J. Venereal Disease Inform., 26:239:1945. « J. Venereal Disease Inform., 31:208:1950.
* Paper read at the annual meeting of the

Canadian Pub. Health Assoc, 1947.

124 DIFCO MANUAL

BACTO

DEXTROSE STARCH AGAR (B66)

DEHYDRATED

Proteose Peptone No. 3, Difco 15 g

Bacto-Dextrose 2 g

Soluble Starch, Difco 10 g

Sodium Chloride 5 g

Disodium Phosphate 3 g.

Bacto-Gelatin 20 g

Bacto-Agar 10 g

Bacto-Dextrose Starch Agar is recommended as a complete solid medium for
the propagation of pure cultures of Neisseria gonorrhoeae. This highly nutritious
medium without any additions will also support excellent growth of a large
number of pathogenic organisms such as the meningococcus, streptococcus and
pneumococcus. This medium is far superior to the ordinary infusion media for
the cultivation of these discriminating bacteria. Bacto-Dextrose Starch Agar, in
half concentration, is recommended as a Stock Culture Agar for the maintenance
of cultures of gonococcus, meningococcus and others not capable of splitting
starch.

In the development of a simple medium for the cultural detection of the
gonococcus, it was considered that luxuriant growth of all strains of N. gonor-
rhoeae was a requisite. Bacto-Dextrose Starch Agar, a nutritious medium with-
out enrichment, fulfilled this requirement satisfactorily, giving luxuriant growth
of freshly isolated fastidious strains of the gonococcus. It was soon shown, how-
ever, on this highly nutritious medium that in mixed cultures, as were en-
countered especially in chronic cases of gonorrhoea, extraneous forms developed
too rapidly and overgrew the gonococcus. For this reason, an enriched Chocolate
Agar prepared with Bacto-Proteose No. 3 Agar as discussed on page 116 or
Bacto-G C Medium Base, page 122, is recommended for the isolation of the
gonococcus.

For the cultivation of the gonococcus, it is imperative to have the incubation
atmosphere saturated with moisture. Satisfactory conditions can be obtained
if the plates of Dextrose Starch Agar are incubated in a closed container, which
contains cotton, a towel or a sponge saturated with water. A can with a suitable
cover, Novy jar, desiccator or any other convenient sized container capable of
retaining the moisture is entirely satisfactory. About 200 ml. of water added in
this manner is ample for a container of one or two gallons capacity. Plates in-
cubated under these conditions will give a luxuriant growth of many gonococci
when identically inoculated plates incubated in the ordinary manner in the
incubator show no growth. If the culture requires carbon dioxide for growth
this may be supplied as indicated under Bacto-Proteose No. 3 Agar, page 118.
Carbon dioxide is recommended for isolation, but is not generally necessary,
in the presence of abundant moisture, for growth of isolated strains.

Swancara^ described a method of obtaining partial carbon dioxide tension in
individual tubes by placing a cotton plug just over the medium following inocu-
lation. The percussion tip from a match is removed and the match broken and
placed on this plug. The tube is then sealed with a rubber stopper or screw cap.
The match head is then ignited by applying heat to the outside of the tube.

Nutritional factors so conducive to the growth of the gonococcus proved
equally satisfactory for many other pathogenic bacteria, making Bacto-Dextrose
Starch Agar, without added enrichment, a most excellent medium for the propa-
gation of streptococci, pneumococci, meningococci and others. For mass growth
of these organisms Bacto-Dextrose Starch Agar is recommended as being superior

DEHYDRATED CULTURE MEDIA 125

to many highly enriched media with complicated formulae and methods of
preparation.

Bacto-Dextrose Starch Agar prepared in half strength is an excellent medium
for maintaining stock cultures of the gonococcus. In tubes of this medium the
gonococcus generally remains viable for 6-8 weeks at 37°G. This medium cannot
be recommended to carry stock cultures of organisms capable of attacking
starch; some streptococci, for example, produce sufficient acid from the starch
in the medium to make it unsatisfactory for this purpose. Bacto-Stock Culture
Agar or Bacto-Cooked Meat Medium as discussed on page 98 and 85, is recom-
mended for the maintenance of starch hydrolyzing organisms.

To rehydrate the medium suspend 65 grams of Bacto-Dextrose Starch Agar in
1000 ml. of cold distilled water, and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). For carrying stock cultures the medium is
prepared in the same manner, except that only 32.5 grams of Bacto-Dextrose
Starch Agar are used in 1000 ml. of cold distilled water. The medium normally
contains a flocculent precipitate which in no way detracts from the value of the
medium. Final reaction of the medium will be pH 7.3.

Since best results are obtained with a solid medium having a moist surface,
Dextrose Starch Agar should be used the same day it is prepared, or if not used
at once, the medium should be melted and allowed to re-solidify just prior to
inoculation. It is recommended that sterile rubber stoppers be substituted for
the cotton plugs as soon as the slants have cooled. Screw-cap tubes also yield
satisfactory results. Cultures of gonococci on plates should be incubated in an
atmosphere saturated with moisture as indicated above.

One pound of Bacto-Dextrose Starch Agar will make 7 liters of medium or 14
liters of half-strength medium.
lAm. J. Med. Tech., 14:214:1948.

BACTO

DEXTROSE AGAR (B67)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Tryptose 10 g.

Bacto-Dextrose 10 g.

Sodium Chloride 5 g.

Bacto-Agar 15 g.

Bacto-Dextrose Agar is recommended as a solid medium for cultivation of a
large variety of organisms and is especially adapted to the preparation of a
Dextrose Blood Agar. In deep tubes, or with removal of oxygen, it will support
good growth of anaerobes.

Dextrose (<i-glucose) is a readily available source of energy, utilized by a large
number of microorganisms. This fact makes Dextrose Agar excellently suited for
the production of early and abundant growth, shortening lag periods of old
cultures and the initiation of growth of bacteria capable of utilizing dextrose.
Bacto-Dextrose Agar contains 1.0 per cent dextrose with a combination of other
ingredients that make an excellent Dextrose Agar capable of supporting growth
of all the more common bacteria and, in addition, such organisms as strepto-
cocci, pneumococci and meningococci. Norton^ recommended an agar medium
containing 0.5 to 1.0 per cent dextrose and about 5 per cent defibrinated blood
as being valuable for the isolation of organisms from pus, as well as being an

126 DIFCO MANUAL

excellent medium for cultivating the meningococcus, gonococcus, Hemophilus
influenzae and H. pertussis.

To rehydrate the medium, suspend 43 grams of Bacto-Dextrose Agar in 1000
ml. cold distilled water and heat to boiling to dissolve the medium completely.
Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). Final reaction of the medium will be pH 7.3.

Since best results are obtained with solid media having a moist surface,
Dextrose Agar should be used the same day as it is prepared. If not used at
once, melt the medium and allow to re-solidify just prior to inoculation.

One pound of Bacto-Dextrose Agar will make 10.5 liters of medium.

1 J. Lab. Clin. Med., 17:585:1932.

BACTO

BREWER ANAEROBIC AGAR (B279)

DEHYDRATED

Bacto- Yeast Extract 5 g

Bacto-Tryptone 5 g

Proteose Peptone No. 3, Difco 10 g

Bacto-Dextrose 10 g

Sodium Chloride 5 g

Bacto-Agar 20 g

Sodium Thioglycollate, Difco 2 g

Sodium Formaldehyde Sulfoxylate . . 1 g

Resazurin, Certified 0.002 g,

Bacto-Brewer Anaerobic Agar is prepared for use with the Brewer anaerobic
cover as described by Brewer.^ This special petri dish cover was designed to
permit surface growth of anaerobes and micro-aerophiles on agar with a low
oxidation-reduction potential without the use of anaerobic jars or other special
apparatus. Brewer Anaerobic Agar is recommended to those laboratories wishing
to use this procedure for anaerobic culture.

To rehydrate the medium, suspend 58 grams of Bacto-Brewer Anaerobic Agar
in 1000 ml. cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in flasks and sterilize in the autoclave for 15 minutes at
15 pounds pressure (121°C.). Dispense the medium in petri dishes using 50-75
ml. of medium in 95 x 15-20 mm. petri dish bottoms. For best results use porous
tops on the dishes containing the medium during solidification in order to ob-
tain a dry surface. Inoculate the surface of the medium by smearing or streak-
ing. Cover the inoculated dish with a sterile Brewer anaerobic petri dish cover.
It is essential that the sealing ring inside the cover makes perfect contact with
the medium. This seal must not be broken before the end of the incubation
period. Poured plates may be made by placing the inoculum in the dish and mix-
ing with the medium before solidification. The final reaction of the medium
will be pH 7.2.

One pound of Bacto-Brewer Anaerobic Agar will make 8.5 liters of medium.

1 Science, 95:587:1942-

DEHYDRATED CULTURE MEDIA 127

BACTO

BREWER ANAEROBIC AGAR (B433)
without Dextrose or Eh Indicator

DEHYDRATED

Bacto-Brewer Anaerobic Agar without dextrose or Eh Indicator has the same
composition as Bacto-Brewer Anaerobic Agar except dextrose and resazurin have
been omited. This carbohydrate-free modification may be used for the Isolation
and growth of anaerobes and may be used as a base for Blood Agar, Upon the
addition of carbohydrates the medium may be employed for fermentation
studies. The addition of 1 per cent of the test carbohydrate is recommended for
these studies.

To rehydrate the medium, suspend 48 grams Bacto-Brewer Anaerobic Agar
without Dextrose or Eh Indicator in 1000 ml. cold distilled water. Heat to
boiling to dissolve the medium completely. Distribute In tubes or flasks and
sterilize in the autoclave for 15 minutes at 15 pounds pressure (121°C.). Final
reaction of the medium will be pH 7.2.

One pound of Bacto-Brewer Anaerobic Agar without dextrose or Eh Indicator
will make 9.4 liters of medium.

BACTO
NUTRIENT AGAR 1.5% (B69)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Peptone 5 g.

Sodium Chloride 8 g.

Bacto-Agar 15 g.

Bacto-Nutrlent Agar 1.5% is recommended as a slightly alkaline Nutrient
Agar, for use in the cultivation of bacteria not requiring a highly nutritious
medium. This medium contains 0.8 per cent sodium chloride, making it well
suited for the addition of blood, ascitic fluid or other enriching fluids. With
added food material, Bacto-Nutrient Agar 1.5% is satisfactory for the cultiva-
tion of pathogenic bacteria.

Bacto-Blood Agar Base, Bacto-Tryptose Blood Agar Base or Bacto-Heart Infu-
sion Agar, as discussed on pages 88, 115 or 87, is recommended as being superior
to Bacto-Nutrient Agar 1.5% for use In making Blood Agar. For general culture
purposes, with or without enrichment, Bacto-Tryptose Agar, Bacto-Proteose No.
3 Agar, Bacto-Dextrose Proteose No. 3 Agar and Bacto-Dextrose Starch Agar, as
discussed on pages 111, 116, 147 and 124, respectively, are recommended as
being more satisfactory than Bacto-Nutrient Agar 1.5%.

To rehydrate the medium, suspend 31 grams of Bacto-Nutrient Agar 1.5% in
1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute In tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). The final reaction of the medium will be pH
7.3. If enrichment Is desired, the freshly sterilized medium Is cooled to 45-50° C.
and sterile blood, ascitic fluid or other sterile serous enrichment is added.

Since best results are obtained on a solid medium having a moist surface,
Nutrient Agar 1.5% should be used the same day as prepared, or, if not used
at once, the medium should be melted and allowed to re-solidify just prior to
inoculation.

One pound of Bacto-Nutrient Agar 1.5% will make 14.6 liters of medium.

128 DIFCO MANUAL

BACTO

LOEFFLER BLOOD SERUM (B70)

DEHYDRATED

Beef Blood Serum 3 parts

Dextrose Broth 1 part

Bacto-Loeffler Blood Serum is employed in the cultural diagnosis of diphtheria.
The medium is of particular value in the determination of pigment production
by bacteria, and of the ability of anaerobes and other microorganisms to attack
protein, and for other special uses. Bacto-Loeffler Blood Serum has been prepared
in dehydrated form for the convenience of those laboratories where adequate
supplies of fresh blood serum are not available.

Bacto-Loeffler Blood Serum is a modification of the horse serum, dextrose
broth medium described by LoefHer in 1887^ for the cultivation of Coryne-
bacterium diphtheriae. In our modern laboratories LoefHer Blood Serum still
retains its important position for the diagnosis of diphtheria. On Loeffler Blood
Serum C. diphtheriae grows luxuriantly and rapidly, developing morphologically
typical organisms, in 12-16 hours, aiding greatly in the diagnosis of diphtheria.

Cleveland and Sanders,^ and Spector^ have used Bacto-Loeffler Blood Serum
in media for the cultivation of Endamoeha histolytica. Thompson* hydrolyzed
Bacto-Loeffler Blood Serum with sodium hydroxide and added this to a citrate
agar for the isolation of C. diphtheriae, on which diphtheria bacilli are stimu-
lated and other throat organisms are inhibited.

To rehydrate the medium, dissolve 80 grams of Bacto-Loeffler Blood Serum in
1000 ml. of warm (42-45°C.) distilled water.

During the dehydration process certain components of the medium become in-
soluble. The removal of this insoluble material by filtration on a Buchner funnel
with the use of filter aid or by centrifugation at high speed for 15-20 minutes
will result in better appearing final slants. The use of tubes that can be sealed
with a tight fitting closure such as a screw cap will produce slants free from
bubbles. It is recommended that the medium be distributed in tubes and coagu-
lated and sterilized in the autoclave as follows:

1. Place not more than three rows of tubes in a slanting position, in wire
baskets, in the autoclave.

2. Close all the ports and the door before turning on the steam to maintain
a mixture of air and steam for coagulation of the medium. If the exhaust of the
autoclave is equipped with an automatic thermo-element type trap, it is necessary
to have this line equipped with a valve which can be closed, thereby making it
possible to maintain a mixture of air and steam in the autoclave during coagula-
tion.

3. Run the pressure as quickly as possible to 15 pounds and hold for 10 minutes
to coagulate the medium.

4. At the end of coagulation, open the lowest port (or valve in thermo-
element type trap line if such is used) and replace the entrapped air with steam,
maintaining constant pressure while replacing the air-steam mixture with live
steam, as any appreciable change in pressure will cause the slants to be filled with
bubbles and render the medium useless.

5. Maintain steam pressure for 15 minutes at 15 pounds (121°C.).

6. After sterilization is completed, the source of steam is cut off. All ports and
the safety valve should be tightly closed as soon as the steam is shut off, allowing
the autoclave to cool slowly. Do not open any ports until the steam pressure
is nil. Final reaction of the medium will be pH 7.2.

The autoclave sterilization methods of Hinkleman^ or Dupray® may be used

DEHYDRATED CULTURE MEDIA

129

with satisfaction. Heise^ has also described a method for the autoclave steriliza-
tion of Loeffler Medium in fruit jars.

Levin^ gives in detail the method of inspissation and sterilization of serum
media using a single chambered autoclave, pressure cooker field autoclave and
a double chambered dressing type autoclave.

Foster and Cohn,^ and Spray and Johnson^^ have described methods which
they use in obtaining bubble-free slants of serum media.

One pound of Bacto-Loeffler Blood Serum will make 5.6 liters of medium.

Bact., 9:179:1924.

iCentr. Bakt., 2:105:1887.

2 Arch. Protistenkunde, 70:223:1930

3 J. Preventive Med., 6:117:1932.

* J. Infectious Diseases, 45:163:1929.
sj. Bact., 8:315:1923.

BJ. Bact

" Bact

Bact

46:223:1943.

50:561:1945.

., 52: 141; 1946.

ADDITIONAL MEDIA

The media listed below have been rather extensively employed in the past for
general or special purposes. Other media have been developed which are con-
sidered superior and serve the purpose more adequately than the older media.
For the present we will continue to carry these media in stock for those labora-
tories where they have been in routine use or where it is desired to continue their
use for comparative purposes.

PRESENT RECOMMENDATIONS

Bacto-Dextrose Infusion Broth Bacto-Dextrose Broth, page 101, or

add 0.5 per cent Bacto-Dextrose to
Bacto-Heart Infusion Broth, page 80

Bacto-Cabbage Infusion Broth Bacto-Brain Heart Infusion, page 77

Bacto-Tryptose Broth, page 102
Bacto-Tryptose Phosphate Broth, page 100

Bacto-Dextrose Heart Agar Bacto-Dextrose Agar, page 125, Bacto-
Dextrose Proteose No. 3 Agar, page 147;
or add 1.0 per cent Bacto-Dextrose
to Bacto-Heart Infusion Agar, page 87

Bacto-Liver Infusion Agar Bacto-Tryptose Agar, page 111

Bacto-Endamoeba Medium, page 97

Bacto-Legumin Trypagar Bacto-Proteose No. 3 Agar, page 116

Bacto-G G Medium Base, page 122
Bacto-Tryptose Agar, page 111
Bacto-Dextrose Starch Agar, page 124

Bacto-Testicular Agar Bacto-Proteose No. 3 Agar, page 116

Bacto-North Gelatin Agar Bacto-G C Medium Base, page 122

Bacto-Dextrose Starch Agar, page 124

Bacto-Cabbage Infusion Agar Bacto-Tryptose Agar, page 111

Bacto-Proteose No. 3 Agar, page 116
Bacto-G G Medium Base, page 122
Bacto-Dextrose Starch Agar, page 124

Bacto-Nutrient Phosphate Agar Bacto-Tryptose Agar, page 111

Bacto-Proteose No. 3 Agar, page 116
Bacto-G G Medium Base, page 122
Bacto-Dextrose Starch Agar, page 1 24
Bacto-Blood Agar Base, page 88

Bacto-Starch Agar Bacto-Dextrose Starch Agar, page 124

Bacto-Proteose No. 3 Agar, page 116
Bacto-G G Medium Base, page 122

Bacto-Brain Liver Heart (Semisolid) . . Bacto-Gooked Meat Medium, page 85

Bacto-Dextrose Starch Agar, page 124

130

DIFCO MANUAL

DIFFERENTIAL MEDIA

Many media have been designed for the separation or differentiation of closely
related organisms or groups of organisms. The differentiation by means of cul-
tural characteristics is of decided value and importance especially where mor-
phology and other biological characteristics are almost identical. The following
dehydrated culture media represent a group of differential media, some of which
are not selective and depend on cultural characteristics for differentiation, while
others are selective and in addition show colonial differences between organisms.
The isolation of members of the enteric group by means of differential media has
been a routine procedure for many years. More recently differential media have
been described for the isolation of other groups of organisms such as diphtheria,
staphylococci and streptococci. Differential media for these isolation purposes are
discussed in this section.

Suggested Schema for the Examination of Stools or Other Material for Bacterial
Incitants of Enteric Disease

Specimen

Enrichment Bacto-Tetrathionate Broth
Bocto-Selenite Broth

Primary Ploting Medio Bocto-MocConkey Agar

Bacto-Desoxycholote Agar
Bacto-S S Agar

Bocto-Desoxychoiote Citrate Agar
Bocto-Bismuth Sulfite Agar

Bacto-Brilliant Green Agar (only for Salmoneilo other than
S. typhoso).

Purification Plate Bacto-MacConkey Agar

Differential Media

Primary Bacto-Russeil Double Sugar Agar

Bacto-Krumwiede Triple Sugar Agar
Bacto-Triple Sugar Iron Agar
Bocto-Kligler Iron Agar
Bacto-Friewer Shaughnessy Medium

Plote for Purity Bocto-MocConkey Agar

Secondory Bacto-Purple Broth Base-fKey Sugars
Bacto-S I M Medium
Bacto-Urea Agar or Bacto-Urea Broth
Bocto-Simmons Citrate Agar
Bacto- Phenol Red Tartrate Agar

Serological Identification

DEHYDRATED CULTURE MEDIA 131

Primary Plating Media

The media listed in this section are used generally in petri dishes for initial
isolation. In most cases the composition of the medium is such that it is selective
for a particular group of organisms.

BACTO
MacGONKEY AGAR (B75)

DEHYDRATED

Bacto-Peptone 17 g.

Proteose Peptone, Difco 3 g.

Bacto-Lactose 10 g.

Bacto-Bile Salts No. 3 1.5 g.

Sodium Chloride 5 g.

Bacto-Agar 13.5 g.

Bacto-Neutral Red 0.03 g.

Bacto-Grystal Violet 0.001 g.

Bacto-MacConkey Agar is a differential plating medium recommended for use
in the detection and isolation of all types of dysentery, typhoid and paratyphoid
bacteria from stool specimens, urine and other materials harboring these organ-
isms. MacConkey Agar is suggested for direct plating of water samples for coli-
form counts in Appendix I of "Standard Methods for the Examination of Water
and Sewage."^ "Standard Methods for the Examination of Dairy Products"^ pre-
fers dehydrated Bacto-MacGonkey Agar for the isolation of pathogenic bacteria
from cheese. MacConkey Agar is also specified in "Diagnostic Procedures and
Reagents"^ of the American Public Health Association.

Over a period of years, particularly in Great Britain, the Neutral Red Bile Salt
Agar of MacConkey* has been quite generally used for differentiating strains of
Salmonella typhosa from members of the coliform group. Bacto-MacConkey
Agar, as modified by the addition of 0.5 per cent sodium chloride, decreasing the
agar content to 1.35 per cent, and by altering the concentrations of bile salts and
neutral red, has the added advantage of supporting excellent growth of all
Shigella and Salmonella strains. It also gives a more clear-cut differential be-
tween these enteric pathogens and the coliform group, making it easier to read
than the original medium. Block and Ferguson^ investigating an outbreak of
Shiga dysentery found MacConkey Agar satisfactory in the isolation of this fas-
tidious strain.

The fact that this medium promotes development of these organisms, and at
the same time differentiates them from lactose fermenting Gram-negative bacilli,
makes it an excellent substrate for the cultural detection of dysentery, typhoid and
other Salmonella organisms in stools and other infected material. Gram-positive
bacteria are inhibited.

About 20 ml. of medium should be poured into previously sterilized petri
dishes to form a relatively thick layer. It is important that the surface of the
medium be quite dry when inoculated; this may be accomplished by allowing
the medium to solidify and to stand for about two hours with the covers of the
plates partially removed.

The medium is inoculated by streaking or smearing with the material under

132 DIFCO MANUAL

investigation. Serial inoculation of several plates is recommended to insure suit-
able distribution of well isolated colonies. After inoculation, the plates should be
incubated at 35-37°C. for 16-18 hours and should be observed at the end of this
period because prolonged incubation may lead to confusion of results.

The differential action of Bacto-MacConkey Agar is clear and distinct. Isolated
colonies of coliform bacteria are brick red in color and may be surrounded by a
zone of precipitated bile. This reaction is due to the action of the acids, produced
by fermentation of lactose, upon the bile salts and the subsequent absorption of
neutral red. Typhoid, paratyphoid and dysentery bacilli do not ferment lactose
and do not greatly alter the appearance of the medium. These colonies, in reality
giving an alkaline reaction, are uncolored and transparent. When growing in
proximity to coliform colonies, they have the appearance of clearing the areas of
precipitated bile. On plates which are not overcrowded the differentiation is
exceptionally distinct. A plate crowded with coli will appear red and opaque,
yet, if not too crowded, typhoid or other lactose non-fermenting organisms may
easily be detected by transmitted light. On such plates they will appear as small
transparent areas against the red background. A plate showing discrete colonies
is to be desired for isolation purposes.

For the cultural detection of typhoid organisms in infected material, it is
recommended that Bacto-MacConkey Agar be used in conjunction with the more
selective media, Bacto-S S Agar and Bacto-Bismuth Sulfite Agar as discussed on
pages 134 and 139.

A procedure designed to show the largest number of pathogens from a speci-
men would be:

A. Streak or smear a large inoculum on one plate of S S Agar and one plate of
Bismuth Sulfite Agar.

B. Streak or smear a light inoculum on one plate of MacConkey Agar.

G. Prepare Bismuth Sulfite Agar poured plates with a 5 ml. and a one drop

inoculum.
D. Enrich for 12-18 hours in Tetrathionate Broth or Selenite Broth, followed by

streaking on one plate of Bismuth Sulfite Agar or Brilliant Green Agar and

one plate of S S Agar.

Saccharose in 1 per cent concentration may be added to isolation media, such
as Bacto-MacConkey Agar to permit the detection of certain members of the
coliform group which ferment saccharose more readily than lactose. This prin-
ciple was described by Holt-Harris and Teague^ and has been employed by many
other bacteriologists. In some laboratories pathogenic significance is assigned to
these organisms, and under such conditions, saccharose should not be added to
the medium.

To rehydrate the medium, suspend 50 grams of Bacto-MacGonkey Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize by autoclaving for 15 minutes at
15 pounds pressure (121°G.). MacGonkey Agar inoculated the same day as re-
hydrated may be used without autoclave sterilization. Under these conditions
the medium need be heated only to boiling to dissolve it completely before pour-
ing into petri plates. The final reaction of the medium will be pH 7.1.

One pound of Bacto-MacGonkey Agar will make 9 liters of medium.

i Standard Methods for the Examination of ^ Diagnostic Procedures and Reagents, 3rd Edi-

Water and Sewage, gth Edition: 228: 1946. tion:24, 195:1950.

2 Standard Methods for the Examination of * J. Hyg., 5:333:1905.

Dairy Products, gth Edition: 166: 1948. ^ Am. J. Pub. Health, 130:42:1940.

*J. Infectious Diseases, 18:596:1916.

DEHYDRATED CULTURE MEDIA 133

BACTO

DESOXYCHOLATE AGAR

DEHYDRATED

A complete discussion of Bacto-Desoxycholate Agar as used for the isolation of
members of the Shigella and Salmonella groups is given on page 63.

BACTO

E.M.B. AGAR (B76)

DEHYDRATED

Bacto-Peptone 10 g

Bacto-Lactose 5 g

Saccharose, Difco 5 g

Dipotassium Phosphate 2 g

Bacto-Agar 13.5 g

Bacto-Eosin Y 0.4 g

Bacto-Methylene Blue 0.065 g

Bacto-E.M.B. Agar, a differential plating medium, is recommended for the
detection and isolation of the Gram-negative intestinal pathogenic bacteria.

The original Eosin Methylene Blue Agar was devised by Holt-Harris and
Teague.^ These workers employed a combination of eosin and methylene blue as
an indicator which gave a sharp and distinct differential between colonies of
lactose fermenting organisms and those which did not ferment lactose. These
authors included saccharose in their medium since certain members of the coli-
form group ferment this carbohydrate more readily than lactose. Colonies of the
coli-aerogenes group were either black or possessed dark centers with transparent
colorless peripheries, while those of typhoid and other organisms which did not
ferment lactose or saccharose remained uncolored. The Eosin Methylene Blue
Agar of Holt-Harris and Teague possessed definite advantages over the Fuchsin
Sulfite Agar of Endo, in that it was more sensitive, more accurate, more stable
and gave an earlier differentiation.

Two years after Holt-Harris and Teague had introduced their new medium,
Levine- described an Eosin Methylene Blue Agar for the purpose of differentiat-
ing the fecal and non-fecal types of the colon-aerogenes group. Levine's medium,
as discussed on page 35, also differentiated the typhoid and other lactose non-
fermenters from the coliform organisms.

Bacto-E.M.B. Agar is a combination of the Levine and the Holt-Harris and
Teague formulae. It contains Bacto-Peptone and phosphate as recommended by
Levine, and retains the two carbohydrates lactose and saccharose, as suggested by
Holt-Harris and Teague. The ratio of the two indicator dyes has been worked out
to give the best differentiation and minimum toxicity. This medium has all the
advantages of the original media referred to above.

It is recommended that for the detection of the more fastidious Shigella and
Salmonella organisms, Bacto-MacConkey Agar, the selective Bacto-S S Agar and
Bacto-Bismuth Sulfite Agar, as discussed on pages 131, 134 and 139, be run in
conjunction with Bacto-E.M.B. Agar.

To rehydrate the medium, suspend 36 grams of Bacto-E.M.B. Agar in 1000 ml.
of cold distilled water and heat to boiling to dissolve the medium completely.
Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). Bacto-E.M.B, Agar inoculated the same day as re-
hydrated may be used without autoclave sterilization. Under these conditions

134 DIFGO MANUAL

the medium need be heated only to boiling to dissolve it completely before pour-
ing into petri plates.

Sterilization reduces the methylene blue, leaving the medium orange color.
The normal purple color of the medium may be restored by gentle shaking. If
the reduced medium is not shaken to oxidize the methylene blue, a dark zone
beginning at the top and extending downward through the medium will gradu-
ally make its appearance. The sterilized medium normally contains a flocculant
precipitate which should not be removed. By cooling to 50° C. and gently agitat-
ing the medium before pouring it into plates, this flocculation will be finely dis-
persed, giving a medium without any objectionable precipitate.

One pound of Bacto-E.M.B. Agar will make 12.6 liters of medium.

* J, Infectious Diseases, 18:596:1916. 2 j, infectious Diseases, 23:43:1918.

BACTO

LEVINE E.M.B. AGAR

DEHYDRATED

Levine E.M.B. Agar has been used for the isolation of enteric pathogens. A
complete discussion of Bacto-Levine E.M.B. Agar is given on page 35.

BACTO

ENDO AGAR

DEHYDRATED

Endo Agar has been used rather extensively in the past for the isolation of
enteric pathogens. A complete discussion of Bacto-Endo Agar is given on page 34.

BACTO

S S AGAR (B74)

DEHYDRATED

Bacto-Beef Extract 5 g.

Proteose Peptone, Difco 5 g

Bacto-Lactose 10 g,

Bacto-Bile Salts No. 3 8.5 g

Sodium Citrate 8.5 g

Sodium Thiosulfate 8.5 g

Ferric Citrate 1 g

Bacto-Agar 13.5 g.

Bacto-Brilliant Green 0.00033 g

Bacto-Neutral Red 0.025 g

Bacto-S S Agar is a highly selective medium recommended for the isolation of
Shigella and Salmonella from stools and other materials suspected of containing
these organisms. Bacto-S S Agar was devised to provide excellent differentiation
of lactose fermenters from lactose non-fermenters, and to give maximum inhibi-
tion of coliform organisms without restriction of the growth of pathogenic Gram-
negative bacilli occurring in specimens. Inasmuch as Bacto-S S Agar is a selective
medium, we recommend that a non-selective medium such as Bacto-MacConkey
Agar be used in conjunction with it. To insure the highest percentage of positive
isolations of all intestinal pathogens, we further recommend the use of Bacto-

DEHYDRATED CULTURE MEDIA 135

Bismuth Sulfite Agar and enrichment in Bacto-Tetrathionate Broth Base or Bacto-
Selenite Broth.

The search for the inciting agents in bacillary intestinal disorders is of vital
importance in the public health laboratory. The value of the detection of typhoid
bacilli in active cases of typhoid fever as well as from carriers has long been recog-
nized, and a number of media are being successfully used for these purposes.
Media suitable for the isolation of Salmonella typhosa have not always proved
entirely satisfactory for the detection of Shigella and other Salmonella organisms
due either to the overgrowth of coliform organisms, or the inability of the medium
itself to support the growth of some particularly fastidious strains.

Bacto-S S Agar gives excellent growth from small inocula of the different
types of Shigella and Salmonella organisms as they occur in fresh fecal specimens.
Strains of Flexner, Newcastle, Schmitz, Shiga, Sonne and alkalescens, as well as
the Salmonella, grow unrestricted. All strains of S. typhosa that have been en-
countered show excellent growth on S S Agar. Coliform organisms are quite
generally inhibited even when heavy inocula are employed.

Shigella, Salmonella and other organisms not fermenting lactose form opaque,
transparent or translucent uncolored colonies, which generally are smooth. The
few lactose fermenting organisms which may develop on the medium are readily
differentiated due to the formation of a red color in the colony. At times, isolated
coliform colonies may not show a definite red color, being pink or nearly colorless
with a pink center. Occasionally an aerogenes type will develop a characteristic
large, white or cream colored opaque and mucoid colony. Some Proteus and
Salmonella types may, under certain conditions, produce black centered colonies.

Hormaeche and Surraco,^ Hardy and co-workers^ and Rose and Kolodny,^ have
reported S S Agar as superior to other media that have been recommended for
isolation of Shigella and Salmonella organisms. Mayfield and Goeber* compared
media for isolation of Shigella organisms and found Bacto-S S Agar to yield the
greatest number of positive isolations. Pots^ and Caudill^ have reported on the
satisfactory use of S S Agar in isolation of Shigella organisms. In their studies
of the acute diarrheal diseases, Mosher, Wheeler, Chant and Hardy'' first enriched
their specimens in Selenite Broth and then plated on Bismuth Sulfite Agar,
S S Agar, MacConkey Agar and Desoxycholate Agar, while Watt and Cummings*
plated directly upon S S Agar. Hormaeche and his co-workers^ used S S Agar
in conjunction with others for isolation of Shigella as the causative agent of
infantile summer diarrhea. Vacarro et al.^*^ employed S S Agar in conjunction
with other plating media in the isolation of Salmonella and Shigella from healthy
carriers, and Neter^^ used it similarly in his study of the Proteus and Paracolo-
bactrum (paracolon bacilli) in feces of healthy infants. McClure and Crossley,^-
using S S Agar and other media, isolated S. newport in an epidemic of food
poisoning, and Cordy and Davis^^ isolated S. morbificans from horses and mules
in an outbreak of salmonellosis. Watt, DeCapito and Morgan^* isolated S. texas
on S S Agar after enrichment in Tetrathionate Broth. For the isolation and typing
of Salmonella and Shigella, Borman, Wheeler and Mickle^^ and Nelson et al.^^
indicate the desirability of plating specimens on S S Agar and other substrates
before and after enrichment. Neter and Clark^'' have reported on the effectiveness
of culture media in isolation of enteric organisms, the usefulness of S S Agar,
especially with other media, being clearly demonstrated by their results.

Bacto-S S Agar is specified for use in "Standard Methods for the Examination,
of Dairy Products"^^ for the isolation of pathogenic bacteria from cheese and in
"Diagnostic Procedures and Reagents" ^^ for the examination of specimens for
evidence of infection with Salmonella and Shigella.

A survey of the methods used in the collection and preservation of stool speci-
mens for the isolation and identification of Salmonella, Shigella and intestinal

136 DIFGO MANUAL

protozoa by Felsenfeld^o showed that generally specimens were promptly plated.
Glycerol-saline was the most frequently used preservative. S S Agar was the most
popular plating medium. It was used especially frequently for direct plating.
There was a decrease in the use of Endo Agar. Desoxycholate Agars were found
to be more frequently used than the Endo Agar. There were more laboratories
using the poured plate of Bismuth Sulfite Agar, MacConkey Agar and Brilliant
Green Agar in 1949 than in 1944. The use of streaked plates of Bismuth Sulfite
Agar showed a decrease during this period if occasional users were not considered.
All but three laboratories were using dehydrated Bismuth Sulfite Agar. He stated
that these changes point toward evaluation of experience and economy in public
health laboratories.

Neter and Brody-^ added saccharose and also saccharose and salicin to Bacto-S
S Agar in studies on the bacteriological diagnosis and epidemiological aspects
of Salmonellosis. The modified media were especially valuable in the follow-up
of proven Salmonella cases and in epidemiological studies, but were not suggested
in routine work since pathogenic paracolons would be discarded by the modified
media. These media would also discard Proteus.

Schaub22.23 jji jigj. study of the differentiation of paracolon bacilli reported
Bacto-S S Agar to give more satisfactory results than the other selective media
tried, in that on S S Agar growth of the paracolon bacilli, regarded as aberrant
coliforms, is inhibited, as is that of the typical coliform bacillus. Certain other
paracolon bacilli grew well. Correlating growth on Bacto-S S Agar with bio-
chemical characteristics, the paracolon bacilli were divided into four large
groups :

Group I — aberrant coliform bacilli, consisting of paracolon Escherichia, para-
colon Aerobacter and paracolon intermediates; growth inhibited on
S S Agar.
Group II — hydrogen-sulfide-producing paracolon bacilli, growth not inhibited
on S S Agar. This group may be divided culturally into three sub-
groups, II-A, II-B and II-C.
Group III — anaerogenic paracolon bacilli; growth not inhibited on S S Agar.
Group IV — malonate-positive paracolon bacilli; growth not inhibited on S S
Agar.

Schaub also used S S Agar as slants in tubes and found that this was a satisfactory
method of demonstrating hydrogen sulfide production. The medium at the base
of the slant became markedly blackened when the surface of the slant was
streaked with a hydrogen suljfide producing organism.

Newman,^* in a study of the detection of food poisoning attributable to dairy
products, used direct streaking on S S Agar as well as enrichment in Tetra-
thionate Broth followed by streaking on S S Agar and Bismuth Sulfite Agar for
the isolation of Salmonellae. In a study of methods to be used as a standard for
(the bacterial examination of puilorum reactors Jungherr, Hall and Pomeroy,^^ in
a committee report showed that in a comparative study of media and enrich-
ments from October, 1946 to February, 1950, Bismuth Sulfite Agar and S S Agar
permitted the highest number of specific isolations of S. puilorum and S. gal-
linarum. These favored selective media suppressed the growth of coliform or-
ganisms. Following enrichment of the specimens in Selenite Broth streaking
on Bismuth Sulfite Agar gave the largest number of positive isolations, followed
t)y S S Agar and then MacConkey Agar. Selenite Broth yielded a higher number
of successful isolations on follow-up media than did Tetrathionate Broth. The
highest percentage of organisms were isolated from the ovary, followed by gall
jbladder, peritoneum, oviduct, intestines and pericardial sac in the order listed.

The outstanding selectivity of Bacto-S S Agar permits the use of heavy inoculaj

DEHYDRATED CULTURE MEDIA 137

which should be evenly distributed over the entire surface of the medium. The
plates are incubated at 35-3 7 °G. for a full 24 hours, at which time at least three
or four of each type of suspected colonies are picked from each plate for
identification.

Bacto-S S Agar inhibits the formation of colonies of contaminating types but
does not destroy them. In the identification procedures it is necessary to have a
pure culture, and, for this reason, only the center of the colony should be picked
for transfer. If the suspected colonies are not well isolated, they should be puri-
fied by subculturing on some non-selective medium such as Bacto-MacConkey
Agar. Suspected colonies are transferred to appropriate differential tube media,
such as Bacto-Kligler Iron Agar, Bacto-Triple Sugar Iron Agar or Bacto-
Krumwiede Triple Sugar Agar, for ascertaining the group to which they belong.
The use of Bacto-Triple Sugar Iron Agar is particularly recommended as a single
medium for separation of the Salmonella and Shigella groups. Salmonella may
be further differentiated by their inability to produce indole from Bacto-Tryptone,
as the experience of recent investigators indicates that indole is not formed by
pathogenic Salmonella. The hydrolysis of urea as demonstrated on Urea Broth
or Urea Agar, as discussed on pages 170 and 171, may be used to identify Proteus
or Paracolobactrum (paracolon) organisms. Agglutination tests for further
identification of isolated strains may be run on organisms from any of these
differential tube media.

Bacto-S S Agar is particularly adapted for the isolation of Shigella and
Salmonella but as this is a selective medium, a non-inhibitive medium such as
Bacto-MacConkey Agar, which permits the growth of even the most fastidious
Gram negative intestinal pathogens, should be run in conjunction with it. If
typhoid is suspected, it is recommended that a poured plate of Bismuth Sulfite
Agar be run on each specimen. Enrichment of the specimen in Tetrathionate
Broth or Selenite Broth followed by streaking on Bismuth Sulfite Agar or S S
Agar is also recommended. This latter procedure is especially desirable in the
isolation of Salmonella.

A procedure designed to show the largest number of pathogens from a speci-
men would be:

A. Streak or smear a large inoculum on one plate of S S Agar and one plate of
Bismuth Sulfite Agar.

B. Streak or smear a light inoculum on one plate of MacConkey Agar.

C. Prepare Bismuth Sulfite Agar poured plates with a 5 ml. and a one drop
inoculum.

D. Enrich for 12-18 hours in Tetrathionate Broth or Selenite Broth followed
by streaking on one plate of Bismuth Sulfite Agar or Brilliant Green Agar
and one plate of S S Agar.

Saccharose in 1 per cent concentration may be added to isolation media, such
as Bacto-S S Agar to permit the detection of certain members of the coliform
group which ferment saccharose more readily than lactose. This principle was de-
scribed by Holt-Harris and Teague^^ and has been employed by many other
bacteriologists. In some laboratories pathogenic significance is assigned to these
organisms, and under such conditions, saccharose should not be added to the
medium.

To rehydrate the medium, suspend 60 grams of Bacto-S S Agar in 1000 ml. of
cold distilled water and heat to boiling to dissolve the medium completely. Do
not sterilize in the autoclave. About 20 ml. of the medium should be poured into
standard petri dishes of 90-100 mm. in diameter. It is important that the surfaces
of the medium be quite dry when inoculated and this may be accomplished by
allowing the medium to solidify and to stand for about 2 hours with the covers

138 DIFCO MANUAL

of the plates partially removed. The final reaction of the medium will be pH 7.0.
One pound of Bacto-S S Agar will make 7.4 liters of the final medium.

1 Apartado De Los Archives Uruguayos De ^^ Am. J. Pub. Health, 36:51:1946.
Medicina, 18:485:1941. "Am. J. Digestive Diseases, 7:229:1944.

2 Public Health Reports, 57:521, 524:1942. is Standard Methods for the Examination of
8 J. Lab. Clin. Med., 27:1081:1942. Dairy Products, 9th Edition: 166: 1948.

* Am. J. Pub. Health, 31:363:1941. ^^ Diagnostic Procedures and Reagents, 3rd Edi-
5 The Lancet, Vol. I, (XXIII) :677: 1942. tion:23:i950.

. Med. Assoc, 119:1402:1942. 20 Pub^ Health Reports, 65:1075:1950.

Health Reports, 56:2415:1944. 21 p^per read at Microbiological Congress, 1950.

* Public Health Reports, 60:1355:1945. 22 j, Bact., 54:1:1947.

8 Am. J. Diseases Children, 66:539:1943. ^3 guU, Johns Hopkins Hospital, 83:367:1948.

10 Rev. Chileana Hyg. Med. Prev., 4:353:1942. ^ J. Milk and Food Tech., 13:226:1950.

^ J. Pediatrics, 26:39:1945. ^ Proc. 22nd Ann. Meet. Northeastern Conf.
^ Can. J. Pub. Health, 36:401:1945. Lab. Workers in Pullorum Disease Control,

Am. Vet. Med. Assoc, 58:20:1946. Burlington, Vermont, June 20-21, 1950.

«J. Am.
'Public 1

"J.
"Pul

iblic Health Reports, 62:806:1947. ^e j^ Infectious Diseases, 18:596:1916.

15 Am, J. Pub. Health, 33:127:1943.

BACTO

DESOXYGHOLATE CITRATE AGAR (B274)

DEHYDRATED

Pork, Infusion from 330 g.

Proteose Peptone No. 3, Difco .... 10 g.

Bacto-Lactose 10 g.

Sodium Citrate 20 g.

Ferric Ammonium Citrate 2 g.

Sodium Desoxycholate 5 g.

Bacto-Agar 13.5 g.

Bacto-Neutral Red 0.02 g.

Bacto-Desoxycholate Citrate Agar, a modification of the original Leifson^
formula, is a selective medium for use in the isolation of enteric pathogens. It is
recommended for use in all cases wherein a selective Desoxycholate Citrate Agar
is specified or desired. The use of Desoxycholate Citrate Agar is recommended
as a plating procedure for examination of specimens for evidence of infection
with Salmonella and Shigella as given in "Diagnostic Procedures and Reagents"^
of the American Public Health Association.

On Desoxycholate Citrate Agar the growth of coliform bacteria is inhibited
or greatly suppressed. Gram-positive bacteria are generally inhibited. Salmonella
and Shigella organisms grow quite unrestricted. The selectivity of this medium
permits the use of fairly heavy inocula without danger of overgrowth of the
Shigella and Salmonella by extraneous organisms. Occasionally, however, coli-
form strains are encountered that persist on Desoxycholate Citrate Agar. Such
strains, if present in large numbers, produce acid from the lactose, precipitate the
bile salt, and give an opaque red medium which makes it diflficult to detect the
pathogens. Distribution of the inoculum over the surface of the medium to give
a sparsely populated section helps to insure against complete masking of the
pathogens by such coliform organisms. Inoculated plates are incubated at 37 °C.
for 24 hours.

Organisms which grow on Desoxycholate Citrate Agar but which do not
ferment lactose produce colorless raised colonies. Salmonella typhosa produces
translucent colonies with a bluish cast. Other Salmonella colonies are large,
opaque and may possess a brownish center. Shigella produce opaque ground-
glass appearing colonies with even margins. Coliform organisms which persist
on Desoxycholate Citrate Agar form raised, even, red colonies that are often
surrounded by a red halo of precipitated bile salt.

DEHYDRATED CULTURE MEDIA 139

For the routine examination of stool and urine specimens it is recommended
that Bacto-MacConkey Agar, a non-selective medium, and Bacto-Bismuth Sulfite
Agar or Bacto-S S Agar be run in conjunction with Bacto-Desoxycholate Citrate
Agar.

Saccharose in 1 per cent concentration may be added to isolation media, such
as Bacto-Desoxycholate Citrate Agar to permit the detection of certain members
of the coliform group which ferment saccharose more readily than lactose. This
principle was described by Holt-Harris and Teague^ and has been employed by
many other bacteriologists. In some laboratories, pathogenic significance is
assigned to these organisms, and under such conditions, saccharose should not be
added to the medium.

To rehydrate the medium, suspend 70 grams of Bacto-Desoxycholate Citrate
Agar in 1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. Do not sterilize the medium in the autoclave. Pour the medium into
petri dishes and allow the surface to dry for two hours with the covers partially
removed before inoculation. The final reaction of the medium will be pH 7.5.

One pound of Bacto-Desoxycholate Citrate Agar will make 6.5 liters of
medium.

1 J. Path. Bact., 40:581:1935- Edition: 212: 1950. „ ^ c

2 Diagnostic Procedures and Reagents, 3rd ^ j. Infectious Diseases, 18:596:1916.

BACTO

BISMUTH SULFITE AGAR (B73)

DEHYDRATED

Bacto-Beef Extract 5 g.

Bacto-Peptone 10 g.

Bacto-Dextrose 5 g.

Disodium Phosphate 4 g.

Ferrous Sulfate 0.3 g.

Bismuth Sulfite Indicator 8 g.

Bacto-Agar 20 g.

Bacto-Brilliant Green 0.025 g.

Bacto-Bismuth Sulfite Agar, a modification of the Wilson and Blair formula,
is a highly selective medium designed especially for the isolation of Salmonella
typhosa from feces, urine, sewage and other materials harboring this organism.
It is of special value in detecting typhoid carriers and in checking cases before
release. It is also satisfactory for the isolation of other members of the Salmonella
group, particularly after preliminary enrichment in Tetrathionate Broth. The use
of Bismuth Sulfite Agar is specified in "Standard Methods for the Examination
of Dairy Products"^ for the isolation of pathogenic bacteria from cheese, and in
"Diagnostic Procedures and Reagents"^ of the American Public Health Associa-
tion for the examination of specimens for evidence of infection with Salmonella
and Shigella.

The early history of the development of Bismuth Sulfite Agar may be found
in the reports of Wilson, ^-^ and Wilson and Blair^'^''^ who clearly demonstrated
the superiority of this type of medium over other media in the isolation of ty-
phoid. The unusually interesting claims of the proponents, and the many enthu-
siastic reports of its successful use by other investigators, stimulated us in an
endeavor to prepare this medium in the dehydrated form. The result of this in-
vestigation was Bacto-Bismuth Sulfite Agar.

Bacto-Bismuth Sulfite Agar closely approaches the ideal medium for the isola-
tion of S, typhosa from feces, urine, sewage and other infectious materials. Upon

140 DIFCO MANUAL

this medium the typhoid organism grows luxuriantly, forming characteristic black
colonies, while the Gram-positive bacteria and members of the coliform group
are inhibited. This unique inhibitory action of Bacto-Bismuth Sulfite Agar toward
Gram-postive and coliform organisms permits the use of a much larger inoculum
than has been possible with other media employed for similar purposes in the
past. The use of larger inocula greatly increases the possibility of recovering the
organisms, especially when they are present in relatively small numbers, such as
may be encountered in the early course of the disease or in the checking of car-
riers and releases.

Cope and Kasper^ increased their positive findings of typhoid from 1.2 to 16.8
per cent among food handlers and from 8.4 to 17.5 per cent among contacts by
the use of Bacto-Bismuth Sulfite Agar. Employing this medium in the routine
laboratory examination of fecal and urine specimens these same authors^ obtained
40 per cent more positive isolations of S. typhosa than were obtained on Endo
Medium. Gunther and Tuft,^° employing various media in a comparative way
for the isolation of typhoid from stool and urine specimens, found Bacto-Bismuth
Sulfite Agar most efficient. Upon this medium they obtained 38.4 per cent more
positives than on Endo, 38 per cent more positives than on Eosin Methylene Blue
Agar, and 80 per cent more positives on Bismuth Sulfite Agar than on the des-
oxycholate media. These workers found Bacto-Bismuth Sulfite Agar to be supe-
rior to Wilson's original medium, being easier to prepare, relatively more stable
and more sensitive. Green and Beard,^^ using Bacto-Bismuth Sulfite Agar in their
studies on the "Survival of E. typhi in Sewage Treatment Plant Processes,"
claimed that this medium so successfully inhibited sewage organisms that their
interference was negligible. Beard^^ stated that such a highly selective medium as
Bismuth Sulfite Agar made possible the study of the survival of typhoid in nature.

Since these earlier references to the use of Bismuth Sulfite Agar, this medium
has been generally accepted as routine for the detection of incitants of enteric
disease. The value of the medium is demonstrated by the many references to the
use of Bismuth Sulfite Agar in scientific publications, laboratory manuals and
texts.

As surface and subsurface colonies on Bismuth Sulfite Agar are strikingly char-
acteristic, it is possible to use the medium both as a smear plate and as a poured
plate in the isolation of ^S". typhosa. Smear plates are prepared by pouring 15-20
ml. quantities of the medium into sterile petri dishes (90 mm.) and allowing the
medium to solidify with the cover removed to obtain a dry surface. In preparing
poured plates the inoculum is placed in the sterile petri dish and the dissolved
medium at 45 °C. is added and mixed in the usual manner.

The following technique is recommended for the isolation of typhoid organ-
isms from fecal specimens.

Method of Inoculation

Streak or Smear Plate

Streak or smear the surface of a plate with a heavy inoculum of the fecal
material in such a way that on some portion of the plate the inoculum will be
light, permitting the development of discrete colonies.

Poured Plate

(a) Transfer about 2 or more grams of the fecal material to a test tube, add
12-15 ml water, and mix well, being careful to break up all the larger particles
of the material. Specimens preserved in glycerol must be diluted with water to
reduce the glycerol content since S. typhosa in poured plates is inhibited in the
presence of 2 per cent glycerol.

DEHYDRATED CULTURE MEDIA 141

(b) Insert a loosely packed cotton plug, about 1 inch long^ into the tube, and
slowly force it down through the fecal mixture by means of a glass rod or pipette,
so that all the gross particles are carried to the bottom of the tube on the cotton
plug, and an opaque fluid rises through the cotton. A second cotton filtration may
be necessary, since it is essential that the supernatant fluid be free from gross par-
ticles. Such solid particles in the medium may support growth of the extraneous
organisms, giving pseudo-blackening which may be mistaken for typhoid colonies.

Some workers may prefer to allow the gross solid particles of fecal suspension
to settle by gravity instead of removing them by filtration with cotton. In such
cases it is not advisable to allow the suspension to stand longer than 30 minutes
in order to obtain a supernatant fluid free from gross particles. Other methods of
preparing fecal suspensions that will give a liquid free from gross solid suspended
material without removing typhoid may also be employed.

(c) Transfer about 5 ml. of the prepared fecal suspension to one petri dish
and 1 drop to a second dish. Add 20 ml. of Bismuth Sulfite Agar, cooled to
45 °C., to each dish, and mix thoroughly. It is necessary to use at least 20 ml. of
the medium to each 5 ml. of inoculum, for dilution of the medium beyond this
point will allow the development of extraneous fecal forms.

(d) Incubate at 37°C. and observe after 24 hours for typical colonies as de-
scribed below. Frequently typical colonies develop within 24 hours incubation;
however, in all cases the plates should be incubated for at least 48 hours to allow
the development of all typhoid strains, before considering the specimen negative.
Specimens containing only a small number of typhoid should show isolated col-
onies from the 5 ml. inoculum, while those specimens containing increasingly
large numbers of typhoid organisms should show isolated colonies from the 1
drop inoculum in the poured plate or on the smear plate.

In the examination of samples of urine, blood, sewage or other material, either
the poured plate or smear method with Bismuth Sulfite Agar may be used. It is
suggested that in examining blood specimens, the specimen first be inoculated
into a tube of broth and after preliminary incubation of 8-12 hours, smeared
onto the Bismuth Sulfite Agar plate.

Description of Colonies

Streak or Smear Plate

The typical discrete surface typhoid colony is black and is surrounded by a
black or brownish-black zone which may be several times the size of the colony.
By reflected light, preferably daylight, this zone exhibits a distinctly characteristic
metallic sheen. Plates heavily seeded with typhoid may not show this reaction
except possibly near the margin of the mass inoculation. In these congested areas,
typhoid frequently appears as small light green colonies. This fact emphasizes the
importance of inoculating plates in such a manner as to have some sparsely popu-
lated areas with discrete typhoid colonies.

Poured Plate

Well isolated subsurface typhoid colonies are circular, jet black and well de-
fined. The size of the black colony may vary from 1 to 4 mm. in diameter de-
pending upon the particular strain, length of incubation and position of the
colony in the agar. Only those colonies growing very close to the surface or on
the surface will show a decided black metallic sheen. Plates containing typhoid
too numerous to permit the development of individual colonies give a black plate
or a plate dotted with black areas. Plates with about three hundred to a thousand
typhoid colonies will exhibit this appearance. When typhoid develops in a plate

142 DIFGO MANUAL

in still larger numbers, typical blackening does not occur and the appearance is
that of a negative plate.

Ordinarily typhoid will develop well isolated colonies showing typical round
jet black colonies with or without sheen, from either the 5 ml. or 1 drop inocula-
tion of cotton-filtered fecal suspension using the poured plate method. However,
the typhoid organisms developing from the specimens containing large numbers
of typhoid may be so numerous that the blackening cannot occur typically and
the plate may appear dotted black or greenish gray. From such heavily seeded
specimens the direct smear on Bismuth Sulfite Agar from feces should demon-
strate typhoid, while the poured plate should give positive results from specimens
containing lesser numbers of typhoid.

Description of Colonies Other Than Typhoid

S. schottmuelleri [Paratyphoid B) and S. enteritidis grow luxuriantly upon
Bacto-Bismuth Sulfite Agar forming black surface and subsurface colonies slightly
more moist, but otherwise similar to those produced by S. typhosa.

S. paratyphi [Paratyphoid A), S. typhimurium, S. choleraesuis and Proteus
morganii develop upon Bacto-Bismuth Sulfite Agar, yielding fiat or only slightly
raised green colonies.

Generally, the members of the dysentery group other than Flexner and Sonne
are inhibited. The Flexner and Sonne strains that do develop upon this medium
produce brownish raised colonies with depressed centers and exhibit a crater-like
appearance.

Coli is usually completely inhibited. Occasionally a strain will be encountered
that will develop small black, brown or greenish glistening surface colonies. This
color is confined entirely to the colony itself and shows no metallic sheen. Like-
wise a few strains of aerogenes may develop on this medium forming raised,
mucoid colonies. These may exhibit a silvery sheen, appreciably lighter in color
than that produced by typhoid. Subsurface colonies of the coliform group, when
they develop, are green or brown in color, generally lenticular in shape, and not
at all to be confused with the typical round black typhoid subsurface colony.
There are some members of the coliform group capable of producing hydrogen
sulfide that may develop on the medium, giving colonies similar in appearance
to typhoid. These may readily be differentiated in that they produce gas from
lactose in differential media — Bacto-Russell Double Sugar Agar, Bacto-Kligler
Iron Agar or Bacto-Triple Sugar Iron Agar, for example. The hydrolysis of urea
as demonstrated on Bacto-Urea Broth or Bacto-Urea Agar, as discussed on pages
170 and 171, may be used to identify proteus or paracolon organisms.

The isolation and purification of S. typhosa for agglutination or fermentation
studies may be readily accomplished by picking characteristic black colonies from
smeared or poured plates of Bismuth Sulfite Agar, and subculturing them upon
Bacto-MacConkey Agar. The purified colonies thus obtained may then be picked
to differential tube media such as Bacto-Russell Double Sugar Agar, Bacto-
Kligler Iron Agar, Bacto-Triple Sugar Iron Agar or other satisfactory differential
media for partial identification. Agglutination tests may be made from the fresh
growth on the differential tube media or from the growth on Nutrient Agar slants
inoculated from the differential media. The growth on the differential tube media
may also be used for inoculating carbohydrate media for fermentation studies. It
is a common practice among many bacteriologists to pick colonies typical of
S. typhosa directly from Bismuth Sulfite Agar onto the differential tube media.
This may be permissible if the colonies are discrete and well isolated, but it must
be remembered that although coliform bacteria are inhibited they are not de-
stroyed by the medium.

DEHYDRATED CULTURE MEDIA 143

It is recommended that Bacto-MacConkey Agar, as discussed on page 131, a
non-selective medium, and Bacto-S S Agar, a selective medium, page 134, sup-
porting luxuriant growth of all Shigella and Salmonella strains, be used in con-
junction with Bacto-Bismuth Sulfite Agar for the routine examination of stool
and urine specimens. Bacto-Tetrathionate Broth Base, page 157, a fluid enrich-
ment medium for Salmonella, is also recommended for use in conjunction with
Bacto-Bismuth Sulfite Agar. Results in our laboratory show that the number of
positive isolations obtained from Tetrathionate Broth enrichment is decidedly
greater when the enriched specimen is plated on Bismuth Sulfite Agar than on
MacConkey Agar or S S Agar. The value of Bismuth Sulfite Agar as a plating
medium subsequent to enrichment was also demonstrated by Hajna and Perry.^^

A procedure designed to show the largest number of pathogens from a speci-
men would be:

A. Streak or smear a large inoculum on one plate of S S Agar and one plate
of Bismuth Sulfite Agar.

B. Streak or smear a light inoculum on one plate of MacConkey Agar.

G. Prepare Bismuth Sulfite Agar poured plates with a 5 ml. and a one drop

inoculum.
D. Enrich for 12-18 hours in Tetrathionate Broth, follow by streaking on one

plate of Bismuth Sulfite Agar and one plate of S S Agar.

A survey of the methods used in the collection and preservation of stool speci-
mens for the isolation and identification of Salmonella, Shigella and intestinal
protozoa by Felsenfeld^* showed that generally specimens were promptly plated.
Glycerol-saline was the most frequently used preservative. S S Agar was the most
popular plating medium. There was a decrease in the use of Endo Agar. Des-
oxycholate Agars were found to be more frequently used than Endo Agar. There
were more laboratories using the poured plate of Bismuth Sulfite Agar, Mac-
Conkey Agar and Brilliant Green Agar in 1949 than in 1944. The use of streaked
plates of Bismuth Sulfite Agar showed a decrease during this period if occasional
users were not considered. All but three laboratories were using dehydrated Bis-
muth Sulfite Agar. He stated that these changes point toward evaluation of
experience and economizing in public health laboratories. Newman,^^ in a study
of the detection of food poisoning attributable to dairy products, used direct
streaking on Bismuth Sulfite Agar as well as enrichment in Tetrathionate Broth
followed by streaking on Bismuth Sulfite Agar and S S Agar for the isolation of
Salmonellae. In a study of methods to be used as a standard for the bacterial ex-
amination of pullorum reactors Jungherr, Hall and Pomeroy^^ in a committee
report showed that in a comparative study of media and enrichments from
October, 1946 to February, 1950, Bismuth Sulfite Agar and S S Agar permitted
the highest number of specific isolations of S. pullorum and S. gallinarium. These
favored selective media suppressed the growth of coliform organisms. Streaking
on Bismuth Sulfite Agar, following enrichment of the specimens in Selenite Broth
gave the largest number of positive isolations, followed by S S Agar and then
MacConkey Agar. Selenite Broth yielded a higher number of successful isolations
on follow-up media than did Tetrathionate Broth. The highest percentage of
organisms were isolated from the ovary, followed by gall bladder, peritoneum,
oviduct, intestines and pericardial sac in the order listed.

To rehydrate the medium, suspend 52 grams of Bacto-Bismuth Sulfite Agar
in 1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. The medium should not be sterilized in the autoclave or by fractional
sterilization, since heating for a longer period than is necessary to dissolve the
medium destroys the selectivity of the medium. A uniformly correct medium may
be obtained at all times merely by dissolving the powder in water. Upon a

144 DIFGO MANUAL

medium prepared in this way, reactions typical of those described by Wilson and
Blair are routinely obtained. The characteristic precipitate present in the medium
should be evenly dispersed by twirling the flask just prior to pouring plates. Best
results are obtained when the medium is dissolved and used immediately. If it is
necessary to prepare the medium several days before using, it should be poured
into plates and stored in a cold moist atmosphere to prevent drying. The melted
medium should not be allowed to solidify in flasks and be remelted. The final
reaction of the medium will be pH 7.7.

One pound of Bacto-Bismuth Sulfite Agar will make 8.7 liters of medium.

1 Standard Methods for the Examination of » Am. J. Pub. Health, 28:1065:1938.
Dairy Products, 9th Edition: 165:1948. ^ ^^ J. Lab. Clin, Med., 24:461:1939.

2 Diagnostic Procedures and Reagents, 3rd Edi- "Am. J. Pub. Health, 28:762:1938.

tion: 212: 1950. ^ J. Am. Water Works Assoc, 30:124:1938.

3J, Hyg., 21:392:1923. 13 J. Lab. Clin. Med., 23:1185:1938.

* Brit. Med. J., 1:1061:1928. i* Public Health Reports, 65:1075:1950.

^ J. Path. Bact., 29:310:1926. ^^ J. Milk and Food Tech., j 3:^26: 1950

Hyg., 26:374:1927. i^Proc. 22. Ann_^ Mtg. N. E. Cqnf. Lab. Work-

Hyg., 31:139:1931- ers P "

Bact., 34:565: 1937- 1950.

Hyg., 31:139:1931. ers Pullorum Disease Control Burlington, Vt.

165:1937-

BACTO

BRILLIANT GREEN AGAR (B285)

DEHYDRATED

Bacto- Yeast Extract 3 g.

Proteose Peptone No. 3, Difco . . 10 g.

Sodium Chloride 5 g.

Bacto-Lactose 10 g.

Saccharose, Difco 10 g.

Bacto-Phenol Red 0.08 g.

Bacto-Brilliant Green 0.0125 g.

Bacto-Agar 20 g.

Bacto-Brilliant Green Agar is a highly selective medium recommended for the
isolation of Salmonella, other than typhosa, directly from stools or other ma-
terials suspected of containing these organisms, or after preliminary enrichment
in Tetrathionate Broth.

The use of a Brilliant Green Agar as a primary plating medium for the isola-
tion of Salmonella was first described by Kristensen, Lester, and Jurgens^ who
reported it useful for the differentiation of "paratyphoid B" from other intestinal
Gram-negative bacilli. Later, Kauffmann^ modified their formula and used the
Brilliant Green Agar in conjunction with a Tetrathionate Broth for the isolation
of Salmonella from stools. Galton and Quan^ increased their positive Salmonella
findings by 164 per cent by the use of Tetrathionate Broth and plating on Bril-
liant Green Agar. Broh-Kahn* and Edwards^ similarly employed the Kauffmann
modification of Brilliant Green Agar with superior results.

Bacto-Brilliant Green Agar is a slight modification of the medium as described
by Kauffmann. The outstanding selectivity of this medium permits the use of
moderately heavy inocula, which should be evenly distributed over the surface.
Inoculation with heavy suspensions of stools or other materials suspected of con-
taining Salmonella usually results in an almost pure culture of these organisms.
Growth of other bacteria is almost completely inhibited. Following incubation at
37°G. for 18-24 hours the plates are examined for typical Salmonella colonies.
These appear as slightly pink-white opaque colonies surrounded by a brilliant red
medium. The few lactose or sucrose fermenting organisms which may develop
on the medium are readily differentiated due to the formation of a yellow-green

DEHYDRATED CULTURE MEDIA 145

colony surrounded by an intense yellow-green zone. Bacto-Brilliant Green Agar
is highly recommended for the isolation of Salmonella. However, it is not suitable
for the isolation of S. typhosa or Shigella organisms. Some strains of S. typhosa
will develop on this medium forming colonies identical to other Salmonella.
Some strains of Proteus may also grow forming red colonies. In the routine exam-
ination of stools, rectal swabs or other materials for the Gram-negative intestinal
pathogens, other primary plating media such as Bacto-S S Agar, Bacto-Bismuth
Sulfite Agar and Bacto-MacConkey Agar, as well as fluid enrichments such as
Bacto-Tetrathionate Broth and Bacto-Selenite Broth, should be used with Bacto-
Brilliant Green Agar.

To rehydrate the medium, suspend 58 grams of Bacto-Brilliant Green Agar in
1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°G.). A longer period of sterilization will tend to de-
crease the selectivity of the medium.

The final reaction of the medium will be pH 6.9.

One pound of Bacto-Brilliant Green Agar will make 7.8 liters of medium.

iBrit. J. Exp. Path., 6:291:1925. * Military Surgeon, 99:770:1946.

3Zeit. Hyg., 117:26:1935. ^Personal Communication, 1947.

"Am. J. Pub. Health, 34:1071:1944.

BACTO

LITMUS LACTOSE AGAR (B81)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Peptone 5 g.

Bacto-Lactose 10 g.

Bacto-Agar 10 g.

Bacto-Litmus 1 g.

Bacto-Litmus Lactose Agar has been used in water bacteriology and in othcf
examinations for the detection of members of the coliform group. This medium
may also be used for the determination of the fermentation of lactose with the
production of acid and gas.

Litmus Lactose Agar, originally described by Wurtz in 1892, is one of the"
oldest of the differential plate media. It has been employed in the past and is still
being used, to a slight extent, in water bacteriology and other examinations for
the detection of members of the coliform group. Upon this medium, colonies of
the lactose fermenting bacteria are surrounded by a red zone which distinguishes
them from colonies of other organisms that either do not change the surrounding
medium or make it more intensely blue due to production of ammonia.

Bacto-Litmus Lactose Agar is prepared according to the generally accepted
formula for this medium; when made up for use it contains 1 per cent of lactose
and 0.1 per cent of Bacto-Litmus.

Bacto- Violet Red Bile Agar as discussed on page 61 is recommended for use
in making coliform counts of water, milk, dairy and other food products. Bacto-
Brilliant Green Bile Agar as discussed on page 52 is also a satisfactory medium
for counts of the coliform bacteria in water.

To rehydrate the medium, suspend 29 grams of Bacto-Litmus Lactose Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°G.). The final reaction of the medium will be pH 7.0.

One pound of Bacto-Litmus Lactose Agar will make 15.6 liters of medium.

146 DIFCO MANUAL

BACTO

PURPLE LACTOSE AGAR (B82)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Peptone 5 g.

Bacto-Lactose 10 g,

Bacto-Agar 10 g.

Bacto-Brom Cresol Purple 0.025 g.

Bacto-Purple Lactose Agar like Litmus Lactose Agar has been used in water
bacteriology and in other examinations for the detection of members of the coli-
form group. This medium may also be used for the determination of the fer-
mentation of lactose with the production of acid and gas.

The disadvantages resulting from the use of litmus in culture media are well
known. Litmus has been replaced quite generally by the more stable and sensitive
sulfonephthalein indicators which are more sensitive to slight changes in hydro-
gen ion concentration.

Bacto-Purple Lactose Agar duplicates the formula of Bacto-Litmus Lactose
Agar except that brom cresol purple has been substituted for the litmus in order
to provide a more satisfactory differential between colonies of the lactose fer-
menting bacteria and those which do not attack lactose. The color change of
this indicator is from blue-purple (alkaline) to yellow (acid). The medium is
used for the same purposes and in the same manner as Litmus Lactose Agar.
Inoculated plates should be observed frequently after the first 18-24 hours in-
cubation to avoid confusing results due to diffusion of the acid through the
medium.

Bacto-Violet Red Bile Agar as discussed on page 61 is recommended for use
in making coliform counts of water, milk, dairy and other food products. Bacto-
Brilliant Green Bile Agar as discussed on page 52 is a most satisfactory medium
for coimts of the coliform bacteria in water.

To rehydrate the medium, suspend 29 grams of Bacto-Purple Lactose Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure ( 121°C.). The final reaction of the medium will be pH 6.8.

One pound of Bacto-Purple Lactose Agar will make 15.6 liters of medium.

BACTO

CONRADI DRIGALSKI AGAR (B83)

DEHYDRATED

Bacto-Peptone 10 g

Bacto-Isoelectric Casein 10 g

Bacto-Lactose 10 g,

Sodium Chloride 5 g

Bacto-Agar 15 g

Bacto-Brom Cresol Purple 0.03 g.

Bacto-Crystal Violet 0.004 g

Bacto-Conradi Drigalski Agar has been rather extensively employed in the past
for the isolation of typhoid and other intestinal pathogens from fecal specimens.
Media described subsequent to Conradi Drigalski Agar have proved more satis-
factory, and are now recommended for this purpose. For the present we will
continue to carry Bacto-Conradi Drigalski Agar for those laboratories where it

DEHYDRATED CULTURE MEDIA 147

has been in routine use, but for the isolation of members of the Salmonella and
Shigella groups we recommend the following media :

Bacto-MacConkey Agar, as discussed on page 131.
Bacto-S S Agar, as discussed on page 134.
Bacto-Bismuth Sulfite Agar, as described on page 139.
Bacto-Tetrathionate Broth, as discussed on page 157.
Bacto-Selenite Broth as discussed on page 158.

The differential restraining action exerted by certain dyes on the growth of
bacteria is a well-known property, and is utilized frequently in culture media.
Crystal violet, for example, when present in a dilution of 1:250,000 in agar
media, inhibits quite generally the development of Gram-positive organisms,
but has no appreciable effect on the growth of Gram-negative bacteria. This
property is employed in the medium of Conradi and Drigalski^ for the isolation
of the Gram-negative intestinal bacteria from contaminated material such as
water, feces, etc.

Bacto-Conradi Drigalski Agar is a dehydrated culture medium, prepared to
duplicate the original formula. Crystal violet is retained because of its selective
action, but the litmus which was originally used as the indicator has been re-
placed with Bacto-Brom Cresol Purple. This indicator is much more satisfactory
than litmus. Colonies fermenting the lactose in the medium are surrounded by
a yellow zone. Bacto-Isoelectric Casein, prepared in our own laboratories, has
been substituted for the Nutrose specified in the original formula.

To rehydrate the medium, suspend 50 grams of Bacto-Conradi Drigalski Agar
in 1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in tubes or flasks and sterilize by autoclaving for 15
minutes at 15 pounds pressure (121°C.). The final reaction of the medium will
be pH 6.8.

One pound of Bacto-Conradi Drigalski Agar will make 9 liters of medium.
^Zeit. Hyg., 39:283:1902.

BACTO

DEXTROSE PROTEOSE NO. 3 AGAR (B68)

DEHYDRATED

Proteose Peptone No. 3, Difco 20 g.

Bacto-Dextrose 2 g.

Sodium Chloride 5 g.

Bacto-Agar 13 g.

Bacto-Dextrose Proteose No. 3 Agar, used with Bacto-Tellurite Blood Solution
is suggested as a selective medium for the detection and isolation of Coryne-
bacterium diphtheriae. The medium is of particular value in the release of
patients and in the detection of diphtheria carriers. This medium inhibits the
streptococci and staphylococci, but permits C. diphtheriae to grow. Without
added tellurite and blood this medium is recommended as a general laboratory
medium containing 0.2 per cent dextrose.

In 1912 Conradi and Troch^ described a selective serum medium, contain-
ing tellurite, used for the isolation of C. diphtheriae. On their medium C. diph-
theriae colonies were coal black due to the reduction of the tellurite. Since then
many methods for the use of potassium tellurite in media have been described
for the isolation and identification of C. diphtheriae. Clauberg^ in 1929 de-
scribed a serum medium containing glycerol and potassium tellurite for growth
of C. diphtheriae from nose and throat smears. Anderson, Happold, McLeod,

148 DIFCO MANUAL

and Thompson^ described a heated-blood agar tellurite medium, having the ad-
vantage of giving a differentiation of gravis, mitis and intermediate strains. Mor-
gan and Marshall^ described a Blood Tellurite Agar which gave a high proportion
of positive results, and also permitted differentiation between gravis and mitis
types. Wilson^ described a blood agar tellurite arsenate selective medium for C.
diphtheriae and used a preserved laked blood. McGuigan and Frobisher^ and
Frobisher^ have used a Cystine Tellurite Blood Agar for isolation of C. diph-
theriae. Hall^ described a medium prepared from Bacto-Cabbage Infusion Agar,
10 per cent defibrinated blood, and potassium tellurite for the isolation and
identification of C. diphtheriae. Many other investigators have described similar
media using tellurite to inhibit Gram-positive organisms and permit the develop-
ment of C. diphtheriae. The advantage of these media lies in the fact that C.
diphtheriae , if present in relatively small numbers, would not be overgrown by
streptococci, staphylococci or other contaminating forms.

Bacto-Dextrose Proteose No. 3 Agar, enriched with Bacto-Tellurite Blood
Solution, produces a selective medium for C. diphtheriae. Staphylococci and
streptococci are generally inhibited, while the diphtheria bacilli develop black
or grayish black colonies in 18-24 hours. Occasionally a strain of staphylo-
coccus is encountered which grows on this medium, producing black colonies.
These, however, may be readily recognized as cocci by microscopic examination.
Confirmation of typical colonies is made by microscopic examination of the
cells and by testing their fermentation reactions. The morphology of C. diph-
theriae grown upon tellurite medium varies somewhat from that obtained upon
Loeffler Medium. On the selective medium the organisms are generally club
shaped, many are barred and only a few show the bipolar staining characteristic
of C. diphtheriae on Loeffler Medium. Confirmation may be made by transfer-
ring the black suspected colony to Loeffler Medium on which typical morphology
can be demonstrated as soon as there has been sufficient growth.

Wilson^ used a preserved laked blood in the preparation of his medium. Neil^
likewise used this preserved laked blood very satisfactorily in his Tellurite Choc-
olate Agar. Bacto-Tellurite Blood Solution, as discussed on page 277 contains
potassium tellurite in blood so that upon the addition of 5 ml. to 100 ml. of sterile
Dextrose Proteose No. 3 Agar a selective medium for C. diphtheriae, will be
obtained. The use of 4 ml. of Bacto-Tellurite Blood Solution per 100 ml. of agar
will give a less selective medium, while 6 ml. will produce a more inhibitive
medium. For ordinary purposes 5 ml. of the Tellurite Blood Solution will give
satisfactory selectivity without marked inhibition of the diphtheria bacillus.

Chocolate tellurite media prepared from Bacto-Dextrose Proteose No. 3 Agar
and Bacto-Tellurite Blood Solution are recommended for a variety of purposes
including the diagnosis of diphtheria, typing of strains and determination of
virulence and are especially useful in release of cases and detection of carriers.
It is recommended that some non-selective medium, Loeffler's for example, be
run in conjunction with the selective tellurite medium for detection of C. diph-
theriae in acute cases.

To rehydrate the medium, suspend 40 grams of Bacto-Dextrose Proteose No. 3
Agar in 1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). Add 5 ml. of Bacto-Tellurite Blood Solution
to each 100 ml. of sterile melted agar at 70-80° C, under aseptic conditions.
Heat at 75-80° C. until it has the appearance of a "chocolate agar" and then
cool to 50° C. Distribute into sterile tubes or plates as desired. For best results
inoculate the medium the same day as prepared.

For a plain Dextrose Agar, use without the addition of Bacto-Tellurite Blood
Solution. Final reaction of the medium without enrichment will be pH 7.4.

DEHYDRATED CULTURE MEDIA 149

One pound of Bacto-Dextrose Proteose No. 3 Agar will make 11.3 liters of
medium.

1 Muench. Wochschr., 59:1652:1912. ^ J. Infectious Diseases, 59:22:1936.

2 Zentr. Bakt., 114:539:1929. ''J. Infectious Diseases, 60:09:1937.
Path. Bact., 34:667:1931. « Am. J. Pub. Health., 29:064:1959.

■i

Hyg., 32:544:1932. ® J- Hyg., 37:552: i937-

Path. Bact., 38:114:1934.

BACTO

MUELLER TELLURITE BASE (B264)

DEHYDRATED

Bacto-Casamino Acids, Technical . . 20 g.

Casein 5 g.

/-Tryptophane 0.05 g.

Potassium Dihydrogen Phosphate . . 0.3 g.

Magnesium Sulfate 0.1 g.

Bacto-Agar 20 g.

Bacto-Mueller Tellurite Base enriched with Bacto-Mueller Tellurite Serum is
recommended for the isolation, differentiation and identification of Corynebac-
terium diphtheriae. The basal medium and sterile enrichment are prepared ac-
cording to the formula given by Mueller and Miller.^

Many selective media containing tellurite have been described for the initial
cultivation of members of the Corynebacterium group. The medium as described
by Mueller and Miller is transparent, requires no heating following the addition
of the sterile tellurite enrichment, and may be prepared from standardized ma-
terials to give a uniform desired growth response. The availability of the compo-
nents of the medium in the form of the dehydrated base and sterilized enrich-
ment is pointed out by the authors making the preparation of the final medium
containing all the nutriments required by the Corynebacterium group and of the
correct selectivity an easy task. A discussion of Bacto-Mueller Tellurite Serum,
the enrichment used in preparing the transparent medium, is described on page
278.

The presence or absence of organisms consistent with the morphological char-
acteristics of C. diphtheriae in a suspected diphtheria case materially assists the
physician in making an accurate diagnosis. This medium was developed after 4
years of practical use of tellurite plate media in the detection of diphtheria
carriers. It has given excellent results in other laboratories in detecting diphtheria
organisms. It permits a differentiation of the mitis, gravis and intermedins types.
As described by Mueller and Miller easily visible confluent growth of either mitis
or gravis types of C. diphtheriae is obtained from a fresh case after 15-18 hours
incubation. Plates showing no growth should be incubated for an additional 24
hours. In 48 hours mitis colonies are 1.0-1.5 mm. in diameter, black and convex
with a glistening surface. Gravis types in contrast show flat irregular colonies with
a dull surface, slate gray in color and 2-3 mm. in diameter. Gravis types seldom
show typical "daisy head" colonies. The size of the colony and degree of darken-
ing increases with length of incubation. Intermedins colonies are pin point in
size in 24 hours and approximately 0.2-0.3 mm. in diameter after 48 hours incu-
bation. The colonies show little darkening but appear brownish gray with a white
background in a good light. Hoffman's bacillus and other diphtheroids grow on
the medium, often resembling mitis types. Cocci, non-diphtheroid bacilli and
yeast are generally inhibited. Cocci, when they do produce colonies, resemble
mitis types. Colonies or growth suggestive of diphtheria bacilli should be ex-

150 DIFGO MANUAL

amined microscopically to determine the presence of organism having Corynebac-
terial characteristics. Mueller and Miller state that the morphology of the various
types of C. diphtheriae on the tellurite medium is entirely uniform. They further
suggest that the diphtheria organisms developing on this medium must be tested
for virulence or toxin production.

About 20 ml. of the final medium are poured into plates. The surface of the
medium must be dry prior to inoculation; this is accomplished by allowing the
medium to cool and solidify with covers removed. The surface of the plate is
then streaked with the swab. A more selective medium for the initial cultivation
of Corynebacteria is described on page 147, prepared with Bacto-Dextrose Pro-
teose No. 3 Agar, enriched with Bacto-Tellurite Blood Solution.

To rehydrate the medium, suspend 45 grams Bacto-Mueller Tellurite Base in
1000 ml. of cold distilled water. Heat to boiling to dissolve the medium com-
pletely. Sterilize in the autoclave for 10 minutes at 10 pounds pressure (116°G).
Cool to 50°C. and add 25 ml. Bacto-Mueller Tellurite Serum. Mix thoroughly
avoiding formation of air bubbles and distribute by pouring 20 ml. quantities in
sterile 95 mm. plates. Final reaction of the medium will be pH 7.6.

One pound of Bacto-Mueller Tellurite Base will make 10.2 liters final medium.

1 J. Bact., 51:743:1946.

BACTO

MANNITOL SALT AGAR (B30)

DEHYDRATED

Bacto-Beef Extract 1 g.

Proteose Peptone No. 3, Difco . . 10 g.

Sodium Chloride 75 g.

<f-Mannitol, Difco 10 g.

Bacto-Agar 15 g.

Bacto-Phenol Red 0.025 g.

Bacto-Mannltol Salt Agar is a selective medium for the isolation of pathogenic
staphylococci. It is prepared according to the formula suggested by Chapman.^
Growth of most bacteria other than staphylococci is inhibited on this medium.

Koch^ reported that on solid media staphylococci were not inhibited by a con-
centration of 7.5 per cent sodium chloride. Chapman^ confirmed this observation
and noted that the addition of 7.5 per cent sodium chloride to Bacto-Phenol Red
Mannitol Agar gave a medium on which staphylococci that coagulated rabbit
plasma grew luxuriantly, producing colonies with yellow zones. Nonpathogenic
staphylococci on the contrary produced small colonies surrounded by red or
purple zones. Other bacteria were generally inhibited, making possible the use of
a heavy inoculation without danger of overgrowth. Chapman recommended
incubation for 36 hours at 37°C. In a study of the resistance of chronic
staphylococcal bovine mastitis to massive penicillin therapy McCuUoch^ stated
that the staphylococci responsible for the mastitis grew well and formed acid in
Phenol Red Mannitol Agar to which 7.0 per cent sodium chloride had been
added. Velilla, Faber and Pelczar* used Bacto-Mannitol Salt Agar for the isola-
tion of coagulase producing staphylococci from milk in bovine mastitis. They
recommended the use of both Bacto-Mannitol Salt Agar and Bacto-Staphylo-
coccus Medium No. 110, to insure maximum recovery of these organisms.

To rehydrate the medium suspend 111 grams Bacto-Mannitol Salt Agar in
1000 ml. cold distilled water, and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes

DEHYDRATED CULTURE MEDIA 151

at 15 pounds pressure (121°C.). The final reaction of the medium will be
pH 7.4.

One pound of Bacto-Mannitol Salt Agar will make 4.0 liters of medium.

1 J. Bact., 50:201:1945. 3 Am. J. Vet. Res., 8:173:1947.

aZentr. Bakt., I Abt. Orig., 149:122:1942. * Am. J. Vet. Res., 8:275:1947.

BACTO

STAPHYLOCOCCUS MEDIUM NO. 110 (B297)

DEHYDRATED

Bacto- Yeast Extract 2.5 g

Bacto-TryTDtone 10 g

Bacto-Gelatin 30 g,

Bacto-Lactose 2 g.

J-Mannitol, Difco 10 g,

Sodium Chloride 75 g.

Dipotassium Phosphate 5 g

Bacto-Agar 15 g

Bacto-Staphylococcus Medium No. 110 is a selective medium for the isolation
of staphylococci. In addition, pigment and coagulase production, Stone's method
for liquefaction of gelatin and fermentation of mannitol may be determined
directly on this medium without the necessity of making transplants for these
conformity tests. These characteristics, and the selective properties of the medium
due to its high sodium chloride content, make it of particular value for the
isolation of staphylococci, especially those suspected of being pathogenic or in-
volved in cases of food poisoning. The medium has the additional advantage
that, with few exceptions, pathogenic strains produce typical orange pigmented
colonies whereas nonpathogenic types produce white colonies, a relationship that
is not obtained on any other medium. The coagulase test should be made using
a subculture of the suspected colony in Brain Heart Infusion or a Brain Heart
Infusion suspension of the organism grown on Heart Infusion Agar slants.

Koch^ reported that staphylococci were not inhibited by a concentration of
7.5 per cent sodium chloride in solid niedia. Chapman^ noted that most bacteria,
other than staphylococci, were inhibited on such media and that pathogenic
staphylococci grew more luxuriantly than did nonpathogenic strains. He suggested
that 7.5 per cent sodium chloride be added to Bacto-Phenol Red Mannitol Agar
as a selective isolation medium for staphylococci. In a study of the resistance of
chronic staphylococcal bovine mastitis to massive penicillin therapy McCulloch^
stated that the staphylococci responsible for the mastitis grew well and formed
acid on Bacto-Phenol Red Mannitol Agar to which 7.0 per cent sodium chloride
had been added. Stone* described a culture medium on which food poisoning
staphylococci gave a gelatinase test, or were "Stone reaction" positive. Chapman,
Lieb and Curcio^ pointed out the nonspecificity of this single test and reported
that in addition to being "Stone reaction" positive, typical food poisoning
staphylococci must produce pigment, coagulate plasma, hemolyze rabbit blood
and ferment mannitol.

Continued study of the isolation of staphylococci by Chapman^ led to the
development of a medium designated as Staphylococcus Medium No. 110. In
addition to being selective for staphylococci due to the high sodium chloride
content, this medium is well suited for pigment formation, may be used for the
determination of the fermentation of mannitol, for the Stone type gelatinase test
and gives a growth satisfactory for the coagulase test. According to Chapman^

152 DIFGO MANUAL

and Chapman and Domingo^ staphylococci incriminated in food poisoning pro-
duce an orange pigment, coagulate plasma, ferment mannitol and give a
positive "Stone reaction" or gelatinase test on Staphylococcus Medium No. 110
when tested at the time of isolation. Velilla, Faber and Pelczar^ suggested the use
of both Mannitol Salt Agar and Staphylococcus Medium No. 110 to insure maxi-
mum recovery of coagulase producing staphylococci from suspected bovine
mastitis.

Evans^° in his studies of coagulase positive staphylococci reported that
pigmentation was much stronger on Staphylococcus Medium No. 110 than on
Infusion Agar and some showed pigment on Chapman's medium but were white
on the Infusion Agar. Newman,ii in a study of the detection of food poisoning
attributable to dairy products, used Staphylococcus Medium No. 100 and Chap-
man Stone Medium for the isolation of staphylococci. The coagulase test was run
to assist in the detection of pathogenic staphylococci. The cultures for the per-
formance of coagulase test were grown in Brain Heart Infusion.

Following streaking or smearing of the specimens on plates of Bacto-Staphylo-
coccus Medium No. 110 the medium is incubated at 30°C.i2 fQj. exactly 48 hours
or for exactly 43 hours at 37°C. The colonies are first observed for signs of the
production of any orange or yellow pigment. Such colonies are picked from the
surface of the medium and emulsified in 0.1-0.2 ml. of Brain Heart Infusion or
Bacto-Tryptose Phosphate Broth for the coagulase test. A drop of brom
cresol purple indicator is added to several of the areas from which typical pig-
mented colonies were removed. Any change in color of the indicator compared
with that of the uninoculated medium is indicative of fermentation of mannitol.
The plate is then flooded with 5 ml. saturated solution of ammonium sulfate kept
in the incubator and let stand 10 minutes. Any clear zone around areas from
which colonies have been picked, or around colonies, are gelatinase positive.

Chapman^^ recently reported that a 20 per cent solution of sulfosalicylic
acid (Chapman-Stone Developer) is superior to saturated ammonium sulfate
solution for the test. The plate is flooded with the solution, and at the end of
10 minutes, a clear zone around the colonies denotes a positive "Stone reaction."
The lytic zones stand out as clear areas in an opaque white background. Chap-
man^* has also described a method of applying confirmation tests directly to
colonies on the medium. Chapman tubes (10 mm. length and diameter) are
sterilized and pressed slightly into the surface of the medium. In the cup thus
formed is added a drop of 0.04 per cent brom thymol blue. Replace cover on
plate and let stand 10 minutes before reading result. The formation of a yellow
color indicated a positive reaction, while a green color denotes a negative reac-
tion. In a similar manner the "Stone reaction" may be determined by using a
19 mm. tube, placing it around the colony to be tested. Add 1 ml. of Chapman
Stone Developer (20 per cent sulfosalicylic acid) or saturated Ammonium Sul-
fate solution. Examine after standing 10 minutes. A clear zone around the colony
denotes a positive "Stone reaction."

To determine the coagulative power, use a 1 6-24 hour culture of the suspected
organism in Brain Heart Infusion or a Brain Heart Infusion suspension of a
16-24 hour culture on a slant of Heart Infusion Agar. Two drops of the culture
or suspension of the organism is added to 0.5 ml. of Bacto-Coagulase Plasma So-
lution. Incubate at 37 °C. Most coagulase positive staphylococci will clot Bacto-
Coagulase Plasma Solution within one hour. However, readings should be made
after two and three hours of incubation before discarding as negative. A positive
culture will show a definite clot often in 20-30 minutes. Chapman clearly states
that with few exceptions those colonies that produce pigment, coagulate plasma,
ferment mannitol and give a gelatinase reaction are food poisoning staphylococci.
According to Chapman^^ this type of staphylococcus may be isolated from only

DEHYDRATED CULTURE MEDIA 153

about 6 per cent of the population. A complete discussion of Bacto-Coagulase
Plasma is given on page 330.

To rehydrate the medium, suspend 149 grams of Bacto-Staphylococcus
Medium No. 110 in 1000 ml. cold distilled water and heat to boiling to dissolve
the medium completely. Distribute in tubes or flasks and sterilize in the autoclave
for 15 minutes at 15 pounds pressure (121°C.). When cool enough to handle,
but hotter than the usual pouring temperatures, disperse the precipitate by gentle
agitation, avoiding air bubbles and pour into plates. If plates are not used when
poured, keep in refrigerator in sealed cans. For field work the medium may be
boiled for 5 minutes without further sterilization and poured into the plates,
preferably while still hot. The final reaction of the medium will be pH 7.0.

One pound of Bacto-Staphylococcus Medium No. 110 will make 3 liters of
medium.

^ Zentr. Bakt. I Abt. Orig., 149:122:1942. ^ J. Bact., 51:405:1946.

2 J. Bact., 50:201:1945. 8 Am. J. Vet. Res., 8:275:1947.

3 Am. J. Vet. Res., 8:173:1947- ^^ J- Bact., 55:793:1948-

* Proc. See. Exp. Biol. Med., 33:185:1935. ^ J. Milk and Food Tech., 13:226:1950.

5 Food Research, 2:349:1937- ^ J- Bact., 53:367:1947-.

^ J. Bact., 51:409:1946. ^ Personal Communication.

'Trans. N. Y. Academy Sciences, 9:52:1946. ^^ J. Bact., 63:147:1952.

BACTO

CHAPMAN STONE MEDIUM (B313)

DEHYDRATED

Bacto- Yeast Extract 2.5 g.

Bacto-Tryptone 10 g.

Bacto-Gelatin 30 g.

£?-Mannitol, Difco 10 g.

Sodium Chloride 55 g.

Ammonium Sulfate 75 g.

Dipotassium Phosphate 5 g.

Bacto-Agar 15 g.

Bacto-Chapman Stone Medium is a selective medium for the isolation of
staphylococci, prepared according to the formula described by Chapman.^ This
medium is similar to Bacto-Staphylococcus Medium No. 110 except that am-
monium sulfate is included in the medium and the amount of sodium chloride
reduced to 5.5 per cent. Flooding the plate with ammonium sulfate solution is
therefore unnecessary for the determination of Stone's method for the liquefac-
tion of gelatin.

Sodium chloride is used as the selective agent in this medium. A complete
discussion of the use of high concentrations of this salt and details for the isola-
tion of staphylococci are given on page 151 under Bacto-Staphylococcus Medium
No. 110. In the examination of specimens, they are streaked on the surface of
the medium and incubated 48 hours at 30° G. Any yellow or orange colonies,
surrounded by a clear zone, which also ferment mannitol and are coagulase posi-
tive, are likely to be those of food poisoning or pathogenic staphylococci. White
or nonpigmented colonies, even though there may be a decided clear zone, are not
significant.

Colonies showing any pigment production are picked from the surface of the
medium and emulsified in 0.1-0.2 ml. of Brain Heart Infusion or Tryptose Phos-
phate Broth for the performance of the coagulase test. A drop of brom cresol
purple indicator is added to several areas from which typical pigmented colonies
were removed. Any change in color of the indicator compared with that of the
uninoculated medium is indicative of the fermentation of mannitol. To determine

154 DIFGO MANUAL

coagulase production, violently shake the colonies emulsified in Brain Heart In-
fusion for 15 minutes. Add 0.2 ml. of Bacto-Coagulase Plasma Solution. Incubate
at 37°C. for one hour. Most coagulase positive staphylococci will form a clot
within one hour; however, readings should be made after 2 and 3 hours incuba-
tion before being considered as negative. A complete discussion of Bacto-Co-
agulase Plasma is given on page 330.

Newman,^ in a study of the detection of food poisoning attributable to dairy
products, used Chapman Stone Medium and Staphylococcus Medium No. 110
for the isolation of staphylococci. The coagulase test was run to assist in the
detection of pathogenic staphylococci. The cultures for the performance of the
coagulase test were grown in Brain Heart Infusion.

To rehydrate the medium, suspend 20.2 grams of Bacto-Chapman Stone
Medium in 100 ml. cold distilled water and heat to boiling to dissolve the
medium completely. Distribute in tubes or flasks and sterilize in the autoclave
for 10 minutes at 15 pounds pressure (121°C.). For field work, or if the medium
is to be inoculated the same day as prepared, sterilization in the autoclave may
be omitted, heating the medium to effect complete solution is sufficient. Pour into
plates while the medium is hot, avoiding formation of bubbles. The medium
after solidification should be opaque and white. Final reaction of the medium will
be pH 7.0.

One pound of Bacto-Chapman Stone Medium will make 2.2 liters of medium.

1 Food Research, 13:100:1948.

* J. Milk and Food Tech., 13:226:1950.

BACTO

MITIS SALIVARIUS AGAR (B298)

DEHYDRATED

Bacto-Tr>'ptose 10 g

Proteose Peptone No. 3, Difco . 5 g,

Proteose Peptone, Difco 5 g

Bacto-Dextrose 1 g

Saccharose, Difco 50 g

Dipotassium Phosphate 4 g

Trypan Blue - 0.075 g

Bacto-Crystal Violet 0.0008 g

Bacto-Agar 15g

Bacto-Mitis Salivarius Agar is prepared according to the formula described by
Chapman^'- for the isolation of Streptococcus mitis, S. salivarius and enterococci.
Some bacteriologists refer to these organisms as "Streptococcus viridans" and
"nonhemolytic streptococci," respectively, because of their alpha and gamma
hemolysis on Blood Agar prepared from Bacto-Heart Infusion Agar or Bacto-
Tryptose Blood Agar Base. The final medium, containing Bacto-Chapman Tel-
lurite Solution, is highly selective for these organisms, making possible their isola-
tion from grossly contaminated specimens such as feces or exudates from
different body cavities.

Different methods have been employed for the isolation of streptococci and
enterococci from mixed cultures. Snyder and Lichstein^ and Lichstein and Sny-
der* used sodium azide as inhibiting agents for Gram-negative bacteria including
proteus. Chapman^ described a Tellurite Medium and an Azide Medium for
isolation of S. salivarius and S. mitis.

Chapman,^'2 continuing his studies, reported a complete and detailed method
for the isolation and testing for the pathogenicity of fecal streptococci. Decimal

DEHYDRATED CULTURE MEDIA 155

dilutions of the specimen are prepared and 0.01 ml. amounts spread by a glass
spreader, over the surface of the Mitis Salivarius Agar containing tellurite. Plates
are incubated for exactly 24 hours at 37 °C. S, mitis produces small or minute
blue colonies. Some S. mitis colonies may be more easily distinguished with a
longer incubation. S. salivarius produces blue, smooth or rough "gum drop" colo-
nies 1-5 mm. in diameter, depending on the number of colonies on the plate.
Enterococci form colonies dark blue or black in color, shiny, slightly raised, 1-2
mm. in diameter. These organisms, few of which are pathogenic, may be readily
differentiated from S. mitis and S. salivarius, particularly when viewed by re-
flected light. Beta hemolytic streptococci resemble S. mitis. Other types of
streptococci have not been studied on this medium. Chapman^ reported that
Erysipelothrix rhusiopathiae produce colorless circular convex colonies. He also
reported that when coliform organisms do grow they produce brown colonies;
however, generally they are not only inhibited but are actually killed. Spreaders
are rarely observed. Molds grow after 2 days incubation. Using this medium
Chapman was able to demonstrate pathogenic streptococci in about 95 per cent
of fecal specimens from chronic invalids. Pathogenicity of these streptococci was
determined culturally according to the method described by Chapman^ -^ using
hexylresorcinol.

Bacto-Mitis Salivarius Agar, to which Bacto-Chapman Tellurite Solution has
been added, duplicates the medium as described by Chapman.^-^ Comparative
tests have shown this medium to be satisfactory for the isolation of streptococci
and enterococci from grossly contaminated specimens such as body secretions and
excretions.

To rehydrate the medium, suspend 90 grams of Bacto-Mitis Salivarius Agar
in 1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Sterilize in the autoclave for 15 minutes at 15 pounds pressure (121°G.).
Cool to 50-55 °C. and just prior to pouring the plates add exactly 1.0 ml. of
Bacto-Chapman Tellurite Solution (discussed in detail on page 277). Prepare
plates with 25 ml. medium per 95 mm. diameter plate. Do not heat the medium
after the addition of the Tellurite Solution. Final reaction of the medium will
be pH 7.0.

One pound of Bacto-Mitis Salivarius Agar will make 5 liters of medium.

1 Am. J. Digestive Diseases, 13:105:1946. * J. Bact., 42:653:1941.

2 Trans. N. Y. Acad. Sciences, 10:45:1947. ^ J. Bact., 48:113:1944.
'J. Infectious Diseases, 67:113:1940. * Personal Communicatioa.

BACTO

AZIDE BLOOD AGAR BASE (B409)

DEHYDRATED

Bacto-Tryptose 10 g.

Bacto-Beef Extract 3 g.

Sodium Chloride 5 g.

Sodium Azide 0.2 g.

Bacto-Agar 15 g.

Bacto-Azide Blood Agar Base is a selective medium for the isolation of strepto-
cocci from stools, sewage and other specimens. It is also suggested as a selective
medium for the isolation of staphylococci. The medium may be employed with
the addition of blood permitting the production of typical hemolytic reactions by
the streptococci, or may be used without added blood.

The addition of sodium azide to culture media as a selective agent has been
suggested by a number of investigators. Edwards^ in 1933 used a liquid medium

156 DIFCO MANUAL

containing crystal violet and sodium azide as a selective broth in the isolation of
mastitis streptococci. Hartman^ reported the value of sodium azide as a selective
agent for the isolation of streptococci causing mastitis. Bryan, Devereux, Hirschey
and Corbett^ reported that sodium azide in a concentration of 1 : 5,000 was a
better selective preservative for milk cultures and gave more accurate results for
the microscopic and Hotis tests for Streptococcus mastitis than 1 : 50,000 brilliant
green. Snyder and Lichstein* and Lichstein and Snyder^ reported that sodium
azide in 0.01 per cent concentration in blood agar prevented the swarming of
proteus, and permitted the isolation of streptococci from known mixtures of
bacteria. The common Gram-negative bacteria were inhibited on this medium.
Packer^ modified Edwards' medium and prepared an Infusion Blood Agar con-
taining 1 : 15,000 sodium azide and 1 : 500,000 crystal violet for the study of bovine
mastitis and recommended it as a selective medium for the cultural examination
of pathogenic streptococci from human sources.

Mallmann, Botwright and Churchill" in studying the selective bacteriostatic
effect of slow oxidizing agents reported that sodium azide exerted a bacteriostatic
effect on Gram-negative bacteria and permitted the growth of Gram-positive
organisms. Packer^ used 1:15,000 sodium azide and 1:500,000 crystal violet in
a Blood Agar prepared with 1.5 per cent Bacto-Tryptose for the selective isola-
tion of Erysipelothrix rhusiopathiae. "Standard Methods for the Examination of
Dairy Products"^ specifies the use of sodium azide in agar media prepared with
Bacto-Tryptose for the isolation of pathogenic bacteria from cheese.

Sodium azide has also been used in liquid media for the detection of fecal
streptococci in stools, sewage, swimming pools and drinking water supplies. See
Bacto-S F Medium, page 46, Bacto-B A G G Broth, page 47, and Bacto-Azide
Dextrose Broth, page 48 for the discussion of these media.

To rehydrate the medium, suspend 33 grams of Bacto-Azide Blood Agar Base
ia 1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in tubes or flasks and sterilize in the autoclave for 15
minutes at 15 pounds pressure (121°C.). In the preparation of Azide Blood
A.gar, cool the sterile melted medium to 45-50°C. and add 5 per cent sterile
defibrinated blood, under aseptic conditions. Distribute in tubes or flasks.

One pound of Bacto-Azide Blood Agar Base will make 13.7 liters of medium.

i J. Comp. Path. Therap., 46:211:1933. « J. Bact., 42:138:1941.

3 Milchw. Forsch., 18:116:1936. ' J. Infectious Diseases, 69:215: 1941.

3 North Am. Vet., 20:424:1939. * J. Bact., 46:343:1943.

* J. Infectious Diseases, 67:113:1940. 8 Standard Methods for the Examination of

^}. Bact., 42:653:1941. Dairy Products, 9th Edition: 165: 1948.

Differential Liquid Enrichments

The use of selective enrichment media is a recommended procedure for aiding
in the isolation of Salmonella. Preliminary inoculation of the suspected sample
into liquid enrichments, followed by incubation for 18 hours before inoculation
on suitable plating media, materially increases the percentage of isolations of
Salmonella.

DEHYDRATED CULTURE MEDIA 157

BACTO

TETRATHIONATE BROTH BASE (B104)

DEHYDRATED

Proteose Peptone, Difco 5 g.

Bacto-Bile Salts 1 g.

Calcium Carbonate 10 g.

Sodium Thiosulfate 30 g.

Bacto-Tetrathionate Broth Base is a selective liquid enrichment medium
employed in the isolation of Salmonella typhosa and other members of the
Salmonella (paratyphoid) group from feces, urine, water, sewage and infected
materials such as eggs and other foodstuffs. Its use is particularly indicated in
the early stages of enteric disorders, in determining the release of patients, and in
detecting carriers of typhoid and paratyphoid organisms. The use of Tetrathio-
nate Broth is specified in "Standard Methods for the Examination of Dairy
Products"^ for the isolation of bacteria from cheese. "Diagnostic Procedures and
Reagents"^ modify the formula by the addition of Brilliant Green to the enrich-
ment as used in the examination of specimens for Salmonella and Shigella.

The credit for discovering the usefulness of a Tetrathionate Broth for enrich-
ing typhoid and the paratyphoid group is ascribed to Mueller^ who demonstrated
clearly that it inhibited or killed the coliform organisms and permitted typhoid
and the paratyphoids to grow almost unrestrictedly. Mueller obtained pure
cultures of typhoid by incubating mixtures containing few typhoid organisms and
infinite numbers of coli in his Tetrathionate Enrichment.

Kauffmann,*'^ using a modified Mueller's broth claimed to have increased
his positive isolations of S. typhosa over 30 per cent and of other members
of the Salmonella group from 100 to 700 per cent over that possible by direct
streaking onto plate media. Schaeffer,^ using Tetrathionate Broth also demon-
strated the greater efficiency of Tetrathionate Enrichment by detecting four
times as many typhoid and paratyphoid positive fecal specimens as could be
found by direct plating. Further demonstrations of the usefulness of Tetra-
thionate Enrichment were made by Jones,^ Ruys^ and by Szper,^ who effected a
technique for its use in examining large volumes of water and sewage material.
Newman^*^ in a study of the detection of food poisoning attributable to dairy
products, used Tetrathionate Broth followed by streaking to Bismuth Sulfite Agar
and S S Agar for the isolation of Salmonella.

The prepared broth is inoculated by adding 1-3 grams of stool, sewage, urine
or other infected material to 10 ml. of the medium and mixing with a swab,
glass rod or pipette to suspend the particulate matter. Many workers find it
advantageous to pass a loosely packed cotton plug down through the inoculated
broth to carry the coarser particles of fecal material to the bottom of the tube.
The inoculated medium is then incubated for 12-24 hours, and streaked out
upon Bacto-MacConkey Agar and Bacto-Bismuth Sulfite Agar. The inoculated
plates are incubated at 37°C. Examine the MacConkey Agar plates after 18-24
hours and the Bismuth Sulfite Agar plates at 24-48 hours for typical typhoid or
paratyphoid colonies. Confirm typical colonies on Bacto-Triple Sugar Iron Agar
or by other suitable means. In cases of suspected Salmonella infections other than
typhoid, some bacteriologists prefer to use Bacto-Brilliant Green Agar as a plating
medium after enrichment because of its selective reaction for these organismr.

To rehydrate the medium, suspend 4.6 grams of Bacto-Tetrathionate Broth
Base in 100 ml. of distilled water, and heat to boiling. Cool below 45 °C. Add
2.0 ml. of iodine solution.* Shake to mix, and tube in 10 ml. quantities, in sterile

*The iodine solution is prepared by dissolving 6 g. iodine crystals and 5 g. potassium iodide in
20 ml. of water.

158 DIFCO MANUAL

tubes, exercising care to obtain an even distribution of the insoluble material.
Do not heat after the iodine has been added. The complete medium containing
iodine should be used the day it is prepared, the base medium without the iodine
can be stored indefinitely after sterilization.

One pound of Bacto-Tetrathionate Broth Base will make 9.8 liters of base
medium.

1 Standard Methods for the Examination of ^Zeit. Hyg., 117:26:1935-36.

Dairy Products, gth Edition: 165:1948. « Zentr. Bakt., I Abt., Orig., 133:458:1935.

3 Diagnostic Procedures and Reagents, 3rd Edi- "^ J. Path. Bact., 42:455:1936.

tion: 212: 1950. 8 Brit. Med. J., 1:606:1940.

3 Compt. rend. soc. biol., 89:434:1923. ^ Comp. rend. soc. biol., 118:1675:1935.

*Zentr. Bakt., I Abt., Orig., 113:148:1930-31. ^o J. Milk and Food Tech., 13:226:1950.

BACTO

SELENITE BROTH (B275)

DEHYDRATED

Bacto-Tryptone 5 g.

Bacto-Lactose 4 g.

Disodium Phosphate 10 g.

Sodium Selenite 4 g.

Bacto-Selenite Broth is recommended as an enrichment medium in the
isolation of Salmonella typhosa and other members of the Salmonella group
from feces, urine and infected tissues. The formula of this medium is essentially
the same as that of Selenite F Broth described by Leifson.^ Selenite Broth, for
the enrichment of enteric pathogens, is described in "Diagnostic Procedures and.
Reagents."^

Handel and Theodorascu according to Guth^ observed that Escherichia coli
was much more susceptible to the toxicity of sodium selenite than was S. typhosa.
Guth^ confirmed the observations of these authors and employed sodium selenite
SIS a selective agent in an agar medium and in an enrichment broth for the
isolation of S. typhosa from feces. Leifson^ extended Guth's observations and
developed a Selenite Agar and a Selenite Broth for use in the isolation of typhoid
and paratyphoid bacilli from feces and urine and found the broth enrichment
to offer the greater promise.

Leifson showed that the Selenite Broth was not sufficiently toxic to inhibit
fecal coli and enterococci completely. However, the colon bacilli were reduced
in numbers during the first 8-12 hours and thereafter increased rapidly. The
typhoid bacilli on the other hand multiplied fairly rapidly from the start. Proteus
and pyocyaneus were not inhibited. Dysentery and alcaligenes were inhibited.
In the Selenite Broth the growth behavior of coli and typhoid in the presence of
feces or urine was similar to that of pure cultures.

Leifson observed that the selenite medium functioned most efficiently under
reduced oxygen tension. To provide optimal conditions, the broth was distributed
in tubes to give a depth of 2 inches or more. Using the enrichment under optimal
conditions Leifson was able to isolate many more typhoid and paratyphoid
organisms than by direct plating without primary enrichment.

In a survey of methods used for the collection and preservation of stool speci-
mens for the isolation and identification of Salmonella, Shigella and intestinal
protozoa, Felsenfeld* reported an increasing number of laboratories using the
Selenite Broth as an enrichment. In a study of methods to be used as a standard
for the bacterial examination of pullorum reactors, Jungherr, Hall and Pomeroy^
in a committee report showed that in a comparative study of media and enrich-
ments, from October, 1946 to February, 1950, Bismuth Sulfite Agar and S S Agar

DEHYDRATED CULTURE MEDIA 159

permitted the highest number of specific isolations of S. pullorum and S. galli-
narum. These favored selective media suppressed the growth of coliform organ-
isms. Following enrichment of the specimens in Selenite Broth streaking on
Bismuth Sulfite Agar gave the largest number of positive isolations, followed by
S S Agar and then MacConkey Agar. Selenite Broth yielded a higher number of
successful isolations on follow-up media than did Tetrathionate Broth. The
highest percentage of organisms were isolated from the ovary, followed by gall
bladder, peritoneum, oviduct, intestines and pericardial sac in the order listed.

For the examination of fecal specimens Selenite Broth is inoculated by adding
1-2 ml. of fecal suspension to tubes of 10-15 ml. of medium. After thorough
mixing, the tubes are incubated at 37°C. for 18-24 hours. For the examination
of infected tissues 1-2 grams of material are mascerated in 10-15 ml. Selenite
Broth by means of a sterile pipette or stirring rod before incubation. For exam-
ination of urine the Selenite Broth should be prepared in double strength and
tubed in 5-7.5 ml. amounts. This broth is inoculated with an equal volume of
urine sample and is incubated as described above. After incubation a loopful of
the enriched culture is streaked on one plate of Bismuth Sulfite Agar and a
similar amount is streaked on one plate of S S Agar. These plates are incubated
at 37 °C. and are examined after 18-24 hours. The Bismuth Sulfite Agar plate
should be incubated for 48 hours before being discarded as negative.

To rehydrate the medium, suspend 23 grams Bacto-Selenite Broth in 1000 ml.
of distilled water and heat to boiling. Distribute in sterile culture tubes to give a
depth of medium of at least 2 inches. Avoid excessive heating. Do not sterilize in
the autoclave. The final reaction of the medium will be pH 7.0.

One pound of Bacto-Selenite Broth will make 19 liters of medium.

1 Am. J. Hyg., 24:423:1936. * Public Health Reports, 63:1075:1950.

2 Diagnostic Procedures and Reagents, 3rd Edi- ^ Proc. 22nd Ann. Mtg. N. E. Conf. Lab. Work-
tion:25:i950. ers Pullorum Disease Control, Burlington, Vt.,

8 Centr. Bakt. I Abt. Orig., 77:487:1916. 1950.

Differential Tube Media

Differential tube media afford the bacteriologist a simple and effective cultural
means of identifying pure cultures of bacteria. Both solid media and liquid media
have been devised for this purpose. Many of the solid differential tube media
contain one or more fermentable carbohydrates and a suitable indicator for the
detection of acid or alkali production; some contain substances rich in sulfur
and an indicator to detect hydrogen sulfide formation; still others contain a
combination of both. The solid tube media containing fermentable carbohydrates
permit the study of both aerobic and anaerobic dissimilation processes by the
bacteria under study. Screw capped tubes are not satisfactory for solid differen-
tial media as pointed out by Marcus and Greaves^ unless the caps be placed on
the tubes loosely, or replaced by cotton plugs. The changes produced in the
media by an organism or group are characteristic, thus differentiating it from
other strains or groups. Differential semisolid media are used to demonstrate
motility and certain biochemical characteristics of microorganisms. Liquid differ-
ential tube media, in general, are employed in studying the fermentation reac-
tions of pure cultures of bacteria, or their ability to utilize certain nutriments.

1 J. Lab. Clin. Med., 36:134:1950.

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162 DIFCO MANUAL

BACTO

RUSSELL DOUBLE SUGAR AGAR (B84)

DEHYDRATED

Bacto-Beef Extract 1 g.

Proteose Peptone No. 3, Difco . . 12 g.

Bacto-Lactose 10 g.

Bacto-Dextrose 1 g.

Sodium Chloride 5 g.

Bacto-Agar 15 g.

Bacto-Phenol Red 0.025 g.

Bacto-Russell Double Sugar Agar is widely employed in the primary identi-
fication of Gram-negative enteric pathogenic organisms, particularly the colon-
typhoid-salmonella-dysentery group. It distinguishes the dextrose-acid, dextrose-
gas, lactose-acid and lactose-gas forming organisms. Russell Double Sugar Agar
may be used to aid in the identification of pure cultures of colonies picked from
primary plating media such as MacConkey Agar, S S Agar, Bismuth Sulfite Agar
and others.

Bacto-Russell Double Sugar Agar conforms to the original formula of Russell^
except that phenol red replaces litmus, and Proteose Peptone No. 3 is utilized
in place of Bacto-Peptone. The phenol red indicator gives exceptionally clear-
cut brilliant reactions on both sides of the neutral point. Alkaline reactions turn
the indicator red, and acid reactions change it to yellow. Investigations have also
demonstrated that faster and clearer reactions are secured in the medium pre-
pared with Proteose Peptone No. 3.

Russell Double Sugar Agar is used in tubes which are slanted so as to provide
a deep butt. Inoculation is made from isolated colonies or pure cultures by smear-
ing over the surface of the slant and stabbing the butt. After suitable incubation,
the production of acid under aerobic and under anaerobic conditions, on the slant
and in the butt, respectively, can be detected by changes in color of the indicator.
Gaseous fermentation is indicated by splitting of the agar or formation of bubbles
in the butt.

Organisms capable of fermenting dextrose but not lactose, Salmonella typhosa
for example, will show an initial acid slant in short incubation periods. As the
dextrose is utilized, the reaction under aerobic conditions reverts and becomes
alkaline. Under anaerobic conditions, in the butt of the tube, these same organisms
are not capable of causing a reversion of the reaction, and remain acid.

After 24-48 hours incubation a properly inoculated tube showing a red or
cerise slope and a yellow butt with or without gas formation indicates fermenta-
tion of the dextrose. Some strains of typhoid may require as long as 30-40 hours
to produce a characteristic alkaline slant. A tube showing a yellow slant and butt
with or without gas indicates fermentation of the lactose. A tube showing no
change indicates that neither dextrose nor lactose has been fermented. See the
table on page 161 for the reactions of various bacteria on Bacto-Russell Double
Sugar Agar and other differential tube media.

To rehydrate the medium, suspend 44 grams of Bacto-Russell Double Sugar
Agar in 1000 ml. of cold distilled water and heat to boiling to dissolve the
medium completely. The solution is distributed in tubes which are stoppered
with cotton plugs or loosely fitting caps. Sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). The tubes should be slanted so as to give a deep
butt when solid. The final reaction of the medium will be pH 7.4.

One pound of Bacto-Russell Double Sugar Agar will make 10.3 liters of
medium.
^ J. Med. Research, 25:217:1911.

DEHYDRATED CULTURE MEDIA 163

BACTO

SACCHAROSE MANNITOL AGAR (B87)

DEHYDRATED

Bacto-Beef Extract 1 g.

Proteose Peptone No. 3, Difco ... 12 g.

Saccharose, Difco 10 g.

^-Mannitol, Difco 1 g.

Bacto-Agar 15 g.

Bacto-Phenol Red 0.03 g.

Bacto-Saccharose Mannitol Agar is a tube medium employed in the differenti-
ation of pure cultures of Gram-negative intestinal bacteria. This medium permits
the determination of the formation of acid and gas from saccharose and mannitol.
It is employed to aid in the identification of pure cultures of colonies picked from
primary plating media such as MacConkey Agar, S S Agar, Bismuth Sulfite Agar
and others.

Russell^ made a noteworthy contribution toward the identification of the
Gram-negative intestinal bacterial rods by the introduction of his Dextrose Lac-
tose Agar. Kendall and Ryan^ introduced a new agar medium containing sac-
charose and mannitol, two important sugars in the differentiation and identifica-
tion of members of the intestinal group. Their Saccharose Mannitol Agar is used
in the same manner as Russell's and, when employed in conjunction with it, per-
mits observations of the fermentation reactions of the bacteria on four sugars by
using only two media.

Bacto-Saccharose Mannitol Agar duplicates the original formula of Kendall
and Ryan except that phenol red has replaced Andrade indicator. The phenol
red indicator responds rapidly to slight changes in reaction due to growth of
inoculated organisms. The tubes are inoculated with pure cultures by smearing
the surface of the slant and stabbing into the butt. On this medium cultural
reactions are clear-cut and readings may be made after 18-24 hours incubation
at 37°G. A red slant and yellov/ butt, with or without gas formation, indicates
fermentation of the mannitol. A yellow slant and butt, with or without gas, indi-
cates that either saccharose alone or both sugars have been fermented. A tube
remaining unchanged, or becoming cerise, indicates that neither sugar has been
attacked. For a table showing typical cultural reactions on this and other differ-
ential tube media, see page 161.

To rehydrate the medium, suspend 39 grams of Bacto-Saccharose Mannitol
Agar in 1000 ml. of cold distilled water and heat to boiling to dissolve the
medium completely. The solution is distributed in tubes which are stoppered with
cotton plugs or loosely fitting caps. Sterilize in the autoclave for 15 minutes at
15 pounds pressure (121°C.). Allow the tubes to solidify in a slanting position
to provide a generous butt. The final reaction of the medium will be pH 7.5.

One pound of Bacto-Saccharose Mannitol Agar will make 11.6 liters of
medium.

"•J. Med. Research, 25:217:1911. " ]. Infectious Diseases, 24:400:1919.

164 DIFCO MANUAL

BACTO

KRUMWIEDE TRIPLE SUGAR AGAR (B85)

DEHYDRATED

Bacto-Beef Extract 1 g.

Proteose Peptone No. 3, Difco . . 12 g.

Bacto-Lactose 10 g.

Saccharose, Difco 10 g.

Bacto-Dextrose 1 g.

Sodium Chloride 5 g.

Bacto-Agar 15 g.

Bacto-Phenol Red 0.025 g.

Bacto-Krumwiede Triple Sugar Agar is a differential medium employed in the
primary identification of Gram-negative enteric pathogens. This medium permits
the determination of the formation of acid and gas from dextrose, lactose and
saccharose. It is employed to aid in the identity of pure cultures of colonies
picked from primary plating media such as MacConkey Agar, S S Agar, Bismuth
Sulfite Agar and others.

Bacto-Krumwiede Triple Sugar Agar is prepared in accordance with the for-
mula of Krumwiede and Kohn,^ except in the choice of indicators. In place of
the Andrade indicator originally recommended, the more stable and sensitive
sulfonephthalein indicator, phenol red, is used. In addition to lactose and dex-
trose, as contained in Russell's Double Sugar Agar, this medium contains sac-
charose. This saccharose-containing medium has the advantage over Russell's
medium in giving an earlier and more accurate differentiation between the so-
called slow lactose fermenting strains of the coliform group and the Salmonella
group. The explanation for this increased rapidity of differential is that the slow
lactose fermenters attack the saccharose with greater avidity than they do lactose
under aerobic conditions. Salmonella are not influenced by the saccharose. There-
fore, with the Triple Sugar Agar many of the organisms giving a doubtful or
atypical Salmonella reaction on Russell's medium can promptly be excluded,
as a rule, as only slow fermenters of lactose.

Like Russell's medium this Triple Sugar Agar is a differential tube medium
and inoculations are made from pure cultures by smearing the surface of the
slant and stabbing the butt. A properly inoculated tube showing, after incubation,
a red or cerise slope and a yellow butt, with or without gas formation, indicates
fermentation of the dextrose. Some strains of intestinal pathogens require 30-40
hours to produce an alkaline slope. A tube showing a yellow slant and butt, with
or without gas, indicates fermentation of the saccharose, or lactose, or both. A
tube showing no change indicates that none of the sugars has been fermented.
See the table on page 161 for the reactions of various intestinal bacteria on differ-
ential media.

To rehydrate the medium, suspend 54 grams of Bacto-Krumwiede Triple
Sugar Agar in 1000 ml. of cold distilled water and heat to boiling to dissolve the
medium completely. The solution is distributed in tubes which are stoppered
with cotton plugs or loosely fitting caps. Sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°G.). Allow the tubes to solidify in a slanting position
so as to provide a deep butt. The final reaction of the medium will be pH 7.4.

One pound of Bacto-Krumwiede Triple Sugar Agar will make 8.4 liters of
medium.

ij. Med. Research, 37:225:1917.

DEHYDRATED CULTURE MEDIA 165

BACTO

KLIGLER IRON AGAR (B86)

DEHYDRATED

Bacto-Beef Extract 3 g

Bacto-Yeast Extract 3 g

Bacto-Peptone 15 g

Proteose Peptone, Difco 5 g

Bacto-Lactose 10 g

Bacto-Dextrose 1 g

Ferrous Sulfate 0.2 g

Sodium Chloride 5 g

Sodium Thiosulfate 0.3 g,

Bacto-Agar 12 g

Bacto-Phenol Red 0.024 g

l^acto-Kligler Iron Agar is a most useful differential tube medium in the study
of the Gram-negative intestinal microorganisms. It combines the principles of
Russell Double Sugar Agar and Lead Acetate Agar into one medium which per-
mits a differentiation of the Gram-negative rods both on the basis of their ability
to ferment dextrose or lactose, and on their ability to produce hydrogen sulfide.
It differentiates the lactose-splitting organisms from the lactose nonfermenters,
distinguishes Salmonella typhosa from the other Salmonella and the Shigella
groups and differentiates S. paratyphi (paratyphoid A) from S. schottmuelleri
and S. enteritidis. Kligler Iron Agar is recommended to identify further pure
cultures of colonies picked from primary plating media such as MacConkey Agar,
S S Agar, Bismuth Sulfite Agar and others.

Kligler's^ original medium was a soft Nutrient Agar containing dextrose,
Andrade indicator and lead acetate. While experimenting with this medium and
other combinations, Kligler discovered that Russell's medium containing Andrade
indicator and lead acetate gave a good differentiation, and later^ recommended
it as being satisfactory for differentiation of the typhoid, paratyphoid and dysen-
tery groups. Bailey and Lacey^ made a study of such a medium in an attempt to
simplify it and to select a more suitable indicator. They found that phenol red
was particularly adaptable and recommended that it be used as the indicator of
hydrogen ion concentration. A similar medium, including saccharose and incorpo-
rating Bacto-Tryptone as a nutrient, with ferrous sulfate and thiosulfate as the
indicator of hydrogen sulfide production, was developed by Sulkin and Willett.'*
They found such a medium to be unique in its ability to give rapid clear-cut
reactions. A complete discussion of this medium is given under Bacto-Triple
Sugar Iron Agar, page 166.

Bacto-Kligler Iron Agar is prepared with phenol red as an indicator of the
production of acid, and ferrous sulfate as an indicator of hydrogen sulfide pro-
duction. This combination of ingredients gives sensitive, distinct clear-cut reac-
tions.

For typical cultural reactions in 18 hours, it is recommended that tubes of
Kligler Iron Agar be inoculated heavily with growth from a solid culture medium
by smearing over the surface of a slant and stabbing in the butt. If inoculated
from a suspension of organisms, or from broth culture, typical reactions of hydro-
gen sulfide production, and reversion, may not be obtained until 36-40 hours
at 37°C. To obtain true differential cultural reactions of this medium it is neces-
sary to have a pure culture. In inoculating directly from isolation media such as
MacConkey Agar, S S Agar or Bismuth Sulfite Agar plates, select well isolated
colonies and pick only the very center of the colony. If there is any question as
to the ability to obtain a pure culture from a certain colony, it is recommended

166 DIFGO MANUAL

that the suspicious colony be purified by streaking on MacConkey Agar before
inoculating into Kligler Iron Agar. This procedure is always recommended to
insure culture purity when picking from poured plates of Bismuth Sulfite Agar.
It is often possible to detect contaminated cultures on Kligler Iron Agar slants,
and when this is the case it is necessary to isolate the organism in pure culture
before its typical cultural reaction can be determined.

Organisms capable of fermenting dextrose but not lactose, S. typhosa for exam-
ple, will show an initial acid slant in short incubation periods. As the dextrose is
utilized, the reaction under aerobic conditions reverts and becomes alkaline.
Under anaerobic conditions, in the butt of the tube, these same organisms are not
capable of causing a reversion of the reaction, and remain acid.

The fermentation reactions in Kligler Iron Agar are similar to those in Russell
Double Sugar Agar, i.e., a red slant and yellow butt with or without gas indicates
fermentation of the small quantity of dextrose, a yellow slant and butt with or
without gas formation indicates fermentation of the lactose, while a tube showing
no change indicates that neither dextrose nor lactose has been attacked. In addi-
tion to these fermentation reactions, Kligler Iron Agar indicates whether or not
hydrogen sulfide is produced. This is shown by a blackening of the medium.
Freshly prepared media will give the best reactions. For characteristic reactions
of some of the enteric organisms on this medium see the table on page 161.

To rehydrate the medium, suspend 55 grams of Bacto-Kligler Iron Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. The solution is distributed in tubes which are stoppered with cotton plugs
or loosely fitting caps. Sterilize by autoclaving for 15 minutes at 15 pounds pres-
sure (121°C.). The reaction of the medium after sterilization v/ill be pH 7.4.
Allow the tubes to solidify in a slanting position in a manner which will give a
generous butt. Best reactions are obtained on freshly prepared media.

One pound of Bacto-Kligler Iron Agar will make 8.2 liters of medium.

-^Am. J. Pub. Health, 7:1042:1917. 3 j. Bact., 13:183:1927-

a J. Exp. Med., 28:319:1918. 4 J. Lab. Clin. Med., 25:649:1940.

BACTO

TRIPLE SUGAR IRON AGAR (B265)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto- Yeast Extract 3 g.

Bacto-Peptone 15 g.

Proteose Peptone, Difco 5 g.

Bacto-Lactose 10 g.

Saccharose, Difco 10 g.

Bacto-Dextrose 1 g.

Ferrous Sulfate 0.2 g.

Sodium Chloride 5 g.

Sodium Thiosulfate 0.3 g.

Bacto-Agar 12 g.

Bacto-Phenol Red 0.024 g.

Bacto-Triple Sugar Iron Agar is recommended as a medium for use in the
identification of Gram-negative enteric pathogens in the routine examination of
stools. It is of especial value when used in conjunction with MacConkey Agar,
S S Agar, Bismuth Sulfite Agar, Brilliant Green Agar, E.M.B. Agar and Endo
Agar. This medium indicates the ability of an organism to ferment lactose, sac-
charose and dextrose with formation of acid and gas, and also its ability to pro-
duce hydrogen sulfide. These characteristics are employed in most schema for
identifying members of the Salmonella-S higella groups.

DEHYDRATED CULTURE MEDIA 167

In 1911 Russell^ described the use of two sugars in an agar medium to differ-
entiate Gram-negative organisms of intestinal origin. The ability of members of
this group to produce hydrogen sulfide was recognized as a valuable character-
istic and to detect its presence lead or iron salts have been added to the Russell
Medium by many investigators. Kligler--^ reported that by adding lead acetate
to Russell Double Sugar Agar, a medium capable of differentiating typhoid, para-
typhoid and dysentery could be obtained. A modification of this medium, Bacto-
Kligler Iron Agar, using phenol red as an indicator of acidity, and iron salts to
detect hydrogen sulfide production, has been used extensively for many years in
the differentiation of enteric organisms. Krumwiede and Kohn'* modified Russell
Double Sugar Agar by the addition of saccharose to the medium. This permitted
an earlier detection of those coliform organisms which ferment lactose slowly,
since many of these organisms attack saccharose more readily than lactose. The
added saccharose also permits the exclusion of certain coliform and proteus
organisms which have the ability to attack saccharose, but not lactose, in a 24-48
hour incubation period.

In 1940 Sulkin and Willett^ described a triple sugar ferrous sulfate medium
for use in the identification of enteric organisms. This medium consisted of a
Beef Extract Tryptone Agar Base to which was added 1 per cent lactose, 1 per
cent saccharose, 0.1 per cent dextrose, 0.02 per cent ferrous sulfate, 0.015 per cent
sodium thiosulfate and brom thymol blue as an indicator of change in reaction.
In our laboratory, independently and concurrently with the work of Sulkin and
Willett, we prepared a similar medium by adding 1 per cent saccharose to Bacto-
Kligler Iron Agar. This latter medium contained phenol red as an indicator.
Bacto-Triple Sugar Iron Agar, so prepared, was distributed at that time to a
number of laboratories for comparative trials. Hajna^ described a similar medium
for the identification of bacteria of the intestinal group. The importance of sac-
charose, as already pointed out, is to eliminate certain saccharose fermenting
bacteria of the paracolon group which ferment lactose slowly as well as certain
proteus organisms capable of fermenting saccharose. Those laboratories especially
interested in recovery of paracolon bacilli or members of the proteus and para-
colon groups which ferment saccharose should use Bacto-Kligler Iron Agar, or
bear in mind when using Bacto-Triple Sugar Iron Agar that these organisms pro-
duce an acid and gas butt with an acid slant.

The pathogenic significance of these saccharose fermenting organisms, mem-
bers of the paracolon and proteus groups, is not clearly defined, as has been
shown by many investigators. Parr'' showed the close relationship of the Morgan
bacillus, paracolons, anaerogenic Escherichia coli and slow lactose fermenting
coliform bacilli to the Shigella and Salmonella groups. Several outbreaks of gas-
troenteritis have been attributed to these normal or aberrant types. Stuart,
Wheeler, Rustigian and Zimmerman^ reported that paracolons are often associ-
ated with, and can cause, mild or acute gastroenteritis of short duration. Neter*
pointed out that the Triple Sugar Iron Agar does not eliminate all saccharose
fermenting strains of proteus and paracolons, and further that the pathogenic
significance of some of these organisms as incitants of diarrheal diseases is not
clearly known. He pointed out that these facts should be taken into consideration
when using the Triple Sugar Iron Agar as a diagnostic medium.

Ewing and Bruner^^ used Triple Sugar Iron Agar as a differential tube medium
in isolation of Salmonella and Shigella for serological classification. Typical
suspicious colonies were picked from the primary media and were inoculated
into Triple Sugar Iron Agar. After overnight incubation at 37°G. the cultures
showing an alkaline slant and acid or acid and gas in the butt were transferred
to the Urea Medium of Christensen.^^ (A complete discussion of this medium,
Bacto-Urea Agar Base, is given on page 171). After incubation for 2-4 hours at

168 DIFCO MANUAt

37°C. a preliminary examination was made and all tubes showing an alkaline
reaction were discarded. The remaining tubes were reincubated and their reac-
tions were generally complete in 24 hours. All cultures showing an alkaline
reaction were Proteus or members of the paracolon-aerobacter group. The Sal-
monella and Shigella cultures failed to produce increased alkalinity in the Urea
Medium. Hydrogen sulfide positive cultures, as determined on the previously
inoculated Triple Sugar Iron Agar, were investigated with Salmonella poly-
valent antiserums, while hydrogen sulfide negative cultures were investigated with
Shigella polyvalent or Salmonella polyvalent antiserums. In a survey of methods
used for the collection and preservation of stool specimens for the isolation and
identification of Salmonella, Shigella and intestinal protozoa Felsenfeld^^ ^t-
ported that the use of Triple Sugar Iron Agar was increasing while the use of
double sugar agar was decreasing. The use of a single differential medium indi-
cates a trend towards standardization of laboratory techniques.

Bacto-Triple Sugar Iron Agar is essentially the formula originally described
by Sulkin and Willett.^ The Bacto-Tryptone has been replaced by a combination
of Bacto-Peptone and Proteose Peptone, Bacto-Yeast Extract has been added, the
agar concentration increased to 1.5 per cent and phenol red used as an indicator
instead of brom thymol blue. On this medium, cultural reactions are clear-cut,
and readings may be made at 18-24 hours incubation. For typical cultural reac-
tions in 18 hours, it is recommended that the medium be inoculated heavily with
growth from a solid culture medium. If inoculated from a suspension of organ-
isms, or from broth culture, typical reactions of hydrogen sulfide production, and
reversion, may not be obtained until 36-40 hours at 37 °C. To obtain true difTer-
ential cultural reactions on this medium it is necessary to have a pure culture.
In inoculating directly from isolation media such as MacConkey Agar, S S Agar
or Bismuth Sulfite Agar plates, select well isolated colonies and pick only the
very center of the colony. If there is any question as to the ability to obtain a
pure culture from a certain colony, it is recommended that the suspicious colony
be purified by streaking on MacConkey Agar before inoculating into Triple Sugar
Iron Agar. This procedure is always recommended to insure culture purity when
picking from poured plates of Bismuth Sulfite Agar. It is often possible to detect
contaminated cultures on Triple Sugar Iron Agar slants, and when this is the case
it is necessary to isolate the organism in pure culture before its typical cultural
reactions can be determined.

The cultural reactions on Bacto-Triple Sugar Iron Agar are similar to those
obtained on Kligler Iron Agar, but to it is added the information of the ability
of an organism to ferment saccharose. A table of typical reactions on this and
other differential media is given on page 161. Results obtained on Triple Sugar
Iron Agar, as on Kligler Iron Agar, constitute presumptive evidence only, and
must be confirmed biochemically and serologically.

To rehydrate the medium, suspend 65 grams of Bacto-Triple Sugar Iron Agar
in 1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. The solution is distributed in tubes which are stoppered with cotton plugs
or loosely fitting caps. Tube and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). Slant in such a manner as to allow a generous butt.
The final reaction of the medium will be pH 7.4.

One pound of Bacto-Triple Sugar Iron Agar will make 6.6 liters of medium.

^ J. Med. Research, 25:217:1911. ' Bact. Rev., 3:1:1939-

3 Am. J. Pub. Health, 7:1042:1917. * J. Bact., 45:101:1943.

J. Exp. Med., 28:319:1918. * J. Bact., 50:609:1945.

'J;

Med. Research, 37:225:1917. 10 Am. J. Clin. Path., 17:1:1947.

"25:649:1c

s J. Lab. Clin. Med., 25:649:1940. ^1 J. Bact., 52:461:1946.

« J. Bact., 49:516:1945. "Public Health Reports, 65:1075:1950.

DEHYDRATED CULTURE MEDIA 169

BACTO

LEAD ACETATE AGAR (B88)

DEHYDRATED

Bacto-Peptone 15 g.

Proteose Peptone, Difco 5 g.

Bacto-Dextrose 1 g.

Lead Acetate 0.2 g.

Sodium Thiosulfate 0.08 g.

Bacto-Agar 15 g.

Bacto-Lead Acetate Agar, like Russell's medium, is used primarily as a tube
medium for the differentiation of the various Gram-negative intestinal bacteria.

Certain bacteria possess the ability of liberating hydrogen sulfide from pro-
teins or their split products. This property has been widely used in culture media
for differentiating and identifying members of the Gram-negative intestinal
group of bacteria, as well as for the identification of other microorganisms.
Orlowski^ noted that Salmonella typhosa could be distinguished from the coli-
form organisms by culturing them in a medium containing lead acetate, an indi-
cator of hydrogen sulfide production. Jordan and Victorson^ showed further that
tS". paratyphi (paratyphoid A) and S. schottmuelleri (paratyphoid B) could be
distinguished on the basis of hydrogen sulfide production by growing them in a
lead acetate medium. S. paratyphi produced no browning, whereas S, schott-
muelleri gave a definite browning of the medium within 1 8-24 hours after inocu-
lation.

Bacto-Lead Acetate Agar was developed after a careful study of the literature
on the subject, and after considerable research to obtain a medium which would
give an accurate differentiation. The modification finally developed was suggested
by R. S. Spray. Unlike many other formulae, this medium shows no inhibition
due to the toxicity of lead. The non-toxicity of Bacto-Lead Acetate Agar for cer-
tain bacteria is confirmed in "Pure Culture Study of Bacteria."^ See the table
on page 161 showing reactions of some of the intestinal bacteria on this and other
differential media.

Bacto-Lead Acetate Agar is sensitive in its cultural reactions, and can be used
for plating when it is desired to demonstrate the relative number of strong hydro-
gen sulfide producing colonies. Its usual method of use is in tubes. Pure cultures
of Gram-negative microorganisms isolated from MacConkey Agar, Bismuth Sul-
fite Agar, Eosin Methylene Blue Agar or other plating media should be streaked
upon the surface of the slant and stabbed into the butt of the Lead Acetate Agar.
With this procedure, surface browning can be observed, as well as browning along
the line of puncture. Since the medium contains dextrose it will also indicate gas
production from this carbohydrate by the presence of bubbles in the butt of the
tube. For the determination of hydrogen sulfide production in a medium free
from dextrose, see Bacto-Peptone Iron Agar, as discussed on page 181.

Morrison and Tanner* used Bacto-Lead Acetate Agar in their study of hydro-
gen sulfide production by the thermophilic bacteria from water. They found the
medium well adapted to the determination of this characteristic. Spray^ em-
ployed it with success in his studies on semisolid media for the cultivation and
identification of the sporulating anaerobes.

To rehydrate the medium, suspend 36 grams of Bacto-Lead Acetate Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. The solution is dispensed into tubes and sterilized in the autoclave for
15 minutes at 15 pounds pressure (121°C.). The tubes are slanted to allow for a
generous butt. The final reaction of the medium will be pH 6.6.

170 DIFCO MANUAL

One pound of Bacto-Lead Acetate Agar will make 12.6 liters of medium.

1 Dissert. St. Petersburg, 1897. * J. Bact., 7:343:1922.

2 J. Infectious Diseases, 21:554:1917. ^ J. Bact., 32:135:1936.

3 Pure Culture Study of Bacteria, i:No. 8:1933.

BACTO

UREA BROTH (B272)

DEHYDRATED

Bacto- Yeast Extract 0.1 g.

Monopotassium Phosphate 9.1 g.

Disodium Phosphate 9.5 g.

Urea, Difco 20 g.

Bacto-Phenol Red 0.01 g.

Bacto-Urea Broth is recommended for the identification of the genus Proteus.
It is prepared according to the formula of Stuart, Van Stratum and Rustigian^
and provides all the essential growth factors for Proteus.

The decomposition of urea by members of the Proteus group has been espe-
cially helpful to differentiate these organisms from other Gram negative intestinal
bacteria and to eliminate them from further study in various schema for the
identification of the Gram negative intestinal pathogens. Using their strongly
buffered medium, and under proper conditions, Stuart, Van Stratum and Rusti-
gian, reported that Proteus could be differentiated from all other members of
the enteric group by its ability to produce sufficient ammonia to give a reaction
more alkaline than pH 8.1 after 12-48 hours incubation at 37 °C. These investi-
gators pointed out that a large number of organisms other than Proteus were
capable of urease production and would give a positive test in weakly buffered
media. They also noted that by decreasing the amount of buffer in their standard
medium to one tenth or one hundredth that of the original concentration, the
time of incubation for identification of Proteus could be decreased from 12-48
hours to 2-4 hours. Rustigian and Stuart^ used urea decomposition as a limiting
characteristic for the identification of Proteus strains from other members of the
family Enter obacteriaceae. Ferguson and Hook^ also reported that urease pro-
duction, as indicated by the Rustigian and Stuart method, was an excellent means
of differentiating between members of the Proteus and Salmonella groups.

According to the procedure recommended by Stuart, Van Stratum and Rusti-
gian^, the filter sterilized medium is distributed under aseptic conditions in
approximately 3 ml. amounts in tubes 14 mm. inside diameter and 125 mm. long.
Inoculation is made with a straight needle from 18-24 hour agar slant cultures,
and the tubes are incubated at 37° C. When possible, reactions are recorded after
8, 12, 24 and 48 hours incubation. A positive urease reaction (hydrolysis of urea)
is indicated by a change in color from yellow (pH 6.8) to a red to a cerise color
(pH 8.1 or more alkaline).

To rehydrate the medium, dissolve 38.7 grams Bacto-Urea Broth, Dehydrated,
in 1000 ml. of distilled water, filter sterilize and distribute under aseptic condi-
tions in 3 ml. amounts into sterile plugged 14 x 125 mm. tubes. Final reaction
of the medium will be pH 6.8.

One pound of Bacto-Urea Broth, Dehydrated, will make 11.7 liters of medium.

ij. Bact., 49:437:1945. ^J- Lab. Clin. Med., 28:1715:1943.

aProc. Soc. Exp. Biol. Med., 53:241:1943.

DEHYDRATED CULTURE MEDIA 171

BACTO

UREA BROTH CONCENTRATE (B280)
(Filter Sterilized Solution)

Bacto-Urea Broth is also supplied in a filter sterilized concentrated solution as
Bacto-Urea Broth Concentrate. The dehydrated medium is recommended for
those laboratories using large amounts of medium and equipped with filter
sterilizing apparatus. For laboratories requiring only small amounts of medium
Bacto-Urea Broth Concentrate is recommended.

Bacto-Urea Broth Concentrate is a filter sterilized concentrated solution of
Bacto-Urea Broth, 10 ml. being sufficient for the preparation of 100 ml. final
medium. It is especially recommended for use by those laboratories desiring to
save time, or requiring smaller quantities of medium.

To prepare the medium from Bacto-Urea Broth Concentrate, add the contents
of one 10 ml. tube of Bacto-Urea Broth Concentrate to 90 ml. of sterile cold
distilled water, under aseptic conditions. The distilled water should be sterilized
in the autoclave for 15 minutes at 15 pounds pressure (121°C.). The water must
be cooled to below 55 °C. before adding Bacto-Urea Broth Concentrate. This
complete medium is distributed under aseptic conditions in 3 ml. amounts into
sterile plugged 14 x 125 mm. tubes.

One 10 ml. tube of Bacto-Urea Broth Concentrate will make 100 ml. of
medium.

BACTO

UREA AGAR BASE (B283)

DEHYDRATED

Bacto-Peptone 1 g.

Bacto-Dextrose 1 g.

Sodium Chloride 5 g.

Monopotassium Phosphate 2 g.

Urea, Difco 20 g.

Bacto-Phenoi Red 0.012 g.

Bacto-Urea Agar Base is recommended for use in the preparation of a differ-
ential medium for the detection of Proteus and most members of paracolon
aerobacter and paracolon intermediate groups. It aids in the elimination of these
cultures from further study in schema for the identification of intestinal patho-
gens.

Christensen^ considered the well buffered liquid Urea Medium described by
Rustigian and Stuart,^ as discussed under Bacto-Urea Broth above, as being
suited only for the identification of Proteus. However, other Gram negative in-
testinal bacteria which are capable of splitting urea cannot do so in this medium
because of the small amount of nutritive material and increased amount of buffer
present. In order to overcome these limitations, Christensen^ devised the Urea
Agar Medium in which he included peptone and dextrose and reduced the buffer
content. His medium supported a more vigorous growth of many of the Gram-
negative enteric bacilli and readily permitted observation of urease production
by Proteus and members of the paracolon aerobacter and paracolon intermediate
groups.

Ewing^ used Urea Agar as a differential medium in the examination of many
cultures from stool specimens and confirmed the findings of Christensen. Ewing
and Bruner* utilized the urease reaction as a screening medium in the selection

172 DIFCO MANUAL

of Salmonella and Shigella cultures for serologic classification. Typical colonies
suspected of being pathogens were picked from primary plating media into Triple
Sugar Iron Agar. AH tubes showing acid and gas in the slant and butt were dis-
carded. Transfers were made from tubes showing an alkaline slant and acid or
acid and gas butt onto Urea Agar. A preliminary reading was made at the end
of 2-4 hours at 37°C. and all tubes showing alkaline reactions were discarded.
The tubes were reincubated and reactions were generally complete in 24 hours.
All cultures producing an alkaline reaction were Proteus or members of the para-
colon aerobacter group. Salmonella and Shigella cultures failed to produce any
increase in alkalinity in the medium. Hydrogen sulfide positive cultures, as
determined on the previously inoculated Triple Sugar Iron Agar, were investi-
gated with Salmonella polyvalent antisera, while hydrogen sulfide negative
cultures were investigated with Shigella polyvalent or Salmonella polyvalent
antisera. Urea Agar is used to determine the decomposition of urea by organ-
isms in the examination of specimens for Salmonella and Shigella according to
the method given in "Diagnostic Procedures and Reagents"^ of the American
Public Health Association.

Bacto-Urea Agar Base, when prepared for use, gives reactions similar to those
described by Christensen^ and Ewing and Bruner.* Proteus attacks the urea
rapidly and after 2-4 hours incubation the color change, due to ammonia pro-
duction, has penetrated deep into the medium. After 24 hours incubation the
entire butt of the tube is alkaline in reaction. Urease positive paracolons, in con-
trast, hydrolyze urea much more slowly, showing only slight penetration of the
alkaline reaction into the butt of the medium in 6 hours and requiring 3 to 5
days to change the reaction of the entire butt. According to Christensen^ most
paracolon aerobacter and paracolon intermediate cultures are urease positive and
paracolon Escherichia cultures are urease negative. Salmonella and Shigella
species fail to produce any trace of alkalinity on this medium. Urea Agar may
also be used to show urease production by other organisms. Some Gram positive
cocci and diphtheroids, as well as certain pigment producing members of the
pyocyaneous group and others give a positive reaction on this medium.

To prepare the medium, dissolve 29 grams of Bacto-Urea Agar Base, Dehy-
drated, in 100 ml. of distilled water. Filter sterilize this concentrated base. Dis-
solve 15 grams Bacto-Agar in 900 ml. distilled water by boiling and sterilize in
the autoclave for 15 minutes at 15 pounds pressure (121°C.). Cool to 50-55°C.
and add 100 ml. filter sterilized concentrated Bacto-Urea Agar Base, under
aseptic conditions. Mix thoroughly and distribute in sterile tubes. Slant the tubes
so as to have a butt of about 1 inch in depth and a slant of about 1.5 inches in
length. After solidification the slants are heavily inoculated by spreading growth
from an agar culture over the entire surface. Do not inoculate into the butt. Final
reaction of the medium will be pH 6.8.

Bacto-Urea Agar Base cannot be sterilized by heat because of the presence of
urea. The complete Urea Agar Medium must be slanted before the medium
solidifies to avoid the necessity of remelting the agar and causing hydrolysis of
the urea in the medium.

One pound of Bacto-Urea Agar Base, Dehydrated, will make 15.6 liters of
medium.

^ J. Bact., 52:461:1946. * Am. J. Clin. Path., 17:1:1947.

3 Proc. Soc. Exp. Biol. Med., 47:108:1941. ^Diagnostic Procedures and Reagents, 3rd Edi-

*J. Bact., 51 =433: 1946. tion: 227: 1950.

DEHYDRATED CULTURE MEDIA 173

BACTO

UREA AGAR BASE CONCENTRATE (B284)

(Filter Sterilized Solution)

Bacto-Urea Agar Base is also supplied in a filter sterilized concentrated solu-
tion as Bacto-Urea Agar Base Concentrate. The dehydrated medium is recom-
mended for those laboratories using large amounts of medium and equipped with
filter sterilizing apparatus. For laboratories requiring only small amounts of
medium Bacto-Urea Agar Base Concentrate is recommended.

Bacto-Urea Agar Base Concentrate is a filter sterilized concentrated solution
of Bacto-Urea Agar Base, 10 ml. being sufficient for the preparation of 100 ml.
final medium. It is especially recommended for use by those laboratories desiring
to save time, or requiring smaller quantities of medium.

To prepare the medium from Bacto-Urea Agar Base Concentrate, dissolve 1.5
grams of Bacto-Agar in 90 ml. distilled water by boiling. Sterilize in the autoclave
for 15 minutes at 15 pounds pressure (121°C.). Cool to 50-55°C. and add the
contents of one tube of Bacto-Urea Agar Base Concentrate (10 ml.), under
aseptic conditions. Mix thoroughly and tube as indicated above.

One tube of Bacto-Urea Agar Base Concentrate will make 100 ml. of complete
medium.

BACTO
S I M MEDIUM (B271)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Peptone 30 g.

Peptonized Iron, Difco 0.2 g.

Sodium Thiosulfate 0.025 g.

Bacto-Agar 3 g.

Bacto-S I M Medium was devised for use as a routine medium in the cultural
identification of members of the Salmonella and Shigella groups, showing hydro-
gen sulfide production, indole production and motility in the same tube. These
characteristics, along with other biochemical reactions, are of prime importance
in the cultural identification of members of the Gram-negative enteric group.

Orlowski^ noted that Salmonella typhosa could be distinguished from the coli-
form organisms by culturing them in a medium containing lead acetate, an indi-
cator of hydrogen sulfide production. Jordan and Victorson- showed further that
S. paratyphi (paratyphoid A) and S. schottmuelleri (paratyphoid B) could be
distinguished on the basis of hydrogen sulfide production by growing them in a
Lead Acetate Medium.

Semisolid media, as described by Hiss,^ Hesse,'* Jackson and Melia,^ Tittsler
and Sandholzer^ and others, have been employed quite extensively in the deter-
mination of motility by bacteria. Sulkin and Willett,^ in Bacto-Triple Sugar Iron
Agar, used 1 per cent agar to demonstrate motility or lack of motility in addition
to hydrogen sulfide production and carbohydrate fermentation by members of
the Salmonella and Shigella groups. They called attention to the "brush-like"
growth or motility of the typhoid organisms. Friewer and Shaughnessy^ used the
fermentation of lactose, hydrogen sulfide production and motility in a Lead
Acetate Semisolid Agar as a screening medium in the isolation of enteric patho-
gens from stool culture. Sosa^ described a peptone medium with a low agar con-

174 DIFGO MANUAL

centration to determine motility, and stated that indole determination could be
made using the Ehrlich reagent in this medium.

In the development of Bacto-S I M Medium it was determined that 0.0025 per
cent sodium thiosulfate added to the semisolid 3 per cent Bacto-Peptone medium
produced results comparable to those secured when hydrogen sulfide was deter-
mined in a 1 per cent Bacto-Peptone solution, using lead acetate paper strips as
an indicator of hydrogen sulfide. Smaller quantities of sodium thiosulfate did
not give a satisfactory response in the semisolid medium, while larger quantities
obscured motility and also permitted some false reactions. Any blackening along
the line of inoculation is considered as a positive hydrogen sulfide reaction. Hy-
drogen sulfide producing organisms generally give a positive reaction in 18-24
hours.

Motile cultures in Bacto-S I M Medium show diffuse growth or turbidity away
from the line of inoculation. Hydrogen sulfide reactions are intensified by motile
cultures. Cultures vary in the amount of motility as pointed out by Friewer and
Shaughnessy.® Strains of Proteus and Salmonella often show diffuse growth
throughout the entire medium, while S. typhosa is not as actively motile. In
Bacto-S I M Medium motility of typical members of the enteric group is demon-
strated in 18-24 hours, or less, incubation at 37 °C. Green and co-workers^*^ used
Bacto-S I M Medium in 1-2 ml. amounts in 75 x 10 mm. tubes and reported
the detection of motility by a large series of cultures following incubation at
37°C. for 90 to 120 minutes.

Bacto-Tryptone has been used universally in the test for indole production. It
has been demonstrated that Bacto-S I M Medium gives parallel indole production
in comparison with a 1 per cent Bacto-Tryptone solution after 18-24 hours in-
cubation at 37 °C. The oxalic acid paper test for indole production may be used
with this medium. This test has the advantage of making it possible to observe
motility, hydrogen sulfide production and indole formation after any desired
incubation period. Oxalic acid paper for testing is prepared by soaking filter paper
in a saturated oxalic acid solution, drying and then cutting the paper into strips.
For the performance of the test, after the medium has been inoculated, suspend
a strip of the paper in the mouth of the tube so that the strip projects one-half
inch below the cotton plug holding it in place. The paper must not become wet
as this may contaminate the medium. Indole production is shown by the formation
of a pink color on the paper during incubation.

If desired, other tests for indole may be employed; the Kovacs or Ehrlich test
gives good results. When using these tests hydrogen sulfide and motility readings
should be made before testing for indole. The recommended procedure for the
Kovacs' test is to overlay the medium with 2 ml. of chloroform without agitation,
followed by the direct addition of 2 ml. of Kovacs' reagent. A pink to deep red
color is formed in the chloroform layer if indole is present. In a negative test no
color is formed in the chloroform layer. Kovacs' reagent consists of 75 ml. amyl
alcohol, 25 ml. concentrated hydrochloric acid and 5 grams /?-dimethylamino-
benzaldehyde. The indole test may also be performed in accordance with the
"Manual of Methods of Pure Culture Study of Bacteria," ^^ of the Society of
American Bacteriologists. To perform this test, add about 2 ml. of ethyl ether to
each tube. Shake gently, but do not break the agar. Let stand several minutes and
then add about 1 ml. Ehrlich reagent by dropping it down the side of the tube so
that it spreads out as a layer between the ether and the medium. The formation
of a purplish red color at the interface of the two liquids within 5 minutes indi-
cates indole production. In negative reactions, after the Ehrlich reagent has been
in contact with the medium for a period longer than 5 minutes, a slight pink
color may be observed on the surface of the medium. This should not be mistaken
for a positive reaction, since in a positive test the color must appear within 5

DEHYDRATED CULTURE MEDIA 175

minutes at the interface of the ether and the Ehrlich reagent. Ehrlich reagent
consists of 1 gram of ^-dimethylaminobenzaldehyde, 95 ml. ethyl alcohol (95
per cent) and 20 ml. of concentrated hydrochloric acid.

Bacto-S I M Medium is especially recommended as an aid in the routine con-
firmation of members of the Salmonella and Shigella groups following presump-
tive evidence as obtained on the differential tube media (Russell Double Sugar
Agar, Krumwiede Triple Sugar Agar and Triple Sugar Iron Agar) . As with other
differential media, it is necessary to use pure cultures for inoculation. Generally,
an incubation period of 18-2.4 hours or less is sufficient to give reactions by typi-
cal organisms in this group. Occasionally an atypical culture may be encountered
which will fail to produce hydrogen sulfide, indole or motility in 18-24 hours
incubation. Typical cultural reactions of the pathogenic Gram negative enteric
bacteria are:

S. typhosa Salmonella Shigella

Motility -|- 4- —

Hydrogen sulfide -|~ — —

Indole — — ±:

To rehydrate the medium, suspend 36 grams Bacto-S I M Medium in 1000 ml.
cold distilled water and heat to boiling to dissolve the medium completely. Dis-
tribute the medium in tubes to a depth of about 3 inches and sterilize in the
autoclave for 15 minutes at 15 pounds pressure (121°C.). The tubes of medium
are solidified in a vertical position. Inoculate with a single stab using a straight
needle through the center and to a depth of about two-thirds of the medium.
Incubate for 18-24 hours at 37 °C. and observe for motility, hydrogen sulfide
and indole, as indicated above. Final reaction of the medium will be pH 7.3.

One pound of Bacto-S I M Medium will make 12.6 liters of medium.

1 Dissert. St. Petersburg, 1897. 8 Tech. Bull. Reg. Med. Tech., 5:1:1944.

a J. Infectious Diseases, 21:554:1917. » Rev. Inst. Bact., "Dr. Carlos G. Malbran",

3 J. Exp. Med., 2:677:1897. 11:286:1943.

* Zeit. Hyg. Infektionskrank., 58:441:1908. 1° J. Bact., 62:347: 1951.

s J. Infectious Diseases, 6:194:1909. ii Manual of Methods of Pure Culture Study of

8 J. Bact., 31:575:1936. Bacteria, Leaflet V, page 6, 7th Editioa, 1939.

'J. Lab. Clin. Med., 25:649:1940.

BACTO

TERGITOL-7 AGAR (B455)

DEHYDRATED

Proteose Peptone No. 3, Difco . . 5 g.

Bacto- Yeast Extract 3 g.

Bacto-Lactose 10 g.

Bacto- Agar 15 g.

Tergitol 7 0.1 ml.

Bacto Brom Thymol Blue 0.025 g.

Bacto-Tergitol 7 Agar is a selective medium for Escherichia coli and members
of the coliform group, prepared according to the formula given by Chapman.^
Chapman 2,3 reported that the addition of triphenyltetrazolium chloride (TTG)
to this medium permitted the confirmation of E. coli after 10 hours incubation,
and also that this medium gave excellent results in the cultivation of Monilia
and other fungi.

Chapman^ reported that the addition of Tergitol 7 to an agar medium consist-
ing of Proteose Peptone No. 3, Bacto-Yeast Extract, Lactose and Brom Thymol
Blue permitted imrestricted development of all coliform organisms and inhibited

176 DIFGO MANUAL

the development of Gram-negative spore formers as well as Gram-positive
microorganisms. Escherichia produce yellow colonies surrounded by yellow
zones; Aerobacter produce large mucoid colonies, usually surrounded by yellow
zones; paracoli and other lactose non-fermenting organisms produce colonies
usually surrounded by blue zones, on this medium. Proteus and other organisms
have little tendency to form spreading colonies. Counts of coliform organisms
on Tergitol 7 Agar were found to be 30 per cent higher than on other selective
media for members of this group. Tergitol 7 Agar is inoculated by smearing
the surface wdth the specimen using a bent glass rod. As much as 0.1 ml. of
inoculum may be used per plate if the surface of the medium is dry. Pour plates
do not give satisfactory results.

The addition of 40 mg. of TTC to a liter of sterile Tergitol 7 Agar permitting
the confirmation of E. coli after 10 hours incubation was described by Chapman. ^
E. coli does not reduce the dye while other coliform organisms rarely fail to do
so. Surface colonies of E. Coli on this medium are greenish yellow surrounded
by a yellow halo while other coliform surface colonies are dark red. Readings can
be made following incubation at 37°C. for 10 hours. Chapman^ also reported
that Tergitol 7 Agar with added TTC (40 mg. per liter) gave a selective
medium for the isolation of Monilia and other fungi. Monilia growing on this
medium produce white circular convex entire colonies about 1 mm. in diameter
in 24-48 hours. The colonies may appear pale blue because of the color of the
medium. Yeasts produce red colonies. Since the medium permits the develop-
ment of coliform organisms this fact must be taken into consideration in the
isolation of Monilia from specimens. Chapman* also used Tergitol 7 in a modi-
fied Sabouraud Maltose Agar, for the isolation and differentiation of Monilia
and other fungi. This medium is described in detail on page 239, under Bacto-
Sabouraud Maltose Agar.

To rehydrate the medium, suspend 33 grams of Bacto-Tergitol 7 Agar in
1000 ml. of cold distilled water. Heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 lbs. pressure (121°C.). Final reaction of the medium will be pH 6.9.

1 J. Bact., 53:504:1947. * Trans. New York Acad. Sci. Series II,

BAm. J. Pub. Health, 41:1381:1951. 14:254:1952.

8 In press.

BACTO

MOTILITY SULFIDE MEDIUM (B450)

DEHYDRATED

Bacto-Beef Extract 3 g.

Proteose Peptone No. 3, Difco .... 10 g.

/-Cystine, Difco 0.2 g.

Ferrous Ammonium Citrate 0.2 g.

Sodium Citrate 2 g.

Sodium Chloride 5 g.

Bacto-Gelatin 80 g.

Bacto-Agar 4 g.

Bacto-Motility Sulfide Medium is a semisolid medium suitable for determining
motility and the production of hydrogen sulfide from /-cystine. It is prepared
according to the formula given by Hajna,^ and used in the rapid method of
differentiation and identification of bacteria of the intestinal group as described
by him. 2 As pointed out by Hajna,^ this medium is the semisolid agar-gelatin
medium of Edwards and Bruner^ modified to permit observation of motility
and simultaneous hydrogen sulfide production from /-cystine. Hydrogen sulfide

DEHYDRATED CULTURE MEDIA 177

reactions on this medium may differ from the reactions usually obtained by a
group of organisms since it contains free 1 -cystine which may give a positive
reaction by organisms considered negative by classical methods.

Bacto-Motility Sulfide Medium is used in the proposed rapid method of
differentiating and identifying bacteria of the intestinal group described by
Hajna," to detect the capacity of an organism to produce hydrogen sulfide from
1 -cystine, and to determine motility. Motility is evidenced by presence of
diffuse growth away from tlie line of inoculation, non-motile organisms growing
only along the line of inocu'iation. Hydrogen sulfide producing organisms show
a blackening of the medium, blackening being confined to the inoculated portion
of the medium with non-mo ti'e organisms, but diffusing throughout the medium
with motile organisms. In this procedure the medium is dispensed in 4 ml.
amounts in 13 x 100 mm. tubes, sterilized and cooled in a verticle position. The
suspected colony on the primary plating medium is picked with a straight needle
and inoculated in succession, without securing additional inoculum, into Bacto-
Motility Sulfide Medium, to a depth of about a quarter of the column, and then
into Triple Sugar Iron Agar and H Broth. The Motility Sulfide Medium is in-
cubated at 30°G. overnight.

The ability of the organism to hydrolyze urea is determined following observa-
tion of motility and hydrogen sulfide production. The Motility Sulfide Medium
culture is overlaid with 1 ml. of Eacto-Urea Broth, and incubated at 37 °C., for
not more than 6 hours. A positive urease reaction is indicated by a reddish purple
color forming in the Urea Broth.

To rehydrate the medium, suspend 104 grams Bacto-Motility Sulfide Medium
in 1000 ml. cold distilled water. When the medium is thoroughly wetted, care-
fully heat to boiling to dissolve the medium completely. The medium requires
nearly constant agitation during the heating process. Dispense in 4 ml. amounts
in 13 X 100 mm. tubes and sterilize in the autoclave for 15 minutes at 10 pounds
pressure (117°C.). Final reaction of the medium will be pH 7.3.

One pound of Bacto-Motility Sulfide Medium will make 4.3 liters of medium.

iPub. Health Lab., 8:36:1950.
2 Pub. Health Lab., 9:23:1951.
^Univ. Ky. Cir., 54:1942.

BACTO

H BROTH (B451)

DEHYDRATED

Bacto-Peptone 5 g.

Bacto-Tryptone 5 g.

Bacto-Beef Extract 3 g.

Bacto-Dextrose 1 g.

Sodium Chloride 5 g.

Dipotassium Phosphate 2.5 g.

Bacto-H Broth is recommended for the preparation of the "H" agglutination
antigen as used in the differentiation and identification of members of the
Salmonella group. It is prepared according to the formula described by Hajna
and Damon.^ This medium is used in the rapid method of differentiating and
identifying bacteria of the intestinal group as described by Hajna.^ The produc-
tion of the "O" type antigen and the elaboration of indole is also shown on this
medium. Hajna^ reported that the combination of Bacto-Peptone and Bacto-
Tryptone in this medium has proven excellent in every way.

Serological analysis of the "H" and "O" antigens has been used as a practical

178 DIFGO MANUAL

means in schema for identification of organisms. Hajna^ has used "H" and "O"
agglutination together with other characteristics and serological reactions for
the identification of Salmonella. In this method, typical colonies are picked from
the primary plating medium, S S Agar, or Bismuth Sulfite Agar plates, and
inoculated into tubes of semisolid agar, Triple Sugar Iron Agar and finally into
H Broth, without securing further inoculum. The inoculated tubes are then
incubated at 30 °C. overnight. Cultures showing reactions on the semi-solid agar
and Triple Sugar Iron Agar suggestive of Salmonella typhosa or other "Sal-
monella like organisms" are examined for "H" antigen. Place 0.02 ml. of a 1-5
dilution of Edwards mixed polyvalent Salmonella "H" Serum in the bottom of
an agglutination tube. Then remove 1 ml. of the H Broth culture and add 1 ml.
of 0.6 per cent formalinized saline, mix thoroughly and place in a water bath at
50° C. for 1 hour, but not to exceed two hours. A flocculant agglutination in the
tube is an indication of the presence of Salmonella "H" antigen. The individual
"H" antigens are detected by specific H antisera, using the tube agglutination
technique. The presence of "O" antigens is determined by an initial slide
agglutination test using the H Broth and polyvalent "O" antiserum followed by
specific "O" antiserum. The balance of the H Broth is used for the indole test
by adding 0.5 ml. of Kovacs' reagent to the tube. A dark red color in the surface
layer indicates a positive test; a yellow color indicates a negative test.

To rehydrate the medium, dissolve 21.5 grams Bacto-H Broth in 1000 ml
cold distilled water. Distribute in 4 ml. amounts in 13 x 100 mm. tubes. Sterilize
in the autoclave for 15 minutes at 10 pounds pressure (117°C.). The final re-
action of the medium will be pH 7.2.

One pound of Bacto-H Broth will make 21.1 liters of medium.

iPub. Health Rep., 65:116:1950.
2 Pub. Health Lab., 9:23:1951.
8 Personal Communication, 1951.

FRIEWER SHAUGHNESSY MEDIUM

BACTO

FRIEWER SHAUGHNESSY BASE NO. 1 (B310)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Peptone 30 g.

Bacto-Agar 3 g.

Bacto-Brom Thymol Blue 0.01 g.

BACTO

FRIEWER SHAUGHNESSY BASE NO. 2 (B311)

DEHYDRATED

Bacto-Lactose 10 g.

Lead Acetate 0.05 g.

Bacto-Friewer Shaughnessy Medium prepared according to the formula of
Friewer and Shaughnessy^ is recommended for use as a primary screening
medium in the laboratory diagnosis of enteric infections. The medium is prepared
in two parts, Bacto-Friewer Shaughnessy Base No. 1 and Bacto-Friewer
Shaughnessy Base No. 2. A solution of each base is prepared separately and mixed
before sterilization to secure the complete medium.

DEHYDRATED CULTURE MEDIA 179

A Lead Semisolid Agar was developed by Friewer and Shaughnessy^ as a
screening medium, in the preliminary classification of the Salmonella-Shigella
group. The determination of motility, considered by some workers to be most
helpful as an early step in the classification of the group, is achieved by the use
of a semisolid agar. This Lead Semisolid Agar also contained lactose and brom
thymol blue in addition to lead acetate. It was possible, therefore, with this
single medium to determine motility, the production of acid or acid and gas from
lactose and the production of hydrogen sulfide. These characteristics, i.e., motility,
lactose fermentation and hydrogen sulfide production, are essential criteria in
practically all methods of classification of members of the enteric group.

Using this medium under practical conditions, colonies may be picked from
any primary plating medium such as Bacto-S S Agar, Bacto-Bismuth Sulfite Agar
or Bacto-MacConkey Agar, or inoculation may be made from a broth or agar
subculture from these media to the Friewer Shaughnessy Medium.

If there is any question as to the purity of the suspected colony, it should be
inoculated into a broth or peptone tube and streaked on a plating medium such
as Bacto-MacConkey Agar or Bacto-E.M.B. Agar, to establish its purity, before
proceeding with the screening test. A stab inoculation, using a straight wire, and
extending down the center of the medium to approximately one-half its depth, is
most satisfactory.

After 12-24 hours incubation at 37°C. the tubes are read for the following
information :

1. Motility. A motile culture is characterized by a diffusion of the growth
from the line of inoculation. Some cultures, such as certain members of the
Proteus and Salmonella groups, are actively motile, producing complete diffusion
throughout the medium. Other cultures, as for example most strains of
Salmonella typhosa, are less actively motile, and growth extends out from the
line of inoculation without giving the appearance of dilTuse turbidity. This
difference in the degree of motility is an aid in the choice of subsequent tests, for
the cultures with moderate diffusion from the line of inoculation plus a hydrogen
sulfide reaction can be selected for special tests for S. typhosa.

Shigella organisms are non-motile and grow only along the line of inoculation.
Strictly aerobic rods and cocci grow only on the surface of the medium and are
readily differentiated from the non-motile facultative anaerobes such as members
of the Shigella group.

2. Hydrogen Sulfide. The production of a brown or black color is an indica-
tion that hydrogen sulfide has been produced. Motility accentuates the diffusion
of the hydrogen sulfide, whereas in a non-motile culture the brown or black
coloration is confined to the line of inoculation.

3. Lactose Fermentation. When lactose is fermented, the medium in the
growth area develops a yellow color. Formation of gas is evidenced by gas bubbles
in the medium or at the surface. The intensity of the lead sulfide reaction is not
great enough to mask the color change.

The following key, suggested by Friewer and Shaughnessy,^ may be used as a
guide for the identification of the pathogenic Gram-negative bacilli. Serological
reactions are used for final confirmation.

J Fermentation of Lactose.

A. Acid and Gas— DISCARD.

B. Acid fermentation, motile culture — DISCARD.

C. Acid fermentation, non-motile culture (see non-motile cultures, III,

below).

II Surface Growth Reactors. (No growth along line of stab, growth only at
surface).

180 DIFCO MANUAL /

Inoculate a Dextrose Broth tube.

Acid fermentation — Suspect Shigella and proceed as in III B.
No reaction in dextrose — DISCARD.

(Alkaligines and pseudomonas groups. An occasional pseudo-
monas strain produces a slight reaction in dextrose).

III Non-motile types.

A. Gram-stain all non-motile types.

Gram-positive types — DISCARD.

B. Gram-negative types — Proceed with tests for the Shigella group. (An

occasional Sonne strain produces fermentation of lactose rather
rapidly. For this reason all non-motile, Gram-negative types are
put through suitable tests for this species).

IV Motile cultures.

A. Moderate motility, hydrogen sulfide production.

Proceed with tests for S. typhosa.

B. Active motility with or without hydrogen sulfide production. Inocu-

late Bacto-Peptone or Bacto-Tryptone tube for indole test, and fer-
mentation tubes of Mannitol and Saccharose Broths.

1. Mannitol fermenter, no fermentation of saccharose, indole nega-

tive.

a. Mannitol fermented (with acid and gas), no hydrogen
sulfide production — S. paratyphi and a few other Salmonella
types.

b. Hydrogen sulfide production.

(I) Mannitol fermented with acid and gas — Proceed with

tests for Salmonella.
(II) Mannitol fermented with acid only — Proceed with tests
for S. typhosa.

c. Indole positive and/or saccharose fermenting types (coli-
aerogenes, proteus groups) — DISCARD.

2. Mannitol non-fermenting types (Morgan's bacillus and other

proteus groups) — DISCARD.

To prepare 200 ml. of the medium, suspend 7.2 grams of Bacto-Friewer
Shaughnessy Base No. 1 in 100 ml. of cold distilled water and heat to boiling to
dissolve the medium completely. To a second flask containing 100 ml. distilled
water, add 2.1 grams Bacto-Friewer Shaughnessy Base No. 2, and warm to 45°C.
When Base No. 2 is in complete solution add to the hot solution of Base No. 1.
Caution: To avoid a precipitate in the medium, it is necessary to have Base No.
1 in complete solution and hot when the warm solution of Base No. 2 is added.
Distribute in tubes to a depth of about 3 inches. Sterilize in the autoclave for 15
minutes at 15 pounds pressure (121°C.). Allow the sterile tubes to solidify in
an upright position. Final reaction of the medium will be pH 7.5.

One pound of Bacto-Friewer Shaughnessy Base No. 1 will make 12.5 liters of
medium.

One pound of Bacto-Friewer Shaughnessy Base No. 2 will make 43.2 liters
of medium.

lAm. J. Clin. Path., Tech. Sect., 8:1:1944.
3 Personal Communication, 1947.

DEHYDRATED CULTURE MEDIA 181

BACTO

PEPTONE IRON AGAR (B89)

DEHYDRATED

Bacto-Peptone 15 g.

Proteose Peptone, Difco 5 g.

Ferric Ammonium Citrate 0.5 g.

Dipotassium Phosphate 1 g.

Sodium Thiosulfate 0.08 g.

Bacto-Agar 15 g.

Bacto-Peptone Iron Agar is a medium designed for use as an indicator of by-
drogen sulfide production by microorganisms.

Levine and co-workers/'^ in their studies on the reactions in the colon group of
bacteria, described a medium containing Proteose Peptone, Difco and ferric
citrate as being particularly satisfactory for the detection of hydrogen sulfide.
They further showed that such a medium served to differentiate the strains which
were Voges-Proskauer negative, methyl red positive and citrate positive from
other members of the colon-aerogenes group.

Levine reported that the ferric citrate was a much more sensitive indicator of
hydrogen sulfide production than was lead acetate. Their medium gave definite
clear-cut reactions within 12 hours.

Bacto-Peptone Iron Agar is a modification of Levine's original formula, in
which Bacto-Peptone has been included with Proteose Peptone and the more
soluble ferric ammonium citrate is used in place of ferric citrate. The medium
in tubes is inoculated by the stab method. Blackening of the medium indicates
the production of hydrogen sulfide. The reaction is intense and takes place quite
rapidly. Bacto-Peptone Iron Agar is not toxic and may be used in plates as well
as in tubes. Plates are convenient when it is desired to estimate the number of
hydrogen sulfide producers.

Tittsler and Sandholzer^ compared Bacto-Peptone Iron Agar with Lead Ace-
tate Agar for the detection of hydrogen sulfide and found that the Bacto-Peptone
Iron Agar had the advantage of giving earlier reactions and more clear-cut results.

Bacto-S I M Medium as discussed on page 173 may be recommended as a
more sensitive medium for the detection of hydrogen sulfide. This is a semisolid
medium and, in addition to indicating early clear-cut hydrogen sulfide producton,
is also recommended for motility determinations and indole production.

To rehydrate the medium, suspend 36 grams of Bacto-Peptone Iron Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in tubes and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). The final reaction of the medium will be pH 6.7.

One pound of Bacto-Peptone Iron Agar will make 12.6 liters of medium.

^ Proc. Soc. Exp. Biol. Med., 29:1022:1932.
2 Am. J. Pub. Health, 24:505:1934.
'Am. J. Pub. Health, 27:1240:1937.

182 DIFCO MANUAL

BACTO

SIMMONS CITRATE AGAR (B91)

DEHYDRATED

Magnesium Sulfate 0.2 g.

Monoammonium Phosphate 1 g.

Dipotassium Phosphate 1 g.

Sodium Citrate 2 g.

Sodium Chloride 5 g.

Bacto-Agar 15 g.

Bacto-Brom Thymol Blue 0.08 g.

Bacto-Simmons Citrate Agar is a medium capable of differentiating between
fecal coli and members of the aerogenes group on the basis of citrate utilization.
It may also be used to differentiate certain members of the Salmonella group.
Bacto-Simmons Citrate Agar is prepared according to the formula of Simmons.^

The differential between fecal coli and aerogenes as described by Koser- is
based upon the inability of fecal coli to develop in a medium containing inorganic
ammonium salts as the only source of nitrogen with citrate as the sole source
of carbon, whereas strains of aerogenes grow quite unrestrictedly. Likewise,
Salmonella typhosa, S. paratyphi and Shigella can be differentiated from S.
5chottm,uelleri, S. enteritidis and S. typhimurium, the latter group being able to
utilize citrate in such a medium while the former cannot.

This principle was first employed in a liquid medium by Koser. The liquid
medium had the disadvantage of appearing turbid when large inocula were
used even when no growth ensued. This observation led Simmons^ to devise a
solid medium which obviates the disadvantage of turbidity as a criterion of
growth.

Simmons' Medium is essentially Koser's Medium to which brom thymol blue
and 1.5 per cent agar have been added. It is a valuable aid in differentiating
certain members of the Salmonella group from S. paratyphi, S. typhosa and
Shigella. The medium is usually prepared as agar slopes, inoculated by stab and
streak, and incubated at 37°G. Some workers, however, prefer to use the medium
in petri dishes. On such a solid medium, growth is indicated very clearly by
colony formation and is usually accompanied by a color change of the indicator
due to acid or alkali production. Strains of aerogenes grow luxuriantly upon Sim-
mons Citrate Agar, producing alkali and changing the medium from its initial
green to deep blue in 24-48 hours. Fecal coli either do not grow at all upon this
medium or grow so sparsely that no change in reaction is apparent. S. schott-
muelleri, S. enteritidis and S. typhimurium, which develop on the medium, pro-
duce alkali as indicated by a deep prussian blue color.

Simmons also recommended its use for isolating and identifying certain fungi
and fungi imperfecti.

To rehydrate the medium, suspend 24.2 grams of Bacto-Simmons Citrate Agar
in 1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in tubes or flasks and sterilize by autoclaving for 15 minutes
at 15 pounds pressure (121° C). Allow the tubed medium to cool in a slanting
position. The final reaction of the medium will be pH 6.8.

One pound of Bacto-Simmons Citrate Agar will make 18.7 liters of medium.

1 J. Infectious Diseases, 39:209:1926. ^ J. Bact., 8:493:1923.

DEHYDRATED CULTURE MEDIA 183

BACTO

MALONATE BROTH (B395)

DEHYDRATED

Ammonium Sulfate 2 g.

Dipotassium Phosphate 0.6 g.

Monopotassium Phosphate 0.4 g.

Sodium Chloride 2 g.

Sodium Malonate 3 g.

Bacto-Brom Thymol Blue 0.025 g.

Bacto-Malonate Broth is liquid medium prepared with materials of known
chemical composition, with ammonium sulfate and sodium malonate as the only
source of nitrogen and carbon. It is prepared according to the formula described
by Leifson^ and used for the differentiation of members of the AerobacteT'
Escherichia group.

The ability of members of the Aerobacter group to utilize malonate as a source
of carbon in a medium of known chemical composition, and the inability of
members of the Escherichia group to grow in this medium was pointed out by
Leifson. A pH indicator, brom thymol blue, was incorporated in the medium.
Aerobacter, utilize malonate as a source of energy, produce an alkaline reaction,
and change the color of the medium to blue. Escherichia, not capable of utilizing
malonate, fail to grow, leaving the medium unchanged.

To rehydrate the medium, dissolve 8 grams Bacto-Malonate Broth in 1000 ml.
distilled water. Sterilize in the autoclave for 15 minutes at 15 pounds pressure
(121°C.). Avoid introduction of carbon and nitrogen from other sources. Final
reaction of the medium will be pH 6.7.

One pound of Bacto-Malonate Broth will make 56.7 liters of medium.
ij. Bact., 26:329:1933.

BACTO

PHENOL RED TARTRATE AGAR (B90)

DEHYDRATED

Bacto-Peptone 10 g.

Sodium Potassium Tartrate 10 g.

Sodium Chloride 5 g.

Bacto-Agar 15 g.

Bacto-Phenol Red 0.024 g.

Bacto-Phenol Red Tartrate Agar is a solid tube medium valuable in the
differentiation and identification of members of the Salmonella (paratyphoid)
group. Brown, Duncan and Henry^ observed that the members of the paratyphoid
group varied in their ability to attack sodium tartrate, and incorporated this
principle in a medium for subdividing the group. Jordan and Harmon^ claimed
that the medium of Brown, Duncan and Henry failed to give sharp differentiation,
and devised a medium which possessed the advantage of being more definite in
its differentiation. On this medium an acid reaction is produced by Salmonella
typhimurium, S. enteritidis, S. choleraesuis, S. ab or tivo equina, S. typhosa,
Escherichi coli and Proteus vulgaris strains, while the S. schottmuelleri and S.
paratyphi strains produce an alkaline reaction.

Bacto-Phenol Red Tartrate Agar duplicates the medium of Jordan and
Harmon. It is used unslanted, and is inoculated by stabbing-. Observations are
made at 24 and 48 hour intervals. An acid reaction is mdicated by the develop-

184 DIFCO MANUAL

merit of a distinct yellow color in the lower portion of the tube, the surface zone
remaining red.

To rehydrate the medium, suspend 40 grams of Bacto-Phenol Red Tartrate
Agar in 1000 ml. of cold distilled water and heat to boiling to dissolve the
medium completely. The solution is distributed in tubes which are stoppered
with cotton plugs or loosely fitting caps. Sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°G.). Allow the tubes to cool in an unslanted position.
The medium will have a final reaction of pH 7.6.

One pound of Bacto-Phenol Red Tartrate Agar will make 11.3 liters of
medium.

^J. Hyg., 23:1:1924. ^ J. Infectious Diseases, 42:238:1928.

BACTO

MOTILITY TEST MEDIUM (B105)

DEHYDRATED

Bacto-Tryptose 10 g.

Sodium Chloride 5 g.

Bacto-Agar 5 g.

Bacto-Motility Test Medium, a modification of the formula of Tittsler and
Sandholzer,^ as suggested by Darby,^ is a semisolid medium employed in the
testing of bacteria for motility. The sterile medium is inoculated by stabbing
through the center of the medium and is incubated at the proper temperature
for the organism under consideration and examined at the end of 8, 24 and 48
hours. Motility is manifested macroscopically by a diffuse zone of growth spread-
ing from the line of inoculation. Certain species of motile bacteria will show
diffuse growth throughout the entire medium, while others may show diffusion
from one or two points only, appearing as nodular outgrowths along the stab.
Tittsler and Sandholzer reported that tubes incubated for one day gave identical
results with the hanging drop method and that incubation for two days permitted
them to demonstrate motility in an additional 4 per cent of the cultures tested.

Bacto-S I M Medium as discussed on page 173 is also recommended for
determination of motility. In addition to motility, hydrogen sulfide production
and the production of indole may be determined on Bacto-S I M Medium.

To rehydrate the medium, suspend 20 grams of Bacto-Motility Test Medium
in 1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in tubes and sterilize by autoclaving for 15 minutes at
15 pounds pressure (121°G.). Allow the medium to cool with the tubes in an up-
right position. The medium will have a final reaction of pH 7.2.

One pound of Bacto-Motility Test Medium will make 22.7 liters of medium.

*J. Bact.j 31:575:1936. 2 Personal Communication.

BACTO

NITRATE BROTH (B268)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Peptone 5 g.

Potassium Nitrate 1 g.

Bacto-Nitrate Broth is recommended for testing the ability of bacteria to reduce
nitrate to nitrite, a characteristic which often has differential value. The medium

DEHYDRATED CULTURE MEDIA 185

is prepared according to the formula published in "Pure Culture Study of
Bacteria"^ of the Society of American Bacteriologists.

For determining the ability of an organism to reduce nitrate to nitrite, tubes of
medium, prepared from Bacto-Nitrate Broth, are inoculated with a pure culture
of the strain under examination. The tubes are then incubated at 37°C. for 12
to 24 hours. The medium is tested for the presence of nitrites by adding a few
drops each of sulfanilic acid and a-naphthylamine reagent solutions. The sul-
fanilic acid reagent is prepared by dissolving 8 grams of sulfanilic acid in 1000 ml.
5N acetic acid. The a-napththylamine reagent consists of 5 grams of «-naph-
thylamine dissolved in 1000 ml. 5N acetic acid. A distinct pink or red color
indicates the presence of nitrite reduced from original nitrate. If an organism
grows rapidly and reduces nitrate actively, it is suggested that the test for nitrite
be performed at an early incubation period since the reduction may be carried
beyond the nitrite stage. The test must always be controlled by comparison with
an uninoculated tube of medium.

To rehydrate the medium, dissolve 9 grams of Bacto-Nitrate Broth in 1000 ml.
distilled water. Distribute in tubes and sterilize in the autoclave for 15 minutes
at 15 pounds pressure ( 121°C.). The final reaction of the medium will be pH 7.0.

One pound of Bacto-Nitrate Broth will make 50 liters of medium.

1 Pure Culture Study of Bacteria, i2:Leaflet 11:8:1944.

BACTO

NITRATE AGAR (B106)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Peptone 5 g.

Potassium Nitrate 1 g.

Bacto-Agar 12 g.

Bacto-Nitrate Agar is a dehydrated medium for use in testing nitrate reduc-
tion by bacteria. It is prepared according to the formula of the "Manual of
Methods" of the Society of American Bacteriologists.

Nitrate reduction by microorganisms is a valuable criterion in the differentia-
tion and identification of various types of bacteria. Certain bacteria reduce the
nitrates to nitrites only, while others are capable of further reduction to free
nitrogen or even ammonia. Furthermore, many aerobic bacteria can grow under
anaerobic conditions in the presence of nitrates from which they derive their
oxygen.

Tubes of sterile slanted medium are inoculated by streaking over the surface of
the slant, and stabbing into the butt. Incubate at 37°C. Examine the cultures on
various days for gas production which will be indicated by splitting of the agar.
Test for nitrates with sulfanilic acid and a-naphthylamine reagent solutions. The
sulfanilic acid reagent is prepared by dissolving 8 grams of sulfanilic acid in 1000
ml. 5N acetic acid. The a-naphthylamine reagent consists of 5 grams of c-naph-
thylamine dissolved in 1000 ml. 5N acetic acid. A few drops of each reagent
are put into the tube to be tested. A distinct pink or red color indicates the
presence of nitrite reduced from original nitrate. The test should always be con-
trolled by comparing with an uninoculated tube of the medium which has been
kept under the same conditions as the inoculated tubes. The evolution of gas in
a nitrate medium containing no sugar or fermentable substance is a definite indi-
cation of reduction to free nitrogen.

To rehydrate the medium, suspend 21 grams of Bacto-Nitrate Agar in 1000

186 DIFCO MANUAL

ml. of cold distilled water and heat to boiling to dissolve the medium completely.
Distribute in tubes and sterilize in the autoclave for 15 minutes at 15 pounds
pressure (121°G.). The final reaction of the medium will be pH 6.8.
One pound of Bacto-Nitrate Agar will make 21 liters of medium.

BACTO

PHENOL RED BROTH BASE (B92)

DEHYDRATED

Bacto-Beef Extract 1 g.

Proteose Peptone No. 3, Difco . . 10 g.

Sodium Chloride 5 g.

Bacto-Phenol Red 0.018 g.

Bacto-Phenol Red Broth Base, and the complete carbohydrate* media de-
scribed below, are used in fermentation studies for the cultural identification of
pure cultures of microorganisms. The fermentative properties of bacteria are
valuable criteria in their identification, and may be determined by culturing the
organisms in a suitable medium containing the appropriate fermentable substance.
A satisfactory basic medium for determining the fermentation reactions of micro-
organisms must be capable of supporting growth of the organisms under study,
and free from fermentable carbohydrates which could give erroneous interpreta-
tions. It must be stable, uniform in composition, give distinct reactions and yield
accurate results.

Bacto-Phenol Red Broth Base is an excellent substrate for streptococci,
pneumococci, meningococci and gonococci, as well as for other less fastidious
types. The cultural value of the medium can be greatly improved for some of the
more delicate strains by the addition of a small amount (0.1-0.2 per cent) of
Bacto-Agar. A medium containing this small quantity of agar may be used to
best advantage by heating it to the boiling point to drive out the dissolved air and
cooling it below 40° C, without excessive agitation, just previous to inoculation.
Such a procedure also makes the medium sufficiently oxygen-free for propagation
of the obligate anaerobes as well as microaerophiles. Bacto-Phenol Red Broth
Base with 0.5 per cent selected carbohydrate and 0.15 per cent agar is suggested
as a satisfactory medium for the fermentation determinations as given in Diag-
nostic Procedures and Reagents.^ Some bacteriologists, determining the fer-
mentation reactions of gonococci, may prefer to use 0.8 per cent Bacto-Agar and
add 5 per cent sterile fresh rabbit serum to the sterile Phenol Red Broth con-
taining the selected carbohydrate, as discussed under Bacto-Proteose No. 3 Agar,
page 116.

For the determination of fermentative properties of members of the enteric
group of bacteria, Bacto-Purple Broth Media are recommended, as discussed on
page 190. These media have the same nutrients, but have a slightly more acid
reaction, and brom cresol purple is employed as an indicator.

With the exception of the carbohydrate, which has been omitted, Bacto-Phenol
Red Broth Base is a complete basic medium prepared with phenol red as an
indicator of changes in reaction. This product makes it possible to prepare as
much or as little medium as is required, adding to different portions any ferment-
able substance in any concentration desired. The concentration of carbohydrate
generally employed for testing the fermentation reactions of bacteria is 0.5 or
1.0 per cent. Some investigators prefer to use 1.0 per cent rather than 0.5 per
cent to insure against reversion of the reaction due to depletion of the carbo-

*The term carbohydrate, as used here, includes carbohydrates, polyhydric alcohols, glucosides and
other fermentable carbon compounds.

DEHYDRATED CULTURE MEDIA 187

hydrate by some microorganisms. An entire series of carbohydrate broths may
thus be made up readily, conveniently, and economically.

To rehydrate the medium, dissolve 16 grams of Bacto-Phenol Red Broth Base
in 1000 ml. of distilled water. To this solution the test carbohydrate is added
in proper quantity, and, when solution is complete, the medium is distributed
in fermentation tubes. Sterilize by autoclaving for not more than 15 minutes
at 15 pounds pressure ( 121°C.) . The minimum amount of heat required for com-
plete sterilization is to be desired. By packing the tubes loosely in the autoclave to
allow free circulation of steam, the time required may be appreciably shortened,
provided the temperature in the autoclave is actually 121°G. The final reaction
of the medium, without added carbohydrate, will be pH 7.4.

The addition of some carbohydrates may result in an acid reaction. In this
case it is suggested that O.IN sodium hydroxide be added drop by drop to restore
the original color, taking care not to obtain too deep red or cerise color.

One pound of Bacto-Phenol Red Broth Base will make 28.3 liters of medium.

An extensive number of carbohydrates, polyhydric alcohols and glucosides,
Difco, are available (see page 291) for use with Bacto-Phenol Red Broth Base.
There is also available a selected list of filter-sterilized ampuled solutions of
some of these carbohydrates, each ampul containing 10 ml. of a 10 per cent
solution, or 1 gram of carbohydrate (see page 292).

^Diagnostic Procedures and Reagents 3rd Edition: 107: 1950.

BACTO

PHENOL RED CARBOHYDRATE BROTHS

DEHYDRATED

Complete fermentation media, prepared with Bacto-Phenol Red Broth Base
and 0.5 per cent of the more frequently used carbohydrates, are available. The
dehydrated media included in this group are:

BACTO-PHENOL RED DEXTROSE BROTH (B93)
BACTO-PHENOL RED LACTOSE BROTH (B94)
BACTO-PHENOL RED MANNITOL BROTH (B97)
BACTO-PHENOL RED SACCHAROSE BROTH (B95)
BACTO-PHENOL RED MALTOSE BROTH (B96)

To rehydrate the media, dissolve 21 grams of the Bacto-Phenol Red Carbo-
hydrate Broth in 1000 ml. distilled water. The complete media are sterilized as
described above, using the minimum amount of heat necessary for sterilization,
for not more than 15 minutes at 15 pounds pressure (121°C.). The final reaction
of these media will be pH 7.4.

One pound of each complete Bacto-Phenol Red Carbohydrate Broth will make
21.6 liters of medium.

BACTO

PHENOL RED AGAR BASE (B98)

DEHYDRATED

Bacto-Beef Extract 1 g.

Proteose Peptone No. 3, Difco . . 10 g.

Sodium Chloride 5 g.

Bacto-Agar 15 g.

Bacto-Phenol Red 0.025 g.

188 DIFCO MANUAL

Bacto-Phenol Red Agar Base and the complete carbohydrate* media described
below, are used in fermentation studies for the cultural identification of pure
cultures of microorganisms. While liquid media are generally employed in study-
ing the fermentation reactions of microorganisms, many bacteriologists prefer a
solid medium for this purpose. The solid media employed usually contain one
per cent of the selected carbohydrate and an indicator of reaction. They are
dispensed in tubes, slanted, and inoculated by smearing over the surface of the
slant and stabbing into the butt.

The advantages claimed for a solid fermentation medium are that it permits
observ^ation of the fermentation reactions under both aerobic and anaerobic con-
ditions, that gas formation is indicated by splitting of the agar or accumulation of
gas bubbles in the base, and that deep tubes can provide sufficiently anaerobic
conditions for the development of the obligately anaerobic bacilli.

Bacto-Phenol Red Agar Base is particularly well adapted to the study of
fermentation reactions of microorganisms. This medium supports excellent growth
of many fastidious bacteria. The basal medium is free from fermentable carbo-
hydrates which could give erroneous interpretation. With the exception of the
carbohydrate, which has been omitted, it is a complete medium prepared with
phenol red as an indicator of changes in reaction. Bacto-Phenol Red Agar Base
permits the user to prepare as much or as little medium as he requires, adding
to different portions any fermentable substance desired (usually one per cent of
the test carbohydrate being added). An entire series of carbohydrate agars may
thus be made up readily, conveniently and economically.

Tubes of the sterile medium are inoculated by smearing over the surface of
the slant and stabbing into the butt. Obligately anaerobic bacteria may be inocu-
lated into the melted medium previously cooled to 45 °C. and subsequently allow-
ing it to solidify. After incubation, fermentation will be denoted by a change
in the color of the medium from red to canary yellow. Gas formation is indi-
cated by the collection of gas bubbles in the base, or by splitting of the agar.

To rehydrate the medium, suspend 31 grams of Bacto-Phenol Red Agar Base
in 1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. To this solution the selected carbohydrate is added. When the solution is
complete the medium is distributed in tubes which are stoppered with cotton
plugs or loosely fitting caps and sterilized in the autoclave for not more than 15
minutes at 15 pounds pressure (121°C.). The minimum amount of heat required
for sterilization is to be desired. By packing tubes loosely in the autoclave to
allow free circulation of the steam, the time required for sterilization may be ap-
preciably shortened, provided the temperature in the autoclave is actually 121 °C.
Some workers may prefer to add the carbohydrate in the form of a sterile solu-
tion to the melted sterile base, and then dispense the complete medium into
sterile tubes. This latter procedure is particularly recommended where only slow
operating autoclaves are available. In either case the sterile tubed medium con-
taining the carbohydrate is allowed to cool in a slanting position so as to provide
a slope and generous butt. The final reaction of the medium, without added
carbohydrate, will be pH 7.4.

The addition of some carbohydrates may result in an acid reaction. In this
case, it is suggested that O.IN sodium hydroxide be added drop by drop to restore
the original color, taking care not to obtain too deep red or cerise color.

One pound of Bacto-Phenol Red Agar Base will make 14.6 liters of medium.

An extensive number of carbohydrates, polyhydric alcohols and glucosides,
Difco, is available (see page 291) for use in conjunction with Bacto-Phenol Red
Agar Base. There is also available a selected list of filter-sterilized ampuled solu-

*The term carbohydrate, as used here, includes carbohydrates, polyhydric alcohols, glucosides and
other fermentable substances.

DEHYDRATED CULTURE MEDIA 189

tions of some of these carbohydrates, each ampul containing 10 ml. of a 10 per
cent solution, or 1 gram of the carbohydrate (see page 292).

BACTO

PHENOL RED CARBOHYDRATE AGARS

DEHYDRATED

Complete fermentation media, prepared with Bacto-Phenol Red Agar Base
and 1 per cent of the more frequently used carbohydrates, are also available. The
dehydrated media included in this group are:

BACTO-PHENOL RED DEXTROSE AGAR (B99)
BACTO-PHENOL RED LACTOSE AGAR (BlOO)
BACTO-PHENOL RED MANNITOL AGAR (B103)
BACTO-PHENOL RED SACCHAROSE AGAR (BlOl)
BACTO-PHENOL RED MALTOSE AGAR (B102)

To rehydrate the media, suspend 41 grams of the Bacto-Phenol Red Carbo-
hydrate Agar in 1000 ml. cold distilled water and heat to boiling to dissolve the
media completely. The complete media are sterilized, as described above, using
the minimum amount of heat necessary, for not more than 15 minutes at 15
pounds pressure (121°C.). The final reaction of these media will be pH 7.4.

One pound of each complete Bacto-Phenol Red Carbohydrate Agar will make
12.6 liters of medium.

BACTO

PURPLE BROTH BASE (B227)

DEHYDRATED

Bacto-Beef Extract 1 g.

Proteose Peptone No. 3, Difco . . 10 g.

Sodium Chloride 5 g.

Bacto-Brom Cresol Purple 0.015 g.

Bacto-Purple Broth Base is recommended for the preparation of carbohydrate
broths used in fermentation studies for the cultural identification of pure cultures
of microorganisms, particularly members of the enteric group. We have prepared
Bacto-Purple Broth Base, containing the indicator brom cresol purple, and
adjusted to a final reaction of pH 6.8, for the convenience of bacteriologists desir-
ing carbohydrate differential broths of slightly acid reaction and containing a
sensitive sulfonephthalein indicator capable of demonstrating minute change in
reaction. It is free from fermentable carbohydrates which could give erroneous
results.

The concentration of carbohydrate generally employed for testing the fer-
mentation reactions of bacteria is 0.5 or 1.0 per cent. Some investigators prefer
to use 1.0 per cent rather than 0.5 per cent to insure against reversion of the
reaction due to depletion of the carbohydrate by some microorganisms. Tubes of
Purple Lactose Broth and Purple Saccharose Broth should be tightly stoppered
during the incubation period for fermentation studies of the enteric group, to
avoid reversion of reaction.

To rehydrate the medium, dissolve 16 grams of Bacto-Purple Broth Base in
1000 ml. of distilled water. To this solution the selected carbohydrate is added in
the desired quantity and when solution is complete the medium is distributed in
fermentation tubes. Sterilize by autoclaving for not more than 15 minutes at 15

190 DIFGO MANUAL

pounds pressure (121°C.). The minimum amount of heat required for steriliza-
tion is to be desired, so as to avoid hydrolysis of the carbohydrate. By packing
the tubes loosely in the autoclave to allow free circulation of steam, the time
required may be appreciably shortened, provided the temperature in the auto-
clave is actually 121 °C. The final reaction of the medium without added carbo-
hydrate, will be pH 6.8.

The addition of some carbohydrates may result in an acid reaction. In this case
it is suggested that O.IN sodium hydroxide be added drop by drop to restore
original color.

One pound of Bacto-Purple Broth Base will make 28.3 liters of medium.

BACTO

PURPLE AGAR BASE (B228)

DEHYDRATED

Bacto-Beef Extract 1 g.

Proteose Peptone No. 3, Difco ... 10 g.

Sodium Chloride 5 g.

Bacto-Agar 15 g.

Bacto-Brom Cresol Purple 0.02 g.

Bacto-Purple Agar Base is recommended for the preparation of carbohydrate
agars used in fermentation studies for the cultural identification of pure cultures
of microorganisms, particularly members of the enteric group. Although broth
media are generally employed in studying the fermentation reactions of micro-
organisms, many bacteriologists prefer a solid medium for this purpose. Bacto-
Purple Agar Base is available for those bacteriologists preferring a fermentation
medium of slightly acid reaction (pH 6.8). This medium supports luxuriant
growth of organisms and with the indicator brom cresol purple gives clear-cut
reactions from slight changes in the pH of the medium. This medium is free
from fermentable carbohydrates which could give erroneous results.

To rehydrate the medium, suspend 31 grams of Bacto-Purple Agar Base in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. To this solution add 5-10 grams of the test carbohydrate, and, when
dissolved, dispense into culture tubes and sterilize in the autoclave for not more
than 15 minutes at 15 pounds pressure (121°C.). The final reaction of the
medium without added carbohydrate will be pH 6.8.

The addition of some carbohydrates may result in an acid reaction. In this
case, it is suggested that O.IN sodium hydroxide be added drop by drop to restore
the original color.

One pound of Bacto-Purple Agar Base will make 14.6 liters of medium.

BACTO

SNYDER TEST AGAR (B247)

DEHYDRATED

Bacto-Tryptone 20 g.

Bacto-Dextrose 20 g.

Sodium Chloride 5 g.

Bacto-Agar 20 g.

Bacto-Brom Cresol Green 0.02 g.

Bacto-Snyder test Agar is prepared for the colorimetric diagnosis of caries
activity as described by Snyder.^'^ The method is based on acid production in a

DEHYDRATED CULTURE MEDIA 191

carbohydrate medium by acidogenic microorganisms from the buccal cavity, and
is evidenced by a change in color of the indicator, brom cresol green, from a blue-
green to a yellow color. The test gives excellent correlation with the Lactobacillus
plate count and with the clinical picture, and provides a simple reliable method
for ordinary clinical use in the diagnosis of caries activity.

The following method of procedure and interpretation of results are suggested
by Snyder.

Procedure

The saliva specimens are obtained by having the patient chew a small piece of
paraffin for three minutes, during which time the saliva is collected in sterile
test tubes or bottles. The time for collecting specimens is preferably before
breakfast and before the teeth are brushed, otherwise just before lunch or dinner.
The specimens of saliva are then shaken vigorously and 0.2 ml. of the saliva
added by means of a sterile pipette to tubes of melted Snyder test Agar. The
medium is melted by placing the tubes in a boiling water bath for 10 minutes
and then cooling at 45 °C. The medium and inoculum are mixed and then
allowed to solidify by standing at room temperature for one-half hour. The
inoculated solidified tubes are incubated at 37°G. for 72 hours. If a thermo-
statically controlled incubator is not available, a thermos bottle may be substi-
tuted, as described by Appleton^ and Grossman.* Briefly stated, the method is to
take a quart sized thermos bottle of reliable make, and place a mercury ther-
mometer through the center of the cork, sealing it in securely. The thermos bottle
is then filled half full of warm water previously adjusted to 43°C. (110°F.) as
determined by the thermometer. The bottle is then closed with the stopper and
shaken to warm the inner walls of the container. When the temperature of the
water in the thermos bottle reaches 38°C. (100°F.) the culture tubes may be
placed inside, care being taken to avoid wetting the cotton plugs, or screw caps.

Observations

Examine the tubes daily for three days and record changes in color compared
with an uninoculated control tube. Observation of the color changes is facilitated
by means of reflected light, with the tubes held against a white background. The
color will change from the bluish-green of the control to yellow.

Positive: Change in color so that green is no longer dom-
inant is recorded as -\-+ to -f + -f -f.

Negative: No change in color or only slight deviation, but
green still dominant is recorded 0 to +.

Interpretation

Caries Activity Hours Incubation

24 48 72

Marked Positive

Moderate Negative Positive

Slight Negative Negative Positive

Negative Negative Negative Negative

The interpretation of laboratory data as given above with clinical activity
depends upon experience and understanding of several factors:

1. The data indicate only what is happening at the time
the specimen was collected.

2. At least two specimens collected within 2-4 days must
be obtained to establish a base-line or reference point.

192

DIFCO MANUAL

3. Only when two or more specimens have been cultured
can any reliability or prediction be obtained.

4. The clinician must study enough cases by use of periodic
laboratory data to establish in his own mind the value or
significance for the purpose intended.

Snyder^ tabulated the correlation between the Snyder colorimetric test and
Lactobacillus counts on specimens of saliva collected routinely:

Colorimetric Change (hours)

No. Lactobacilli

No.
Spec.

per ml. Saliva

Pos.

% Pos.

Pos.

% Pos.

Pos. % Pos.

0
0-100
100-1000
1000-10,000

348

157

105

2
0
5
6

0.6
0.0
3.2
5.7

6.3
11.9
30.0
65.7

85 24.5

32 54.3

111 70.0

99 94.3

10,000-20,000
20,000-50,000
50,000-100,000
100,000-

138
264
245
497

231

13.0
22.2
29.4
46.6

105
229
221
476

71.2
86.8
90.3
95.5

131 95.0
260 98.7
243 99.3
494 99.3

To rehydrate the medium, suspend 65 grams Bacto-Snyder test Agar in 1000
ml. cold distilled water and heat to boiling to dissolve the medium completely.
Distribute in tubes and sterilize in the autoclave for 15 minutes at 15 pounds
pressure (121°C.). Final reaction of the medium will be pH 4.8.

One pound of Bacto-Snyder Test Agar will make 7 liters of medium.

1 J. Dental Res., 19:349:1940. * Grossman: Root Canal Therapy, and Edition:

3 J. Ana. Dental Assoc, 28:44:1941. 273:1946.

8 Dental Items Int., 49:589:1937. ^ Personal Communication, 1948.

BACTO

LITMUS MILK

DEHYDRATED

(B107)

Bacto-Skim Milk 100 g.

Bacto-Litmus 0.75 g.

Bacto-Litmus Milk is recommended for propagating and carrying stock cul-
tures of the lactic acid bacteria associated with dairy products, and also for
determining the action of bacteria, upon milk.

Milk has been used for the propagation and study of microorganisms since
the beginning of bacteriology. In addition to its being an excellent substrate for
propagating the lactic acid bacteria commonly associated with dairy products,
milk may be employed as a differential medium for bacteria on the basis of
lactose fermentation, caseolysis, and casein coagulating properties.

The usefulness of milk in the study of bacteria is increased by the addition of
suitable indicators. Among the many indicators employed litmus has perhaps
enjoyed the widest usage. Litmus has the advantage of being readily reduced by
certain bacteria. This reduction of the litmus is useful as a differential aid.

To rehydrate the medium, dissolve 105 grams of Bacto-Litmus Milk in 1000
ml. of distilled water. Distribute in tubes or flasks as desired and sterilize by auto-
claving for 15 minutes at 15 pounds pressure (121°C.). During the sterilization
period the litmus is reduced to the colorless leuco base, taking on color as the

DEHYDRATED CULTURE MEDIA

193

medium cools and absorbs oxygen. Overheating during sterilization results in the
carmelization of the milk sugar. This is to be avoided since this resulting dis-
coloration may give an atypical appearance of the sterile medium and may be
reflected in the appearance of growing cultures. The final reaction of the medium
will be pH 6.8.

One pound of Bacto-Litmus Milk will make 4.3 liters of medium.

BACTO

ULRIGH MILK (B251)

DEHYDRATED

Bacto-Skim Milk 100 g.

Bacto-Methylene Blue 0.005 g.

Bacto-Chlor Phenol Red 0.015 g.

Bacto-Ulrich Milk is recommended for propagating and carrying stock cul-
tures of the lactic acid bacteria associated with dairy products, and for deter-
mining the action of bacteria in general on milk. Ulrich,i in 1944, described an
indicator system for use to replace litmus in Litmus Milk. His system consists of
a mixture of chlor phenol red (dichlorophenolsulfonphthalein) and methylene
blue. Chlor phenol red is a hydrogen ion indicator, yellow at pH 4.8 and more
acid reactions, and pink to red at pH 6.5 and more alkaline reactions. Methylene
blue in the concentration employed is used to indicate changes in the oxidation-
reduction potential (Eh) in the medium, being blue when oxidized and colorless
when reduced, by growth of organisms and when heated to boiling, or when first
removed from the autoclave following sterilization. The combination of the two
indicators in milk gives information similar to Litmus Milk, plus indication of
alkalinity and acidity in the lower (reduced) portion of the tube.

In his descriptions of the color changes occurring in his medium due to the
growth of various types of lactic bacteria Ulrich used the Ridgeway^ color classi-
fication. The table showing the Ridgeway color description, the Munsell^ color
system, and the Inter-Society Color Council and National Bureau of Standards
terminology, which uses familiar terms, is given below to show typical color
changes of some of the more common biological reactions in Ulrich Milk. Slight
variations in color, due to the variable alteration of the milk during sterilization,
and to changes in pH or Eh of the growing cultures, may occur.

Ulrich Description
(Ridgeway)

Munsell Color
Description

*ISCC— NBS
Terminology

Uninoculated
Medium

Pale Glaucous Green

5.0 B6/1 to 5.0 B7/1

Medium Bluish-Gray

Slight acid

Yellowish Glaucous

10.0 GY8/4 to 10.0 GY8/2

Pale Yellow-Green

Acid with
Reduction

Ivory Yellow

10.0 YR9/2 to 10.0 YR9/4

Pale Yellow-Orange

Alkaline

Alkaline with
Reduction

Pale Russian Blue

Pale Pinkish
Cinnamon

5.0 P7/6 to 5.0 RP7/4 to 5.0
RP7/6

5.0 RP8/2 to 5.0 RP8/4

10.0 P3/10 to 10.0 P3/8

Pale Bluish-Purple passing
through various graduations
of Light Red-Purple and

Pale Purplish-Pink into Pole-
Pink to Strong Red-Purple
(Transparent)

Alkaline with
Peptonization

Clear, Transparent
Red

^We are greatly indebted to Dr. H. J. Conn, Biological Stain Commission, Geneva,
New York, and to Prof. F. L. Dimmick, Hobart College, Geneva, New York, for
this color terminology.

194 DIFGO MANUAL

Ulrich Milk, when first taken from the autoclave after sterilization, is nearly
white in color but quickly develops a bluish-gray top layer with a pink under
portion. As the methylene blue oxidizes the blue color extends progressively
downward until the entire medium is uniformly bluish-gray. In contrast. Litmus
Milk takes on a pink coloration.

When inoculated with lactic acid producing organisms Ulrich Milk first
changes to a pale pink in the subsurface areas, the upper layer remaining blue.
The depth of the top layer will depend on the rate of reduction of the methylene
blue by the growing organism against air oxidation. As more acid is produced
the blue top layer changes to a pale yellow-green and the pink portion fades to
white and then becomes a pale yellow-orange. If the medium does not become
coagulated, or if acid is produced slowly, 60 per cent of the tube may be yellow-
ish-green in color. If the medium is coagulated the greenish zone is limited to a
narrow band or collar at the top because of the decreased oxidation. In Litmus
Milk, as acid is produced, the pink color develops, and as the litmus is reduced,
especially in the lower portion of the tube, the medium becomes white.

Proteolytic organisms, or those producing alkali, do not generally coagulate
milk. In Ulrich Milk they change the lower portion of the tube from blue to
white and then to pink. If peptonization occurs the medium becomes transparent
and reddish-purple in color.

To rehydrate the medium, dissolve 100 grams of Bacto-Ulrich Milk in 1000
ml. distilled water. Distribute into tubes or flasks as desired and sterilize for 15
minutes at 15 pounds pressure (121°C.). Overheating during sterilization results
in the carmelization of the milk sugar. This is to be avoided since this resulting
discoloration may give an atypical appearance to the sterile medium which may
be further reflected in the appearance of growing cultures. The final reaction of
the medium will be pH 6.5.

One pound of Bacto-Ulrich Milk will make 4.5 liters of medium.

1 Science, 99:352:1944. s Munsell Color Company, Inc., Bedtimore,

3 Color Standards and Color Nomenclature, 1912. Maryland.

ADDITIONAL MEDIA

The media listed below have been rather extensively employed in the past.
Recently other media have been developd which are considered superior and
serve the purpose more adequately than the older media. For the present we will
continue to carry these media in stock for those laboratories where they have
been in routine use, or where it is desired to continue them for comparative
purposes.

PRESENT RECOMMENDATIONS

Bacto-B.T.B. Lactose Agar Bacto-Mannitol Salt Agar, page 150

Bacto-Crystal Violet Agar Bacto-Staphylococcus Medium No. 110, page 151

Bacto-Stone Gelatin Agar Bacto-Chapman Stone Medium, page 153

Bacto-Purple Milk Bacto-Litmus Milk, page 192

(

DEHYDRATED CULTURE MEDIA 195

STERILITY TEST MEDIA

The media listed in this section are recommended for sterility testing. Included
are the media specified by the National Institute of Health in its circular "Cul-
ture Media for the Sterility Test" 2nd. Rev. February 5, 1946. Media for sterility
testing according to the "Compilation of Regulations for Tests and Methods of
Assay and Certification of Antibiotic Drugs," Federal Security Agency, Food and
Drug Administration and U. S. Pharmacopeia and the National Formulary are
also described in this section.

BACTO

FLUID THIOGLYGOLLATE MEDIUM (B256)

DEHYDRATED

Bacto-Yeast Extract 5 g.

Bacto-Casitone 15 g.

Bacto-Dextrose 5 g.

Sodium Chloride 2.5 g.

/-Cystine, Difco 0.75 g.

Thioglycollic Acid 0.3 ml.

Bacto-Agar 0.75 g.

Resazurin, Certified 0.001 g.

Bacto-Fluid Thiogly col late Medium conforms to the formula specified by
the National Institute of Health^ for the sterility testing of biologicals and for the
sterility testing of antibiotics according to the method of the "Compilation of
Regulations for Tests and Methods of Assay and Certification of Antibiotic
Drugs," Federal Security Agency, Food and Drug Administration. The medium
is prepared according to the formula given for Thioglycollate Medium in the
U.S. Pharmacopeia^ and the National Formulary.* It may be recommended as
a liquid medium for the cultivation of anaerobes. The suitability of Bacto-Fluid
Thioglycollate Medium for the sterility testing of surgical catgut sutures has been
reported by Clock.^

The advantages of a small amount of agar in liquid media used for the
cultivation of anaerobes has been pointed out by a number of investigators.
Hitchens^ demonstrated that broth containing 0.1 per cent agar was particularly
well suited for the growth of both aerobes and anaerobes. In such a medium
anaerobes grew well without any seal or other special precautions. Falk, Bucca
and Simmons^ pointed out the advantages of the use of small quantities of agar
(0.06-0.25 per cent) in the detection of contaminants in the sterility testing of
biologicals.

In 1898 Trenkmann^ first reported the aerobic growth of anaerobes in the
presence of alkaline sulfide. Quastel and Stephenson^ reported that the presence
of a small amount of compound containing an -SH group permitted "aerobic"
growth of Clostridium sporogenes in a Tryptic Digest Broth. The -SH group
could be supplied by cysteine, thioglycollic acid or glutathione.

The value of combining a small amount of agar and a reducing substance was
demonstrated by Brewer.^^ He showed that in a liquid medium containing 0.05
per cent agar, anaerobes grew equally well in the presence or absence of sodium

196 DIFGO MANUAL

thioglycollate. He noted the value of media containing thioglycollic acid and
agar particularly in the initiation of growth of anaerobes from catgut sutures.
Marshall, Gunnison and Luxen^^ reported that the Thioglycollate Medium of
Brewer was satisfactory for the cultivation of anaerobes, and permitted the
growth of organisms in the presence of mercurial preservative. Nungester, Hood
and Warren^2 ^nd Portwood^^ confirmed the neutralization of the bacteriostatic
effect of mercurial compounds by sodium thioglycollate. Malin and Finn^* re-
ported that it has been observed that the commonly used medium containing
thioglycollate is inhibitory to some organisms in the presence of carbohydrate.

The National Institute of Health in 1941 ^^ specified the use of two Thiogly-
collate Media in sterility testing. The Brewer formula was prepared from infusion
of meat and 1 per cent peptone. The Linden formula contained 2 per cent
peptone and 0.2 per cent yeast extract. Each medium, in addition to 0.1 per
cent sodium thioglycollate, contained potassium phosphate, sodium chloride,
dextrose, agar and 1 : 500,000 methylene blue as an Eh indicator. In a study of
media used for sterility testing Christensen^^'^'^ reported that methylene blue as
used in the media was toxic for a number of organisms, and suggested the use
of resazurin as an Eh indicator. Sodium chloride and potassium phosphate were
toxic for some organisms encountered in sterility tests. Sodium thioglycollate, if
used in concentrations not greater than 0.05 per cent, was only slightly toxic
and in this concentration adequately neutralized the toxicit) of mercurial pre-
servatives used in biologicals. A reaction of pH 7.0 was suggested as being opti-
mum for the sterility test medium. Many suggestions on the media for sterility
testing leading to the present revised formula, as given by the National Institute
of Health,^ were carefully investigated and summarized by Pittman.^^

Bacto-Fluid Thioglycollate Medium is prepared according to the latest revision
as given by the "Compilation of Regulations for Tests and Methods of Assay and
Certification of Antibiotic Drugs," Federal Security Agency, Food and Drug
Administration, and conforms also to the specifications of the Federal Register
of the Food and Drug Administration,^ the U. S. Pharmacopeia^ and the Na-
tional Formulary.* This medium is carefully tested for its growth-promoting
abilities, oxidation-reduction qualities and its suitability to arrest mercurial stasis.
The medium supports the growth of aerobic as well as anaerobic organisms in a
cotton plugged tube without a special seal or other manipulation. Tubes 20 x 150
mm. containing 15 ml. of medium give the best ratio of surface exposed to depth
of medium for all types of organisms. Following autoclave sterilization the
medium should be cooled promptly to 25 °C. and kept at 20-30° C, preferably
in the dark, until ready for inoculation. If 30 per cent of the upper layer has a
pink color (oxidized resazurin) the medium should be heated in a boiling water
or steam bath to drive off absorbed oxygen. Do not reheat the medium more than
once, as continued reheating gives rise to toxicity.

In testing the sterility of solutions containing one of the customary mercury
salts in 1:10,000 dilution as a preservative, 15 ml. of Bacto-Fluid Thioglycollate
Medium will inactivate the mercurial preservative contained in 3 ml. of the
solution. When testing preparations containing other preservatives, the amount
of Fluid Thioglycollate Medium used must be sufficient to dilute the inoculum
beyond the bacteriostatic limits of the preservative. Using 1 ml. of inoculum of
biologicals or other preparations containing various preservatives, the minimum
quantities of culture media to use are given in the accompanying table.

Following inoculation the contents of the container must be thoroughly mixed
and mixed again when the first or 48 hour sterility test reading is made.

To rehydrate the medium, suspend 29.5 grams of Bacto-Fluid Thioglycollate
Medium in 1000 ml. cold distilled water, and heat to boiling to dissolve the
medium completely. Distribute in tubes or flasks and sterilize in the autoclave

DEHYDRATED CULTURE MEDIA 197

Minimum Amount of Culture

Media per 1 ml. of

Inoculum

Preservative

Concentration

Biological Minimum Volume of

Preservative

Product

Culture Medium

Phenol

0.5 per cent

Serums and
vaccine

40 ml.

Cresol, U.S.P.

0.35 per cent

Serum

60 ml.

Merthiolate

1:10,000

Toxoids

1:35,000

Human Plasma

10 ml.

Phenylmercuric Acetate

1:50,000

Human Plasma

1:100,000

Normal saline

10 ml.

Phenylmercuric Borate

1:50,000

Human Plasma

1:100,000

Normal saline

10 ml.

Phenylmercuric Nitrate

1:50,000

Human Plasma

1:100,000

Normal saline

10 ml.

Merthiolate and Phenol

1:10,000 and

0.2 per cent

Serum

20 ml.

Chlorobutanol

0.5 per cent

40 ml.

Formalin

0.4 per cent

40 ml.

for 18-20 minutes at 15-17 pounds pressure (121-123°C.). Cool quickly to
25°C. The final reaction of the medium will be pH 7.1.

One pound of Bacto-Fluid ThioglycoUate Medium will make 15.4 liters of
medium.

^ National Institute of Health Circular : Culture
Media for the Sterility Test, and Revision,
Feb. 5, 1946.

2 Compilation of Regulations for Tests and
Methods of Assay and Certification of Anti-
biotic Drugs, Federal Security Agency, Food
and Drug Administration.

3 Pharmacopeia of the United States, XIV Re-
vision: 758: 1950.

* National Formulary, 9th Edition: 768: 1950.

6 J. Lab. Clin. Med., 32:1153:1947.

<• J. Infectious Diseases, 29:390:1951.

' J. Bact., 37:121:1939.

sCentr. Bakt. I., Abt., 23:1038:1898.

1940.

;d. 43:672:1940.

^ Biochem. J., 20:1125:1926.

^0 J. Am. Med. Assoc, 115:;

^ Proc. Soc. Exp. Biol. Mec

12 Proc. Soc. Exp. Biol. Med. 52:287:1943.

^ J. Bact., 48:255:1944.

1* J. Bact., 62:349:1951.

^ National Institute of Health. Fluid Thiogly-
coUate Medium for the Sterility Test, Dec.
30, 1941-

M J. Bact., 48:256:1944-

" Paper Read at New York Meeting of Am.
Pub. Health Assoc, 1944.

18 J. Bact., 51:19:1946.

BACTO

BREWER THIOGLYGOLLATE MEDIUM

DEHYDRATED

(B236)

BACTO

LINDEN THIOGLYGOLLATE MEDIUM

DEHYDRATED

(B237)

These media formerly specified by the National Institute of Health have been
used in the past for testing of sterility or other purposes. For those laboratories
desiring to continue to use these media Bacto-Brewer ThioglycoUate Medium
and Bacto-Linden ThioglycoUate Medium are carried in stock.

198 DIFGO -MANUAL

BACTO

N.I.H. THIOGLYGOLLATE BROTH (B257)

DEHYDRATED

Bacto-Yeast Extract 5 g

Bacto-Casitone 15 g

Bacto-Dextrose 1 g

Sodium Chloride 2.5 g

/-Cystine, Difco 0.05 g

Thioglycollic Acid 0.3 ml

Bacto-N.I.H. Thioglycollate Broth is prepared according to the formula for
Broth Medium for sterility tests as specified by the National Institute of Health.^
This medium may be substituted for Bacto-Fluid Thioglycollate Medium in the
sterility testing of certain biological products that are turbid or otherwise cannot
be cultured satisfactorily in Fluid Thioglycollate Medium because of its viscosity.
Bacto-N.I.H. Thioglycollate Broth conforms to the specifications for the alternate
fluid medium for sterility tests as given in the U. S. Pharmacopeia^ and the
National formulary.^

In testing solutions for sterility, Bacto-N.I.H. Thioglycollate Broth should be
used in Smith fermentation tubes and be heated in boiling water or on a steam
bath just prior to use to drive off dissolved oxygen. When testing the sterility
of solutions containing preservatives, the preservative must be inactivated, or
sufficient medium used to dilute the inoculum beyond the bacteriostatic limits of
the preservative. Bacto-N.I.H. Thioglycollate Broth contains thioglycollate to
neutralize the bacteriostatic effect of mercurial preser\'atives. The table on page
197 under Bacto-Fluid Thioglycollate Medium indicates minimum quantities of
medium to use in testing the sterility of solutions containing other preservatives.

To rehydrate the medium, suspend 24 grams Bacto-N.I.H. Thioglycollate
Broth in 1000 ml. cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in Smith fermentation tubes and sterilize in the auto-
clave for 18-20 minutes at 15-17 pounds pressure (121-123°C.). The final
reaction of the medium will be pH 7.1.

One pound of Bacto-N.I.H. Thioglycollate Broth will make 18.9 liters of
medium.

1 National Institute of Health Circular: Culture ^ Pharmacopeia of the United States, XIV Re-
Media for the Sterility Test, and Revision, vision: 759:i95o.
Feb. 5, 1946. s >Tational Formulary, 9th Edition: 769: 1950.

BACTO

N.I.H. AGAR MEDIUM (B258)

DEHYDRATED

Bacto-Yeast Extract 5 g.

Bacto-Casitone 15 g.

Bacto-Dextrose 1 g.

Sodium Chloride 2.5 g.

/-Cystine, Difco 0.05 g.

Bacto-Agar 15 g.

Bacto-N.I.H. Agar Medium is prepared according to the formula for the
Agar Medium as specified by the National Institute of Health Circular for Cul-
ture Media for Sterility Test.^ It is recommended for use when a solid agar
medium is needed for the maintenance of cultures isolated in connection with the
sterility testing of biological products. It may also be used as a solid medium for
sterility testing.

U-^

DEHYDRATED CULTURE MEDIA 199

Bacto-N.I.H. Agar Medium contains no thioglycoliate. If the medium is to be
used to test the sterility of a biological product containing a mercurial preserva-
tive, 0.05 per cent sodium thioglycoliate or 0.03 per cent thioglycoUic acid should
be added.

To rehydrate the medium, suspend 38.5 grams of Bacto-N.I.H. Agar Medium
in 1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 18-20
minutes at 15-17 pounds pressure ( 121-123 °G.). The final reaction of the
medium will be pH 7.1.

One pound of Bacto-N.I.H. Agar Medium will make 11.7 liters of medium.

1 National Institute of Health Circular: Culture
Media for the Sterility Test, and Revision,
Feb. 5, 1946.

BACTO

THIOGLYGOLLATE MEDIUM (B363)
without Dextrose

DEHYDRATED

Bacto- Yeast Extract 5 g.

Bacto-Gasitone 15 g.

Sodium Chloride 2.5 g.

/-Cystine, Difco 0.25 g.

ThioglycoUic Acid 0.3 ml.

Bacto-Agar 0.75 g.

Bacto-Methylene Blue 0.002 g. ^-

Bacto-Thioglycollate Medium without Dextrose is a sugar-free basal medium
containing thioglycoUic acid and 0.075 per cent Bacto-Agar and to which carbo-
hydrate can be added for studying the fermentation reactions of anaerobic micro-
organisms. The medium contains thioglycoUic acid and Bacto-Agar which gives
and maintains a low Eh, permitting the growth of the strictest anaerobes without
special seal, making the medium ideally suited for fermentation studies of anaer-
obes. Similar media prepared without Eh indicator and without Dextrose or Eh
Indicator are described below.

The value of small amounts of agar in liquid media used for the cultivation
of anaerobes and microaerophiles as well as aerobes has been pointed out by
Kitchens^ and others. The use of alkaline sulfides and of sulfhydryl compounds
such as thioglycoliate, cysteine and glutathione for the reduction of the Eh poten-
tial of culture media for the propagation of anaerobes was first described by
Trenkmann^ and Quastel and Stephenson.^ Later Brewer"^ combined the use of
thioglycoliate with agar in liquid media for anaerobic culture.

The presence of the thioglycoliate in the medium will also inactivate any
mercurial that might be carried over with the inoculum, as was demonstrated
by Marshall, Gunnison and Luxen.° Methylene blue serves as an indicator of Eh.
Most organisms, including anaerobes, will show earlier and more vigorous growth
in the presence of a carbohydrate.

The sterile medium should not be stored in the refrigerator, especially if the
medium is to be used for the cultivation of anaerobes. The amount of oxidation
in the medium is shown by the depth of the color zone of the Eh indicator. Tubes
showing about one-third or more oxidized methylene blue are not satisfactory for
anaerobe cultivation and should be heated to the boiling point in steam or hot
water to drive off dissolved gases and rapidly cooled prior to inoculation. The
medium should be heated but once in this manner.

To rehydrate the medium, suspend 24 grams Bacto-Thioglycollate Medium

200 DIFCO MANUAL

without Dextrose in 1000 ml. cold distilled water and heat to boiling to dissolve
the medium completely. Add selected carbohydrate, distribute in tubes and
sterilize in the autoclave for 15 minutes at 15 pounds pressure (121°C.). Final
reaction of the medium will be pH 7.2.

One pound of Bacto-Thiogly collate Medium without Dextrose will make 19
liters of medium.

1 J. Infectious Diseases, 29:390:1921. * J. Am. Med. Assoc, 115:598:1940.

2Centr. Bakt., I Abt., 23:1038:1898. ^ Proc. Soc. Exp. Biol. Med., 43:672:1940.

3 Biochem. J., 20:1125:1926.

BACTO

THIOGLYGOLLATE MEDIUM (B430)
without Indicator

DEHYDRATED

Bacto-Thioglycollate Medium without Indicator has the same composition as
Bacto-Thioglycollate Medium without Dextrose, described above except that
it contains 0.5 per cent Bacto-Dextrose and no methylene blue as an indicator
of Eh.

To rehydrate the medium, suspend 29 grams Bacto-Thioglycollate Medium
without Indicator in 1000 ml. cold distilled water and heat to boiling to dis-
solve the medium completely. Distribute in tubes or flasks and sterilize in the
autoclave for 15 minutes at 15 pounds pressure (121°C.). Final reaction of the
medium will be pH 7.2.

One pound of Bacto-Thioglycollate Medium without Indicator will make
15.6 liters of medium.

BACTO

THIOGLYGOLLATE MEDIUM (B432)
without Dextrose or Indicator

DEHYDRATED

Bacto-Thioglycollate Medium without Dextrose or Indicator has the same
composition as Bacto-Thioglycollate Medium without Dextrose as described
on page 199 except that it does not contain methylene blue as an indicator of Eh.

To rehydrate the medium, suspend 24 grams Bacto-Thioglycollate Medium
without Dextrose or Indicator in 1000 ml. cold distilled water and heat to
boiling to dissolve the medium completely. Add selected carbohydrate, distribute
in tubes and sterilize in the autoclave for 15 minutes at 15 pounds pressure
(121°C.). Final reaction of the medium will be pH 7.2.

One pound of Bacto-Thioglycollate Medium without Dextrose or Indicator
will make 19 liters of medium.

BACTO

SABOURAUD LIQUID MEDIUM (B382)

DEHYDRATED

Neopeptone, Difco 10 g.

Bacto-Dextrose 20 g.

Bacto-Sabouraud Liquid Medium is recommended as a liquid medium for the
cultivation of yeast, molds and aciduric and acidophilic bacteria. This medium

DEHYDRATED CULTURE MEDIA 201

is particularly well suited for sterility test procedures for the detection of fungi.
It is prepared according to the formula specified in the U. S. Pharmacopeia^ and
National Formulary^ as used in sterility test procedures. The acid reaction of the
final medium, pH 5.7, is inhibitive to a large number of bacteria, but particu-
larly well suited for the cultivation of fungi and acidophilic microorganisms.
Neopeptone is used in the preparation of this medium since this peptone is
particularly well suited for the cultivation of fungi.

To rehydrate the medium dissolve 30 grams Bacto-Sabouraud Liquid Medium
in 1000 ml. of distilled water. Distribute in tubes or flasks and sterilize in the
autoclave for 15 minutes at 15 pounds pressure (121°C.). Final reaction of the
medium will be pH 5.7.

One pound of Bacto-Sabouraud Liquid Medium will make 15.1 liters of
medium.

1 Pharmacopeia of the United States, XIV 2 National Formulary, gth Edition: 769: 1950.
Revision : 760 : 1 950.

BACTO

A G MEDIUM (B316)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto- Yeast Extract 3 g.

Bacto-Malt Extract 3 g.

Proteose Peptone No. 3, Difco 20 g.

Bacto-Dextrose 5 g.

Bacto-Agar 1 g.

Ascorbic Acid 0.2 g.

Bacto-A C Medium is recommended for use in controlling sterility of prod-
ucts in the process of manufacture and for testing sterility of solutions and other
materials not containing mercurial preservatives. For the sterility testing of bio-
logicals and solutions containing mercurials as a preservative, Bacto-Fluid Thio-
glycoUate Medium, page 195, should be employed.

Bacto-A C Medium is an infusion-free medium possessing unique growth-
promoting properties for both aerobic and anaerobic microorganisms, making
possible an early and voluminous growth. It is recommended as a general
culture medium for the propagation of anaerobes, micro-aerophiles and aerobes.
Bacto-A C Medium does not exhibit the toxicity shown by media containing
sodium thioglycollate for some organisms as reported by Christensen,^ and Malin
and Finn.2

Reed and Orr^ obtained excellent growth of all species of Clostridium on
Bacto-A C Medium. Schneiter, Dunn and Caminita* in their studies on the
bacterial content of air samples reported Proteose Extract Agar, the same com-
position as A C Medium with 0.85 per cent sodium chloride and 1.7 to 2.4 per
cent Bacto-Agar added to be satisfactory for the growth of staphylococci and
sporulates, and superior to all other media tested for the growth of streptococci.
Bailey et al.^ employed A C Medium in assaying for potency of streptomycin
products using Clostridium perfringens as a test organism and reported excellent,
rapid results. Schneiter and Kolb^ used Bacto-A C Medium for the growth of
Bacillus anthracis and related mesophilic aerobic bacilli in their studies of the
heat resistance of these organisms from hair and bristles. They reported that the
medium permitted the distinctive cottonball appearance of the anthrax colonies.
Kolb and Schneiter^ used Bacto-A G Medium to test the viability of B. anthracis
following exposure to methyl bromide to test the efficiency of this compound as
a germicidal and sporicidal agent.

202 DIFCO MANUAL

For best grovs^h of anaerobes and most aerobes, A C Medium should be dis-
tributed in tubes or flasks to give a depth of at least 70 mm. of medium. For
obligately aerobic bacteria shallower layers permitting aeration of the substrate
are desirable.

To rehydrate the medium, suspend 35 grams of Bacto-A G Medium in 1000
ml. of cold distilled water and heat to boiling to dissolve the medium completely.
Distribute in tubes or bottles to give the desired depth of medium and sterilize
in the autoclave for 15 minutes at 15 pounds pressure (121°C.). If the medium
is not used the same day it is prepared and sterilized, it is advisable to place it in
flowing steam or in a boiling water bath to drive off dissolved gases and then cool
before inoculating. The final reaction of the medium will be pH 7.3.

One pound of Bacto-A G Medium will make 13 liters of medium.

1 Paper read at New York Meeting Am. Pub. * Pub. Health Reports, 60:789:1945.

Health Assoc, 1944. ^Personal Communication, 1947.

' J. Bact., 62:349:1951. ^ Pub. Health Reports, Sup. No. 207, June, 1948.

«J. Bact., 45:309:1943- ^J- Bact., 59:401:1950.

BACTO

A G BROTH (B317)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto- Yeast Extract 3 g.

Bacto-Malt Extract 3 g.

Proteose Peptone No. 3, Difco 20 g.

Bacto-Dextrose 5 g.

Ascorbic Acid 0.2 g.

Bacto-A C Broth is particularly suitable for use as a sterility test broth in the
detection of obligately aerobic contaminants in biologicals and other products.
Sterility tests on materials containing mercurial preservative should be made in
Bacto-Fluid Thiogly collate Medium, as discussed on page 195.

Bacto-A G Broth is recommended as a general culture m_edium for the propa-
gation of pathogenic and saprophytic microorganisms. It has the same formula
as Bacto-A G Medium, as discussed on page 201, except that the small amount of
agar has been omitted. This medium with the addition of 0.85 per cent sodium
chloride and 1.8 to 2.5 per cent Bacto-Agar was reported by Schneiter, Dunn and
Caminita^ to be a superior medium for bacteriological examination of air, espe-
cially for recovery of streptococci.

To rehydrate the medium, dissolve 34 grams of Bacto-A G Broth in 1000 ml.
of distilled water. Distribute in tubes or other suitable containers and sterilize in
the autoclave for 15 minutes at 15 pounds pressure (121°G.). The final reaction
of the medium will be pH 7.2.

One pound of Bacto-A G Broth will make 13 liters of medium.
1 Pub. Health Reports, 60:789:1945.

BACTO

MALT EXTRACT BROTH

DEHYDRATED

Bacto-Malt Extract Broth is recommended as a sterility test medium for the
detection of yeasts and molds. This acid medium supports early and luxuriant

DEHYDRATED CULTURE MEDIA 203

growth of these organisms from small inocula. A complete discussion of Bacto-
Malt Extract Broth is given on page 242.

BACTO-PENASE

Bacto-Penase and Bacto-Penase Concentrate are potent penicillinase prepara-
tions recommended for the inactivation of penicillin in the sterility testing of
penicillin or penicillin products. A complete discussion of these products are
given on pages 283 and 284.

MEDIA FOR
MICROBIOLOGICAL ASSAYS

MEDIA FOR THE ASSAY OF ANTIBIOTICS

The Difco dehydrated media described in this section are prepared especially
for the assay of the potency of antibiotics. The use of dehydrated media corre-
sponding to the specified formula is permitted for the assay of antibiotics accord-
ing to the method prescribed by the "Compilation of Regulations for Tests and
Methods of Assay and Certification of Antibiotic Drugs," Federal Security
Agency, Food and Drug Administration and the United States Pharmacopeia.
Included in this section are media conforming to their specifications.

BACTO

PENASSAY BROTH (B243)

DEHYDRATED

Bacto-Beef Extract 1.5 g.

Bacto- Yeast Extract 1.5 g,

Bacto-Peptone 5 g.

Bacto-Dextrose 1 g.

Sodium Chloride 3.5 g.

Dipotassium Phosphate 3.68 g.

Monopotassium Phosphate 1.32 g.

Bacto-Penassay Broth duplicates the formula of the medium recommended
by Schmidt and Moyer^ for use in the serial dilution method for assaying peni-
cillin. It also corresponds to the formula of the broth prescribed in "Tests and
Methods of Assay for Antibiotic Drugs,"^ Federal Security Agency, Food and
Drug Administration, for the preparation of the inoculum in the cylinder assay
of penicillin, or in the tube dilution method of penicillin assay; in the turbi-
dimetric assay of streptomycin; for the preparation of the suspension of Sarcina
lutea (PCI 1001) in the cylinder assay of aureomycin and chloramphenicol; in
the turbidimetric assay of bacitracin. Waksman and Lechevalier^ used Bacto-
Penassay Broth for the turbidimetric assay of neomycin with Escherichia coli
^9637 as the test organism.

Schmidt and Moyer^ used the serial dilution method for assaying ethereal
solutions and untreated culture liquor of penicillin as a check on the results
obtained by the cylinder plate method. Serial dilution methods may also be used
in assaying for penicillin in body fluids of individuals under treatment.

Essentially the serial dilution methods are based upon determinations of the

204 DIFCO MANUAL

lowest concentration of antibiotic capable of inhibiting growth of the test organ-
ism in the Penassay Broth. The approximate amount of antibiotic present in an
unknown is determined by comparing its activity in parallel with that of a
standard preparation of known potency. A quantity of the sample to be tested
is added to the first of a series of tubes containing broth previously inoculated
with the test organism. Further dilutions are made from this tube. Comparable
dilutions of the standard are also made. All tubes are thoroughly shaken and
incubated at 30°C. for 18 or 40 hours. More definite readings may be made in
40 hours than in 18. The highest dilution in which no growth appears is the end
point of the series, and the dilution of the unknown which gives such an end
point contains the same amount of antibiotic as that in the end point tube of
the standard series.

It is sometimes difficult to obtain a sharp end point in the serial dilution
methods of penicillin or streptomycin assay. Also, the amount of antibiotic re-
quired is usually much larger in these methods than in the turbidimetric pro-
cedure. Foster* described a turbidimetric method for penicillin assay and claimed
that it was the most accurate method available. McMahan^ offered a 3-4 hour
turbidimetric procedure based on the same procedure used in the microbiological
assay of vitamins and considered it more precise than the cup plate method. Lee,
Foley, Epstein and Wallace^ modified the method of Foster and Woodruff^ and
obtained results in 90 minutes instead of four hours.

The turbidimetric method is based on the proportional inhibition of the
growth of the test organism in the liquid medium as a function of the antibiotic
concentration. The growth measurements are made turbidimetrically and com-
pared with a standard curve obtained by running various concentrations of a
standard antibiotic in parallel with the unknown.

To rehydrate the medium, dissolve 17.5 grams of Bacto-Penassay Broth in
1000 ml. distilled water. When used in the serial dilution method, distribute in
200 ml. quantities in 500 ml. Erlenmeyer flasks. For carrying tests organisms, as
described by the Food and Drug Administration,^ distribute in 10 ml. quantities
in tubes. For the turbidimetric procedure, distribute as required by the specific
procedure used. The medium is sterilized for 15 minutes at 15 pounds pressure
(121°C.). The final reaction of the medium will be pH 7.0.

One pound of Bacto-Penassay Broth will make 25.8 liters of medium.

ij. Bact., 47:199: 1944- * J- Biol. Chem., 144:285:1942.

2 The Compilation of Tests and Methods of ^ j. giol. Chem., 153:249:1944.
Assay for Antibiotic Drugs, Federal Security e j. Biol. Chem., 152:485:1944.
Agency, Food and Drug Administration. 'J. Bact., 46:187:1943-

3 Science, 1 09 : 305 : 1 949.

BACTO

PENASSAY BASE AGAR (B270)

DEHYDRATED

Bacto-Beef Extract 1.5 g.

Bacto- Yeast Extract 3 g.

Bacto-Peptone 6 g.

Bacto-Agar 15 g.

Bacto-Penassay Base Agar is prepared according to the formula specified in
"The Compilation of Tests and Methods of Assay for Antibiotic Drugs,"i
Federal Security Agency, Food and Drug Administration, for antibiotic assay. It
is recommended as a base layer in the cylinder assay of penicillin; for the cylinder
assay of aureomycin; for the plate assay of bacitracin and for maintaining cul-

DEHYDRATED CULTURE MEDIA 205

tures of Micrococcus pyogenes var. aureus (PCI 1203) in the turbidimetric assay
of bacitracin.

The discovery of penicillin^ and of streptomycin^ and the widespread use of
these and other antibiotics in the treatment of many bacterial infections neces-
sitated the development of methods for their detection and assay. A number of
methods for the microbiological assay of antibiotic drugs have been described.
These include serial dilution methods with liquid or solid media, turbidimetric
methods and the Oxford cup or cylinder plate method.

The cylinder plate method for the assay of penicillin was first described by
Abraham et al.* and later modified by Foster and Woodruff^ and by Schmidt
and Moyer.^ This method depends upon the diffusion of the antibiotic material
from steel or porcelain cups placed upon agar plates which have been seeded with
the test organism. Inhibition of growth of the organism occurs in the proximity
of the cup, and the diameter of the inhibited zone varies with the concentration
of the antibiotic in the material being tested. This method is commonly employed
for assaying commercial penicillin preparations and is also adapted for the detec-
tion of penicillin in body fluids of patients under penicillin treatment.

The cylinder plate method has given uniform results which may be duplicated.
Continued study of this method of penicillin assay showed that a modification of
the Schmidt and Moyer formula^ resulted in more accurate readings; also, that
if dextrose is present in the seed layer but omitted from the base layer the zones
of inhibition are more clear-cut and defined. These modifications were adopted
by the Food and Drug Administration^ as standard for penicillin assay using
Micrococcus pyogenes var. aureus as the test organism.

Plates are prepared the same day samples are to be tested, using the cylinder
method of assay for antibiotics by adding 21 ml. of sterile Bacto-Penassay Base
Agar to sterile petri dishes (100 x 20 mm.). After the base layer has solidified
it is overlaid with 4 ml. of Bacto-Penassay Seed Agar previously inoculated with
a carefully standardized dilution of the test organism.

To rehydrate the medium, suspend 25.5 grams Bacto-Penassay Base Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in 500 ml. quantities in one-liter Erlenmeyer flasks and steri-
lize in the autoclave for 15 minutes at 15 pounds pressure (121°C.). The final
reaction of the medium will be pH 6.6.

One pound of Bacto-Penassay Base Agar will make 17.7 liters of medium.

1 The Compilation of Tests and Methods of ^ PfQc. Soc. Exp. Biol. Med., 55:66:1944.
Assay for Antibiotic Drugs, Federal Security * Lancet, 2:177:1941.

Agency, Food and Drug Administration. ^ j_ Bact., 46:187:1943.

2 Brit. J. Exp. Path., 10:226:1929. a j. Bact., 47:199:1944.

BACTO

PENASSAY SEED AGAR (B263)

DEHYDRATED

Bacto-Beef Extract 1.5 g.

Bacto- Yeast Extract 3 g.

Bacto-Casitone 4 g.

Bacto-Peptone 6 g.

Bacto-Dextrose 1 g.

Bacto-Agar 15 g.

Bacto-Penassay Seed Agar conforms to the medium specified by "The Com-
pilation of Tests and Methods of Assay for Antibiotic Drugs,"i Federal Security
Agency, Food and Drug Administration, for antibiotic assay. It is recommended

206 DIFCO MANUAL

as a seed layer and for carrying stock cultures of Micrococcus pyogenes var.
aureus (PCI 209P) in penicillin cylinder assay; for maintaining test organism,
Bacillus suhtilis (ATCC ?^6633) in streptomycin assay; for seed layer in aureo-
mycin cylinder assay and for maintaining cultures of M. pyogenes var. aureus
(PCI 209P) for the turbidimetric assay of aureomycin; for seed and base layer
and for maintaining cultures of Sarcina lutea (PCI 1001) in chloramphenicol
cylinder assay; for seed layer in bacitracin plate assay.

In the assay of antibiotics by the cylinder method it has been found that for
penicillin, aureomycin and bacitracin, better results are obtained with a small
amount of dextrose in the seed layer and no dextrose in the medium used for the
base layer. For the assay of these antibiotics the use of Bacto-Penassay Seed Agar
has resulted in more clear-cut zones of inhibition. A layer of Bacto-Penassay Base
Agar is allowed to solidify in plates, and is overlaid with about 4 ml. of Penassay
Seed Agar (100 x 20 mm. plate) containing a carefully standardized inoculum
of the test organism. The plate is tilted to obtain an even distribution of the
inoculum on the surface. Following solidification of the seed layer, the plate is
ready to receive the cylinders and antibiotic solutions under assay.

Formerly, the base agar was used for both the base and seed layers; however,
it was found that the zones of inhibition were not as sharply defined as was
desired. The use of Bacto-Penassay Seed Agar as the seed layer has resulted in a
more clear-cut zone of inhibition.

Plates are prepared the day samples are to be tested by adding 21 ml. of
Penassay Base Agar to sterile petri dishes (100 x 20 mm.). After the base layer
has solidified it is overlaid with 4 ml. of Bacto-Penassay Seed Agar, inoculated
with the test organism.

To rehydrate the medium, suspend 30.5 grams of Bacto-Penassay Seed Agar
in 1000 ml. of cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in 100 ml. quantities in 300 ml, Erlenmeyer flasks. Sterilize
in the autoclave for 15 minutes at 15 pounds pressure (121°C.). The final reac-
tion of the medium will be pH 6.6.

One pound of Bacto-Penassay Seed Agar will make 14.8 liters of medium.

1 The Compilation of Tests and Methods of
Assay for Antibiotic Drugs, Federal Security
Agency, Food and Drug Administration.

BACTO

YEAST BEEF AGAR (B244)

DEHYDRATED

Bacto-Beef Extract 1.5 g.

Bacto- Yeast Extract 3 g.

Bacto-Peptone 6 g.

Bacto-Dextrose 1 g.

Bacto- Agar 15 g.

Bacto-Yeast Beef Agar is a slight modification of the medium recommended
by Schmidt and Moyer^ for use in the cylinder plate procedure for assaying
penicillin as described by Abraham, et al.^

As a result of their extensive use of the cylinder plate method, Schmidt and
Moyer made a number of observations and refinements which improved the
technique of penicillin assay by this method. They claimed that the cylinder
plate method was a more practical procedure than the serial dilution method,
that it is more adaptable for assay of a large number of specimens, that samples
need not be sterile, as is necessary in the serial dilution method, and that more

DEHYDRATED CULTURE MEDIA 207

potent penicillin preparations can be assayed with fewer dilutions than in the
serial dilution technique.

More recently the Food and Drug Administration^ recommended modifica-
tions of this medium for use in the cylinder plate method. Bacto-Penassay Base
Agar and Bacto-Penassay Seed Agar used in this procedure are discussed on
pages 204 and 205.

Plates are prepared the day previous to the actual testing of penicillin samples
by adding 22 ml. of Yeast Beef Agar to petri dishes (100 x 20 mm.) which then
stand at room temperature for 24 hours before being overlaid with 3 ml. of Yeast
Beef Agar previously inoculated with the test organism, Micrococcus pyogenes
var. aureus (P209).

To rehydrate the medium, suspend 26.5 grams of Bacto-Yeast Beef Agar in
1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in 500 ml. quantities in one liter Erlenmeyer flasks. Sterilize in
the autoclave for 15 minutes at 15 pounds pressure (121°C.). The final reaction
of the medium will be pH 6.6.

One pound of Bacto-Yeast Beef Agar will make 17.5 liters of medium.

1 J. Bact., 47:199:1944. Assay for Antibiotic Drugs, Federal Security

2 Lancet, 2:177:1941. Agency, Food and Drug Adininistration.
* The Compilation of Tests and Methods of

BACTO

MYCIN ASSAY BROTH (B295)

DEHYDRATED

Bacto-Beef Extract 5 g.

Bacto-Peptone 10 g.

Sodium Chloride 2.5 g.

Bacto-Mycin Assay Broth duplicates the formula of the medium recommended
by Price, Nielsen and Welch^ for their serial dilution method of assay of strepto-
mycin in body fluids.

The authors found that the turbidimetric method in use for assaying strepto-
mycin in aqueous or buffered solution gave erroneous results when used for
testing body fluids because of the stimulating properties of these fluids for the
test organism. Also, that the equipment and material required for the agar
cylinder plate method may not be readily available to the small laboratory. For
these reasons a study was made to determine which organism would lend itself
to use in a simple serial dilution procedure for determining streptomycin in body
fluids. They found that Bacillus circulans was the most sensitive to streptomycin
and gave reproducible results.

Essentially, the test consists in preparing various dilutions of the fluid under
test in sterile Bacto-Mycin Assay Broth. A streptomycin of known potency is used
as a standard and is serially diluted in a similar manner. The tubes are inoculated
with the test organism and incubated at 37°C. over night. The concentration of
streptomycin in the unknown is determined by comparing the end point in the
unknown with that in the standard.

To rehydrate the medium, dissolve 17.5 grams of Bacto-Mycin Assay Broth in
1000 ml. cold distilled water. Distribute in tubes and sterilize in the autoclave
for 15 minutes at 15 pounds pressure (121°G.). The final reaction of the medium
will be pH 7.9.

One pound of Bacto-Mycin Assay Broth will make 25.9 liters of medium.

1 Science, 1 03 : 56 : 1 946.

208 DIFCO MANUAL

BACTO

STREPTOMYCIN ASSAY AGAR (B277)

DEHYDRATED

Bacto-Beef Extract 1.5 g.

Bacto- Yeast Extract 3 g.

Bacto-Peptone 6 g.

Bacto- Agar 15 g.

Bacto-Streptomycin Assay Agar was originally developed with the coopera-
tion of Dr. Phillip S. Skell, University of Illinois, for the assay of streptothrycin.
Later, this medium was found to give excellent results for the streptomycin
assay, using the cylinder plate technique with Bacillus subtilis (ATCC ?$^6633)
as the test organism. The use of this medium assures well-defined zones of inhibi-
tion of growth of the test organism.

Plates are prepared the same day samples are to be tested by adding 21 ml.
of Bacto-Streptomycin Assay Agar to petri dishes (100 x 20 mm.). After this base
layer has solidified, it is overlaid with 4 ml. of Streptomycin Assay Agar previ-
ously inoculated with spores of the test organism.

To rehydrate the medium, suspend 25.5 grams Bacto-Streptomycin Assay Agar
in 1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in 500 ml. quantities in one-liter Erlenmeyer flasks. Sterilize in
the autoclave for 15 minutes at 15 pounds pressure (121°C.). The final reaction
of the medium will be pH 8.0.

One pound of Bacto-Streptomycin Assay Agar will make 17.7 liters of medium.

BACTO

MYCIN ASSAY AGAR (B281)

DEHYDRATED

Bacto-Beef Extract 3 g.

Bacto-Peptone 5 g,

Bacto- Agar 15 g.

Bacto-Mycin Assay Agar duplicates the formula of the medium specified by
the Food and Drug Administration^ for assay of streptomycin by the cylinder
plate method. The use of this medium assures well defined zones of inhibition of
the test organism.

The general principles used for the assay of penicillin have proved practical
for the assay of streptomycin. However, different antibiotics require different test
organisms and a slight change in the composition of the medium employed. The
Food and Drug Administration has specified that the medium used in the assay
of streptomycin should have an alkaline reaction and that the test organism be
Bacillus subtilis (ATCC ?^6633).

Plates are prepared the same day samples are to be tested by adding 21 ml. of
Mycin Assay Agar to petri dishes (100 x 20 mm.). After this base layer has solid-
ified it is overlaid with 4 ml. of Mycin Assay Agar previously inoculated with
spores of the test organism.

To rehydrate the medium, suspend 23 grams of Bacto-Mycin Assay Agar in
1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in 500 ml. quantities in one-liter Erlenmeyer flasks. Sterilize in

DEHYDRATED CULTURE MEDIA 209

the autoclave for 15 minutes at 15 pounds pressure (121°C.). The final reaction
of the medium is pH 7.9.

One pound of Bacto-Mycin Assay Agar will make 19.6 liters of medium.

1 The Compilation of Tests and Methods of
Assay for Antibiotic Drugs, Federal Security
Agency, Food and Drug Administration.

BACTO

TYROTHRIGIN ASSAY BROTH (B415)

DEHYDRATED

Bacto-Tryptone 10 g.

Bacto-Dextrose 5 g.

Disodium Phosphate 1-6 g,

Bacto-Tyrothricin Assay Broth is prepared according to the U. S. Pharmacopeia
formula^ for the inoculum broth used in the assay of tyrothricin. The test or-
ganism, streptococcus sp., Lancefield group D, strain No. H69D5 (ATCC ^9854)
is carried in stock on Blood Agar prepared with Bacto-Tyrothricin Assay Agar.
Inoculation is made from the slant into Bacto-Tyrothricin Assay Broth and tubes
are incubated at 37°C. for 18-20 hours. The cultures are examined micro-
scopically at this time to check their purity. Bacto-Tyrothricin Assay Broth is
also recommended as a buffered Dextrose Broth for the cultivation of a large
variety of saprophytic and pathogenic microorganisms.

In the assay of tyrothricin by the U. S. Pharmacopeia method, 80 ml. of the
sterile Tyrothricin Assay Broth is enriched with 20 ml. of a filter sterilized 5
per cent solution of bovine serum albumin at pH 6.8-7.2 to give the assay
medium. To 100 ml. of this mixture is added 1 ml. of an 18-20 hour culture of
the test organism. The Tyrothricin Reference Standard, and the sample under
assay properly diluted, are mixed with propylene glycol diluting solution in
5 ml. amounts to give 10 tubes with 0.0, 0.1, 0.15, etc., to 0.5 microgram tyro-
thricin. To each tube containing 5 ml. of the mixture of tyrothricin and propylene
glycol is added 5 ml. of the 1:100 dilution of the test organism in the assay
medium. Tubes are prepared in triplicate. The tubes are thoroughly shaken and
incubated at 37 °G., for 16-20 hours. The amount of growth is determined by any
suitable photometric method. The average value of the readings of each set of
triplicate tubes is used to obtain a single value for each tube. Prepare a standard
curve for the U. S. P. Tyrothricin Reference Standard by plotting the average
density measurements against the concentration of tyrothricin in micrograms per
ml. Similarly, prepare a curve from the average values obtained from the tyro-
thricin being assayed. From the average density of the control tubes, determine
the photometric reading equivalent to one-half the density of the controls and
interpolate this point on both of the above curves. Intercepts drawn from these
points to the base line will indicate the quantity of tyrothricin.

To rehydrate the medium, dissolve 16.6 grams Bacto-Tyrothricin Assay Broth
in 1000 ml. of distilled water. Distribute in tubes and sterilize in the autoclave
for 15 minutes at 15 pounds pressure (121°C.). Avoid excessive heat during
the sterilization period and cool the medium rapidly. Final reaction of the
medium will be pH 7.0. To prepare the complete liquid medium for the assay of
tyrothricin add 20 ml. of a 5 per cent filter sterilized solution of bovine serum
albumin, adjusted to pH 6.8-7.2 with normal sodium hydroxide solution to each
80 ml. of sterile Bacto-Tyrothricin Assay Broth cooled to 45 °G., or less.

One pound of Bacto-Tyrothricin Assay Broth will make 27.4 liters medium.

1 Pharmacopeia of the United States, XIV Re-
vision: 650: 1950.

210 DIFGO MANUAL

BACTO

TYROTHRIGIN ASSAY AGAR (B414)

DEHYDRATED

Bacto-Tryptone 5 g.

Bacto-Beef Extract 3 g.

Bacto-Agar 15 g.

Bacto-Tyrothrlcin Assay Agar is recommended for carrying stock cultures of
streptococcus sp., Lancefield group D, strain No. H69D5 (ATCC ^9854) em-
ployed in the assay of tyrothricin according to the procedure given by U. S.
Pharmacopeia.^ This medium is also suggested as a solid medium for the culti-
vation of a large variety of microorganisms.

In the assay of tyrothricin the test organism is carried in stock on a Blood Agar
prepared by adding 2 per cent sterile defibrinated human, horse or rabbit blood
to sterile melted and cooled Tyrothricin Assay Agar. The complete medium is
allowed to solidify in a slanted position. If Blood Agar is to be prepared for
purposes other than carrying cultures of streptococcus sp., Lancefield group D,
strain No. H69D5 for tyrothricin assay, it is suggested that 0.5 per cent sodium
chloride be added to the medium to make it isotonic, in order to prevent
hemolysis of the blood cells.

To rehydrate the medium, suspend 23 grams of Bacto-Tyrothricin Assay Agar
in 1000 ml. of cold distilled water. Heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°G.). To prepare Blood Agar, cool the sterile melted
agar to 45-50° C. and add the desired sterile defibrinated blood under aseptic
conditions. Final reaction of the medium will be pH 6.8.

One pound of Bacto-Tyrothricin Assay Agar will make 19.7 liters of medium.

1 Pharmacopeia of the United States, XIV Re-
vision: 650: 1950.

BACTO

POLYMYXIN BASE AGAR (B462)

DEHYDRATED

Bacto-Casitone 17 g.

Bacto-Soytone 3 g.

Bacto-Dextrose 2.5 g.

Sodium Chloride 5 g.

Dipotassium Phosphate 2.5 g.

Bacto-Agar 20 g.

Bacto-Polymyxin Base Agar is prepared according to the formula specified
by "The Compilation of Tests and Methods of Assay for Antibiotic Drugs,"^
Federal Security Agency, Food and Drug Administration for base layer in the
cylinder assay of polymyxin B. This medium is also used for carrying stock cul-
tures of the test organism. Brucella bronchiseptica (ATCC ^4617), and pre-
paring the inoculum in the polymyxin B. assay.

To rehydrate the medium, suspend 50 grams Bacto-Polymyxin Base Agar in
1000 ml. distilled water and heat to boiling to dissolve the medium completely.
Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). Final reaction of the medium will be pH 7.3.

One pound of Bacto-Polymyxin Base Agar will make 9.1 liters of medium.

*The Compilation of Tests and Methods of
Assay for Antibiotic Drugs. Federal Security
Agency, Food and Drugs Administration.

DEHYDRATED CULTURE AIEDIA 211

BACTO

POLYMYXIN SEED AGAR (B463)

DEHYDRATED

Bacto-Casitone 17 g.

Bacto-Soytone 3 g.

Bacto-Dextrose 2.5 g.

Sodium Chloride 5 g.

Dipotassium Phosphate 2.5 g.

Bacto-Agar 12 g.

Tween 80 10 g.

Bacto-Polymyxin Seed Agar is prepared according to the formula specified by
"The Compilation of Tests and Methods of Assay for Antibiotic Drugs,"^
Federal Security Agency, Food and Drug Administration for the seed layer in the
cylinder assay of Polymyxin B. This medium has the same composition as Bacto-
Polymyxin Base Agar, as described above, except the agar content has been re-
duced to 1.2 per cent and contains 1.0 per cent Tween 80. In the assay of
Polymyxin B, Brucella bronchiseptica ATCG 4617 is the test organism using this
method.

To rehydrate the medium, suspend 52 grams Bacto-Polymyxin Seed Agar in
1000 ml. distilled water and heat to boiling to dissolve the medium completely.
Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). Final reaction of the medium will be pH 7.3.

One pound of Bacto-Polymyxin Seed Agar will make 8.7 liters of medium.

1 The Compilation of Tests and Methods of
Assay for Antibiotic Drugs, Federal Security
Agency, Food and Drug Administration.

BACTO

FLUID THIOGLYCOLLATE MEDIUM

DEHYDRATED

Fluid ThioglycoUate Medium is specified in the Compilation of Tests and
Methods of Assay for Antibiotic Drugs, Federal Security Agency, Food and Drug
Administration in the sterility testing of antibiotics. A complete discussion of this
medium is given on page 195.

BACTO-PENASE

212 DIFCO MANUAL

MEDIA FOR THE ASSAY
OF VITAMINS AND AMINO ACIDS

The Difco dehydrated media described in this section are prepared especially
for use in the microbiological assay of vitamins and amino acids. Two types of
media are employed for this purpose, one for carrying the culture in stock or
preparing the inoculum, the other for assay. The latter type of media contain all
the factors necessary for growth of the test organism except one essential
ingredient.

Each medium for assay purposes is free from the essential growth requirement
factor for which the medium is recommended. When this factor is added, in
specified increasing concentrations, the growth response of the particular test
organism is measured either acidimetrically or turbidimetrically. In the per-
formance of the various tests the basal medium is prepared and is generally
tubed in 5 ml. amounts. To one series of tubes are added graded quantities of
the known essential ingredient. To the second similar series are added varying
amounts of the material under investigation. Included in each series of tubes is
one containing only distilled water and the basal medium. This tube acts as a
blank control. The tubes of each series are then made up to 10 ml. with distilled
water.

Great care to avoid contamination of media or glassware must be taken in
microbiological assay procedures. Extremely small amounts of foreign material
may be sufficient to give erroneous results. Scrupulously clean glassware free from
detergents and other chemicals must be used. For example, in the assay of
Vitamin B12, as small a quantity as 0.01 millimicrogram will give rise to a
definite growth response. The importance of clean glassware in the assay of this
vitamin was stressed by the U. S. Pharmacopeia Vitamin Bjg Study Panel, since
they stated that glassware for this purpose required special handling, and as
many as twelve rinses appeared to be necessary for satisfactory results.

For the successful execution of these procedures all conditions of the assay
must be adhered to meticulously. In the description of each medium points of
prime importance for successful assays have been stressed.

BACTO

MICRO ASSAY CULTURE AGAR (B319)

DEHYDRATED

Bacto-Yeast Extract 20 g.

Proteose Peptone No. 3, Difco 5 g.

Bacto-Dextrose 10 g.

Monopotassium Phosphate 2 g.

Sorbitan Monooleate Complex 0.1 g.

Bacto-Agar 10 g.

Bacto-Micro Assay Culture Agar is recommended for carrying stock cultures of
Lactobacilli and other test organisms used for microbiological assay. This medium
is also recommended for the general cultivation of Lactobacilli and many other
microorganisms.

In the microbiological assay of vitamins and other essential nutriments, the

DEHYDRATED CULTURE MEDIA 213

organism employed is an essential part of the test. In addition to selecting the
proper strain, the previous environment, the age of the culture and size of the
inoculum are of prime importance in the satisfactory performance of the test.
Micro Assay Culture Agar has proved to be an excellent medium in which to
carry stock cultures of Lactobacilli and other test organisms used for microbio-
logical assay work, while Micro Inoculum Broth is recommended for the prepara-
tion of the inoculum.

Stock cultures of the test organism are prepared by stab inoculation. It is de-
sirable to prepare stock cultures at least in triplicate at monthly intervals. One of
the transfers is saved for the preparation of stock cultures, and the others used
to prepare inoculum in Micro Inoculum Broth, for assay, as needed. Stock
cultures following incubation at 35-3 7 °C. for 24-48 hours should be kept in the
refrigerator at 2-6°C.

To rehydrate the medium, suspend 47 grams of Bacto-Micro Assay Culture
Agar in 1000 ml. of cold distilled water and heat to boiling to dissolve the
medium completely. The medium is then distributed in 10 ml. quantities in
tubes of 16-20 mm. diameter. Sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). The tubes are then allowed to cool in an upright posi-
tion. Final reaction of the medium will be pH 6.7.

One hundred grams of Bacto-Micro Assay Culture Agar will make 2.1 liters
of medium.

BACTO

MICRO INOCULUM BROTH (B320)

DEHYDRATED

Bacto- Yeast Extract 20 g.

Proteose Peptone No. 3, Difco 5 g.

Bacto-Dextrose 10 g.

Monopotassium Phosphate 2 g.

Sorbitan Monooleate Complex 0.1 g.

Bacto-Micro Inoculum Broth is recommended for the cultivation of Lacto-
bacilli used in microbiological assays. It is of particular value in the preparation
of the inoculum for these tests.

Bacto-Micro Inoculum Broth may be employed in preparing the inoculum of
Lactobacilli used in microbiological assay. This medium eliminates the necessity
of using various basal media for this purpose. Subcultures of Lactobacilli are
made from Bacto-Micro Assay Culture Agar into 10 ml. tubes of Bacto-Micro
Inoculum Broth and incubated for 24 hours at 37°C. The culture is then centri-
fuged, washed with sterile isotonic sodium chloride solution, and diluted before
use as inoculum. It is essential that the directions given in the discussion of
each assay medium be followed in minute detail, since the age, preparation and
size of the inoculum are most important factors in obtaining a satisfactory assay.
Although other media and methods may be used successfully for carrying the
cultures and preparing the inocula, we feel that uniformly good results will be
obtained if the methods described under each medium are followed.

To rehydrate the medium, dissolve 37 grams of Bacto-Micro Inoculum Broth
in 1000 ml. distilled water. Distribute in 10 ml. quantities in tubes of 16-20
mm. diameter. Sterilize in the autoclave for 15 minutes at 15 pounds pressure
(121°C.). Final reaction of the medium will be pH 6.7.

One hundred grams of Bacto-Micro Inoculum Broth will make 2.7 liters of
medium.

214 DIFCO MANUAL

BACTO

NEUROSPORA CULTURE AGAR (B321)

DEHYDRATED

Bacto- Yeast Extract 5 g.

Proteose Peptone No. 3, Difco 5 g.

Bacto-Maltose 40 g.

Bacto-Agar 15 g.

Bacto-Neurospora Culture Agar was developed for the cultivation of Neuro-
spora to be used in microbiological assays as for example for the assays of pyri-
doxine and choline. It is recommended that the spores from a 48 hour culture of
Neurospora be used as an inoculum in such assays. This medium is also recom-
mended as a nearly neutral medium with 4 per cent maltose, for the cultivation
of a large variety of fungi. A selective medium for fungi may be prepared by the
addition of penicillin and streptomycin as discussed under Bacto-Sabouraud
Dextrose Agar page 238.

To rehydrate the medium, suspend 65 grams of Bacto-Neurospora Culture
Agar in 1000 ml. cold distilled water and heat to boiling to dissolve the medium
completely. Distribute in tubes and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). Final reaction of the medium will be pH 6.7.

One hundred grams of Bacto-Neurospora Culture Agar will make 1.5 liters of
medium.

BACTO

RIBOFLAVIN ASSAY MEDIUM (B325)

DEHYDRATED

Photolyzed Peptone 22 g,

Yeast Supplement 2 g,

Bacto-Dextrose 20 g,

Sodium Acetate 1.8 g.

/-Cystine, Difco 0.2 g,

Dipotassium Phosphate 1 g.

Monopotassium Phosphate 1 g.

Magnesium Sulfate 0.4 g,

Sodium Chloride 0.02 g

Ferrous Sulfate 0.02 g

Manganese Sulfate 0.02 g.

Bacto-Riboflavin Assay Medium is a complete dehydrated medium for the
microbiological assay of riboflavin. It is free from riboflavin but contains all other
factors necessary for the growth of Lactobacillus casei e 7469 ATCC. The addi-
tion of riboflavin, in certain increasing concentrations, gives a growth response by
JL. casei e 7469 which may be measured acidimetrically or turbidimetrically. This
medium is a slight modification of the medium described by Snell and Strong^.
It is prepared according to the specifications given in the U. S. Pharmacopeia,^
National Formulary^ and the Official Method of Analyses of the Association of
Official Agricultural Chemists* except that it contains 2 per cent anhydrous
dextrose in the basal medium instead of 6 per cent. Additional dextrose may
be added if desired, however, in our experience this causes carmelization during
the preparation of the medium with adverse effects on the assay procedure. The
lower concentration of dextrose has given parallel results.

Best results, using Bacto-Riboflavin Assay Medium, have been obtained through
the use of the following procedure:

DEHYDRATED CULTURE MEDIA 215

Stock cultures of L. casei e 7469 are prepared by stab inoculation into 10 ml.
of Bacto-Micro Assay Culture Agar. After 24-48 hours incubation at 35-37°C.,
the stock cultures are kept in the refrigerator. Transplants are made at monthly
intervals, in triplicate. Inoculum for assay is prepared by subculturing from a
stock culture of L. casei e 7469 into 10 ml. of Bacto-Micro Inoculum Broth.
Following incubation for 24 hours at 35-37° C, the culture is centrifuged, under
aseptic conditions, and the supernatant liquid decanted. The cells are resuspended
in 10 ml. sterile isotonic sodium chloride. The cell suspension is then diluted 1-20
with sterile isotonic sodium chloride. One drop of this latter suspension is then
used to inoculate each of the assay tubes.

Bacto-Riboflavin Assay Medium may be used for both turbidimetric and
acidimetric determinations. Turbidimetric readings should be made after 18-24
hours incubation at 35-3 7 °C., whereas acidimetric determinations are best made
after 72 hours incubation at 35-3 7 °C. We have found the most effective assay
range, using Bacto-Riboflavin Assay Medium, to be between 0.025 microgram
and 0.15 microgram riboflavin.

The concentrations of riboflavin required for the preparation of the standard
curve may be prepared by dissolving 0.1 gram of riboflavin in 1000 ml. of
distilled water by heating, giving a stock solution of 100 micrograms per ml.
Dilute the stock solution by adding 1 ml. to 999 ml. distilled water. Use 0.0, 0.25,
0.5, 0.75, 1.0, 1.5, 2.0 and 3 ml. of the diluted stock solution per tube. The stock
solution of riboflavin used for preparing the standard curve is stable for 2 months
when stored at 2-6° C. under toluene.

To rehydrate the basal medium, suspend 48 grams of Bacto-Riboflavin Assay
Medium in 1000 ml. of distilled water and heat to boiling for 2-3 minutes. The
flask should be well shaken to distribute evenly the slight precipitate which forms.
Five (5) ml. of the medium are added to each tube in the preparation of the
tubes for the standard curve and to each tube containing material under assay.
For the assay, each tube must contain 5 ml. of rehydrated medium, increasing
amounts of the standard or the unknown and sufficient distilled water to give a
total volume of 10 ml. per tube. The tubes are then autoclaved for 10 minutes
at 15 pounds pressure (121°C.). Oversterilization of the medium will give un-
satisfactory results.

One hundred grams of Bacto-Riboflavin Assay Medium will make 4 liters of
final medium.

ilnd. Eng. Chem. Anal. Ed., 11:346:1939. s National Formulary, 9th Edition: 763: 1950.

8 Pharmacopeia of the United States Revision * A.O.A.G., 7th Edition: 779:1950.
XIV: 753: 1 950.

216 DIFCO MANUAL

BACTO

NIACIN ASSAY MEDIUM (B322)

DEHYDRATED
Bacto- Vitamin Free Pyridoxine Hydrochloride . . 0.0004 g,

Riboflavin 0.0004 g

j&-Aminobenzoic Acid, Difco . 0.0001 g

Biotin 0.0000008 g

Dipotassium Phosphate 1 g

Monopotassium Phosphate 1 g

Magnesium Sulfate 0.4 g

Sodium Chloride 0.02 g

Ferrous Sulfate 0.02 g

Manganese Sulfate 0.02 g

Casamino Acids > 12 g

Bacto-Dextrose 40 g

Sodium Acetate 20 g,

/-Cystine, Difco 0.4 g,

^^/-Tryptophane 0.2 g,

Adenine Sulfate 0.02

Guanine Hydrochloride 0.02 g,

Uracil ^ 0.02 g,

Thiamine Hydrochloride . . . 0.0002 g,
Calcium Pantothenate 0.0002 g,

Bacto-Niacin Assay Medium is a complete dehydrated medium for the assay
of nicotinic acid or nicotinamide (niacin). It is free from nicotinic acid and its
analogs, but contains all the other factors necessary for the growth of Lacto-
bacillus arabinosus 17-5 ATCC 8014. The addition of nicotinic acid or its analogs
in specified increasing concentrations gives a growth response by L. arabinosus
17-5 which may be measured acidimetrically or turbidimetrically. Bacto-Niacin
Assay Medium is prepared according to the formula described by Snell and
Wright^ and modified by Krehl, Strong, and Elvehjem^ and Barton-Wright.^ The
formula duplicates that listed in the U. S. Pharmacopeia,* and National Formu-
lary^ and the Official Method of Analyses of the Association of Official Agricul-
tural Chemists.*

Best results, using Bacto-Niacin Assay Medium, are obtained through the use
of the following procedure:

Stock cultures of L. arabinosus 17-5 are prepared by stab inoculation of
Bacto-Micro Assay Culture Agar. Following incubation at 35-3 7 °G. for 24-48
hours, the tubes are kept in the refrigerator. Transplants are made at monthly
intervals. Inoculum for assay is prepared by subculturing from a stock culture of
L. arabinosus 17-5 into a tube containing 10 ml. of Bacto-Micro Inoculum Broth.
After 24 hours incubation at 35-3 7 °C., the cells are centrifuged, under aseptic
conditions, and the supernatant liquid decanted. The cells are resuspended in 10
ml. sterile isotonic sodium chloride. The cell suspension is then diluted 1-100
with sterile isotonic sodium chloride. The suspension should be just faintly cloudy.
One drop of this suspension is then used to inoculate each of the assay tubes.

It is essential that a standard curve be set up for each separate assay since
conditions of autoclaving, temperature of incubation, etc., which influence the
standard curve readings, caimot be duplicated exactly from time to time. The
standard curve is obtained by using niacin at levels of 0.0, 0.05, 0.1, 0.2, 0.3, 0.4,
and 0.5 micrograms niacin per assay tube (10.0 ml.). Bacto-Niacin Assay Me-
dium may be used for both turbidimetric and acidimetric analyses. Turbidimetric
readings should be made after 16-18 hours incubation at 35-37°C. Acidimetric
determinations are best made following 72 hours incubation at 35-37°C. We have
found the most effective assay range, using Bacto-Niacin Assay Medium, to be
between 0.05 micrograms and 0.3 micrograms niacin.

The concentrations of niacin required for the preparation of the standard
curve may be prepared by dissolving 0.1 gram of niacin in 1000 ml. of distilled
water, giving a stock solution of 100 micrograms per ml. Dilute the stock solution
by adding 1 ml. to 999 ml. distilled water. Use 0.0, 0.5, 1.0, 2, 3, 4 and 5 ml. per
tube. The stock solution of niacin used for preparing the standard curve is stable
for 2 months when stored at 2-6° C. under toluene.

DEHYDRATED CULTURE MEDIA 217

To rehydrate the basal medium, suspend 75 grams of Bacto-NIacin Assay
Medium in 1000 ml. distilled water and heat to boiling for 2-3 minutes. The
slight precipitate which forms should be evenly distributed by shaking. Five (5)
ml. of the medium are added to each tube in the preparation of the tubes for the
standard curve and to each tube containing material under assay. For the assay,
each tube must contain 5 ml. of rehydrated medium, increasing amounts of the
standard or the unknown and sufficient distilled water to give a total volume of
10 ml. per tube. The tubes are then autoclaved for 10 minutes at 15 pounds pres-
sure (121°C.). Oversterilization of the medium will give unsatisfactory results.

One hundred grams of Bacto-Niacin Assay Medium will make 2.6 liters of
final medium.

ij. Biol. Chem., 139:675: i94i' Revision: 737: 1950.

2 Ind. Eng. Chem. Anal. Ed., 15:471:1943. ^ National Formulary, 9th Edition: 746:1950.

s Biochem. J., 38:314:1944- « A.O.A.G., 7th Edition:782: 1950.

* Pharmacopeia of the United States, XIV

BACTO

THIAMINE ASSAY MEDIUM (B326)

DEHYDRATED

Photolyzed Peptone 22 g. Niacin 0.0002 g.

Bacto-Vitamin Free Pyridoxine Hydrochloride . . 0.0002 g.

Casamino Acids 5 g. j&-Aminobenzoic Acid, Difco 0.0002 g.

Bacto-Dextrose 40 g. Folic Acid 0.0000005 g.

Sodium Acetate 15 g. Biotin . 0.0000008 g.

/-Cystine, Difco 0.2 g. Dipotassium Phosphate 1 g.

Adenine Sulfate 0.02 g. Monopotassium Phosphate 1 g.

Guanine Hydrochloride 0.02 g. Magnesium Sulfate 0.4 g.

Uracil 0.02 g. Sodium Chloride 0.02 g.

Riboflavin 0.0002 g. Ferrous Sulfate 0.02 g.

Calcium Pantothenate 0.0002 g. Manganese Sulfate 0.02 g.

Bacto-Thiamine Assay Medium is a complete dehydrated medium for the
assay of thiamine. It is free from thiamine but contains all the other factors
necessary for the growth of Lactobacillus jermentum 36 ATCC 9833. The addi-
tion of thiamine, in certain increasing concentrations, gives a growth response
which may be measured turbidimetrically. Bacto-Thiamine Assay Medium is
prepared according to the formula given by Sarett and Cheldelin.^

The following procedure is recommended for the use of Bacto-Thiamine Assay
Medium:

Stock cultures of the test organism, L. jermentum 36, are prepared by stab
inoculation of Bacto-Micro Assay Culture Agar. After 24—48 hours incubation
at 35-3 7 °G., the tubes are kept in the refrigerator. Transplants are made at
monthly intervals, in triplicate.

Inoculum for assay ijs prepared by subculturing from a stock culture to 10 ml.
of Bacto-Micro Inoculum Broth. After 16-18 hours incubation at 35-3 7 °C., the
cells are centrifuged, under aseptic conditions, and the supernatant liquid is de-
canted. The cells are resuspended in 10 ml. sterile isotonic sodium chloride. One-
half (0.5) ml. of this cell suspension are then added to 100 ml. of sterile saline.
One drop of this suspension is then used to inoculate the assay tubes.

The tubes for the standard curve, which should be set up each time an assay
is run, contain 0.0, 0.005, 0.01, 0.015, 0.02, 0.03, 0.04 and 0.05 microgram of
thiamine hydrochloride per tube (10 ml.). It is essential that a standard curve
be run with each assay since conditions of heating, temperature of incubation,
etc., which influence the standard curve readings cannot be duplicated exactly

218 DIFGO MANUAL

from time to time. Using Bacto-Thiamine Assay Medium we have found the
most effective assay range to be between 0.005 and 0.03 microgram thiamine.

The concentrations of thiamine required for the preparation of the standard
curve may be prepared by dissolving 0.1 gram of thiamine in 1000 ml. of distilled
water, giving a stock solution of 100 micrograms per ml. Dilute the stock solution
by adding 1 ml. to 99 ml. of distilled water. This solution is further diluted by
adding 1 ml. to 99 ml. distilled water to give the final solution. Use 0.0, 0.5, 1.0,
1.5, 2.0, 3, 4 and 5 ml. of this final solution per tube. The stock solution of
thiamine used for preparing the standard curve is stable for 2 months when stored
at 2-6° C. under toluene.

After 20-24 hours incubation at 35-3 7 °G., L. jermentum 36 is capable of
utilizing the pyrimidine and thiazole moieties of the thiamine molecule. It is
essential, therefore, that the growth response be measured turbidimetrically prior
to this time. The tubes should be incubated at 35-3 7 °C. for not longer than
16-18 hours, and then placed in the refrigerator for 15-30 minutes in order to
stop growth. The growth can then be measured by any suitable nephelometric
method.

To rehydrate the basal medium, suspend 85 grams of Bacto-Thiamine Assay
Medium in 1000 ml. distilled water and heat to boiling for 2-3 minutes. The
slight precipitate which forms should be evenly distributed by shaking. Five (5)
ml. of the medium are added to each tube in the preparation of the tubes for the
Standard curve and to each tube containing material under assay. For the assay,
each tube must contain 5 ml. of rehydrated medium, increasing amounts of the
standard or the unknown and sufficient distilled water to give a total volume of
10 ml. per tube. The tubes should not be autoclaved because of possible destruc-
tion of thiamine, but are steamed at 100°C. for 15 minutes. Overheating of the
medium will give unsatisfactory results.

One hundred grams of Bacto-Thiamine Assay Medium will make 2.2 liters of
final medium.

1 J. Biol. Chem., 155:153:1944.

DEHYDRATED CULTURE MEDIA 219

BACTO

PANTOTHENATE ASSAY MEDIUM (B323)

DEHYDRATED
Bacto- Vitamin Free Niacin 0.002 g

Casamino Acids 12 g,

Bacto-Dextrose 40 g

Sodium Acetate 20 g

/-Cystine, Difco 0.2 g

^/-Tryptophane 0.2 g

Adenine Sulfate 0.02 g

Guanine Hydrochloride 0.02 g

Uracil 0.02 g

Thiamine Hydrochloride .... 0.002 g
Riboflavin 0.002 g

Pyridoxine Hydrochloride . . . 0.004 g
j&-Aminobenzoic Acid, Difco 0.0002 g,

Biotin . 0.0000008 g

Dipotassium Phosphate 1 g,

Monopotassium Phosphate 1 g

Magnesium Sulfate 0.4 g.

Sodium Chloride 0.02 g,

Ferrous Sulfate 0.02 g

Manganese Sulfate 0.02 g

Bacto-Pantothenate Assay Medium is a complete dehydrated medium for
the assay of pantothenic acid. It is free from pantothenic acid or pantothenate
but contains all the other factors necessary for the growth of Lactobacillus
arabinosus 17-5 ATCG 8014. The addition of pantothenate, in specified increas-
ing concentration, gives a linear growth response by L. arabinosus 17-5 which
may be measured acidimetrically or turbidimetrically. Bacto-Pantothenate Assay
Medium is a slight modification of the medium described by Skeggs and Wright.^

The following procedure is recommended for the use of Bacto-Pantothenate
Assay Medium :

Stock cultures of the test organism, L. arabinosus 17-5, are prepared by stab
inoculation of Bacto-Micro Assay Culture Agar. Following incubation at 35-
37°C. for 24-48 hours, the tubes are kept in the refrigerator. Transplants are
made at monthly intervals. Inoculum for assay is prepared by subculturing from
a stock culture of L. arabinosus 17-5 into a tube containing 10 ml. of Bacto-Micro
Inoculum Broth. Following incubation for 24 hours at 35-3 7 °C., the cells are
centrifuged, under aseptic conditions, and the supernatant liquid decanted. The
cells are resuspended in 10 ml. sterile isotonic sodium chloride. The cell suspen-
sion is then diluted 1-100 with sterile isotonic sodium chloride. The suspension
should be just faintly cloudy. One drop of this suspension is then used to inoculate
each of the assay tubes.

It is essential that a standard curve be set up for each separate assay since
conditions of autoclaving, temperature of incubation, etc., which influence the
standard curve readings, cannot be duplicated exactly from time to time. The
standard curve is obtained by using calcium pantothenate at levels of 0.0, 0.02,
0.04, 0.06, 0.08, 0.1, 0.12 and 0.2 microgram per assay tube (10 ml.). Bacto-
Pantothenate Assay Medium may be used for both turbidimetric and acidimetric
analysis. Turbidimetric readings should be made after 18-24 hours at 35-3 7 °C.
Acidimetric determinations are made after 72 hours incubation at 35-37°C. We
have found the most effective assay range, using Bacto-Pantothenate Assay
Medium, to be between 0.02 microgram and 0.12 microgram calcium pan-
tothenate.

The concentrations of calcium pantothenate required for the preparation of
the standard curve may be prepared by dissolving 0.1 gram of calcium pan-
tothenate in 1000 ml. of distilled water, giving a stock solution of 100 micrograms
per ml. Dilute the stock solution by adding 1 ml. to 99 ml. of distilled water.
This solution is further diluted by adding 4 ml. to 96 ml. distilled water to give
the final solution. Use 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 5 ml. of this final solu-
tion per tube. The stock solution of calcium pantothenate used for preparing the
Standard curve is stable for 2 months when stored at 2-6° G. under toluene.

220 DIFCO MANUAL

To rehydrate the basal medium, suspend 75 grams of Bacto-Pantothenate
Assay Medium in 1000 ml. distilled water, and heat to boiling for 2-3 minutes.
The slight precipitate which forms should be evenly distributed by shaking. Five
(5) ml. of the medium are added to each tube in the preparation of the tubes
for the standard curve and to each tube containing material under assay. For the
assay, each tube must contain 5 ml. of rehydrated medium, increasing amounts
of the standard or the unknown and sufficient distilled water to give a total
volume of 10 ml. per tube. The tubes are then autoclaved for 10 minutes at 15
pounds pressure (121°C.). Oversterilization of the medium will give unsatisfac-
tory results.

One hundred grams of Bacto-Pantothenate Assay Medium will make 2.6 liters
of final medium.
1 J. Biol. Chem., 156:21:1944.

BAGTO-BIOTIN ASSAY MEDIUM (B419)

Bacto-Biotin Assay Medium has the same composition as Bacto-Pantothenate
Assay Medium described immediately above, except that biotin has been omitted
and 0.002 g. calcium pantothenate included.

Bacto-Biotin Assay Medium is a complete dehydrated medium for the assay of
biotin. It is free from biotin but contains all the other factors necessary for the
growth of Lactobacillus arabinosus 17-5 ATCC 8014. The addition of biotin in
specified increasing concentrations gives a growth response by L. arabinosus 17-5
which may be measured acidimetrically or turbidimetrically.

The following procedure is recommended for the use of Bacto-Biotin Assay
Medium:

Stock cultures of the test organism, L. arabinosus 17-5, are prepared by stab
inoculation of Bacto-Micro Assay Culture Agar. After 24-48 hours incubation at
35-3 7 °C., the tubes are stored in the refrigerator. Transplants are made at
monthly intervals, in triplicate.

Inoculum for assay is prepared by subculturing from a stock culture of L.
arabinosus 17-5 to 10 ml. of Bacto-Micro Inoculum Broth. After 16-24 hours
incubation at 35-3 7 °C., the cells are centrifuged under aseptic conditions, and
the supernatant liquid decanted. The cells are resuspended in 10 ml. sterile
isotonic sodium chloride solution. The cell suspension is then diluted 1-100
with sterile isotonic sodium chloride. One drop of this latter suspension is then
used to inoculate each of the assay tubes (10 ml.).

The concentrations of biotin required for the preparation of the standard curve
may be prepared by dissolving 0.1 gram of biotin in 1000 ml. of distilled water
(100 micrograms per ml.). Dilute this stock solution by adding 2 ml. to 98 ml.
of distilled water. This solution is diluted by adding 1 ml. to 999 ml. distilled
water giving a solution of 2 millimicrograms of biotin per ml. This solution is
further diluted by adding 5 ml. to 95 ml. distilled water giving a final solution of
0.1 millimicrogram of biotin per ml. Use 0.0, 0.25, 0.5, 1.0, 2, 3, 4 and 5 ml. of
this final solution. The last tube is prepared by adding 0.5 ml. of the standard
solution containing 2 millimicrograms of biotin per liter. The stock solution of
biotin used for preparing the standard curve is stable for 2 months when stored
at 2-6°C. under toluene.

DEHYDRATED CULTURE MEDIA 221

Bacto-Biotin Assay Medium may be used for both turbidimetric and acidi-
metric analyses. Turbidimetric readings should be made after 16-20 hours at
35-3 7 °C. Acidimetric determinations are made after 72 hours incubation at 35-
37°C. The most effective assay range, using Bacto-Biotin Assay Medium, has been
found to be between 0.025 millimicrogram and 0.5 millimicrogram biotin.

To rehydrate the medium, suspend 75 grams of Bacto-Biotin Assay Medium
in 1000 ml. distilled water, and heat to boiling for 2-3 minutes. The slight pre-
cipitate which forms should be evenly distributed by shaking. Five (5) ml. of the
medium are added to each tube in the preparation of the tubes for the standard
curve and to each tube containing material under assay. For the assay each tube
must contain 5 ml. of rehydrated medium, increasing amounts of the standard
or the unknown, and sufficient distilled water to give a total volume of 10 ml.
per tube. The tubes are then autoclaved for 10 minutes at 15 pounds pressure
(121°tll.). Oversterilization of the medium will give unsatisfactory results.

One hundred grams of Bacto-Biotin Assay Medium will make 2.6 liters of
final medium.

BACTO

Bi2 ASSAY MEDIUM USP (B457)

DEHYDRATED

Bacto- Vitamin Free Biotin 0.000008 g.

Casamino Acids 15 g. Niacin 0.002 g.

Tomato Juice 10 g. />-Aminobenzoic Acid, Difco . 0.002 g.

Bacto-Dextrose 40 g. Calcium Pantothenate 0.001 g.

Bacto-Asparagine 0.2 g. Pyridoxine Hydrochloride . . . 0.004 g.

Sodium Acetate 20 g. Pyridoxal Hydrochloride .... 0.004 g.

Ascorbic Acid 4 g. Pyridoxamine Hydrochloride 0.0008 g.

/-Cystine, Difco 0.4 g. Folic Acid 0.0002 g.

^/-Tryptophane 0.4 g. Monopotassium Phosphate 1 g.

Adenine Sulfate 0.02 g. Dipotassium Phosphate 1 g.

Guanine Hydrochloride 0.02 g. Magnesium Sulfate 0.4 g.

Uracil 0.02 g. Sodium Chloride 0.02 g.

Xanthine 0.02 g. Ferrous Sulfate 0.02 g.

Riboflavin 0.001 g. Manganese Sulfate 0.02 g.

Thiamine Hydrochloride .... 0.001 g. Sorbitan Monooleate Complex . . 2 g.

Bacto-Bi2 Assay Medium USP is prepared according to the formula given in
the United States Pharmacopeia.^ It is free from Vitamin B^g but contains all
the other factors necessary for the growth of Lactobacillus leichmannii ATCC
7830. The addition of Vitamin B^a to this medium in specified increasing concen-
trations gives a growth response by the test organism which may be measured
acidimetrically or turbidimetrically.

The following procedure, or the procedure specified by USP is recommended
for the preparation of the inoculum for the assay of Vitamin 6^2 using Bacto-Bi2
Assay Medium USP:

Stock cultures of L. leichmannii 7830 are prepared by stab inoculation of 3 or
more tubes of Bacto-Micro Assay Culture Agar. Transfers should be made bi-
monthly for stock culture. Before using a culture in the assay, make at least 10
successive transfers of the culture in Bacto-Micro Assay Culture Agar in a two-
week period. Incubate 24-48 hours at 35-37°C. Prepare fresh stab cultures every
second day and do not use them for preparing the inoculum if more than 4 days
old. Inoculum for assay is prepared by subculturing from a 24-48 hour stock
culture of L. leichmannii 7830 to 10 ml. of Bacto-Micro Inoculum Broth. After
16-24 hours incubation at 35-37°G., the cells are centrifuged under aseptic con-

222 DIFCC MANUAL

ditions and the supernatant liquid decanted. Suspend the cells from this culture in
10 ml. of sterile B12 Assay Medium USP in single strength, prepared by dis-
solving 4.75 grams of Bacto-Bi2 Assay Medium USP in 100 ml. of distilled
water. Centrifuge and decant the supernatant liquid. Again, suspend the cells in
10 ml. of the medium, centrifuge and decant the supernatant liquid. Repeat this
process a third time. Finally, resuspend the cells in 10 ml. of the sterile single
strength medium. Add 0.1 ml. of this suspension to 10 ml. of sterile suspension
medium and mix. The cell suspension so obtained is the inoculum. Inoculate
each tube aseptically with one drop of the inoculum. The use of such a diluted
inoculum gives more uniform results than a heavier inoculum.

Using Bacto-B22 Assay Medium USP the most effective range has proved to
be between 0.01 millimicrograms and 0.1 millimicrograms.

The concentration of Vitamin B12 required for the preparation of the standard
curve may be prepared by adding the contents of one ampul containing 20
micrograms per ml. (1 ml.) in 99 ml. of 25 per cent alcohol in distilled water
giving the stock solution of 200 millimicrograms per ml. Further dilutions are
made as follows:

A. Add 1 ml. stock solution to 99 ml. distilled water
(1 ml. = 2 millimicrograms)

B. Add 2.5 ml. of A to 97.5 ml. distilled water

(1 ml. = 0.05 millimicrograms).

C. Add 1 ml. of A to 99 ml. distilled water
(1 ml. =z 0.02 millimicrograms).

Use 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5 ml. dilution C per tube and for
the last tube use 5 ml. dilution B. If preferred, Vitamin Bjg may be supplied in
the form of a 0.1 per cent trituration of crystalline Vitamin B12 with man-
nitol (1 gram = 1 mg. Vitamin B12). Dissolve 0.2 grams in 1000 ml. dis-
tilled water to obtain the stock solution of 200 millimicrograms per ml. The
stock solution of Vitamin Bjg is stable for 2 months if stored at 2-6° C. under
toluene.

Acidimetric determinations are made electrometrically using pH 7.0 as the end
point after 72 hours incubation at 30-37°C., but held constant to within 0.5°. We
recommend an incubation temperature of 35-3 7 °G. Turbidimetric determina-
tions are made after 20-24 hours incubation. The standard curve is then con-
structed from the values obtained.

To rehydrate the basal medium, dissolve 95 grams of Bacto-B^g Assay Medium
USP in 1000 ml. of distilled water, and heat to boiling for 2-3 minutes. The
slight precipitate which forms should be evenly distributed by shaking. Five (5)
ml. of the medium are added to each tube in the preparation of the tubes for the
standard curve and to each tube containing material under assay. For the assay,
each tube must contain 5 ml. of rehydrated medium, increasing amounts of the
standard or the unknown and sufficient distilled water to give a total volume
of 10 ml. per tube. Tubes are sterilized by autoclaving for 5 minutes at 15 lbs.,
pressure (121°G.). Overheating of the medium will give unsatisfactory results.

One hundred grams of Bacto-Bi2 Assay Medium USP will make 2.1 liters final
medium.

1 Pharmacopeia of the United States, Third Supplement, XIV, Revision: 15:1951.

DEHYDRATED CULTURE MEDIA 223

BACTO

G. S. VITAMIN Bio AGAR (B399)

DEHYDRATED

Bacto- Vitamin Free Calcium Pantothenate 0.002 g

Casamino Acids 10.0 g. Niacin 0.002 g

Bacto-Soytone, Vitamin Free . . 5.0 g. Pyridoxine 0.004 g

Bacto-Dextrose 20.0 g. Pyridoxal 0.004 g

Sodium Acetate 12.0 g. Biotin 0.00000001 g

Ribose Nucleic Acid 1.0 g. <i/-Tryptophane 0.2 g,

Sodium Thioglycollate, Difco . . 1.7 g. Potassium Sulfate 20.0 g

/-Cystine, Difco 0.2 g. Monopotassium Phosphate .... 1.0 g,

Adenine Sulfate 0.0176 g. Dipotassium Phosphate 1-0 g

Guanine Hydrochloride .... 0.0124 g. Magnesium Sulfate 0.4 g,

Uracil 0.01 g. Sodium Chloride 0.02 g

Xanthine 0.01 g. Ferrous Sulfate 0.02 g

Folic Acid 0.001 g. Manganese Sulfate 0.02 g

Riboflavin 0.002 g. Sorbitan Monooleate Complex . 1.0 g

Thiamine Hydrochloride .... 0.002 g. Bacto- Agar 15.0 g

Bacto-C. S. Vitamin B22 Agar is prepared according to the formula given by
Cohen and Bennett.^ This medium is suggested for the assay of Vitamin Bjg with
Lactobacillus leichmannii ^1^1 ATCG as the test organism. The addition of a
solution of crystalline Vitamin 6^2 in specified increasing amounts to cylinders
or disks placed on this medium produces proportionately larger zones of growth
by L, leichmannii ^191.

The following procedure is recommended for the use of Bacto-G. S. Vitamin
BiaAgar:

Stock cultures of the test organism, L. leichmannii 4797, are prepared by stab
inoculation of Bacto-Micro Assay Culture Agar. Following incubation at
35-3 7 °C. for 24-48 hours, the tubes are kept in the refrigerator. Transplants are
made at two-week intervals. Inoculum for assay is prepared by subculturing from
a stock culture of L. leichmannii 4797 into a tube containing 10 ml. of Bacto-
Micro Inoculum Broth. Following incubation for 16-24 hours at 35-3 7 °C., the
cells are centrifuged, under aseptic conditions, and the supernatant liquid de-
canted. The cells are resuspended in 10 ml. sterile isotonic sodium chloride,
centrifuged, the supernatant liquid discarded and made up to 10 ml. volume
with sterile isotonic sodium chloride. Under aseptic conditions 10 ml. of washed
cells are added per 1000 ml. of sterile melted Bacto-C. S. Vitamin B12 Agar at
45-50 °C. Into each of 4 sterile flat bottom petri dishes (95 mm.) are poured
25 ml. of the inoculated medium and allowed to solidify. When the surface is
dry 6 sterile penicillin assay cups or 13 mm. filter paper disks are distributed
evenly on the surface of the medium.

For the preparation of the standard, prepare sterile solutions of Vitamin B-^g
containing 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 micrograms per ml. Use 0.2-0.3 ml. of
the standard B12 solutions per cup. When paper disks are used saturate the pads
with 0.1 ml. of the above solutions under aseptic conditions. The use of a syringe
and needle or a capillary pipette is suggested as a convenience in restricting the
solutions to the pad. One or more duplicate plates per test may be employed
using 4 to 6 disks per plate and modifying the concentration of Vitamin B12
as desired. Using Bacto-C. S. Vitamin B12 Assay Medium experience has shown
that the most effective assay range is between 0.1 microgram and 0.8 microgram.

The concentrations of Vitamin Bj^2 required for the preparation of the standard
curve may be prepared by dissolving 1.6 g. of a 0,1 per cent trituration of crystal-
line Vitamin B12 with mannitol (1 gram = 1 mg. Vitamin 'Q-^^) ^^ 1000

224 DIFCO MANUAL

ml. distilled water. This is the stock solution A containing 1.6 micrograms per
ml. To obtain solution B containing 0.8 micrograms dilute 10 ml. of A with 10
ml. of distilled water. To obtain solution C containing 0.4 micrograms dilute
10 ml. of B with 10 ml. distilled water. To obtain solution D containing 0.2
micrograms dilute 10 ml. of C with 10 ml. distilled ^vater. To obtain solution E
containing 0.1 micrograms dilute 10 ml. of D with 10 ml. distilled water. To
obtain solution F containing 0.05 micrograms dilute 10 ml. of E with 10 ml.
distilled water. These solutions are stable for 2 months when stored at 2-6° C.
under toluene.

In determining the Vitamin B;l2 content of unknown materials, the assay
samples are appropriately diluted and similarly applied. In the assay of some
preparations, better defined, clearer cut zones may be obtained by the addition of
0.5 to 1.0 per cent of sodium chloride to Bacto-C. S. Vitamin B^o Agar, since
added sodium chloride, with some unknown samples, makes the assay more
specific for vitamin B^o- Sodium chloride, under these conditions, must be added
to the medium in preparing the standard curve. Incubate inoculated plates at
35-3 7 °C. for 24 hours and measure size of zone of growth produced. The diame-
ter of the growth zone is indicative of the amount of Vitamin B^o present.

To rehydrate the medium, suspend 90 grams Bacto-C. S. Vitamin B^g Agar in
1000 ml. of cold distilled water. Heat to boiling to dissolve the medium com-
pletely. Distribute in flasks and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C,).

One hundred grams of Bacto-C. S. Vitamin B12 Agar are sufficient for 1.1
liters of medium.

1 Paper read at American Chemical Society,
Philadelphia, 1950.

DEHYDRATED CULTURE MEDIA 225

BACTO

FOLIC ACID ASSAY MEDIUM (B318)

DEHYDRATED

Bacto- Vitamin Free Riboflavin 0.002 g

Casamino Acids 12 g

Bacto-Dextrose 40 g

Sodium Citrate 20 g

/-Cystine, Difco 0.2 g

^/-Tryptophane 0.2 g

Adenine Sulfate 0.02 g

Guanine Hydrochloride 0.02 g

Uracil . . 0.02 g

Thiamine Hydrochloride .... 0.002 g.
Pyridoxine Hydrochloride . . . 0.004 g.

Niacin 0.002 g

/>-Aminobenzoic Acid, Difco 0.0002 g

Biotin 0.0000008 g

Calcium Pantothenate 0.0004 g

Dipotassium Phosphate 1 g

Monopotassium Phosphate 1 g

Magnesium Sulfate 0.4 g

Sodium Chloride 0.02 g

Ferrous Sulfate 0.02 g

Manganese Sulfate 0.02 g

Bacto-Folic Acid Assay Medium is a complete dehydrated medium for the
assay of folic acid. It is free from folic acid but contains all the other factors
necessary for the growth of Streptococcus lactis R. 8043 ATCG [Streptococcus
jecalis R.) . The addition of folic acid in specified increasing concentrations gives
a growth response by S. lactis R. 8043 after 18 hours incubation at 35-37°C.
which may be measured turbidimetrically. Bacto-Folic Acid Assay Medium is
prepared according to the formula described by Capps, Hobbs and Fox,^ modi-
fied by the use of sodium citrate instead of sodium acetate.

The following procedure for the preparation of the inoculum and performance
of the test is recommended for the assay of folic acid using Bacto-Folic Acid
Medium :

Stock cultures of S. lactis R. 8043 are prepared by stab inoculation of the
Bacto-Micro Assay Culture Agar. Following incubation at 35-37°C. for 24-48
hours, the tubes are stored in the refrigerator. Transplants are made at monthly
intervals. Inoculum for assay is prepared by subculturing from a stock culture of
S. lactis R. 8043 into a tube containing 10 ml. of the Bacto-Micro Inoculum
Broth. After 24 hours incubation at 35-3 7 °C., the cells are centrifuged, under
aseptic conditions, and the supernatant liquid decanted. The cells are resuspended
in 10 ml. of sterile isotonic sodium chloride. The cell suspension is then diluted
1-100 with sterile isotonic sodium chloride. The suspension should be just faintly
cloudy. One drop of this latter suspension is then used to inoculate each of the
assay tubes.

The concentrations of folic acid required for the construction of the standard
curve may be prepared by dissolving 0.1 gram folic acid in 1000 ml. of distilled
water (100 micrograms per ml.). Add 0.05N sodium hydroxide drop by drop to
effect complete solution of the folic acid, being careful not to get more alkaline
than pH 7.0. Dilute the stock solution by adding 1 ml. to 999 ml. distilled water.
This solution is diluted further by adding 4 ml. to 996 ml. distilled water. Use
0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.25 and 2.5 ml. of this final solution per tube. The
stock solution of folic acid used for preparing the standard curve is stable for 2
months when stored at 2-6° C. under toluene.

Following inoculation, tubes are incubated at 35-37°C. for 18-24 hours and
placed in the refrigerator for 15-30 minutes in order to stop growth. The growth
can then be measured by any suitable nephelometric method, and the curve con-

226 DIFCO MANUAL

structed from the values obtained. Acidimetric determinations of growth have
not been found satisfactory. Using Bacto-Folic Acid Assay Medium, we have
found the most effective assay range to be between the levels of 0.002 and 0.01
microgram folic acid per tube (10 ml.).

To rehydrate the basal medium, suspend 75 grams of Bacto-Folic Acid Assay
Medium in 1000 ml. distilled water, and heat to boiling for 2-3 minutes. The
slight precipitate which forms should be evenly distributed by shaking. Five (5)
ml. of the medium are added to each tube in the preparation of the tubes for the
standard curve and to each tube containing material under assay. For the assay,
each tube must contain 5 ml. of rehydrated medium, increasing amounts of the
standard or the unknown and sufficient distilled water to give a total volume of
10 ml. per tube. The tubes are autoclaved for 10 minutes at 15 pounds pressure
(121°C). Do not sterilize longer than specified above. One hundred grams of
Bacto-Folic Acid Assay Medium will make 2.6 liters of final medium.
ij. Bact., 55:869:1948.

BACTO

PYRIDOXINE ASSAY MEDIUM (B324)

DEHYDRATED

Saccharose, Difco 30 g

Biotin 0.000008 g

Ammonium Tartrate 10 g

Sodium Dihydrogen Citrate 4 g

Monopotassium Phosphate 5 g

Magnesium Sulfate 1 g

Sodium Chloride 0.2 g

Calcium Chloride 0.2 g

Ferric Chloride 0.01 g

Zinc Sulfate 0.004 g

Bacto-Pyridoxine Assay Medium is a complete dehydrated medium for the
microbiological assay of pyridoxine. It is patterned after the medium described
by Stokes, Larsen, Woodward and Foster^ and modified by Barton-Wright^.
Bacto-Pyridoxine Assay Medium is free from pyridoxine but contains all other
factors necessary for growth of Neurospora sitophila 299 ATCG 9276. The addi-
tion of pyridoxine, in specified concentrations, gives increasing growth response
by N. sitophila 299 which may be determined gravimetrically.

The test is run according to the method described by Stokes, Larsen, Wood-
ward and Foster^ and modified by Barton- Wright. ^ Remove one loop of spores
from a 48-hour culture of A^. sitophila 299 on Bacto-Neurospora Culture Agar
and suspend in 100 ml. sterile saline. Add one drop of this spore suspension to
each flask. Incubate at 30° C. for 5 days. At the end of the incubation period
steam the flasks at 100°G. for 5 minutes. Remove all the mycelium from the flask
using a stiff wire needle or glass rod, press dry between paper towels, and roll into
a small pellet. Dry the pellets at 100°C. for 2 hours in a vacuum. A glazed porce-
lain spot plate is convenient for handling the mycelium during drying and weigh-
ing. A standard curve is then constructed from the weights obtained and the
unknown determined by interpolation.

It is essential that a standard curve be constructed each time as assay is run
since conditions of autoclaving, temperature of incubation, etc., which influence
the standard curve readings, cannot be duplicated exactly from time to time. The
standard curve is obtained by using pyridoxine at levels of 0.0, 0.2, 0.4, 0.6, 0.8
and 1.0 microgram per assay flask (10 ml.). Using Bacto-Pyridoxine Assay

DEHYDRATED CULTURE MEDIA 227

Medium the most effective assay range has proved to be between 0.2 and 0.8
microgram pyridoxine.

The concentrations of pyridoxine required for the preparation of the standard
curve may be prepared by dissolving 0.1 gram of pyridoxine in 1000 ml. of dis-
tilled water, giving a stock solution of 100 micrograms per ml. Dilute the stock
solution by adding 1 ml. to 499 ml. distilled water. Use 0.0, 1.0, 2, 3, 4 and 5 ml.
per tube. This stock solution of pyridoxine used for preparing the standard curve
is stable for 2 months when stored at 2-6°C. under toluene.

To rehydrate the basal medium, dissolve 50 grams of Bacto-Pyridoxine Assay
Medium in 1000 ml. of distilled water. Distribute in 5 ml. quantities in 50 ml.
Erlenmeyer flasks. Five (5) ml. of the medium are added to each flask in the
preparation of the flasks for the standard curve and to each flask containing
material under assay. For the assay, each flask must contain 5 ml. of rehydrated
medium, increasing amounts of the standard or the unknown and sufflcient dis-
tilled water to give a total volume of 10 ml. per flask. Sterilize in the autoclave
for 15 minutes at 15 pounds pressure (121°C.). The flasks are then ready for
inoculation. Oversterilization of the medium will give unsatisfactory results.

One hundred grams of Bacto-Pyridoxine Assay Medium will make 4 liters of
final medium.

ij. Biol. Chem., 150:17:1943. 2 Analyst, 70:283:1945.

BACTO

CHOLINE ASSAY MEDIUM (B460)

DEHYDRATED

Saccharose, Difco 40 g.

Ammonium Nitrate 2 g.

Biotin 0.00001 g.

Potassium Sodium Tartrate 11.4 g.

Monopotassium Phosphate 2 g.

Magnesium Sulfate 1 g.

Sodium Chloride 0.2 g.

Calcium Chloride 0.2 g.

Sodium Borate 0.0007 g.

Ammonium Molybdate 0.0005 g.

Ferrous Sulfate 0.0011 g.

Cuprous Chloride 0.0003 g.

Manganese Sulfate 0.00011 g.

Zinc Sulfate 0.0176 g.

Bacto-Choline Assay Medium is a complete dehydrated medium for the micro-
biological assay of choline. It is a slight modification of the medium described by
Horowitz and Beadle.^ Bacto-Choline Assay Medium is free from choline but
contains all the other factors necessary for the growth of Neurospora crassa 34486
ATCG. The addition of choline in specified increasing concentrations gives a
growth response by N. crassa 34486 which may be determined gravimetrically.

The following procedure for the preparation of the inoculum and performance
of the test is recommended for the assay of choline using Bacto-Choline Assay
Medium:

Remove one loop of spores from a 48-hour culture of A^. crassa 34486 on Bacto-
Neurospora Culture Agar and suspend in 100 ml. sterile saline. Add one drop of
this spore suspension to each flask. Incubate at 25 °C. for 3 days. At the end of
the incubation period, steam the flasks at 100°C. for 5 minutes. Remove all the
mycelium from the flask using a stiff wire needle or glass rod, press dry between

228 DIFGO MANUAL

paper towels, and roll into a small pellet. Dry the pellets at 100°C. in a vacuum
oven for 2 hours. A glazed porcelain spot plate is convenient for handling the
mycelium during drying and weighing. Weigh to the nearest 0.5 mg. A standard
curve is then constructed from the weights obtained and the unknown determined
by interpolation. In the assay for choline, 125 ml, Erlenmeyer flasks containing
a total volume of 20 ml. medium each are used.

It is essential that a standard curve be constructed each time an assay is run
since conditions of autoclaving, temperature of incubation, etc., which influence
the standard curve readings, cannot be duplicated exactly from time to time. The
standard curve is obtained by using choline at levels of 0.0, 2.5, 5.0, 10, 20, 30,
40 and 50 micrograms per assay flask (20 ml.). Using Choline Assay Medium the
most effective assay range has proved to be between 2.5 and 30 micrograms
choline.

The concentration of choline required for the preparation of the standard curve
may be prepared by dissolving 1.0 gram choline in 1000 ml. distilled water. This
is the stock solution (1000 micrograms per ml.). Dilute the stock solution by
adding 1 ml. to 99 ml. distilled water. Use 0.0, 0.5, 1.0, 2, 3, 4, and 5 ml. per
tube. The stock solution is stable for 2 months when stored at 2-6° C. under
toluene.

To rehydrate the basal medium, dissolve 57 grams of Bacto-Choline Assay
Medium in 1000 ml. of distilled water. Ten (10) ml. of the medium are added
to each flask in the preparation of the flasks for the standard curve and to each
flask containing material under assay. For the assay, each flask must contain 10
ml. of rehydrated medium, increasing amounts of the standard or the unknown
and sufficient distilled water to give a total volume of 20 ml. per flask. Sterilize
in the autoclave for 10 minutes at 15 pounds pressure (121°C.). The flasks are
then ready for inoculation. Oversterilization of the medium will give unsatisfac-
tory results.

One hundred grams of Bacto-Choline Assay Medium will make 3.5 liters of
final medium.
1 J. Biol. Chem., 130:325:1943.

DEHYDRATED CULTURE MEDIA

229

BACTO

GF ASSAY MEDIUM

DEHYDRATED

(B456)

Bacto-Vitamin Free

Casamino Acids 10

Bacto-Dextrose 50

Sodium Acetate 40

Ammonium Chloride 6

/-Cystine, Difco 0.2

Cysteine 0.2

^//-Tryptophane 0.2

^//-Alanine 0.2

Glycine 0.2

Adenine Sulfate 0.02

Guanine Hydrochloride 0.02

Uracil 0.02

Xanthine 0.02

Thiamine Hydrochloride .... 0.001

Pyridoxine Hydrochloride . . 0.002 g

Pyridoxamine Hydrochloride . 0.006 g,

Pyridoxal Hydrochloride . . . 0.0006 g,

Calcium Pantothenate 0.001 g.

Riboflavin 0.001 g

Nicotinic Acid 0.002 g,

p-Aminobenzoic Acid, Difco . 0.0002 g

Biotin 0.000002 g

Folic Acid 0.00002 g

Monopotassium Phosphate .... 1.2 g,

Dipotassium Phosphate 1.2 g,

Magnesium Sulfate 0.4 g.

Sodium Chloride 0.02 g

Ferrous Sulfate 0.02 g,

Manganese Sulfate 0.04 g

Bacto-CF Assay Medium is a complete dehydrated medium for the assay of
citrovorum factor. It is free from citrovorum factor but contains all the other
factors necessary for the growth of Leuconostoc citrovorum 8081 ATCC. The
addition of citrovorum factor in specified increasing concentrations gives a growth
response by L. citrovorum 8081 which may be measured acidimetrically or turbi-
dimetrically. Bacto-CF Assay Medium is a modification of the formula given by
Sauberlich and Baumann^ and William F. Faloon.-

The following procedure is recommended for the use of Bacto-CF Assay
Medium :

Stock cultures of the test organism, L. citrovorum 8081, are prepared by stab
inoculation of Bacto-Micro Assay Culture Agar. After 24-48 hours incubation at
35-3 7 ''C, the tubes are kept in the refrigerator. Transplants are made at monthly
intervals, in triplicate.

Inoculum for assay is prepared by subculturing from a stock culture to 10 ml.
of Bacto-Micro Inoculum Broth. After 16-24 hours incubation at 35-37°C., the
cells are centrifuged under aseptic conditions, and the supernatant liquid de-
canted. Suspend the cells from the culture in 10 ml. of sterile isotonic sodium
chloride. The cell suspension is then diluted 1-100 with sterile isotonic sodium
chloride solution. One drop of this latter suspension is used to inoculate the assay
tubes (10 ml.).

The concentration of citrovorum factor required for the construction of the
standard cur\'e may be prepared by dissolving the contents of 1 ampul leucovorin
(citrovorum factor) in 1000 ml. distilled water. This is the stock solution (3000
micrograms per ml.). Dilute the stock solution by adding 4 ml. to 96 ml. distilled
water. Dilute this solution further by adding 1 ml. to 99 ml. distilled water. Use
0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4 and 5 ml. per tube. The stock solution is stable
for 2 months when stored at 2-6° C, under toluene.

Bacto-CF Assay Medium may be used for both acidimetric and turbidimetric
analysis. Acidimetric determinations are made after 72 hours incubation at
35-37°C. Turbidimetric readings should be made after 20-24 hours incubation

230

DIFCO MANUAL

at 35-3 7°C. The most effective assay range, using Bacto-CF Assay Medium, has
been found to be between 0.6 and 4.8 millimicrograms of citrovorum factor.

To rehydrate the medium, suspend 110 grams of Bacto-CF Assay Medium in
1000 ml. distilled water, and heat to boiling for 2-3 minutes. The slight precipi-
tate which forms should be evenly distributed by shaking. Five (5) ml. of the
medium are added to each tube in the preparation of the tubes for the standard
curve and to each tube containing material under assay. For the assay, each tube
must contain 5 ml. of rehydrated medium, increasing amounts of the standard or
the unknown, and sufficient distilled water to give a total volume of 10 ml. per
tube. The tubes are then autoclaved for 10 minutes at 15 pounds pressure
(121°C.). Oversterilization of the medium will give unsatisfactory results.

One hundred grams of Bacto-CF Medium will make 1.8 liters final medium,

^ J. Biol. Chem., 176:165:1948. 2 Personal Communication, 1951.

AMINO ACID ASSAY MEDIA

Media for the microbiological assay of the amino acids, leucine, methionine,
lysine and isoleucine have been prepared. A complete description of each medium
is given in the individual discussions. The formulation of the media per liter is
as follows :

Bacto-Dextrose 50 g,

Sodium Acetate 40 g,

Ammonium Chloride 6 g

^/-Alanine 0.4 g

i-Arginine Hydrochloride .... 0.484 g

Bacto-Asparagine 0.8

i-Aspartic Acid 0.2

/-Cystine, Difco 0.1

/-Glutamic Acid 0.6

Glycine 0.2

/-Histidine Hydrochloride ... 0.124 g

^/-Phenylalanine 0.2 g

/-Proline 0.2 g

£?/-Serine 0.1 g

^/-Threonine 0.4 g

J/-Tryptophc ne 0.08 g

/-Tyrosine 0.2 g

^f/- Valine 0.5 g

Adenine Sulfate 0.02 g

Guanine Hydrochloride 0.02 g.

Uracil 0.02 g.

Xanthine 0.02 g.

Thiamine Hydrochloride .... 0.001 g.

Pyridoxine Hydrochloride . . . 0.002 g.

Pyridoxamine Hydrochloride 0.0006 g.

Pyridoxal Hydrochloride . . . 0.0006 g.

Calcium Pantothenate 0.001 g.

Riboflavin 0.001 g.

Nicotinic Acid 0.002 g.

p-Aminobenzoic Acid, Difco . 0.0002 g.

Biotin 0.000002 g.

Folic Acid 0.00002 g.

Monopotassium Phosphate .... 1.2 g.

Dipotassium Phosphate 1.2 g.

Magnesium Sulfate 0.4 g.

Ferrous Sulfate 0.02 g.

Manganese Sulfate 0.04 g.

Sodium Chloride 0.02 g.

BACTO

LEUCINE ASSAY MEDIUM

DEHYDRATED

(B421)

The composition of Bacto-Leucine Assay Medium is as shown above, but in
addition it contains 0.5 g. of /-lysine hydrochloride, 0.2 g. c?/-methionine and 0.5
g. £?/-isoleucine. Bacto-Leucine Assay Medium, prepared according to the formula
given by Steel, Sauberlich, Reynolds and Baumann,^ is a complete dehydrated
medium for the assay of /-leucine and c?/-leucine. It is free from leucine but con-
tains all the other growth factors and amino acids necessary for the growth of
Leuconostoc mesenteroides P-60 ATCC 8042. The addition of /-leucine or <i/-leu-
cine in specified increasing concentrations gives a growth response by L. meseri'
teroides P-60, which may be measured acidimetrically or turbidimetrically.

DEHYDRATED CULTURE MEDIA 231^

The following procedure is recommended for the use of Bacto-Leucine Assay
Medium :

Stock cultures of the test organism, L. mesenteroides P-60, are prepared by
stab inoculation of Bacto-Micro Assay Culture Agar. After 24—48 hours incuba-
tion at 35-3 7 °C., the tubes are stored in the refrigerator. The transplants are
made at monthly intervals, in triplicate.

Inoculum for assay is prepared by subculturing from a stock culture to 10 ml.
of Bacto-Micro Inoculum Broth. After 16-24 hours incubation at 35-3 7 °C., the
cells are centrifuged under aseptic conditions, and the supernatant liquid de-
canted. The cells are resuspended in 10 ml. sterile isotonic sodium chloride solu-
tion. The cell suspension is diluted 5-100 with sterile isotonic sodium chloride.
One drop of this latter suspension is used to inoculate each of the assay tubes
(10 ml.).

Bacto-Leucine Assay Medium may be used for both turbidimetric and acidi-
metric analyses. Turbidimetric readings should be made after 16-20 hours at
35-3 7 °C. Acidimetric determinations are made after 72 hours incubation at
35-3 7 °C. The most effective assay range, using Bacto-Leucine Assay Medium,
has been found to be between 20 micrograms and 80 micrograms /-leucine.
(20-160 micrograms of fi?/-leucine).

The concentrations of leucine required for the construction of the standard
curve may be prepared by dissolving 2.0 grams of /-leucine (4.0 g. ^//-leucine) in
1000 ml. distilled water giving a stock solution of 2000 micrograms of /-leucine
per ml. Dilute the stock solution by adding 1 ml. to 99 ml. distilled water. Use
0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4 and 5 ml. per tube. The stock solution of leucine
used for preparing the standard curve is stable for 2 months when stored at
2-6°G. under toluene.

To rehydrate the medium, suspend 105 grams of Bacto-Leucine Assay Medium
in 1000 ml. distilled water, and heat to boiling for 2-3 minutes. The slight pre-
cipitate which forms should be evenly distributed by shaking. Five (5) ml. of
the medium are added to each tube in the preparation of the tubes for the stand-
ard curve and to each tube containing material under assay. For the assay, each
tube must contain 5 ml. of rehydrated medium, increasing amounts of the stand-
ard or the unknown, and sufficient distilled water to give a total volume of 10 ml.
per tube. The tubes are autoclaved for 10 minutes at 15 pounds pressure
(121°G.). Oversterilization of the medium will give unsatisfactory results.

One hundred grams of Bacto-Leucine Assay Medium will make 1.9 liters of
final medium.
*J. Biol, Chem., 177:533:1949.

232 DIFCO MANUAL

BACTO

METHIONINE ASSAY MEDIUM (B423)

DEHYDRATED

The composition of Bacto-Methionine Assay Medium is as shown above, but
in addition it contains 0.5 g. /-lysine hydrochloride, 0.5 g. isoleucine and 0.5 g.
cf/-leucine. Bacto-Methionine Assay Medium, prepared according to the formula
given by Steel, Sauberlich, Reynolds and Baumann,^ is a complete dehydrated
medium for the assay of c?/-methionine. It is free from methionine but contains
all the other growth factors and amino acids necessary for the growth of Leu-
conostoc mesenteroides P-60 ATCC 8042. The addition of c?/-methionine in speci-
fied increasing concentrations gives a growth response by L. mesenteroides P-60
which may be measured acidimetrically or turbidimetrically.

The following procedure is recommended for the use of Bacto-Methionine
Assay Medium:

Stock cultures of the test organism, L. mesenteroides P-60 are prepared by stab
inoculation of Bacto-Micro Assay Culture Agar. After 24-48 hours incubation at
35-37°C., the tubes are stored in the refrigerator. Transplants are made at
monthly intervals, in triplicate.

Inoculum for assay is prepared by subculturing from a stock culture to 10 ml.
of Bacto-Micro Inoculum Broth. After 16-24 hours incubation at 35-37°C., the
cells are centrifuged under aseptic conditions, and the supernatant liquid de-
canted. The cells are resuspended in 10 ml. sterile isotonic sodium chloride solu-
tion. The cell suspension is diluted 5-100 with sterile isotonic sodium chloride.
One drop of this latter suspension is used to inoculate each of the assay tubes
(10 ml.).

The concentrations of methionine required for the preparation of the standard
curve may be prepared by dissolving 1.2 grams ^/-methionine in 1000 ml. dis-
tilled water. This is the stock solution (1200 micrograms per ml.). Dilute the
stock solution by adding 1 ml. to 99 ml. distilled water. Use 0.0, 0.5, 1.0, 1.5,
2.0, 2.5, 3.0, 4 and 5 ml. per tube. The stock solution is stable for 2 months when
stored at 2-6° G. under toluene.

Bacto-Methionine Assay Medium may be used for both turbidimetric and
acidimetric analyses. Turbidimetric readings should be made after 16-20 hours
at 35-37°C.; acidimetric determinations are made after 72 hours incubation. The
most effective assay range, using Bacto-Methionine Assay Medium, has been
found to be between 6 and 48 micrograms c?/-methionine.

To rehydrate the medium, suspend 105 grams of Bacto-Methionine Assay
Medium in 1000 ml. distilled water, and heat to boiling for 2-3 minutes. The
slight precipitate which forms should be evenly distributed by shaking. Five (5)
ml. of the medium are added to each tube in the preparation of the tubes for the
standard curve and to each tube containing material under assay. For the assay,
each tube must contain 5 ml. of rehydrated medium, increasing amounts of the
standard or the unknown, and sufficient distilled water to give a total volume of
10 ml. per tube. The tubes are autoclaved for 10 minutes at 15 pounds pressure
(121°C.). Oversterilization of the medium will give unsatisfactory results.

One hundred grams of Bacto-Methionine Assay Medium will make 1.9 liters
of final medium.

ij. Biol. Chem., ?95:533:i949.

DEHYDRATED CULTURE MEDIA 233

BACTO

LYSINE ASSAY MEDIUM (B422)

DEHYDRATED

The composition of Bacto-Lyslne Assay Medium is as shown above, but in
addition it contains 0.2 g. of cf/-methionine, 0.5 g. c?/-isoleucine and 0.5 g. c^Meu-
cine. Bacto-Lysine Assay Medium, prepared according to the formula given by
Steel, Sauberlich, Reynolds and Baumann,^ is a complete dehydrated medium
for the assay of /-lysine. It is free from lysine but contains all the other growth
factors and amino acids necessary for the growth of Leuconostoc mesenteroides
P-60 ATCC 8042. The addition of /-lysine in specified increasing concentrations
gives a linear growth response by L. mesenteroides P-60, which may be measured
acidimetrically or turbidimetrically.

The following procedure is recommended for the use of Bacto-Lysine Assay
Medium :

Stock cultures of the test organism, L. mesenteroides P-60 are prepared by stab
inoculation of Bacto-Micro Assay Culture Agar. After 24-48 hours incubation at
35-3 7 °C., the tubes are stored in the refrigerator. Transplants are made at
monthly intervals, in triplicate.

Inoculum for assay is prepared by subculturing from a stock culture to 10 ml.
of Bacto-Micro Inoculum Broth. After 16-24 hours incubation at 35-3 7 °C., the
cells are centrifuged under aseptic conditions, and the supernatant liquid de-
canted. The cells are resuspended in 10 ml. sterile isotonic sodium chloride
solution. The cell suspension is diluted 5-100 with sterile isotonic sodium
chloride. One drop of this latter suspension is used to inoculate each of the assay
tubes (10 ml.).

The concentrations of lysine required for the preparation of the standard curve
may be prepared by dissolving 6.0 grams /-lysine in 1000 ml. distilled water. This
is the stock solution (6000 micrograms per ml.). Dilute the stock solution by
adding 1 ml. to 99 ml. distilled water. Use 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4 and 5
ml. per tube. The stock solution is stable for 2 months when stored at 2-6° G.
under toluene.

Bacto-Lysine Assay Medium may be used for both turbidimetric and acidi-
metric analyses. Turbidimetric readings should be made after 16-20 hours at
35-37°C.; acidimetric determinations are made after 72 hours incubation. The
most effective assay range, using Bacto-Lysine Assay Medium, has been found to
be between 30 micrograms and 240 micrograms of /-lysine.

To rehydrate the medium, suspend 105 grams of Bacto-Lysine Assay Medium
in 1000 ml. distilled water, and heat to boiling for 2-3 minutes. The slight pre-
cipitate which forms should be evenly distributed by shaking. Five (5) ml. of the
medium are added to each tube in the preparation of the tubes for the standard
curve and to each tube containing material under assay. For the assay, each tube
must contain 5 ml. of rehydrated medium, increasing amounts of the standard
or the unknown, and sufficient distilled water to give a total volume of 10 ml.
per tube. The tubes are autoclaved for 10 minutes at 15 pounds pressure
(121°C). Oversterilization of the medium will give unsatisfactory results.

One hundred grams of Bacto-Lysine Assay Medium will make 1 .9 liters of final
medium.

ij. Biol. Chem., 177:533 = 1 949-

234 DIFCO MANUAL

BACTO

ISOLEUCINE ASSAY MEDIUM (B437)

DEHYDRATED

The composition of Bacto-Isoleucine Assay Medium is as shown above, but in
addition, it contains 0.2 g. of c?/-methionine, 0.5 g. of c?/-leucine and 0.5 g. /-lysine.
Bacto-Isoleucine Assay Medium, prepared according to the formula by Steel,
Sauberlich, Reynolds and Baumann,^ is a complete dehydrated medium for the
assay of cf/-isoleucine. It is free from isoleucine but contains all the other growth
factors and amino acids necessary for the growth of Leuconostoc mesenteroides
P-60 ATCC 8042. The addition of df/-isoleucine in specified increasing concentra-
tions gives a growth response by L. mesenteroides P-60, which may be measured
acidimetrically.

The following procedure is recommended for the use of Bacto-Isoleucine Assay
Medium:

Stock cultures of the test organism, L. mesenteroides P-60, are prepared by
stab inoculation of Bacto-Micro Assay Culture Agar. After 24—48 hours incuba-
tion at 35-3 7 °C., the tubes are stored in the refrigerator. Transplants are made
at monthly intervals, in triplicate.

Inoculum for assay is prepared by subculturing from a stock culture to 10 ml.
of Bacto-Micro Inoculum Broth. After 16-24 hours incubation at 35-3 7 °C., the
cells are centrifuged under aseptic conditions, and the supernatant decanted. The
cells are resuspended in 10 ml. sterile isotonic sodium chloride solution. The cell
suspension is diluted 5-100 with sterile isotonic sodium chloride. One drop of
this latter suspension is used to inoculate the assay tubes (10 ml.).

It is essential that a standard curve be constructed each time an assay is run,
since conditions of autoclaving, temperature of incubation, etc., which influence
the standard curve readings, cannot be duplicated exactly from time to time. A
standard curve is obtained by using ^/-isoleucine at levels of 0.0, 12.0, 24, 36, 48,
60, 72, 96 and 120 micrograms per assay tube (10 ml.). The concentrations of
isoleucine required for the construction of the standard curve may be prepared
by dissolving 2.4 grams of ^/-isoleucine in 1000 ml. distilled water. This is the
stock solution (2400 micrograms per ml.). Dilute the stock solution by adding
1 ml. to 99 ml. distilled water. Use 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4 and 5 ml. per
tube. The stock solution is stable for 2 months when stored at 2-6° G. under
toluene.

Tubes are incubated at 35-3 7 °G. for 72 hours following inoculation. The
growth response is then measured by acidimetric determinations. The most effec-
tive assay range, using Bacto-Isoleucine Assay Medium, has been found to be
between 12 and 96 micrograms cf/-isoleucine.

To rehydrate the medium, suspend 105 grams of Bacto-Isoleucine Assay
Medium in 1000 ml. distilled water, and heat to boiling for 2-3 minutes. The
slight precipitate which forms should be evenly distributed by shaking. Five
(5) ml. of the medium are added to each tube in the preparation of the tubes
for the standard curve and to each tube containing material under assay. For
the assay, each tube must contain 5 ml. of rehydrated medium, increasing
amounts of the standard or the unknown and sufficient distilled water to give a
total volume of 10 ml. per tube. The tubes are autoclaved for 10 minutes at 15
pounds pressure (121°G.). Oversterilization of the medium will give unsatis-
factory results.

One hundred grams of Bacto-Isoleucine Assay Medium will make 1.9 liters of
final medium.

1 J. Biol. Chem., i77:333:»949«

DEHYDRATED CULTURE MEDIA 235

BAGTO-ARGININE ASSAY MEDIUM (B466)
BAGTO-GYSTINE ASSAY MEDIUM (B467)
BAGTO-TYROSINE ASSAY MEDIUM (B468)
BAGTO-PHENYLALANINE ASSAY MEDIUM (B469)

These media have been just recently developed for the microbiological assay of
Arginine, Cystine, Tyrosine and Phenylalanine. They are similar in composition
to the media described immediately above, being free of the amino acid
specified for assay, but containing all other growth factors and nutriments
necessary for the growth of Leuconostoc mesenteroides P-60 ATCC 8042. The
addition of the amino acid under assay in specified increasing concentrations
gives a growth response by L. mesenteroides which may be measured acidimetri-
cally or turbidimetrically. Complete directions for the assay is given on the
package label.

To rehydrate these media, dissolve 105 grams in 1000 ml. distilled water and
heat to boiling for 2-3 minutes. The slight precipitate which forms should be
evenly distributed by shaking. Five (5) ml. of the medium are added to each
tube in the preparation of the tubes for the standard curve and to each tube con-
taining material under assay. For the final assay each tube must contain 5 ml.
of rehydrated medium, increasing amounts of the standard or the unknown and
sufficient distilled water to give a total volume of 10 ml. per tube. The tubes are
autoclaved for 10 minutes at 15 pounds pressure (121°C.). Oversterilization of
the medium will give unsatisfactory results.

One hundred grams of each of these media will make 1 .9 liters of final medium.

BACTO

TRYPTOPHANE ASSAY MEDIUM (B327)

DEHYDRATED

Bacto- Vitamin Free Riboflavin 0.0004 g,

/^-Aminobenzoic Acid, Difco . 0.0002 g

Niacin 0.0002 g

Biotin 0.0000008 g

Dipotassium Phosphate 1 g

Monopotassium Phosphate 1 g

Magnesium Sulfate 0.4 g,

Sodium Chloride 0.02 g

Ferrous Sulfate 0.02 g

Manganese Sulfate 0.02 g

Casamino Acids 12

Bacto-Dextrose 40 g,

Sodium Acetate 20 g

/-Cystine, Difco 0.2 g

Adenine Sulfate 0.02 g

Guanine Hydrochloride 0.02 g.

Uracil 0.02 g,

Thiamine Hydrochloride . . . 0.0002 g.

Calcium Pantothenate .... 0.0002 g,

Pyridoxine Hydrochloride . . 0.0004 g

Bacto-Tryptophane Assay Medium is a complete dehydrated medium for the
assay of /-tryptophane or c?/-tryptophane. It is free from tryptophane but contains
all the other factors necessary for the growth of Lactobacillus arabinosus 17-5
ATCC 8014. The addition of tryptophane in certain concentrations gives a linear
growth response by L. arabinosus 17-5, which may be measured acidimetrically.
Bacto-Tryptophane Assay Medium is prepared according to the formula de-
scribed by Greene and Black.^

The following procedure is recommended for the assay of tryptophane using
the dehydrated medium:

Stock cultures of L. arabinosus 17-5 are prepared by stab inoculation of Bacto-
Micro Assay Culture Agar. Following incubation at 35-3 7 °C. for 24^8 hours,
the tubes are stored in the refrigerator. Transplants are made at monthly inter-

236 DIFCO MANUAL

vals. Inoculum for assay is prepared by subculturing from a stock culture of
L. arabinosus 17-5 into a tube containing 10 ml. of Bacto-Micro Inoculum Broth.
After 24 hours incubation at 35-3 7 °C., the cells are centrifuged under aseptic
conditions, and the supernatant liquid decanted. The cells are resuspended in 10
ml. of sterile isotonic sodium chloride. The cell suspension is then diluted 1-100
with sterile isotonic sodium chloride. The suspension should be just faintly
cloudy. One drop of this latter suspension is then used to inoculate each of the
assay tubes.

The concentrations of tryptophane required for the preparation of the standard
curve may be prepared by dissolving 0.1 gram of /-tryptophane (0.2 g. t/Z-trypto-
phane) in 1000 ml. of distilled water giving a stock solution of 100 micrograms of
/-tryptophane per ml. Dilute this stock solution by adding 4 ml. to 96 ml. dis-
tilled water. Use 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 5 ml. per tube. This stock
solution of tryptophane used for preparing the standard curve is stable for 2
months when stored at 2-6° C. under toluene.

Following inoculation, tubes are incubated at 35-3 7 °C. for 72 hours and are
assayed by acidimetric methods. Using Bacto-Tryptophane Assay Medium, we
have found the most effective assay range to be between 2 and 10 micrograms of
/-tryptophane. (4 and 20 micrograms c?/-tryptophane).

To rehydrate the basal medium, suspend 75 grams of Bacto-Tryptophane Assay
Medium in 1000 ml. distilled water and heat to boiling for 2-3 minutes. The
slight precipitate which forms should be evenly distributed by shaking. Five (5)
ml. of the medium are added to each tube in the preparation of the tubes for
the standard curve and to each tube containing material under assay. For the
assay, each tube must contain 5 ml. of rehydrated medium, increasing amounts
of the standard or the unknown and sufficient distilled water to give a total
volume of 10 ml. per tube.

The tubes are sterilized by autoclaving for 10 minutes at 15 pounds pressure
(121°G.). Oversterilization of the medium will give unsatisfactory results.

One hundred grams of Bacto-Tryptophane Assay Medium will make 2.6 liters
of final medium.

1 J. Biol. Chem., 155:1:1944'

DEHYDRATED CULTURE MEDIA 237

MEDIA FOR MYCOLOGY

The study of yeasts and molds is one of the oldest branches of microbiology.
The importance of this group of microorganisms in many fields is being recog-
nized. Particular mention may be made of the role of yeasts and molds in relation
to sanitary control, in the production of antibiotics and other industrial fermenta-
tions, as the causative agents of infections in animals and plants, the deterioration
of fabrics and many other applications.

The value of selective media for the initial cultivation of pathogenic fungi
particularly has been demonstrated by numerous investigators. They have shown
that many fungi prefer neutral or slightly alkaline rather than acid reactions for
early and luxuriant growth. Earlier media for fungi generally relied on the acid
reaction to make the medium less suited for the growth of many bacteria. More
recently developed media and modifications use neutral or slightly alkaline reac-
tions, antibiotics, bile salts and dyes as selective agents against bacteria without
effecting the growth of fungi. The results obtained with these selective media
make their consideration important by any laboratory interested in the isolation
and cultivation of fungi.

Plant diseases are exceedingly important from an economic point of view. The
great majority of these diseases are undoubtedly due to infections by molds, and
other fungi, viruses and bacteria. For the purpose of assisting the plant pathologist
and mycologist in his studies incident to the investigation and control of the plant
diseases, we have developed in our laboratories a number of dehydrated culture
media. The formulae chosen for this group of media have been devised and
selected with the cooperation of scientists engaged in active research in problems
of plant pathology. Since no universal standard formulae prevail, selections have
been based on the general usefulness of the media. The developments and applica-
tions of pure culture methods from a phytopathological point of view have
aroused interest in the study of systematic mycology. The use of dehydrated cul-
ture media brings into such work the development of definite standards of uni-
formity and comparability.

The media listed in this section are employed for the cultivation, isolation and
identification of these microorganisms. Some are recommended for pathogenic
fungi, while other media are primarily for the cultivation of non-pathogenic
yeasts and molds.

238 DIFCO MANUAL

BACTO

SABOURAUD DEXTROSE AGAR (B109)

DEHYDRATED

Neopeptone, Difco 10 g.

Bacto-Dextrose 40 g.

Bacto-Agar 15 g.

Bacto-Sabouraud Dextrose Agar is a modification of the Dextrose Agar
described by Sabouraud.^ Comparative tests have shown that Neopeptone, Difco
is a most satisfactory source of nitrogen for the development of fungi. It is
recommended for the cultivation and growth of fungi, particularly those associ-
ated with skin infections.

The majority of molds are not pathogenic, but some are true parasites, pro-
ducing a number of common diseases such as ringworm, favus and various other
hair and skin lesions. Internal infections of the lung and lymphatics may also be
traced to molds. Bacto-Sabouraud Dextrose Agar is particularly adapted for the
cultivation and identification of such molds, especially those infecting the skin.

For the primary isolation of fungi from scales and crusts, Ch'in^ suggests
the addition of 0.015 per cent potassium tellurite, or 0.05 per cent copper sulfate
to this medium in order to suppress the growth of bacteria. Emmons and Ash-
burn^ used a Sabouraud Dextrose Agar containing 1 per cent Neopeptone and 2
per cent dextrose for the isolation of Histoplasma capsulatum from rats. Emmons
and Hollaender* used Sabouraud Dextrose Agar prepared with Neopeptone for
growing Trichophyton gypseum asteroides in their studies on mutation of the
dermatophytes induced by ultraviolet irradiation. Robinson and Kotcher^ used
Sabouraud Dextrose Agar containing 20 units penicillin and 40 units dihydro-
streptomycin hydrochloride per ml. of medium for the isolation of Histoplasma
from dogs. Kotcher, Robinson and Miller^ in a study of media for the recovery
of H. capsulatum from tissues of artificially infected rats reported that the highest
percentage recovery of the organism was from spleen on Sabouraud Dextrose
Agar. Serowy and Jung^ used Bacto-Sabouraud Dextrose Agar in their study of
the Microspora and called attention to the suitability of this medium for the
cultivation of Microspora and other pathogenic fungi, as well as the ease with
which this medium may be prepared and used.

The addition of antibiotics to acid as well as neutral media for the isolation of
pathogenic fungi has proven especially satisfactory. Generally 20 units penicillin
and 40 micrograms streptomycin or dihydrostreptomycin per ml. medium are
added to the sterile melted medium at 45-50 °C. under aseptic conditions. These
desired concentrations of penicillin may be readily obtained by dissolving the
contents of one (1) vial of penicillin containing 100,000 units penicillin in 10
ml. sterile distilled water. Two (2) ml. of this solution are added to one liter of
sterile melted medium at 45-50°C. under aseptic conditions (0.2 ml. per 100 ml.
of medium). To obtain the desired concentration of streptomycin in the same
medium dissolve the contents of a one gram vial of streptomycin (one million
micrograms) in 10 ml. sterile distilled water. One (1) ml. of this solution is
added to 9 ml. sterile distilled water to give a solution containing 10,000 micro-
grams streptomycin per ml. To each liter of medium are added 4 ml. of this
solution to obtain 40 micrograms per ml. (0.4 ml. for 100 ml. medium).

To rehydrate the medium, suspend 65 grams of Bacto-Sabouraud Dextrose
Agar in 1000 ml. of cold distilled water and heat to boiling to dissolve the
medium completely. Distribute in tubes or flasks and sterilize in the autoclave for
15 minutes at 15 pounds pressure (121°C.) The final reaction of the medium will
be pH 5.6.

DEHYDRATED CULTURE MEDIA 239

One pound of Bacto-Sabouraud Dextrose Agar will make 6.9 liters of medium.

lAnn. dermatol. syphilol., 1892-1893. "Pub. Health Reports, 66:1533:1951.

2 Proc. Soc. Exp. Biol. Med., 38:700:1938. ^j Bact., 62:613:1951.

8 Pub. Health Reports. 63:1416:1948. 'Derm. Wschr., 124:663:1951.

* Arch. Dermatol Syphilol., 52:257:1945.

BACTO

SABOURAUD MALTOSE AGAR (BllO)

DEHYDRATED

Neopeptone, Difco 10 g.

Maltose, Difco 40 g.

Bacto-Agar 15 g,

Bacto-Sabouraud Maltose Agar is a modification of the formula suggested by
Sabouraud.i Comparative tests have shown that Neopeptone, Difco is a most
satisfactory source of nitrogen for the development of fungi. This medium is an
excellent substrate for the propagation of molds and yeasts, particularly the
parasitic fungi concerned with skin and scalp lesions.

Bacto-Sabouraud Maltose Agar, like Bacto-Sabouraud Dextrose Agar, contains
no selective agent, and depends entirely on the acid reaction, pH 5.6, for the
selective growth of fungi over bacteria. In the initial cultivation of fungi from
specimens many investigators prefer to use a selective medium such as Bacto-
Littman Oxgall Agar, as discussed on page 240 or Bacto-Brain Heart Infusion
Agar as described on page 90.

Antibiotics may also be added to Sabouraud Maltose Agar to give selective
media, as discussed under Bacto-Sabouraud Dextrose Agar immediately above.

The use of maltose, or the extractives of malt, in media designed for the
cultivation of molds and other fungi is quite universal. Maltose is well adapted
to the nutritional requirements of these organisms.

Frank^ has used Bacto-Sabouraud Maltose Agar successfully in cultivating the
causative organisms of perleche. Davidson, Dawding and Buller^ reported that
Bacto-Sabouraud Maltose Agar was a most satisfactory medium in their studies
of the infections caused by Microsporon audouini, M. lanosum and Trichophyton
gypseum. Davidson and Dawding^ also used this medium in isolating T. gypseum
from a case of tinea barbae. Serowy and Jung^ used Bacto-Sabouraud Maltose
Agar in their study of the Microspora and called attention to the suitability of
this medium for the cultivation of Microspora and other pathogenic fungi, as well
as the ease with which this medium may be prepared and used. A. W. Bengtson^
observed that Sabouraud Maltose Agar could be used to advantage in the isola-
tion and differentiation of Pseudomonas. On this medium the blue pyocyanin
pigment is enhanced making it easy to determine pigment production thereby
detecting Pseudomonas organisms in mixed infections. Sabouraud Dextrose Agar
on the other hand, tends to elicit the production of the pink fluorescene pigment
with suppression of the pyocyanin. Chapman^ modified Sabouraud Maltose Agar
in the preparation of a selective medium for the isolation and identification of
Monilia and other fungi. The medium was prepared by adding 0.1 ml. of Tergitol
7 and 0.0025 per cent Brom Cresol Purple to Sabouraud Maltose Agar at
pH 5.6. The medium was sterilized in the autoclave for 10 minutes and when
cooled to 45-55 °C., 0.3 ml. of Bacto-Chapman Tellurite Solution and 3 ml. of
2,3,5-triphenyltetrazolium chloride (TTC), was added. Surface of the plates
were inoculated followed by incubation at 37°C. for 48 hours. Chapman reported
that the Tergitol 7 inhibited all bacteria except members of the coliform group,
while the potassium tellurite inhibited these organisms. Candida albicans pro-

240 DIFCO MANUAL

duced "off white" circular smooth entire convex to pulvinate colonies about
4 mm. in diameter. Other Candida produce colored colonies ranging from orange
to tan to lilac, often discoloring the medium. Sac char omyces grew in 48 hours
on the medium producing colonies somewhat resembling those of Candida.

To rehydrate the medium, suspend 65 grams of Bacto-Sabouraud Maltose
Agar in 1000 ml. of cold distilled water and heat to boiling to dissolve the
medium completely. Distribute in tubes or flasks, and sterilize in the autoclave for
15 minutes at 15 pounds pressure (121°C.). The final reaction of the medium
will be pH 5.6.

One pound of Bacto-Sabouraud Maltose Agar will make 6.9 liters of medium.

1 Ann. dermatol. syphilol., 1892-1893. ^ Derm. Wschr., 124:665:1951.

2 Arch. Dermatol. Syphilol., 26:451:1932. ^ Personal Communication, 1951.

8 Can. J. Research, 6:1 : 1932. 'Trans. New York Acad. Sci. Series II,

* Arch. Dermatol. Syphilol., 26:660:1932. 14:254:1952.

BACTO

SABOURAUD MALTOSE BROTH (B429)

DEHYDRATED

Neopeptone, Difco 10 g.

Maltose, Difco 40 g.

Bacto-Sabouraud Maltose Broth is a liquid medium recommended for the culti-
vation of fungi. It is acid in reaction and has the same formula as Bacto-
Sabouraud Maltose Agar except agar is omitted. Bacto-Sabouraud Maltose Broth
is also well suited for the detection of fungi in sterility test procedures.

To rehydrate the medium, dissolve 50 grams Bacto-Sabouraud Maltose Broth
in 1000 ml. of distilled water. Distribute in tubes or flasks and sterilize in the
autoclave for 15 minutes at 15 pounds pressure (121°G.). Final reaction will
be pH 5.6.

One pound of Bacto-Sabouraud Maltose Broth will make 9 liters of medium.

BACTO

SABOURAUD LIQUID MEDIUM

DEHYDRATED

Bacto-Sabouraud Liquid Medium is recommended as a liquid medium for the
cultivation of fungi. This medium supports early and luxuriant growth of fungi
from small inocula. A complete discussion of this medium is given on page 200.
A selective liquid medium may be prepared by the addition of penicillin and
streptomycin as described in the discussion on Bacto-Sabouraud Dextrose Agar
page 238.

BACTO

LITTMAN OXGALL AGAR (B294)

DEHYDRATED

Bacto-Peptone 10 g.

Bacto-Dextrose 10 g.

Bacto-Oxgall 15 g.

Bacto-Agar 20 g.

Bacto-Crystal Violet ; 0.01 g.

DEHYDRATED CULTURE MEDIA 241

Bacto-Littman Oxgall Agar with the addition of streptomycin is a selective
medium for the primary isolation of fungi. It is also of especial value for the
cultivation of the dermatophytes.

Littman^ described a selective medium for the primary isolation of fungi.
Crystal violet and streptomycin are used as selective bacteriostatic agents, while
Bacto-Oxgall is used to restrict the spreading of fungus colonies. The medium is
neutral in reaction, which favors the growth of many pathogenic fungi. The
studies of Littman have indicated that this medium offers considerable promise as
a diagnostic tool for the primary isolation of fungi from specimens possessing
a mixed bacterial and fungal flora.

Littman has shown that his medium is especially valuable for culturing the
dermatophytes. Molds and yeasts form non-spreading discrete colonies, easy to
isolate in pure culture. He also suggests that the medium may be used for the
following purposes: estimation of the normal fungal flora of feces, sputum and
other human discharges; evaluation of human disorders of the upper and lower
respiratory and gastrointestinal tract caused by fungi; single cell isolation of
fungi; plate count of viable saprophytic fungi in foodstuffs and air. In a com-
parative study Littman^ compared this medium with Sabouraud Dextrose Agar
using a large variety of pathogenic and saprophytic fungi. On the Littman Oxgall
Agar the majority of fungi tested produced colonies at the end of the first month
of incubation about half the size of the colonies on Sabouraud Dextrose Agar, but
equal in size after 56 days of incubation. He reported the isolation of three times
as many fungi from feces, sputum, skin scrapings and hair on his medium as
were isolated on Sabouraud Dextrose Agar, and four times as many pathogenic
dermatophytes on the selective medium as on the Sabouraud medium. The
selective oxgall agar of Littman is specified in "Diagnostic Procedures and
Reagents"^ of the American Public Health Association for the isolation of
pathogenic fungi.

For inoculation, skin and nail scrapings or infected hairs are placed directly
on the surface of the medium. Exudates, sputa, or fecal suspensions are spread
over the surface with a sterile swab. The selectivity of the medium permits the
use of a heavy inoculum without the danger of overgrowth by bacteria or sapro-
phytic fungi. Plates are incubated at room temperature or preferably at 30 °C.
for four to eight days. Do not incubate at 37°C.

To rehydrate the medium, suspend 55 grams of Bacto-Littman Oxgall Agar in
1000 ml. of cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in flasks and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). Cool to 45-50°C. and add 30 micrograms of strepto-
mycin per ml. of medium. Dispense in 27-30 ml. amounts in sterile petri dishes
100 mm. in diameter or distribute in sterile tubes. Let stand at room temperature
for 6-8 hours before inoculation. Plates or tubes of media may be kept in the
refrigerator for 2-3 weeks without deterioration if placed in sealed containers
to prevent evaporation. A concentration of 30 micrograms per ml. of medium
may be obtained by adding 10 ml. sterile distilled water to a 1 gram (one
million micrograms) bottle of streptomycin or dihydrostreptomycin. One (1)
ml. of this solution is added to 9 ml. sterile distilled water to give a solution con-
taining 10,000 micrograms streptomycin per ml. To each liter of sterile melted
medium at 45-50° C. are added 3 ml. of this solution to obtain 30 micrograms
per ml. (0.3 ml. for 100 ml. medium). Final reaction of the sterile medium will
be pH 7.0.

One pound of Bacto-Littman Oxgall Agar will make 8.2 liters of medium.

1 Science, 106:109:1947. ^ pjagnostlc Procedures and Reagents, 3rd Edi-

2 Tech. Bull., Reg. Med. Tech., 18:409:1948. 1100:452:1950

242 DIFGO MANUAL

BACTO

BRAIN HEART INFUSION AGAR (B418)

DEHYDRATED

Bacto-Brain Heart Infusion Agar is recommended as a solid medium for the
cultivation of fastidious pathogenic bacteria and fungi. A selective Brain Heart
Infusion Agar especially recommended for the isolation of fungi is obtained
by the addition of 20 units penicillin and 40 micrograms of streptomycin per ml.
of sterile melted medium at 45-50°C. A method to obtain these concentrations
of the antibiotics is given in the description of Bacto-Sabouraud Dextrose Agar
page 238. A complete discussion of Bacto-Brain Heart Infusion Agar is given
on page 90.

BACTO

MALT EXTRACT BROTH (B113)

DEHYDRATED

Bacto-Malt Extract Broth is prepared from Bacto-Malt Extract, according to
the directions given by Thom and Church.^ It is recommended as a liquid
medium for the cultivation of yeasts and molds and may be employed as a
sterility test medium to detect the presence of these organisms.

The use of malt and malt extracts for the propagation of yeasts and molds is
quite common. Reddish^ described a culture medium prepared from malt extract
which was a satisfactory substitute for wort. Thom and Church, following the
formula of Reddish, used Bacto-Malt Extract, as a base. The carbohydrates
present in such media are well suited to the growth requirements of fungi, par-
ticularly if the reaction of the media is somewhat acid. Comparative tests have
shown that early and luxuriant growth will be initiated in Bacto-Malt Extract
Broth from inocula of yeasts and molds as small or smaller than those required
for other media, including broths prepared with honey.

For the cultivation of molds, filter paper cones may be dipped into Malt
Extract Broth and sterilized in deep culture dishes containing a layer of the
medium. The molds are seeded upon the moist surface of the sterile cones. Thom
and Church advise a solution of 100 grams of Bacto-Malt Extract Broth in 900
ml. of distilled water.

To rehydrate the medium, dissolve 15 grams of Bacto-Malt Extract Broth in
1000 ml. of distilled water. A more nutritious medium can be obtained by using
more Bacto-Malt Extract Broth per unit of water. Distribute in tubes or flasks
and sterilize in the autoclave for 15 minutes at 15 pounds pressure (121°G.). The
final reaction of the medium will be pH 4.7.

One pound of Bacto-Malt Extract Broth will make 30.2 liters of medium.

^Thorn and Church: The Aspergilli, igaS. 2 Abst. Bact., 3:6:1919.

BACTO

NEUROSPORA CULTURE AGAR

DEHYDRATED

Bacto-Neurospora Culture Agar is recommended as a neutral medium for the
cultivation of fungi. A selective medium for this group of microorganisms may
be prepared by the addition of penicillin and streptomycin as discussed under
Bacto-Sabouraud Dextrose Agar page 238. A complete description of Bacto-
Neurospora Culture Aear is given on page 214.

DEHYDRATED CULTURE MEDIA 243

BACTO

MYGOLOGIGAL AGAR (B405)

DEHYDRATED

Bacto-Soytone 10 g.

Bacto-Dextrose 10 g.

Bacto-Agar 15 g.

Bacto-Mycologlcal Agar is a medium which is neutral in reaction, recom-
mended for the cultivation of fungi. Better growth of many fungi is obtained on
neutral or slightly alkaline media. This medium is also recommended for carry-
ing stock cultures of fungi and for chlamydospore production. A selective medium
for fungi may be prepared from Bacto-Mycological Agar by the addition of peni-
cillin and streptomycin. Generally 20 units of penicillin and 40 micrograms of
streptomycin per ml. of medium are added to the sterile melted medium at
45-50° C. under aseptic conditions. A method to obtain these concentrations of
the antibiotics is given in the description of Bacto-Sabouraud Dextrose Agar page
238. This medium is also well suited as a Dextrose Agar for the cultivation of a
large variety of; :;aprophytic and pathogenic microorganisms.

To rehydrate ihe medium, suspend 35 grams of Bacto-Mycological Agar in
1000 ml. of cold distilled water. Heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure. Final reaction of the medium will be pH 7.0.

One pound of Bacto-Mycological Agar will make 13 liters of medium.

BACTO

MYGOLOGIGAL BROTH (B406)

DEHYDRATED

Bacto-Soytone 10 g.

Bacto-Dextrose 40 g.

Bacto-Mycological Broth is a liquid medium neutral in reaction recommended
for the cultivation of fungi. A selective liquid medium for fungi may be prepared
by the addition of penicillin (generally 20 units per ml. medium) and strepto-
mycin (40 micrograms per ml. medium) or other selective agents to the sterile
medium at 50° C. or less under aseptic conditions. A method to obtain these
concentrations of the antibiotics is given in the description of Bacto-Sabouraud
Dextrose Agar page 238. Bacto-Mycological Broth is also well suited as a sterility
test medium for the detection of fungi.

To rehydrate the medium, dissolve 50 grams Bacto-Mycological Broth in 1000
ml. distilled water. Distribute in tubes or flasks and sterilize in the autoclave for
15 minutes at 15 pounds pressure (121°G.). Final reaction of the medium will
be pH 7.0.

One pound of Bacto-Mycological Broth will make 9.0 liters of medium.

BACTO BACTO

POTATO DEXTROSE AGAR MALT AGAR

DEHYDRATED DEHYDRATED

Bacto-Potato Dextrose Agar and Bacto-Malt Agar are recommended for the
cultivation of yeasts and molds and for counts of these organisms. A complete
discussion of these media is given on page 64, 65.

244 DIFCO MANUAL

BACTO

WORT AGAR (Bill)

DEHYDRATED

Bacto-Malt Extract 15 g

Bacto-Peptone 0.78 g

Maltose, Technical 12.75 g

Dextrin, Difco 2.75 g,

Glycerol 2.35 g

Dipotassium Phosphate 1 g,

Ammonium Chloride 1 g

Bacto-Agar 15 g,

Bacto-Wort Agar is a medium prepared particularly for the cultivation of
yeasts. The reaction is adjusted so that after sterilization it will be pH 4.8, which
is near the optimum for most yeasts and at the same time will inhibit most
bacterial growth.

The study of yeasts is one of the oldest branches of the science of micro-
biology, and it remains today one of the most important. A variety of processes
in industry are performed by yeasts. A study of the pathogenic activities of
yeasts (blastomycetes) has revealed them to be the causative organisms of
such conditions as certain skin ulcers, tumors, infections of the upper and
lower respiratory and gastrointestinal tract etc. As a rule, the yeasts grow well
upon culture media containing dextrose or maltose, particularly if the reaction is
somewhat acid. The formula of Bacto-Wort Agar closely duplicates the composi-
tion of wort, which is a favorable medium for the cultivation of yeasts. Growth-
promoting qualities have been increased by the addition of salts and other nutri-
ments.

Parfitt^ reported satisfactory results with Bacto-Wort Agar in his study of the
influence of culture media on the mold and yeast counts of butter, and in order
to procure comparative yeast and mold counts he suggested that dehydrated
Bacto-Whey Agar, Bacto-Malt Agar or Bacto-Wort Agar should be used.

To rehydrate the medium, suspend 50 grams of Bacto-Wort Agar in 1000 ml.
of cold distilled water and heat to boiling to dissolve the medium completely.
Distribute in tubes or flasks, and sterilize in the autoclave for 15 minutes at
15 pounds pressure (121°C.). The final reaction of the medium will be pH 4.8.

Due to the high acidity of the medium the heating process should be com-
pleted in as short a period of time as possible. Excessive heating of this acid
medium causes a breaking down of the agar, resulting in an inability to solidify
properly when cool. Normally, medium prepared from Bacto-Wort Agar is
soft and is ideal for plating purposes. However, if a medium of solidity satis-
factory for streaking is desired, it can be prepared by using 60 grams of Bacto-
Wort Agar per 1000 ml. of water or including 5 grams Bacto-Agar.

One pound of Bacto-Wort Agar will make 9 liters of medium.

1 J. Dairy Science, 16:141:1933.

BACTO

MALT EXTRACT AGAR (B112)

DEHYDRATED

Some laboratories have used Bacto-Malt Extract Agar for the cultivation of
yeasts and molds. We will continue to carry this medium in stock for those
laboratories where it has been in routine use or where it is desired for compara-
tive purposes.

DEHYDRATED CULTURE MEDIA 245

BACTO

CZAPEK DOX BROTH (B338)

DEHYDRATED

Saccharose, Difco 30 g.

Sodium Nitrate 3 g.

Dipotassium Phosphate 1 g.

Magnesium Sulfate 0.5 g.

Potassium Chloride 0.5 g.

Ferrous Sulfate 0.01 g.

Bacto-Czapek Dox Broth is a liquid medium, nearly neutral in reaction, pre-
pared from materials of known chemical composition and designed for the culti-
vation of fungi and bacteria capable of utilizing inorganic nitrogen. Sodium
nitrate is the sole source of nitrogen, and saccharose serves as a source of carbon
in this medium. Bacto-Czapek Dox Broth is a modification of the Czapek formula
of Doxi prepared according to the directions given by Thom and Raper.^

Media prepared with only inorganic sources of nitrogen and chemically de-
fined compounds as sources of carbon are useful for a variety of microbiological
procedures. They are of principal value in soil microbiology, for the enrichment,
cultivation and identification of soil bacteria and fungi, or for mildew resistance
tests, as well as for other tests wherein a simple chemically defined medium is de-
sired. Czapek Dox Broth will support a moderately vigorous growth of nearly
all saprophytic Aspergilli.^

To rehydrate the medium, dissolve 35 grams of Bacto-Czapek Dox Broth in
1000 ml. distilled water. Distribute in tubes or flasks and sterilize in the autoclave
for 15 minutes at 15 pounds pressure (121°C.). Final reaction of the medium
will be pH 7.3.

One pound of Bacto-Czapek Dox Broth will make 13 liters of medium.

1 U. S. Dept. Ag. Bur. Anim. Ind. Bull., "Thorn and Raper, Manual of the Aspergilli:

120:70:1910. 39:1945-

BACTO

CZAPEK SOLUTION AGAR (B339)

DEHYDRATED

Saccharose, Difco 30 g

Sodium Nitrate 2 g,

Dipotassium Phosphate 1 g,

Magnesium Sulfate 0.5 g,

Potassium Chloride 0.5 g,

Ferrous Sulfate 0.01 g

Bacto-Agar 15 g,

Bacto-Czapek Solution Agar is a solid neutral medium of known chemical
composition with nitrate as the only source of nitrogen for the cultivation of
saprophytic fungi, soil bacteria and other microorganisms. It is prepared accord-
ing to the formula given by Thom and Church.^ This medium is of value in many
microbiological procedures such as the cultivation and identification of fungi,
growth of soil bacteria, mildew resistance tests and others. Thom and Raper^
state that this medium will produce a moderately vigorous growth of nearly all
saprophytic Aspergilli, and yield characteristic mycelia and conidia useful in
comparative studies.

To rehydrate the medium suspend 49 grams of Bacto-Czapek Solution Agar in
1000 ml. cold distilled water. Heat to boiling to dissolve the medium completely.

246 DIFGO MANUAL

Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). Final reaction of the medium will be pH 7.3.
One pound of Bacto-Czapek Solution Agar will make 9.2 liters of medium.

1 Thorn and Church, The Aspergilli, 39:1926. 2 fhom and Raper, Manual of the Aspergilli,

39:1945.

BACTO

MILDEW TEST MEDIUM (B428)

DEHYDRATED

Sodium Nitrate 3 g.

Dipotassium Phosphate 1 g.

Magnesium Sulfate 0.25 g.

Potassium Chloride 0.25 g.

Bacto-Agar 10 g.

Bacto-Mildew Test Medium is recommended for use in all procedures for test-
ing processed fabrics, threads and cordage for mildew proofing. This medium is
also recommended as a solid basal medium of known chemical composition with
sodium nitrate as the sole source of nitrogen to which various sources of carbon
may be added to complete the medium.

To rehydrate the medium, suspend 14.5 grams of Bacto-Mildew Test Medium
in 1000 ml. of cold distilled water. Heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). Final reaction of the medium will be pH 6.8.

One pound of Bacto-Mildew Test Medium will make 31.3 liters of medium.

BACTO

CORN MEAL AGAR (B386)

DEHYDRATED

Corn Meal, Infusion from 50 g.

Bacto-Agar 15 g.

Bacto-Corn Meal Agar is recommended for the production of chlamydospores
by Candida albicans and for the cultivation of phytopathological and other fungi.
The medium is prepared from an infusion of ground yellow corn, and solidified
by the addition of 1.5 per cent Bacto-Agar. A similar medium, with the addition
of 0.2 per cent Bacto-Dextrose, Bacto-Corn Meal Agar with Dextrose, will pro-
duce a more luxuriant growth of some fungi.

To rehydrate the medium, suspend 17 grams of Bacto-Corn Meal Agar in
1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). Final reaction of the medium will be pH 6.0.

One pound of Bacto-Corn Meal Agar will make 26.7 liters of medium.

BACTO

CORN MEAL AGAR (B114)
with Dextrose

DEHYDRATED

Corn Meal, Infusion from 50 g.

Bacto-Dextrose 2 g.

Bacto-Agar 15 g.

DEHYDRATED CULTURE MEDIA 247

Bacto-Corn Meal Agar with Dextrose is recommended for the cultivation of
phytopathological and other fungi. It is prepared from an infusion of ground
yellow com to which is added 0.2 per cent Bacto-Dextrose and 1.5 per cent
Bacto-Agar. A similar medium prepared without added dextrose, Bacto-Corn
Meal Agar, is recommended for chlamydospore production by Candida albicans.

To rehydrate the medium, suspend 19 grams of Bacto-Corn Meal Agar with
Dextrose in 1000 ml. cold distilled water and heat to boiling to dissolve the
medium completely. Distribute in tubes or flasks and sterilize in the autoclave
for 15 minutes at 15 pounds pressure (121°C.). Final reaction of the medium
will be pH 6.0.

One pound of Bacto-Corn Meal Agar with Dextrose will make 23.8 liters of
medium.

BACTO

BEAN POD AGAR (B116)

DEHYDRATED

Green String Beans, Infusion from. . 200 g.
Bacto-Agar 15 g.

Bacto-Bcan Pod Agar is recommended for the cultivation of phytopathological
and other fungi. It is prepared from an infusion of fresh green beans, and solidi-
fied with 1.5 per cent Bacto-Agar.

Edgerton^ reported the value of fresh green bean pod infusion in media for the
cultivation of phytopathological fungi. He described a method of drying green
bean pods and storing the dry bean meal so as to have it available for the prepara-
tion of media at all seasons of the year. Bacto-Bean Pod Agar prepared from
fresh green beans possesses all the growth properties of fresh bean pod infusion,
and eliminates the necessity of preparing and storing the desiccated bean meal.

To rehydrate the medium, suspend 22.5 grams of Bacto-Bean Pod Agar in
1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). Final reaction of the medium will be pH 5.6.

One pound of Bacto-Bean Pod Agar will make 20.1 liters of medium.

1 Phytopathology, 8 : 445 : 1 9 1 8.

BACTO

LIMA BEAN AGAR (B117)

DEHYDRATED

Lima Beans, Infusion from 62.5 g.

Bacto-Agar 15 g.

Bacto-Lima Bean Agar is recommended for the cultivation of phytopathological
and other fungi. It is prepared from an infusion of dry lima beans, and is
solidified by the addition of 1.5 per cent of Bacto-Agar. This medium possesses
all the nutritive properties of an infusion of lima beans and has a final reaction
of pH 5.6, making it well suited for the growth of fungi.

To rehydrate the medium, suspend 23 grams of Bacto-Lima Bean Agar in
1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). Final reaction of the medium will be pH 5.6.

One pound of Bacto-Lima Bean Agar will make 19.7 liters of medium.

248 DIFCO MANUAL

BACTO

PRUNE AGAR (B56)

DEHYDRATED

Prunes, Infusion from 36 g.

Bacto-Agar 15 g.

Bacto-Prune Agar is recommended for the cultivation of phytopathological
and other fungi. In the preparation of this medium the infusion of prunes is
solidified by the addition of 1.5 per cent Bacto-Agar. The final reaction of the
medium is pH 5.6, well suited for the growth of a large number and variety of
fungi.

To rehydrate the medium, suspend 24 grams of Bacto-Prune Agar in 1000 ml.
cold distilled water and heat to boiling to dissolve the medium completely.
Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes at 15
pounds pressure (121°C.). Final reaction of the medium will be pH 5.6.

One pound of Bacto-Prune Agar will make 18.9 liters of medium.

BACTO

W. L. NUTRIENT MEDIUM (B424)

DEHYDRATED

Bacto- Yeast Extract 4 g.

Bacto-Casitone 5 g.

Bacto-Dextrose 50 g.

Monopotassium Phosphate 0.55 g.

Potassium Chloride 0.425 g.

Calciuni Chloride 0.125 g.

Magnesium Sulfate 0.125 g.

Ferric Chloride 0.0025 g.

Manganese Sulfate 0.0025 g.

Bacto-Agar 20 g.

Bacto-Brom Cresol Green 0.022 g.

Bacto-W. L. Nutrient Medium is recommended for use in the control of brew-
ing and industrial fermentation processes. It is prepared according to the formula
described by Green and Gray.^-- The nutrient medium permits the development
of the yeast. Also, in those instances in which the number of yeast cells is com-
paratively small, certain bacteria can be detected. A similar medium, Bacto-W. L.
Differential Medium, containing actidione as a selective agent inhibits the devel-
opment of yeast and molds but permits unrestricted growth of bacteria. Gray^ in
a discussion on the microbiological control for beer quality describes in detail
the value of these two media and the type of information they are able to give.

The microbiological population of beers is an important factor in the scientific
control of brewing and other fermentation industries. Direct microscopic counts
do not give sufficient or specific information. In their study of various fermenta-
tion processes, Green and Gray pointed out the inadequacy of the microscopic
count in fermentation control procedures and made an exhaustive study of the
method of examination of worts, beers and liquid yeast and similar fermentation
products. The application of their method to other fermentation industries was
also shown by these authors. Two media were developed, one containing no
selective agent, and the other, a differential medium, containing, as a selective
agent, the antibiotic actidione, having the ability to inhibit the development of
yeasts without in any way interfering with the development of bacteria generally
encountered in beers. Experimental results indicated that yeasts grew as well on

DEHYDRATED CULTURE MEDIA 249

their nutrient medium in the absence of the selective agent, as on the conven-
tional hopped wort agar.

Green and Gray^ reported that counts of viable baker's yeast may be made on
the Nutrient Medium at pH 5.5. If the reaction is adjusted to pH 6.5, the count
of baker's and distiller's yeast may be made. The differential medium at pH 5.5
permits a reliable count of the bacteria generally encountered in brewing. If the
plates of this medium be incubated under anaerobic conditions, then estimations
of beer cocci and lactic rods will be obtained. Aerobic incubation gives an esti-
mation of the acetic acid rods and termobacteria (very small rods occurring in
wort as described by Linder in about 1900 as Termobacterium lutescens,
iridescens and erythrinum) etc., present in the sample.

For the analysis of baker's yeast and alcohol fermentation mashes the reaction
of the Differential Medium is adjusted to pH 6.5. Plates containing dilutions of
baker's yeast are incubated aerobically, whereas those from alcoholic fermenta-
tion mashes are incubated anaerobically under an atmosphere of carbon dioxide.
With the alcoholic fermentation mashes, roll tubes containing 15 ml. of medium
may be used and, unlike the plates, the roll tubes may be incubated aerobically
instead of anaerobically. In preparing media for the estimation of the number of
bacteria from alcohol mashes, markedly increased growth will be obtained if the
medium be rehydrated in a diluted clarified canned tomato juice, using 400 ml.
of the juice with 600 ml. of distilled water per liter.

In making microbial counts using these media the temperature and time of
incubation will vary depending on the various materials under investigation.
Temperatures of 25 °C. are generally employed with brewing materials, and
30°C. for baker's yeast and alcohol fermentation mash analyses. Incubating
periods run from 2 to 7 days, depending on the flora encountered. Incubation
periods from 10 to 14 days may be used in some cases.

To rehydrate the medium, suspend 80 grams of Bacto-W. L. Nutrient Medium
in 1000 ml. cold distilled water and heat to boiling to dissolve the medium com-
pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). Final reaction of the medium is pH 5.5. The
Nutrient Medium with a final reaction of pH 6.5 is prepared by adding sodium
carbonate according to directions given on the label of each bottle.

One pound of Bacto-W. L. Nutrient Medium will make 5.6 liters of medium.

I Paper read at Am. Soc. of Brewing Chemists ^ \^rallerstein Lab. Comm., 13:357: i950-
Meeting, Detroit, May, 1950, 8 WaUgrstein Lab. Comm., 14:169:1951.

BACTO

W. L. DIFFERENTIAL MEDIUM (B425)

DEHYDRATED

Bacto-W. L. Differential Medium has the same composition as Bacto-W. L.
Nutrient Medium described above except for the addition of 0.004 g. of actidione
per liter. This medium permits unrestricted growth of bacteria and inhibits the
development of yeast and molds.

To rehydrate the medium, suspend 80 grams of Bacto-W. L. Differential
Medium in 1000 ml. cold distilled water and heat to boiling to dissolve the
medium completely. Distribute in tubes or flasks and sterilize in the autoclave
for 15 minutes at 15 pounds pressure (121°C.). Final reaction of the medium is
pH 5.5. The Differential Medium with a final reaction of pH 6.5 is prepared by
adding sodium carbonate according to directions given on the label of each
bottle. Avoid overheating or repeated re-melting of the Differential Medium.

One pound of Bacto-W. L. Differential Medium will make 5.6 liters of medium.

250 DIFGO MANUAL

MEDIA FOR
THE CLASSIFICATION OF YEASTS

These media have been prepared according to the formulae of Wick-
erham^'2,3,4,5 ^^^^ ^^.q recommended for use in classifying yeast according to the
following criteria suggested by him.

1. Colonial characteristics and cell morphology using Bacto-Yeast Morphology
Agar.

2. Carbon assimilation using Bacto-Yeast Nitrogen Base.

3. Nitrogen assimilation using Bacto-Yeast Carbon Base.

4. Vitamin requirements using Bacto-Vitamin Free Yeast Base.

These media will be considered separately, and the composition of each
medium is given on the following page.

BACTO

YEAST MORPHOLOGY AGAR (B393)

DEHYDRATED

Colonial characteristics and cell morphology are determined by a modified
Dalmau technique using Bacto-Yeast Morphology Agar. This medium is com-
posed of ingredients of known composition prepared according to the formula
suggested by Wickerham.

The following technique has been suggested by Wickerham for the determina-
tion of typical colonial characteristics and cell morphology.

Following sterilization, pour the sterile medium into plates to a depth of about
1.5 mm. Allow the plates to stand at room temperature for one or two days
before using to be assured of a dry surface. Inoculation is made using the Dalmau
plate technique, as described by Wickerham and Rettger^. Briefly, this method
consists of making a single streak inoculation near one side of the plate (as from
the relative positions 10 o'clock to 2 o'clock). The inoculum should be light and
taken from a slant culture. In addition to the single streak inoculation, two point
inoculations are made near the other sides of the plate (as at positions 4 o'clock
and 8 o'clock). A central section of the streak inoculation and one of the point
inoculations are covered with cover glasses. With forceps, remove cover glasses
from absolute alcohol, drain momentarily, and burn off the excess alcohol by
passing over a low flame. When the cover glass has cooled, place one edge on
the agar and allow the cover glass to fall across the central portion of the
inoculated streak and a second cover glass over one point inoculation. The plate
is incubated six or seven days at 25 °C. and then observed, using the high-dry
objective. This is an excellent method for studying the hyphae of filamentous
yeasts.

To rehydrate the medium, suspend 35 grams of Bacto-Yeast Morphology Agar
in 1000 ml. of cold, distilled water. Heat to boiling to dissolve the medium
completely. Distribute in tubes or flasks and sterilize in the autoclave for 15
minutes at 15 pounds pressure (121°C.). The final reaction of the medium will
be pH 4.5.

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252 DIFGO MANUAL

BACTO

YEAST NITROGEN BASE (B392)

DEHYDRATED

Media for carbon assimilation tests may be prepared by adding various sources
of carbon to Bacto-Yeast Nitrogen Base. Wickerham^'-'^**'^ reported that some
strains of yeast require the presence of certain vitamins for the assimilation of
carbon. Bacto-Yeast Nitrogen Base is a suitable medium for such studies.

For these demonstrations the tubes of media are inoculated very lightly, then
placed at 25 °C. After 6 to 7 days of incubation, and again at 20 to 24 days, the
tubes are shaken to suspend growth, then they are placed against a white card
bearing lines approximately ^ of a millimeter wide. The lines are drawn with
India ink. If the lines cannot be seen through the culture, or if the lines appear
as diffuse, broad bands, the test is positive. If the lines are distinguishable as such,
the test is negative. The two observations indicate which reactions occur rapidly
and which occur latently.

The medium should be filter sterilized in lOX strength for best results. This is
accomplished by suspending 6.7 grams of Bacto-Yeast Nitrogen Base and 5 grams
of dextrose or an equivalent amount of other carbohydrate in 100 ml. of distilled
water. It may be necessary to warm the distilled water slightly to effect complete
solution of some of the carbohydrates. This lOX strength solution is then filter
sterilized. The lOX strength sterile medium is kept in the refrigerator and used
as needed. The final medium is prepared by pipetting, under aseptic conditions,
0.5 ml. into 4.5 ml. of sterile distilled w^ater in 16 mm. cotton stoppered tubes.
The required number of such water blanks are prepared and autoclaved in
advance. After receiving the lOX strength solution, the tubes are shaken and are
then ready for inoculation.

Some laboratories may prefer to filter sterilize the basal medium and the carbo-
hydrate solution separately. To accomplish this, dissolve 6.7 grams of Bacto-
Yeast Nitrogen Base in 100 ml. distilled water and filter sterilize. Distribute in
0.5 ml. amounts in sterile 16 mm. cotton stoppered tubes. Dissolve sufficient
carbon compound under test for 100 ml. (0.5 gram dextrose) in 90 ml. of dis-
tilled water. Filter sterilize. The final medium is prepared by adding 4.5 ml. of
the sterile carbohydrate solution to each 0.5 ml. of the sterile basal medium under
aseptic conditions and mixing thoroughly by shaking. Final reaction of the basal
medium will be pH 4.5.

BACTO

YEAST CARBON BASE (B391)

DEHYDRATED

The ability of yeasts to assimilate nitrogeni»2,3,4,5 \^ determined by adding
various nitrogen sources to Bacto-Yeast Carbon Base and inoculating with the
test organisms. The inclusion of vitamins in this base was found necessary by
Wickerham as an aid for the utilization of nitrogen-containing compounds by
certain yeasts w^hich cannot assimilate these compounds in the absence of vita-
mins. Bacto-Yeast Carbon Base is a suitable medium for such studies.

Yeasts which have grown on a rich medium may carry a reserve of nitrogen in
the form of protein. Possible errors due to this reserve are eliminated by making
two serial transfers in the complete medium. When the first transfer is seven days
old, the culture is shaken and one loopful is transferred to a second tube of the
complete medium containing the same source of nitrogen. If a positive test is

DEHYDRATED CULTURE MEDIA 253

obtained when the second culture is seven days old, the organism under test
assimilates this particular nitrogen source.

The medium should be filter sterilized in lOX strength for best results. This is
accomplished by dissolving 11.7 grams of Bacto-Yeast Carbon Base and the
nitrogen sources as desired in 100 ml. of distilled water. It may be necessary to
warm the distilled water slightly to efTect complete solution. This lOX strength
solution is then filter sterilized. The lOX strength sterile medium is kept in the
refrigerator and used as needed. The final medium is prepared by pipetting
0.5 ml. into 4.5 ml. of sterile distilled water in a 16 mm. cotton stoppered tube.
The required number of such water blanks are prepared and autoclaved in
advance. After receiving the lOX strength solution, the tubes are shaken, and are
then ready for inoculation.

At present, the most important nitrogen-containing compound being used for
nitrogen assimilation studies is potassium nitrate. This medium may be prepared
by dissolving 0.78 gram potassium nitrate in 100 ml. distilled water containing
11.7 grams of Bacto-Yeast Carbon Base.

Some laboratories may prefer to filter sterilize the basal medium and nitrogen
source solution separately. To accomplish this, dissolve 11.7 grams of Bacto-
Yeast Carbon Base in 100 ml. distilled water and filter sterilize. Distribute in
0.5 ml. amounts in sterile 16 mm. cotton stoppered tubes. Dissolve sufficient
source of nitrogen for 100 ml. (0.078 gram potassium nitrate) in 90 ml. of dis-
tilled water. Filter sterilize. The final medium is prepared by adding 4.5 ml. of
the sterile nitrogen solution to each 0.5 ml. of the sterile basal medium under
aseptic conditions and mixing thoroughly by shaking. The final reaction of the
basal medium will be pH 4.5.

BACTO

VITAMIN FREE YEAST BASE (B394)

DEHYDRATED

Vitamin requirements of yeast may be determined by adding various vitamins
to Bacto-\'itamin Free Yeast Base. This base contains sufficient nitrogen and
carbon sources to permit growth of yeast after the addition of the required
vitamins, as described by Wickerham-^'^'^'-^'S

Because the inoculum may carry a large supply of vitamin reserve, it is neces-
sary to make two serial transfers in a medium containing either no vitamins or
the particular combination to be tested. When the first transfer is seven days old,
the culture is shaken and one loopful is transferred to a second tube of medium
having the same combination of vitamins. This second tube is likewise incubated
for seven days. If growth occurs, the yeast does not require an exogenous source
of whatever vitamins are absent from the medium.

The medium should be filter sterilized in lOX strength for best results. This is
accomplished by dissolving 16.7 grams Bacto-Vitamin Free Yeast Base in 100 ml.
of distilled water containing the desired vitamins. It may be necessary to warm
the distilled water slightly to effect complete solution. This lOX strength solu-
tion is then filter sterilized. The sterile lOX strength solution is kept in the
refrigerator and used as needed. The final medium is prepared by pipetting,
under aseptic conditions, 0.5 ml. into 4.5 ml. of sterile distilled water in 16 mm.
cotton stoppered tubes. The solutions are mixed thoroughly by shaking and are
then ready for inoculation.

Some laboratories may prefer to filter sterilize the basal medium and the
desired vitamins separately. To accomplish this, dissolve 16.7 grams of Bacto-
Vitamin Free Yeast Base in 100 ml. distilled water and filter sterilize. Distribute

254 DIFCO MANUAL

in 0 5 ml. amounts in sterile 16 mm. cotton stoppered tubes Filter sterilize
ufficient amounts of the desired vitainins for 100 ml. of ^^e Hnal medmm m
90 ml. of distilled water. The final medmm is prepared by addmg 4.5 ml. of the
sterile vitamin solution to each 0,5 ml. of the sterile basal medmm under aseptic
conditions and mixing thoroughly by shaking. The final reaction of the basal
medium will be pH 4.5.

iTI S Dent Ae. Tech. Bull., No. 1029:1931. ^ l '^^^■' ^^a'^^^'-^^^^-
^y-Tropicil Med. Hyg., 42:i7^:x939. ^ J- B^^t.. 46:501:1943.

a J. Bact., 52:293:1946.

Ingredients of Culture Media

The choice of the ingredients to be used is most important for the successful
preparation of microbiological culture media. Superior products will yield su-
perior media and will lessen the time-consuming labor incident to their prepara-
tion.

The ingredients listed in this section are prepared expressly for use in making
microbiological culture media. Each product is carefully tested for its usefulness
and suitability for the purpose for which it is intended.

PEPTONES, DIFCO
ENZYMATIC HYDROLYSATES

Peptone prepared expressly for bacteriological purposes was first introduced
by Difco Laboratories in 1914 after many years of preliminary study. The first
of our bacteriological peptones to be produced was Bacto-Peptone. This was
followed several years later by Proteose Peptone, Difco, and in more recent
years other peptones have been added to the group. Since no one peptone is
satisfactory for all microbiological purposes each of these peptones is particularly
suitable for some special purpose or possesses singular characteristics which dis-
tinguish it from other peptones. For this reason it has been the consistent policy
of Difco Laboratories to keep unchanged the method of preparation of its
bacteriological peptones once these products have become established. Slight
modifications or changes in method might readily result in products possessing
entirely different nutritive properties. Bacto-Peptone and other Difco peptones are
prepared today in the same manner as when first developed. Users of Difco
peptones are, therefore, assured of products prepared in a uniform manner at all
times.

It is not improbable that increased knowledge of the minutiae of bacterial
metabolism may make it necessary to prepare additional specialized peptones in
the future, since the formula of a Difco peptone is never modified after once be-
ing adopted. For this reason Difco Laboratories is continuing its investigation, in
cooperation with other bacteriologists, to develop new products which will meet
the new demands. To this end, not only are extensive studies inaugurated and
carried on by Difco Laboratories, but many more are conducted in conjunction
with bacteriologists throughout the world, and we extend to all our active
cooperation to aid in the development of the peptones necessary to meet the
problems encountered in this field.

It has been our consistent effort to make available, to the bacteriologist,
products which are eminently fitted and suitable for specific purposes in view.
Only when these preparations have been tested by extensive and impartial investi-
gation in the hands of laboratory workers are they released for general use.

Difco peptones may be relied upon for biochemical studies, particularly fer-
mentation reactions because of their freedom from fermentable substances which
would interfere with the accuracy of such determinations.

Practical experience, concerned not only with the complexities of bacterial

255

256 DIFGO MANUAL

metabolism and nutrition, but also with the difficulties attendant upon the
separation, classification and recognition of bacterial species by means of specific
cultural reactions, have long demonstrated that no single peptone can be equally
suitable for all the varied ends in view. Indole production, maintenance of
growth, maximum production of soluble toxins, etc., are all definitely influenced
by the nature and character of the peptone and the other nitrogenous constitu-
ents of the culture medium.

Recognizing the essential importance and great practical value of these facts
in relation to bacteriological investigations, Difco Laboratories has for many years
engaged in a consistent and progressive study of this problem, the fruits of which
are represented in the group of products briefly described below. These products
are the results of original, intensive investigations, checked and corroborated by
extensive practical trials in the hands of experienced bacteriologists before such
materials are released for distribution. It is believed that they represent a definite
advance in practical bacteriology and that they cover, as fully as is possible at
present, the field of peptones in their relation to microbiology.

B AGTO-PEPTONE ( B 11 8 )

Bacto-Peptone, first introduced in 1914, has long since become the universal
standard peptone for the preparation of bacteriological culture media. Countless
studies which have been published during the intervening years specify the use
of Bacto-Peptone and constant reference to its use is currently being made in
bacteriological literature.

When Bacto-Peptone was originally developed it was first subjected to trial
in the routine bacteriological examination of water and milk, and was shown to
be well suited for these purposes. It has continually been recommended as the
peptone to be employed for culture media preparation in "Standard Methods of
Water Analysis" and is at the present time included in the Ninth Edition.^ It is
similarly specified for use in a number of special studies of milk and other dairy
products. 2'^ Bacto-Peptone, furthermore, is also suggested for general use in the
preparation of culture media in "Standard Methods"* of the Division of Labora-
tories and Research of the New York State Department of Health.

Bacteriological literature abounds with an increasing number of references
to the use of Bacto-Peptone, not only for the preparation of general culture media,
but also for those employed in special research studies. Marbe and Olariu^ have
employed Bacto-Peptone in combination with Proteose Peptone in their medium
for the elaboration of toxin by Corynehacterium diphtheriae. Veldee^ also em-
ployed a combination of these two peptones in his medium for cultivating strep-
tococcus for scarlet fever toxin. He states that his medium gives a toxin of
uniformly high potency and that it is particularly suitable for the preparation of
purified and tannic acid precipitated toxin. Bacto-Peptone is also used in culture
media for studies of the intestinal bacteria,^ ^° ^^ streptococci,^^ *° ^^ pneumo-
cocci,^^ lactobacilli,26 anaerobes^^'^^'^^ and the fungi. ^^-^^ It has been suggested
for use in media for the Brucella,^--^^ but is now superseded by Bacto-Tryptose
for this purpose. Bacto-Peptone is also specified in media used in studies of bac-
terial metabolism,^* studies of disinfectants and determinations of phenol coeffi-
cients,^^ studies of bacterial luminosity,^^ and the reduction of nitrates. ^^ Al-
though it has been suggested for use in media for detection of hydrogen sulfide
productions^ its use for this purpose has been supplanted by Bacto-Tryptone and
Proteose Peptone. Similarly, for pigment production, Proteose Peptone or Bacto-
Tryptone is more satisfactory than Bacto-Peptone as recommended in earlier
papers. s^'*^

"i:

INGREDIENTS OF CULTURE MEDIA 257

In a study by Morton, Smith and Leberman*^ Bacto-Peptone was reported to
be superior to other peptones in a medium recommended for the isolation and
cultivation of pleuropneumonia-like organisms.

This list is by no means complete or exhaustive but it suffices to indicate the
wide range of usefulness of Bacto-Peptone. In addition to the references men-
tioned many other citations are to be found in the literature.

Bacto-Peptone contains nitrogen in a form which is readily available for bac-
terial growth requirements. It has a high peptone and amino acid content and
only a negligible quantity of proteoses and more complex nitrogenous constituents.
Bacto-Peptone is completely soluble in water and yields sparklingly clear solu-
tions in the concentrations usually employed for culture media. From the very
first, Bacto-Peptone has been standardized to a definite hydrogen ion concentra-
tion of pH 7.0 in a one per cent solution, as generally employed in culture media.

A typical quantitative analysis of Bacto-Peptone is given on page 265.

1 Standard Methods for the Examination of 22 Sixth Annual Year Book (1935-1936) p. 159.
Water and Sewage, 9th Edition: 184: 1946. Suppl., Am. J. Pub. HeaUh, 26: No. 3:1936.

2 Bull. 524, N. Y. Agr. Exp. Sta., 1924. ^^ J. Infectious Diseases, 63:122:1938.

3 J. Dairy Science, 16:277:1933. ^ J. Infectious Diseases, 62:138:1938.
* Standard Methods, Div. Labs. Res. N. Y. 25 j_ Path. Bact., 39:323:1934.

Dept. Health, 1927. 26 Am. J. Pub. Health 28:759:1938.

^ Compt. rend. soc. biol., 118:1673:1935. ^^ Proc. Soc. Exp. Biol. Med., 26:88:1928-29.

^ Public Health Reports, 53:909:1938. 28 j Bact., 20:85:1930.

' J. Infectious Diseases, 24:260:1919. 29 j, Immunol., 18:141:1930.

^ J. Infectious Diseases, 28:384:1921. so j_ Infectious Diseases, 54:35:1934.

^ J. Bact., II :359: 1926. ^Phytopathology, 24:1153:1934.

10 Abst. Bact., 6:34:1922. S2 Sixth Annual Year Book (1935-1936) p. 118.
Bact., 29:349:1935. Suppl. Am. J. Pub. Health, 26:No. 3:1936.

Bact., 32:329:1936. 83 Sixth Annual Year Book (1935-1936) p. 193.
J. Infectious Diseases, 58:225:1936. Suppl., Am. J. Pub. Health, 26:No. 3:1936.

^* J. Am. Water Works Assoc, 30:808:1938. ^ J. Infectious Diseases, 15:417:1914.

1^ J. Am. Water Works Assoc, 30:1821:1938. ^5 ^.m. J. Pub. Health, 14:1043:1924.

"J. Bact., 7:449:1922. 3«J. Gen. Physiol., 8:89:1925.

1'^ J. Infectious Diseases, 39:186:1926. ^ J. Bact., 19:261:1930.

^^ J. Lab. Clin. Med., 17:530:1931-32. ^ J. Bact., 9:235:1924.

^^ J. Lab. Clin. Med., 15:662:1929-30. 89 BJochem. J., 30:1323:1936.

""^ J. Exp. Med., 50:617:1929. ^ J. Bact., 32:533:1936.

"1 J. Bact., 25:527:1938. *i Am. J. Syphilis Gonorrh. Venereal Diseases,

35:361:1951.

PROTEOSE PEPTONE, DIFGO (B120)

Proteose Peptone is particularly adapted for use in culture media for the
production of various bacterial toxins. This peptone is the result of extensive
investigations which had their inception in the fact, developed by practical ex-
perience, that no one peptone was equally useful for all the varied phenomena
included in the biological and biochemical reactions produced by bacteria. As an
illustration of this fact the work carried out on the production of diphtheria toxin
may be cited. Excellent, and indeed luxuriant, growth of Corynebacterium
diphtheriae may be obtained in the usual Veal Infusion Medium containing
Bacto-Peptone, but toxin production is exceedingly slight. Accordingly, over a
period of six years the full time of bacteriologists and chemists in our laboratoriei
was devoted to a study of all phases of diphtheria toxin production before Pro
teose Peptone was selected as the most satisfactory peptone for this purpose.

While the exact chemical composition of diphtheria toxin is still unknowi\
many factors influencing its production have been established. During the early
work on the development of Proteose Peptone and while studying the production
of diphtheria toxin, Bunker^'^.s recognized the importance of a correctly buffered
peptone, the necessity of the optimum initial reaction of the toxin broth, and the
limiting hydrogen ion concentration within which potent toxin could be harvested

258 DIFCOMANUAL

with a reasonable degree of certainty. For example, diphtheria toxin is produced
under practical conditions only when the organism grows as a film on the surface
of liquid media. Media supporting such growth are, therefore, essential. Accord-
ing to Robinson and Rettger^ the toxin factors are probably supplied by the
proteoses and polypeptides. Gibbs and Rettger^ have shown that in sugar-free
media toxin production occurs only when proteoses are present. Toxin produc-
tion, therefore, is definitely dependent upon the composition of the peptones
present in the medium. '

Hazen and Heller^ have shown that diphtheria toxin of high Lf can be pro-
duced in a "bob veal" infusion medium containing 2 per cent Proteose Peptone, ^
0.15 per cent dextrose and 0.3 per cent maltose. Pope^ has also used Proteose
Peptone in his semi-synthetic media for diphtheria toxin production, and Bayne-
Jones^ was able to obtain a diphtheria toxin having more than 300 M.L.D. per
milliliter in a synthetic medium to which he added 2 per cent Proteose Peptone.
Semi-synthetic media containing Proteose Peptone have also been employed by
Kirkbride, Berthelsen and Clark^ and by Wadsworth and Wheeler.i^

Proteose Peptone quickly became established as a suitable peptone for use in
the production of diphtheria toxin, and it is now employed almost universally in
the biological laboratories engaged in the preparation of diphtheria toxin for
antitoxin, toxin-antitoxin and toxoid. In addition to the references already cited,
mention should also be made of the work of Hewitt,^^ Bunney^^ ^nd Povitsky,
Eisner and Jackson.^^

Proteose Peptone is also valuable in the elaboration of other bacterial toxins
as well as that of C diphtheriae. Kirkbride and Wheeler^* used this peptone
successfully for the production of scarlet fever toxin, and Veldee^^ has shown that
a combination of Proteose Peptone and Bacto-Peptone with inorganic salts is an
excellent medium for elaboration of scarlet fever toxin of uniformly high potency.
Nelson^^ used Proteose Peptone for elaboration of toxin by Clostridium hotuli-
num, Kneeland and Dawes^'^ for that of the pneumococcus, and Hanks and
Rettger^s that of Salmonella pullorum.

Proteose Peptone is a satisfactory nutriment for the propagation of many
bacteria as may be seen from the work of Chapman, Lieb, Berens and Curcio,^^
and Dolman and Wilson-^ on the pathogenic staphylococci, Eldering and Ken-
drick^i on Hemophilus pertussis, Tittsler and Lisse22 on S. pullorum, and Scott^*
on Pasteurella multocida. This peptone, furthermore, is a valuable ingredient of
culture media designed for such special biological reactions as the methyl red
and Voges-Proskauer tests,24'25 bacterial fluorescence^^ and pigment production.^^
Many factors account for the suitability of Proteose Peptone for cultivating the
fastidious types of bacteria; among them are the availability of its nitrogenous
components and its buffer range. These factors also aid in establishing favorable
conditions for maintaining the virulence of the cultures and for the elaboration
of bacterial by-products.

A method for the in vitro testing for virulence of C. diphtheriae was described
by Elek^s and also a modification of this method was given by King, Frobisher
and Parsons.2^ King, Frobisher and Parsons^^ and Frobisher, King and Parsons^^
continuing their studies on the in vitro test for virulence of C. diphtheriae simpli-
fied their basal medium, and a comparative study of peptones showed Proteose
Peptone to give the most satisfactory results. Handley^- described a selective
medium for the identification and isolation of C. diphtheriae, and in an exhaus-
tive study of available peptones found that Proteose Peptone, Difco was more
suitable than any other peptone tried for the preparation of his medium. Mc-
Vickar^^ in a study of the factors important for the growth of Histoplasma
capsulatum reported that of the 18 peptones used, Proteose Peptone, Difco gave
the best growth of the organisms.

INGREDIENTS OF CULTURE MEDIA 259

A typical quantitative analysis of Proteose Peptone, Difco is given on page 265.

lAbst. Bact., 1:31:1917. 1^ J. Immunol., 22:283:1932.

2 Abst. Bact., 2:10:1918. 18 J. Bact., 33:533: i937-

8 Abst. Bact., 4:106:1919. 20 J, Immunol., 35:13:1938.

* J. Med. Research, 36:357:1917. 21 Am. J. Pub. Health, 26:506:1936.

^ J. Immunol., 13:323:1927. 22 j. Bact., 15:105:1928.

« J. Bact., 23:195:1932. 23 J, Bact., 20:9:1930.

'^ Brit. J. Exp. Path., 13:218:1932. ^Standard Methods for the Examination of

8 Bayne- Jones : A Textbook of Bacteriology, Water and Sewage, 9th Edition: 230: 1946.

1066:1934. 25 Pure Culture Study of Bacteria, 2:No. 4:1934.

8 J. Immunol., 21:1:1931. 28 j, Bact., 23:135:1932.

10 J, Infectious Diseases, 55:123:1934. 27 j, Bact., 29:223:1935.

11 Biochem. J., 24:983:1930. 28 Brit. Med. J., 1:493:1948.

12 J. Immunol., 20:71:1930. 29 Am. J. Pub. Health, 39:1314:1949-
^ J. Infectious Diseases, 52:246:1933. ^ Am. J. Pub. Health, 40:704:1950.
"J. Immunol., 11:477:1926. si Am. J. CHn. Path., 21:282:1951

15 Public Health Reports, 53:909:1938. ^^ J. Hygiene, 47:102:1949.

i« J. Infectious Diseases, 41:9:1927. ^J. Bact., 62:137:1951.
"J. Exp. Med., 55:735:1932-

PROTEOSE PEPTONE NO. 2, DIFCO (B121)

The development of new peptones designed for specific purposes frequently
leads to their use in new fields of investigation. Thus, Proteose Peptone No. 2,
which is similar in type to Proteose Peptone, was originally developed in our
studies on peptones for use in production of diphtheria toxin. Although well
adapted for this purpose, it was never used extensively because Proteose Peptone,
either in a semi-synthetic medium or in a veal infusion base, became established
as a superior peptone in media used for diphtheria toxin production.

Interest in Proteose Peptone No. 2 was revived by the work of Bunney and
Thomas^ in their study of diphtheria toxin production in semi-synthetic media.
These workers describe the preparation of a uniformly constant, infusion-free
medium composed of Proteose Peptone, Proteose Peptone No. 2, dextrose, maltose
and sodium acetate. This medium was reported as supporting a consistently good
yield of toxin. The work of Bunney and Thomas followed soon after that of
Marbe and Olariu^ who used Bacto-Peptone and Proteose Peptone for a similar
purpose.

1 J. Immunol., 31:95:1936. 2 Compt. rend. soc. biol., 118:1673:1935.

PROTEOSE PEPTONE NO. 3, DIFCO (B122)

Investigating Proteose Peptone No. 2 for various purposes we developed an-
other modification of Proteose Peptone that proved to be excellent for the culti-
vation of many organisms. This peptone was designated as Proteose Peptone No.
3. Studies of the nutritive requirements of fastidious organisms have demonstrated
that a 2 per cent solution of Proteose Peptone No. 3 with 0.05 per cent dextrose
satisfactorily replaces the meat infusion-peptone portion of culture media. The
dextrose is added to supply a uniform amount of utilizable carbohydrate to
replace the variable amount of muscle sugars present in infusions from meat.
The use of Proteose Peptone No. 3 in this manner eliminates all of the laborious
processes of trimming, chopping and infusing meat. For example, a medium
superior to Douglas Agar enriched with whole blood as employed by Carpenter,^'^
is prepared by mixing equal volumes of an autoclaved 2 per cent solution of
Bacto-Hemoglobin with an autoclaved agar medium composed of 4 per cent
Proteose Peptone No. 3, 0.1 per cent dextrose, 1 per cent sodium chloride, 1 per
cent disodium phosphate and 3 per cent agar. Chocolate Agar prepared according

260 DIFGO MANUAL

to this formula is satisfactory for the isolation of Neisseria gonorrhoeae from
gonococcal infection in the male and female. In 2 per cent concentration Proteose
Peptone No. 3 is an excellent carrying fluid for suspending suspected gonococcal
specimens. The suitability of Proteose Peptone No. 3 in the preparation of media
for the isolation of A^. gonorrhoeae from all types of gonococcal infection is amply
demonstrated by the many references to the use of this peptone for this purpose.
Among others Peizer and Steffen,^ Bucca,* Morton and Leberman,^ Landy and
Gerstung,^ Lankford and Skaggs^ and the "Diagnostic Procedures and Reagents"^
of the American Public Health Association used Proteose Peptone No. 3 in media
for gonococcal studies. Continued study of the nutritional requirements of gono-
cocci has shown that a small percentage are exacting in their growth require-
ments. Chocolate Agars, prepared with Bacto-Proteose No. 3 Agar and Bacto-
Hemoglobin, enriched with Bacto-Supplement A or Bacto-Supplement B, have
proved excellent for the isolation of all types of N, gonorrhoeae. The method for
the isolation of this organism is discussed in detail under Bacto-Proteose No. 3
Agar, page 116, or Bacto-G C Medium Base, page 122.

Proteose Peptone No. 3 is well suited for the preparation of media for cultiva-
tion of C orynebacterium diphtheriae. This peptone is employed in the preparation
of Bacto-Dextrose Proteose No. 3 Agar, as discussed on page 147, for isolation of
the diphtheria bacillus. Levin^ used Proteose Peptone No. 3 in media for the
isolation of meningococci and the diphtheria bacillus.

This peptone is well suited for the growth of a large variety of organisms
including streptococci, staphylococci, meningococci, pneumococci, anaerobes and
aerobes. In addition to the media listed above it is employed in Bacto-Phenol Red
Carbohydrate Media discussed on page 187, 189, Bacto-Purple Broth Media,
page 189, 190, and other fermentation media, Bacto-A C Broth and Bacto-A C
Medium, pages 201 and 202.

1 Seventh Annual Year Book (1936-37) p. 125. ^ U. S. Naval Med. Bull., 43:409:1944.
Suppl., Am. J. Pub. Health, 27: No. 3:1937. ^ J. Immunol., 51:269:1945.

2 Bull. Genitoinfectious Diseases, Mass. Dept. '^ Arch. Biochem., 9:265:1946.

Pub. Health, 2: No. 9:1938. ^ Diagnostic Procedures and Reagents, 3rd Edi-

8 Venereal Disease Inform., 23:224:1942. tion: 119: 1950.

* J. Bact., 46:133:1943. "J. Bact., 46:235:1943.

B AGTO-TR YPTONE ( B 1 23 )

Bacto-Tryptone was developed in our search for a peptone particularly suitable
for the elaboration of indole by bacteria. Tests for the presence or absence of
indole as a by-product of bacterial metabolism are of definite value in the identifi-
cation and classification of bacteria. However, unless the culture medium in
which the organisms are grown can be relied upon to support indole production
uniformly, the results of the test are misleading and fallacious as shown by
Tilley.i

Indole production is dependent upon the presence of the tryptophane group
in the medium,^'^.* Bacto-Tryptone is exceptionally well suited for use in tests
for indole production because it is rich in this form of nitrogen. Strong indole tests
are possible after incubation of the cultures for 15 or 16 hours in a 1 per cent
solution of Bacto-Tryptone. The reaction, furthermore, will remain strong even
when cultures are incubated for 4—5 days. The advantages of securing a positive
indole test, particularly after a relatively short incubation period, are obvious.
"Standard Methods for the Examination of Water and Sewage"^ of the American
Public Health Association and the American Water Works Association, and "Pure
Culture Study of Bacteria"^ of the Society of American Bacteriologists, specify
the use of Bacto-Tryptone in demonstrating the presence of indole.

INGREDIENTS OF CULTURE MEDIA 261

A complete discussion of the methods for the determination of indole is given
under Bacto-Tryptone on page 53.

Investigation of the usefulness of Bacto-Tryptone in other fields has demon-
strated a number of industrial applications. The National Canners Association,
in its "Bacterial Standards for Sugar,"^ specified the use of Tryptone media for
the detection of the organisms causing "flat sour" spoilage of canned goods and
also for detecting the presence of "sulfide spoilage" organisms in sugar.^

In his studies of the thermophilic bacteria of milk, Prickett^ developed a
medium containing Bacto-Tryptone which possessed excellent growth-stimulating
properties for these organisms. Bowers and Hucker^ also demonstrated the advan-
tages of culture media containing this peptone for propagating bacteria from
milk. An extensive survey of the use of the Bowers and Hucker medium for plate
counts of milk has been summarized by Yale.^*^ A modification of this medium,
still utilizing Bacto-Tryptone as the peptone, was originally recommended in
"Standard Methods for the Examination of Dairy Products,"^^ Seventh Edition,
1939, of the American Public Health Association for standard plate counts of
milk. The report of Abele^- analyzes in detail the results of extensive comparative
studies made with Tryptone Glucose Extract Milk Agar, and the former standard
Nutrient Agar. The use of the Tryptone medium has been continued since its
original introduction as a medium for the plate count of milk, and is specified in
the current Ninth Edition of "Standard Methods for the Examination of Dairy
Products."^^ The usefulness of Bacto-Tryptone as an ingredient of culture media
for the examination of milk is further shown by its use in the medium for the
total bacterial plate count of certified milk^* in which medium it may be em-
ployed alone or in conjunction with Proteose Peptone.

Among the many other uses for Bacto-Tryptone that may be mentioned, Mc-
Clungi° in a study of anaerobic bacteria utilized a 1 per cent solution of Tryptone
as a basal medium for studying the physiological characteristics of Clostridia.
These organisms grew sparsely in the medium containing only Tryptone, but
developed rapidly in the presence of a utilizable source of carbon. Thus profuse
growth and also increase in hydrogen ion concentration served as criteria for the
utilization and fermentation of the test compound by these organisms. Spray^^
utilized Tryptone for the propagation of anaerobes and McCoy^^ prepared the
antigens for her serological studies of the Clostridia in a 1 per cent Tryptone
Medium containing 0.5 per cent dextrose. Bowers and West^^ used Tryptone in
their studies on the streptococci and pneumococci and Moberly^^ employed this
peptone for the cultivation of Lactobacillus acidophilus. Pringsheim and Pring-
sheim^^ reported that Bacto-Tryptone renders the growth of Porphyridium
cruentum more vigorous and lasting. The best liquid medium for the growth of
this organism is sea water plus one half volume of Beijerinck solution with 0.2
per cent added Bacto-Tryptone. In 3-4 weeks a dense red mucilaginous growth is
formed.

A typical quantitative analysis of Bacto-Tryptone is given on page 265.

1 Am. J. Pub. Health, 11:834:1921. "^ Standard Methods for the Examination of

2 J. Bact., 25:623:1933. Dairy Products, 7th Edition : 21 : 1939.
' Pure Culture Study of Bacteria, 5: No. 3:1947. ^ Am. J. Pub. Health, 29:821:1939.

* Centr. Bakt., I Abt., 76:1:1915. "Standard Methods for the Examination of
8 Standard Methods for the Examination of Dairy Products, 9th Edition: 93: 1948.

Water and Sewage, 9th Edition: 230: 1946. ^* Methods and Standards for the Production of

• Bacterial Standards for Sugar, 1935. Certified Milk, 1 953-54-
''J. Assoc. Official Agr. Chem., 21:457:1938. ^^ J. Bact., 29:189:1935.
«Tech. Bull., 147, N. Y. State Agr. Exp. Sta., i« J. Bact., 32:135:1935.

1928. " J. Bact., 34:321:1937.

»Tech. Bull., 228, N. Y. State Agr. Exp. Sta., is Am. J. Pub. Health, 26:880:1036.

1935- 19 Zentr. Bakt., II Abt. Orig., 96:329:1937.

10 Am. J. Pub. Health, 28:148:1938. 80 j. Ecology, 37:57:1949.

262 DIFGO MANUAL

B AGTO-TR YPTOSE ( B 1 24)

In the course of a series of studies on the growth requirements of the Brucella,
a peptone which was particularly suited for these organisms was developed. This
peptone was designated as Bacto-Tryptose. In the absence of meat infusion,
Bacto-Tryptose is recommended for use in an agar medium with sodium chloride
and dextrose for the propagation of all types of Brucella.'^ With suitable dyes this
medium also proved satisfactory for the isolation and differentiation of Brucella
strains. Many of the irregular results obtained on liver infusion media are elimi-
nated by the use of culture media containing Bacto-Tryptose.

Subsequent investigation of the nutritive properties of Bacto-Tryptose demon-
strated that culture media prepared with this peptone were superior to the meat
infusion peptone media employed for cultivation of the streptococci, pneumo-
cocci, meningococci and other fastidious pathogenic bacteria. A 2 per cent solu-
tion of Bacto-Tryptose with 0.05 per cent dextrose to replace muscle sugars, is an
excellent substitute for the infusions plus peptones generally employed in culture
media for the propagation of many of the organisms which grow with difficulty
even on infusion media. Other ingredients are added as usual to this solution to
make the complete medium.

An agar medium prepared with 2 per cent Bacto-Tryptose and 0.5-0.8 per
cent sodium chloride, without tissue infusion, is an excellent general culture
medium and base for Blood Agar. This peptone is excellently suited for the
preparation of a base for Blood Agar as was reported by Gasman. --^

A medium containing 2 per cent Bacto-Tryptose, 0.5 per cent sodium chloride,
0.3 per cent disodium phosphate and 0.2 per cent dextrose may be used as a
liquid medium for blood culture work. A discussion of Bacto-Tryptose Phosphate
Broth is given on page 100. Excellent growth of many fastidious organisms occurs
in this liquid medium. As in the case with other liquid substrates, the medium
is greatly improved by the addition of 0.1 per cent agar. A medium consisting of
1 per cent Bacto-Tryptose, 0.5 per cent sodium chloride and 0.1 per cent di-
sodium phosphate is an excellent base free from carbohydrate to which a suitable
indicator and appropriate sugars may be added for studying the fermentation
reactions of the pathogenic bacteria.

Tryptose has proved of value in media for the detection of coliform bacteria.
Darby and Mallmann*'^ developed a Lauryl Tryptose Broth that gave a high
colon index and could be used as a presumptive as well as a confirmatory test
for members of the coliform group. This medium was investigated by Levine,^
McCrady'^ and Perry and Hajna^ and is recommended for use in the standard
tests for the coliform group as specified in "Standard Methods for the Examina-
tion of Water and Sewage"^ of the American Public Health Association. A
complete discussion of this medium is given under Bacto-Lauryl Tryptose Broth
on page 39. Perry and Hajna used Bacto-Tryptose in their modified Eijkman
Lactose Medium for the differentiation of Escherichia coli from other coliform
bacteria. This medium is discussed on page 44 under Bacto-Eijkman Lactose
Medium.

The usefulness of Bacto-Tryptose in a large variety of bacteriological studies is
demonstrated by the many references to it in the literature. Among these may be
cited those of Schneiter et al.^° and Wells et al.^^ in the bacteriological examina-
tion of air, Pike^^ ^nd Parker and Callen^^ in their studies of media for the isola-
tion of streptococci and many others.

Bacto-Tryptose was employed in preparing the media used in Castaneda's
double medium method^* for the isolation of Brucella from the blood. A detailed

INGREDIENTS OF CULTURE MEDIA 263

description of this method is given on page 111 under the discussion of Bacto-
Tryptose Agar.

Schuhardt, Rode, Foster and Oglesby,^^ by special techniques, demonstrated
that a few of the numerous samples of Bacto-Tryptose which had been in his
laboratory exhibited some toxicity for certain Brucella abortus strains used in his
laboratory. The particular samples of Bacto-Tryptose possessing this character-
istic had absorbed moisture and had undergone chemical change. Schuhardt^^
in a discussion of this observation stated that "the ease of neutralization of this
toxic factor by blood, serum, agar and other substances tends to make the practi-
cal significance of the toxicity relatively minor. We probably would not have
encountered it had we not been doing extensive tests on the in vitro effect of
sulfonamides on Brucella using decimal dilution inocula." The high productivity
of Bacto-Tryptose Agar, and Bacto-Tryptose used clinically for the isolation and
cultivation of Brucella attests to its value for the primary cultivation of Brucella
as well as other fastidious organisms.

A typical analysis of Bacto-Tryptose is given on page 265.

^ Huddleson: Brucellosis in Man and Animals, ^ Standard Methods for the Examination of
14:1939- Water and Sewage, gth Edition: 193: 1946.

2 J. Bact., 43:33:1942. 10 Am, J. Hyg., 40:136:1944.

'^Am. J. Clin. Path. 17:281:1947. " Am. J. Pub. Health, 36:324:1946.

J. Am. Water Works Assoc, 31:689:1939. 12 Am. J. Hyg., 41:211:1945.

''Am. J. Pub. Health, 31:127:1941. ^ J. Bact., 46:343:1943.

« Am. J. Pub. Health, 31:351:1941. " Proc. Soc. Expl. Biol. Med., 64:114:1947.

"Am. J. Pub. Health, 33:"99:i943- "J- Bact., 57:1:1949.

8 Am. J. Pub. Health, 34:735:1944. i« Personal Communication, 1949.

NEOPEPTONE, DIFGO ( B 11 9 )

Neopeptone is an enzymatic protein digest especially adapted for the prepara-
tion of media to be used in the propagation of organisms usually considered
difficult to cultivate in vitro. Neopeptone has been shown to be particularly well
suited to the growth requirements of many delicate and fastidious bacteria.

Dubos^ obtained growth of pneumococci from small inocula using media pre-
pared with Neopeptone. According to Dawson and his associates^-s.* the use of
Neopeptone facilitates the development of mucoid colonies of the hemolytic
streptococci. Long and Bliss^ consider Neopeptone to have definite advantages
over other peptones in media for the cultivation of the minute beta hemolytic
streptococci. Neopeptone media were also utilized by Lancefield^ in her work
on the classification of the streptococci.

In addition to these studies, Neopeptone has been used with satisfaction for
the propagation of many other pathogenic organisms. Parker'^ used Neopeptone
media for the pneumococcus, and Bailey^ employed it in her medium for differ-
entiation of the gonococcus and meningococcus from other Neisseria. Spray^ used
Neopeptone in his media for classification of the sporulating anaerobes, Hobby^^
employed it in her study of the diphtheria bacilli, and Eldering and Kendrick^^
reported good results with this peptone in cultivating Hemophilus pertussis.
Krumwiede^2 ^sed Neopeptone in media for cultivation of group A streptococci.
Engley and Snyder^^ cultivated Pasteurella tularensis in media prepared with
Neopeptone. Casman,^^ in a comparison of peptones used in the preparation of
fresh beef infusion agar for Blood Agar, reported Neopeptone to be best suited
for inclusion in the infusion base.

This peptone has proved to be of decided value in media for the cultivation of
pathogenic fungi. Growth of these microorganisms is rapid and colonial formation
uniform and typical for the various types. Bacto-Sabouraud Dextrose Agar and
Bacto-Sabouraud Maltose Agar, prepared with Neopeptone, are discussed on
pages 238-240.

264 DIFCO MANUAL

A typical quantitative analysis of Neopeptone, Difco, is given on page 265.

ij. Exp. Med., 52:331:1930- ^ J- Bact., 34:645:1937-

* Proc. Soc. Exp. Biol. Med., 3i:594:i934' ^ J- Bact., 32:135:1936.

'Science, 80:296:1934. 10 J. Infectious Diseases, 57:186:1935.

* J. Infectious Disesises, 62:138:1938. ^ Am. J. Pub. Health, 26:506:1936.
"J. Exp. Med., 60:619:1934. ^ J. Bact., 46:117:1943.

* Proc. Soc. Exp. Biol. Med., 38:473:1938. ^^ j. Bact., 51:573:1946.

'J. Exp. Med., 67:667:1938. "Am. J. Clin. Path., 17:281:1947.

B AGTO-CASITONE ( B259 )

Bacto-Casitone is a pancreatic digest of casein, conforming to the specifications
given by the National Institute of Health^ and the U. S. Pharmacopeia. ^ It is
recommended for use in the preparation of media for sterility testing according
to the National Institute of Health,^ "The compilation of Tests and Methods of
Assay for Antibiotic Drugs,"^ Federal Security Agency, Food and Drug Adminis-
tration, National Formulary,* and the U. S. Pharmacopeia.^ It is also recom-
mended for the preparation of media where an enzymatic hydrolyzed casein is
desired, as for example the liquid medium for the cultivation of Mycobacterium
tuberculosis as described by Dubos and Middlebrook.^

Bacto-Casitone is readily soluble in distilled water giving a clear nearly color-
less solution, neutral in reaction. It has a high tryptophane content making it
satisfactory for the detection of indole production.

1 National Institute of Health Circular: Culture Assay for Antibiotic Drugs, Federal Security
Media for the Sterility Test, 2nd Revision: Agency, Food and Drug Administration.
February 5, 1946. * National Formulary, 9th Edition: 768: 1950.

2 Pharmacopeia of the United States, XIV ^ Pharmacopeia of the United States, XIV
Revision: 075: 1950. Revision:758: 1950.

>The compilation of Tests and Methods of ^ Am. Rev. Tuber., 56:335:1947.

BAGTO-PEPTONIZED MILK (B35)

Bacto-Peptonized Milk, as the name implies, is a skim milk digest. Inasmuch
as this product contains carbohydrates as well as nitrogenous materials it is con-
sidered as a complete medium. Bacto-Peptonized Milk is discussed in detail on
page 75.

BAGTO-SOYTONE (B436)

Bacto-Soytone is an enzymatic hydrolysate of soybean meal prepared under
controlled conditions especially for use in microbiological procedures. It is
recommended for use in media for the cultivation of a large variety of organisms,
including fungi, and is also used in media for microbiological assay. This peptone
contains the naturally occurring carbohydrate of the soybean and is suitable in
all microbiological procedures wherein carbohydrates are not objectionable.
Bacto-Soytone is completely soluble in distilled water in concentrations generally
employed and forms clear solutions with a neutral reaction.

BACTO-PROTONE (B125)

Bacto-Protone was developed in response to a demand for a product high in
proteoses which could be used in preparing varying combinations of proteoses,
peptones and amino acids in studies of bacterial metabolism. About 85 per cent
of the nitrogen in Bacto-Protone is precipitated by saturation with ammonium
sulfate, thus indicating a high content of proteoses. Until pure primary or

INGREDIENTS OF CULTURE MEDIA 265

secondary proteoses are available Bacto-Protone forms the nearest and most
useful substitute.

Being low in readily utilizable nitrogen Bacto-Protone does not, by itself,
support good growth of bacteria. Its usefulness lies in combination with more
readily available forms of nitrogen. W. E. Bunney has suggested its use as a
diluent for maintaining the potency of diluted diphtheria toxin for use in the
Schick test. Studies undertaken by the New York State Health Department^
indicate that marked stability of the toxin dilutions was secured when Bacto-
Protone was used.

A typical quantitative analysis of Bacto-Protone is given below.

IN. Y. State Health Dept., 54th Annual Report: 72: 1933.

Typical Analyses of Peptones

Peptone , c

u O.

Per Cent

Total Nitrogen 16.16

Primary Proteose N . . 0.06

Secondary Proteose N 0.68

Peptone N 15.38

Ammonia N 0.04

Free Amino N (Van

Slyke) 3.20

Amide N 0.49

Mono-amino N 9.42

Di-amino N 4.07

Tryptophane 0.29

Tyrosine 0.98

Cystine (Sullivan) . . . 0.22

Organic Sulfur 0.33

Inorganic Sulfur 0.29

Phosphorus 0.22

Chlorine 0.27

Sodium 1.08

Potassium 0.22

Calcium 0.058

Magnesium 0.056

Manganese nil

Iron 0.0033

Ash 3.53

Ether Soluble Extract 0.37

Reaction, pH 7.0

pH I per cent solution in dis-
tilled water after autoclaving 15
minutes at 121 oC.

npLH

14.37

13.14

13.76

14.33

15.41

0.60

0.20

0.40

0.46

5.36

4.03

1.63

2.83

3.03

7.60

9.74

11.29

10.52

10.72

2.40

0.00

0.02

0.01

0.12

0.05

2.66

4.73

3.70

2.82

1.86

0.94

1.11

1.03

1.23

7.61

7.31

7.46

7.56

4.51

3.45

3.98

4.43

0.51

0.77

0.64

0.73

1.03

2.51

4.39

3.45

4.72

2.99

0.56

0.19

0.38

0.39

0.27

0.60

0.53

0.57

0.63

0.45

0.04

0.09

0.16

0.47

0.97

0.72

0.19

0.27

3.95

0.29

2.77

0.84

0.38

2.84

2.69

2.77

0.45

0.30

0.70

0.30

0.50

0.85

0.06

0.137

0.096

0.117

0.198

0.263

0.118

0.045

0.082

0.051

0.057

0.0002

nil

0.0001

nil

0.0056

0.0104

0.0080

0.0041

0.0023

9.61

7.28

8.45

3.90

2.50

0.32

0.30

0.31

0.30

0.31

6.8

7.2

7.3

6.8

6.7

HYDROLYSATES, ACID

BAGTO-G AS AMINO AGIDS (B230)

Bacto-Casamino Acids is acid hydrolyzed casein recommended for use in the
production of diphtherial toxin and other biological products. Hydrolysis is car-

266 DIFCO MANUAL

ried on until all the nitrogen in the casein is converted to amino acids or other
compounds of relative chemical simplicity. Bacto-Casamino Acids is prepared
according to the method described by Mueller and Miller^ and Mueller and
Johnson.^ This product has a satisfactory sodium chloride content for diphtheria
toxin production^ '^ and has had all but the last traces of iron removed. Bacto-
Casamino Acids is also particularly well suited for nutritional studies, micro-
biological assays, the preparation of so called "synthetic" or chemically defined
media and media used for sulfonamide inhibitor studies.

Mueller^ prepared diphtheria toxin in a medium containing a casein hydroly-
sate as the source of nitrogen. It was shown that the high sodium chloride content
was the limiting factor in the amount of toxin that could be produced in this
medium. Mueller and Miller^ described a method of reducing the sodium chloride
content of the hydrolyzed casein and also for reducing the iron content to a
minimum. Using this hydrolyzed casein supplemented with inorganic salts,
growth accessory factors, cystine, maltose and an optimum amount of iron, 100
flocculating units per ml. of diphtheria toxin were prepared. Bacto-Casamino
Acids duplicates this especially treated hydrolyzed casein. Complete detailed
directions for the preparation of diphtheria toxin are given by these authors. ^

Using this hydrolyzed casein, Bacto-Casamino Acids, Mueller and Miller*
determined the growth requirements for Clostridium tetani. Morton and Gon-
zalez^ employed Bacto-Casamino Acids in the medium used to study the site
of formation of diphtheria toxin. They obtained diphtheria toxin of high potency
using the method described by Mueller and Miller.^

Bacto-Casamino Acids has also been used in media for the testing of disin-
fectants. Wolf^ used Bacto-Casamino Acids or Bacto-Casamino Acids, Technical
in the preparation of his disinfectant test medium. Klarman and Wright^ used
Bacto-Casamino Acids in the semi-synthetic medium for testing disinfectants
described by them. Logan, Tytell, Danielson and Griner^ employed Bacto-
Casamino Acids in the preparation of the medium used for maintaining stock
cultures for the elaboration of Clostridium perfringens alpha toxin. Straus,
Dingle and Finland^ employed a medium containing Bacto-Casamino Acids
in their study of the mechanism of sulfonamide bacteriostasis.

Bacto-Casamino Acids also is well suited for use in media for microbiological
assay. A description of these media with references is given on page 216 to 230.
Bird, Bressler, Brown, Campbell and Emmett^^ employed Bacto-Casamino Acids
in the medium for the assay for folic acid using Lactobacillus casei as the test
organism. This casein hydrolysate has been used with excellent results in media
for the cultivation of Hemophilus pertussis according to the method described
by Hornibrook^^ and by Verwey, Thiele and Sage.^-

The following is an approximate analysis of Bacto-Casamino Acids:

Total Nitrogen 10 per cent

Sodium Chloride 14 per cent

Ash 20 per cent

P as P04 2 per cent

Iron, 3 grams Bacto-Casamino

Acids 15 micrograms

^ J. Immunol., 40:21:1941. ' Soap and Sanitary Chemicals, 21:113:1945.

3 J. Immunol., 40:33:1941. ^ J. Immunol., 51:317:1945.

3 J. Immunol., 37:io3:i939- "J- Immunol., 42:331:1941.

* J. Bact., 43:763:1942. ^0 J. Biol. Chem., 159:631:1945.

0 J. Immunol., 45:63:1942. " Public Health Reports, 54:1847:1939.

« J. Bact., 49:463:1945- "J- Bact., 54:71:1947.

INGREDIENTS OF CULTURE MEDIA 267

BACTO-GAS AMINO ACIDS, TECHNICAL (B231)

Bacto-Casamino Acids, Technical is acid hydrolyzed casein. The hydrolysis is
carried out as in the preparation of Bacto-Casamino Acids, but the sodium
chloride and iron content of this product have not been decreased to the same
extent. Bacto-Casamino Acids Technical is recommended for use in culture media
where amino acid mixtures are required for a nitrogen source and the sodium
chloride content need not be low. It is particularly valuable in studying the
growth requirements of bacteria.

Bacto-Casamino Acids, Technical is prepared according to the method sug-
gested by Mueller^ for use in the preparation o^ diphtheria toxin in a medium
containing nitrogen substances of chemical composition similar to amino acids
in simplicity. Later it was shown that the sodium chloride of the hydrolysate
was the limiting factor in the production of diphtheria toxin and a method was
described by Mueller and Miller^ for the reduction of this material in the
hydrolysate, see Bacto-Casamino Acids discussed above.

Casein hydrolysates prepared according to Mueller's method^ have been used
for many purposes. Tetanus toxin of high potency was produced by Mueller and
Miller^ in a simple medium using this hydrolysate.

Favorite and Williams* used a casein hydrolysate prepared according to the
method of Mueller^ for the production of staphylococcus enterotoxin and alpha
hemolysin. Mueller and Hinton^ used Bacto-Casamino Acids, Technical in a
medium for primary isolation of the gonococcus and meningococcus. Bacto-
Casamino Acids, Technical was used in agar-free media for the isolation of
Neisseria and also in a tellurite medium for the isolation of C orynebacterium,
described by Levin.^ Wolf used Bacto-Casamino Acids or Bacto-Casamino Acids,
Technical in the preparation of a medium for the testing of disinfectants. Mueller
and Miller^ used Bacto-Casamino Acids, Technical in their Tellurite Serum Agar
for a selective medium for diphtheria. This casein hydrolysate has been used
with excellent results in media for the cultivation of Hemophilus pertussis
according to the method described by Homibrook^ and by Verwey, Thiele and
Sage.io

The following is an approximate analysis of Bacto-Casamino Acids, Technical :

Total Nitrogen 8 per cent

Sodium Chloride 37 per cent

Ash 39 per cent

1 J. Immunol., 37:103:1939. a J. Bact., 46:233:1943.

a J. Immunol., 40:21:1941. 7 J. Bact., 49:463:1945.

3 Proc. Soc. Exp. Biol. Med., 43:389:1940- ^ J- Bact., 51:743:1946.

* J. Bact., 41:305:1941. 8 Public Health Reports, 54:1847:1939.

^ Proc. Soc. Exp. Biol. Med., 48:330:1941. 10 J. Bact., 54:71:1947.

BACTO-VITAMIN FREE CASAMINO ACIDS

As the name implies, Bacto- Vitamin Free Casamino Acids is acid hydrolyzed
casein, free from vitamins. It is recommended for use in microbiological assay
rnedia and in studies on the growth requirements of microorganisms. A descrip-
tion of these media with references is given on page 216 to 230. Sarett^ used
Bacto- Vitamin Free Casamino Acids as the acid hydrolyzed casein in his studies
on p-aminobenzoic acid and pteroylglutamic acid as growth factors for Lacto-
bacilli.

Bacto-Vitamin Free Casamino Acids is readily soluble in distilled water,

268 DIFGO MANUAL

yielding a clear colorless solution. The following is a typical analysis of Bacto-
Vitamin Free Casamino Acids:

Total Nitrogen 7 per cent

Sodium Chloride 38 per cent

Ash 41 per cent

* J. Biol. Chem., 171:265:1947.

AMINO ACIDS

A DISCUSSION of the amino acids most commonly used is given here. Additional
amino acids are available as required. Difco amino acids are pure and recom-
mended as standards for chemical and microbiological procedures as well as for
use in media of chemically defined composition for nutritional studies.

BAGTO-ASPARAGINE ( B 144)

Bacto-Asparagine is /-asparagine of exceptional purity. It is recommended as
a particularly useful ingredient of synthetic culture media employed in studies
of bacterial nutrition.

Asparagine is used as a source of organic nitrogen of known chemical com-
position. It is readily available for bacterial energy and growth when used in
culture media. Bacto-Asparagine is widely employed in media for the mass
cultivation of Mycobacterium tuberculosis for use in the preparation of tuber^
culin for the detection and eradication of tuberculosis. Asparagine has also
been used in recent investigations for the growth of tubercle bacilli as reported
by Dubos and Davis,^ and others,
ij. Exp. Med., 83:409:1946.

J/- ALANINE, DIFGO (B182)

Alanine has been used occasionally in culture media as a source of nitrogen
in studies of the metabolism of various organisms. c^/-Alanine, Difco is a highly
purified amino acid suitable for use in studies of this type.

/-GYSTINE, DIFGO (B184)

Cystine is used extensively in synthetic culture media, particularly those
employed in studies of bacterial metabolism. Davis and Ferry^ devised a cystine
medium for use in their studies on diphtheria toxin production, and DuvaP
employed a cystine medium for propagation of Mycobacterium leprae. Shaw^
and Rhamy* used cystine media for the cultivation of Pasteurella tularensis.
Cystine is employed in the media specified by the National Institute of Health,^
for the sterility testing of biologicals, for sterility testing as specified in the
Pharmacopeia^ and National Formulary''' and for the sterility testing of anti-
biotics according to the method of the Food and Drug Administration.^

/-Cystine, Difco is a highly purified amino acid recommended for use in the
preparation of culture media.

1 J. Bact., 4:217:1919. 0 Pharmacopeia of the United States, XIV Re-

^ J. Exp. Med., 12:649:1910. vision: 758: 1950.

Zentr. Bakt., I Abt. Orig., 118:216:1930. ' National Formulary, 9th Edition: 778: 1950.

Am. J. Clin.
National Inst:
Media for tl
Feb. 5j 1946.

* Am. J. Clin. Path., 3:121:1933. ^ Compilation of Tests and Methods of Assay for

* National Institute of Health Circular : Culture Antibiotic Drugs, Federal Security Agency,
Media for the Sterility Test, 2nd Revision: Food and Drug Administration.

INGREDIENTS OF CULTURE MEDIA 269

/.TRYPTOPHANE, DIFGO (B188)

Tryptophane is an amino acid, essential for the growth of many micro-
organisms. Tryptophane is utilized by bacteria in the elaboration of indole and
has, therefore, been employed in media devised for testing for indole production.

Since tryptophane is destroyed by acid hydrolysis, synthetic media employing
acid hydrolysates or chemically pure amino acid mixtures as sources of nitrogen,
require the addition of tryptophane for the growth of most bacteria.

/-TYROSINE, DIFGO (B189)

Tyrosine is an amino acid which has been used occasionally in the preparation
of culture media.

EXTRACTS

BAGTO-BEEF EXTRAGT (B126)

Bacto-Beef Extract is prepared and standardized for use in microbiological
culture media, where it is generally used to replace infusions of meat. It is
standard in its composition and reaction, and does not require adjustment of
reaction or filtration. For many years beef extract media have been recom-
mended as standard for use in the routine bacteriological examination of water,
milk and other materials where it is important to have media of uniform
composition.

Bacto-Beef Extract may be relied upon for biochemical studies, particularly
fermentation reactions because of its freedom from fermentable substances which
would interfere with the accuracy of such determinations.

In culture media, Bacto-Beef Extract is usually employed in concentrations
of 0.3 per cent as in the standard media recommended for water and for milk
analysis.^'2 Concentrations may vary slightly according to the requirements of
individual formula, but do not often exceed 0.5 per cent. In 0.3 per cent con-
centration Bacto-Beef Extract forms brilliantly clear solutions, rich in the nutri-
ments required for bacterial metabolism. After autoclave sterilization this solution
has a reaction of pH 6.8.

In the fields of special research Bacto-Beef Extract has been employed in
media by a number of investigators. Bedell and Lewis^ used it in their medium
for the study of the non-sporulating anaerobes of the intestinal tract, and Tittsler
and Sandholzer* employed it in carbohydrate broths for studying the cultural
characteristics of the Escherichia-Aerobacter intermediates. Hutner^ used a
medium containing Bacto-Beef Extract as a stock broth in his study of the
nutritional needs of the streptococci. Kent^ also employed Bacto-Beef Extract

270 DIFCO MANUAL

in a medium for culturing Agrobacterium tumejaciens in studying the specific
bacteriophage of this organism. Countless other references to the use of this
product are to be found in bacteriological literature.

1 Standard Methods for the Examination of » j. Bact., 36:567:1938.

Water and Sewage, 9th Edition: 1946. * J. Bact., 29:349: 1935.

8 Standard Methods for the Examination of 5 J. Bact., 35:429: 1938.

Dairy Products, 9th Edition: 1948. « Phytopathology, 27:871:1937.

BAGTO-YEAST EXTRACT (B127)

Bacto-Yeast Extract is the water soluble portion of autolyzed yeast. The
autolysis is carefully controlled to preserve the naturally occurring B-complex
vitamins. Bacto-Yeast Extract is prepared and standardized for bacteriological
use. It is an excellent stimulator of bacterial growth and is used in culture media
in place of, or as an adjuvant to, beef extract. In concentrations of 0.3-0.5 per
cent, as it is generally employed, it forms sparklingly clear solutions with a
reaction of pH 6.6.

Bacto-Yeast Extract has been used advantageously in culture media for studies
of the bacteria in milk and other dairy products. Its usefulness for this purpose is
attested in such reports as those of Prickett^ on the thermophilic and thermoduric
bacteria of milk. Since publication of this work, Bacto-Yeast Extract has been
used with increasing frequency in the study of the bacterial flora of milk. Among
the references to its use for this purpose are those of Hucker and Hucker^ on the
number and type of bacteria in commercially prepared infant foods, Breed et al.^
on methods for the examination of thermophiles in dry milk and Downs and his
associates* on methods for the study of these organisms in evaporated and con-
densed milk. Bowers and Hucker^ have also studied the effect of Bacto-Yeast
Extract in media employed for counting bacteria in milk. An increasing number
of references to Bacto-Yeast Extract is to be found in bacteriological literature.
Hutner^ used this product in a stock broth for the streptococci. Nelson and
Werkman''' have used Bacto-Yeast Extract as an ingredient of the medium they
employed for cultivation of Lactobacillus brevis. Werkman and his associ-
^|-gs8,9,io,ii have used Bacto-Yeast Extract media for propagating organisms of
the propionic acid group. Partansky and McPherson^^ ^5^^ Bacto-Yeast Extract
in combination with Bacto-Malt Extract, and Bacto-agar for the growth of molds
in their laboratory method for testing the mold resistant properties of oil paints.

Pringsheim and Robinow^^ in their studies on the cultivation of Caryophanon
latum reported very good growth of the organism in agar media containing
Bacto-Yeast Extract. Some batches of laboratory made autolysates of Bakers
yeast gave excellent results but others were unsatisfactory. They were able to
rely on a medium prepared with 0.5 per cent Bacto-Yeast Extract and 0.5 per
cent Bacto-Peptone, which was very favorable for isolation and maintenance of
the culture. In a survey of ingredients for a medium for the standard plate count
for dairy products, Buckbinder and associates^* compared various yeast extracts
and showed that media prepared with Bacto-Yeast Extract gave a higher count
than similar media prepared with other yeast extracts.

Bacto-Yeast Extract is an excellent source of B-complex vitamins and is often
used to supply these factors in bacteriological culture media. For example, Snell
and Strongly used Bacto-Yeast Extract for the preparation of the yeast supple-
ment in their medium for riboflavin assay. It has proved to be a valuable ingredi-
ent of media used for carrying stock cultures and for preparation of inocula of

INGREDIENTS OF CULTURE MEDIA 271

Lactobacilli for microbiological assay of vitamins. This product is also of value in
the assay of antibiotics. ^^-^^

1 Tech. Bull. 147, N. Y. Agr. Exp. Sta., 1928. 10 Biochem. J., 31 :349:i937-

ch. Bull. 153, N. Y. Agr. Exp. Sta., 1929. ^ J. Bact., 31:595:1936-

Dairy Science, 15:383: 1932. '^ Ind. Eng. Chem., Anal. Edition: 12:443: 1940.

a Tech. Bull. 153, N. Y. Agr. Exp. Sta., 1929. ^ J. Bact., 31 :595:i936

- - 12 In - —

5 Am. J. Pub. Health, 24: 396 :"F934. "Pub. Health Reports : 66: 327 : 1 951.

« J. Bact., 35:429: 1938. 15 ind. Eng. Chem., Anal. Edition: n :346: 1939.

Bact., 31 : 603: 1936. 18 J. Bact., 47:199:1944.

3 J. Dairy Science, 15:383:1932. 12 ind. Eng. Chem., Anal. Editic

* J. Dairy Science, 18:647:1935. 13 j_ Qgn. Microbiol., i : 267: 1947.

5 Am. J. Pub. Health, 24:396:1934. "Pub. Health Reports : 66: 327 : i

_. Bact., 36:201:1933. "Science, 98:69:1943.

8 Biochem. J., 32:1262:1938.

BACTO-MALT EXTRACT (B186)

Bacto-Malt Extract is a useful ingredient of culture media designed for the
propagation of yeasts and molds. This product was used by Thom and Church^
in the preparation of "wort" medium as originally described by Reddish.^ Bacto-
Malt Extract Broth, as discussed on page 242, is a complete dehydrated medium
and duplicates the medium of Thom and Church.

Bacto-Malt Extract is employed in the preparation of Bacto-Malt Agar, a
widely used medium for the detection and isolation of yeast and molds from
dairy products, food and other materials. This medium is discussed in detail on
page 65. Partansky and McPherson^ used Bacto-Malt Extract, in conjunction
with Bacto-Yeast Extract and Bacto-Agar for the cultivation of molds in their
laboratory method for testing mold resistant properties of oil paints.

1 Thom and Church: The Aspergilli, 1926. 3 Ind. Eng. Chem., Anal. Edition: 12:443: 1940.

a Abst. Bact., 3:6:1919.

ENRICHMENTS

The enrichments described in this section are used with liquid or solid basal
media to give the complete formulation. The enrichments are of such a nature
that they cannot be sterilized with the base medium but must be added under
aseptic conditions. Some of the enrichments are sterilized in the autoclave fol-
lowing rehydration while others are supplied as sterile solutions of thermo-
labile complexes. Some contain a selective agent producing a medium designed
for the isolation or cultivation of a certain group of microorganisms. Care must
be taken to use the selective enrichments with the exact base medium specified
since the selective agent is standardized with the designated medium and the use
of a different base would not give the desired results.

B AGTO-HEMOGLOBIN ( B 1 36 )

Bacto-Hemoglobin, prepared from beef blood, is a convenient, readily avail-
able enrichment, which may be sterilized in the autoclave. It is prepared
according to the procedure described by Spray^ and is recommended for use in
media for the cultivation of Neisseria gonorrhoeae. Hemophilus influenzae, H,
ducreyi, Pasteurella tularensis, streptococci, pneumococci, etc.

Bacto-Proteose No. 3 Agar and Bacto-G C Medium Base enriched with Bacto-
Hemoglobin and Bacto-Supplement A or Bacto-Supplement B are recommended
for the cultural isolation of the gonococcus from all types of gonococcal infections
in the male or female. The technique for this procedure is given in detail on

272 DIFGO MANUAL

pages 116 and 122. The medium suggested for the cultural detection of the
gonococcus by "Diagnostic Procedures and Reagents"^ of the American Public
Health Association is Bacto-G C Medium Base enriched with Bacto-Hemoglobin
and Bacto-Supplement B. These media may also be recommended for the isola-
tion of H. influenzae and H. ducreyi. For the isolation of P. tularensis a medium
prepared with Bacto-Hemoglobin and Bacto-Cystine Heart Agar, as discussed
on page 91, is recommended.

Solutions of Bacto-Hemoglobin have the advantage of being sterilizable in the
autoclave. Bacto-Hemoglobin is used solely as an enrichment, and cannot be
employed as an indicator of hemolysis. For use as an enrichment in media, 2
grams of Bacto-Hemoglobin are dissolved in 100 ml. of distilled water. This
solution is best prepared by placing 2 grams of Bacto-Hemoglobin in a dry flask
and adding 100 ml. cold distilled water while the flask is being agitated vigor-
ously. The hemoglobin suspension is shaken intermittently for 10-15 minutes to
break up all aggregates and effect complete solution. It is sterilized in the auto-
clave for 15 minutes at 15 pounds pressure (121°C.). The sterile solution is
added, in equal proportions, to sterile double strength medium. When agar media
are prepared it is important to cool the melted agar and the hemoglobin solution
to 50-60°C. before the solutions are mixed, and the mixture should be agitated
to insure a perfect suspension of hemoglobin throughout the medium. Agar
media enriched with Bacto-Hemoglobin are similar to Chocolate Agar in
appearance. Broth media may be enriched in the same manner except that the
temperature of the mixture may be disregarded.

^ J. Lab. Clin. Med., 16:166:1930. Diagnostic Procedures and Reagents, 3rd Edi-

tion: 30: 107: 1950.

BAGTO-BEEF BLOOD (B137)

Bacto-Beef Blood is whole beef blood which has been desiccated and powdered.
It is an enrichment, capable of being sterilized in the autoclave, duplicating the
dilute whole blood (Preparation No. 3) as described by Spray^ for use in the
cultivation of hemoglobinophilic organisms. Bacto-Beef Blood is used solely as
an enrichment for culture media. It cannot be employed as an indicator of
hemolysis.

Bacto-Proteose No. 3 Agar as discussed on page 116, enriched with Bacto-Beef
Blood, is well suited for propagation of the hemophilic bacteria. This enriched
medium possesses excellent growth-promoting properties. For the cultivation of
the gonococcus, however, Bacto-Hemoglobin should be employed as the enrich-
ment.

When Bacto-Beef Blood is first dissolved in water it has the appearance of
laked blood. After it has been autoclaved there is a tendency for the soft coagu-
lum to settle. It is, therefore, advisable to maintain an even dispersion by gentle
agitation of the flask when the sterilized solution is added to the medium. After
addition of the solution to an agar medium, the mixture should be agitated to
secure an even suspension of the blood in the medium as described below. Agar
media enriched with Bacto-Beef Blood are similar to Chocolate Agar in appear-
ance.

Bacto-Beef Blood is prepared for use by dissolving 2 grams of the powder
in 100 ml. of distilled water. It is then sterilized in the autoclave for 15 minutes
at 15 pounds pressure (121°C.). To prepare a chocolate or enriched agar, mix
equal parts sterile beef blood solution and sterile melted double strength agar,
under aseptic conditions. The temperature of both solutions should be between
50-60° C. at the time of mixing and they should be gently agitated to insure

\
INGREDIENTS OF CULTURE MEDIA 273

a perfect suspension of the blood in the medium without the formation of air
bubbles. Broth media may be enriched in the same manner except that the
temperature of the mixing may be disregarded.

^ J. Lab. Clin. Med., 16:166:1930.

BAGTO-BEEF BLOOD SERUM (B138)

Bacto-Beef Blood Serum is a dehydrated powdered form of fresh beef blood
serum. In a 10 per cent solution it is equivalent to fresh serum.

Bacto-Beef Blood Serum is a useful aid to bacteriologists who have occasional
use for serum media. The use of this product eliminates all the disagreeable and
time-consuming procedures attendant upon the preparation of fresh serum. Bacto-
Beef Blood Serum may be employed with excellent success in the preparation
of many serum media. When it is used in plain 10 per cent solution or as an
ingredient of Loeffler Medium it forms firm, smooth, white slants upon heat
coagulation.

The Beef Blood Penetration Test of the Institute of Paper Chemistry in their
Institute Tentative Method No. 522, November, 1941, specifies the use of a 10
per cent solution of Bacto-Beef Blood Serum in distilled water adjusted to pH
6.6 with a 5 per cent solution of phosphoric acid for the performance of their
test.

BACTO-BEEF SERUM (B260)

Bacto-Beef Serum is filter sterilized fresh normal beef serum. It is recom-
mended as an enrichment for use in bacteriological culture media, and in other
procedures requiring beef serum. Bacto-Beef Serum is supplied in packages of
one-half dozen and one dozen ampuls of 10 ml. each.

BAGTO-HORSE SERUM (B261)

Bacto-Horse Serum is prepared from normal horse serum. It is filter sterilized
and is recommended for use as an enrichment in bacteriological culture media
and in other procedures requiring horse serum. Bacto-Horse Serum is supplied
in packages of one-half dozen and one dozen ampuls of 10 ml. each.

BAGTO-HORSE SERUM SALINE 1-6 (B262)

Bacto-Horse Serum Saline 1-6 is normal horse serum diluted 1 to 6 with
saline. It is filter sterilized and is especially recommended for use in the cultiva-
tion of Endamoeba histolytica according to the procedure discussed on page 97.
Bacto-Horse Serum Saline 1-6 is supplied in packages of one-half dozen and one
dozen ampuls of 10 ml. each.

BAGTO-ASGITIG FLUID (B135)

Bacto-Ascitic Fluid is prepared from serous fluids removed aseptically from
the peritoneal cavity of a number of individuals. It is filter sterilized and
packaged aseptically. The finished product is subjected to regular sterility tests

274 DIFGO MANUAL

and found to be free from demonstrable organisms. It is not guaranteed, how-
ever, to be free from filterable forms of viruses.

Bacto-Ascitic Fluid is used as an enrichment for culture media. The usual
proportion for use is one 10 ml. ampul of Bacto-Ascitic Fluid for each 20 or 30
ml. of sterile medium. To prevent coagulation of the ascitic fluid, the medium
should be cooled to 45-50 °G. before the enrichment is added. Agar media
should be prepared with concentrations of agar sufficiently high to allow for
dilution of the media with the fluid. In order to prevent contamination, great
care should always be taken to observe aseptic precautions when Bacto-Ascitic
Fluid is added to culture media. The enriched media should always be incubated
to insure their sterility before they are used.

Bacto-Ascitic Fluid is supplied in packages of one-half dozen or one dozen
ampuls of 10 ml. each.

BAGTO-DUBOS MEDIUM SERUM (B292)

Bacto-Dubos Medium Serum is filter sterilized fresh beef serum to which is
added 7.5 per cent Bacto-Dextrose. Bacto-Dubos Medium Serum is prepared
for use in the preparation of a liquid medium for the rapid cultivation of
Mycobacterium tuberculosis as discussed on page 108 and 105 under Bacto-TB
Broth Base and Bacto-Dubos Broth Base. Bacto-Dubos Medium Serum is sup-
plied in packages of one-half dozen and one dozen tubes of 20 ml. each.

BAGTO-DUBOS MEDIUM ALBUMIN (B309)

Bacto-Dubos Medium Albumin is a filter sterilized 5 per cent solution of
albumin fraction V from bovine plasma in normal saline and contains 7.5 per
cent Bacto-Dextrose. Bacto-Dubos Medium Albumin is prepared especially for
use in the preparation of a liquid medium for the rapid cultivation of Myco-
bacterium tuberculosis, giving readily dispersible growth as discussed on page
108 and 105 under Bacto-TB Broth Base and Bacto-Dubos Broth Base. Bacto-
Dubos Medium Albumin is a filter sterilized solution supplied in packages of
one-half dozen and one dozen tubes of 20 ml. each.

BAGTO-DUBOS OLEIG SERUM GOMPLEX (B376)

Bacto-Dubos Oleic Serum Complex is a sterile liquid enrichment for use
with Bacto-Dubos Oleic Agar Base for the preparation of a medium for the
isolation and cultivation of Mycobacterium tuberculosis. This enrichment is
similar to Bacto-Dubos Oleic Albumin Complex, prepared according to the
directions given by Dubos and Middlebrook,i except that normal beef serum
is used instead of albumin fraction V.

The complete medium is prepared by adding 20 ml. of Bacto-Dubos Oleic
Serum Complex and 5,000 to 10,000 units of penicillin (25 to 50 units per ml.
medium) to 180 ml. of sterile rehydrated Bacto-Dubos Oleic Agar Base at
50-55 °C. under aseptic conditions. A description of the medium is given on
page 107, Bacto-Dubos Oleic Agar Base. The desired concentration of penicillin
may be readily obtained by dissolving the contents of one vial of penicillin
containing 100,000 units in 10 ml. sterile distilled water. One-half (0.5) ml.
(5,000 units) is added to 200 ml. of the sterile medium at 50-55 °C. under aseptic
conditions to obtain a concentration of 25 units per ml. medium ( 1 ml. to give

INGREDIENTS OF CULTURE MEDIA 275

50 units per ml. medium). The enriched medium is then mixed to obtain an
even distribution of all components, and distributed into tubes or plates as
desired. When solidified in tubes or plates, it is ready for surface inoculation.

Bacto-Dubos Oleic Serum Complex is supplied in packages of one-half dozen
tubes of 20 ml. each.

1 Am. Rev. Tuberculosis, 56:334:1942.

BACTO-DUBOS OLEIC ALBUMIN COMPLEX (B375)

Bacto-Dubos Oleic Albumin Complex is a sterile liquid enrichment for use
with Bacto-Dubos Oleic Agar Base for the preparation of a medium for the
isolation and cultivation of Mycobacterium tuberculosis, as described by Dubos
and Middlebrook.^ The enrichment consists essentially of a 0.05 per cent
solution of alkalinized oleic acid in a 5 per cent solution of albumin fraction V
in normal saline (0.85 per cent sodium chloride solution).

A discussion of the complete medium is given under Bacto-Dubos Oleic Agar
Base, page 107. The medium is prepared by adding 20 ml. of Bacto-Dubos
Oleic Albumin Complex and 5,000 to 10,000 units of penicillin (25 to 50 units
per ml. medium) to 180 ml. of sterile rehydrated Bacto-Dubos Oleic Agar Base
at 50-55 °C. under aseptic conditions. The desired concentration of penicillin
may be readily obtained by dissolving the contents of one vial of penicillin
containing 100,000 units in 10 ml. sterile distilled water. One-half (0.5) ml.
(5,000 units) is added to 200 ml. of the sterile medium at 50-55 °C. under
aseptic conditions to obtain a concentration of 25 units per ml. medium ( 1 ml.
to give 50 units per ml. medium). The enriched medium is then mixed to
obtain an even distribution of all components, and distributed into tubes or
plates as desired. When solidified in tubes or plates, it is ready for surface
inoculation.

Bacto-Dubos Oleic Albumin Complex is supplied in packages of one-half
dozen tubes of 20 ml. each.

1 Am. Rev. Tuberculosis, 56:334:1942.

BACTO-PEIZER TB MEDIUM ENRICHMENT (B401)

Bacto-Peizer TB Medium Enrichm.ent is prepared according to the formula
described by Peizer and Schecter,^ and is used with Bacto-Peizer TB Medium
Base for the preparation of a medium for the isolation and cultivation of Myco-
bacterium tuberculosis. The enrichment consists of fresh egg yolk, glycerol, dex-
trose and malachite green as a selective agent. A complete discussion of the
medium is given on page 110. Bacto-Peizer TB Medium Enrichment is packaged
in 29 ml. amounts, sufficient for the preparation of 129 ml. of complete medium.
It is supplied in packages of one-half dozen vials.
^Am. J. Clin. Path., 20:682:1950.

BACTO-PPLO SERUM FRACTION (B441)

Bacto-PPLO Serum Fraction is a sterile solution recommended as an enrich-
ment in media for the cultivation of PPLO (pleuropneumonia-like organisms).
It is employed in 1 per cent concentration in Bacto-PPLO Agar and Bacto-
PPLO Enrichment Broth as discussed on pages 89 and 82. Bacto-PPLO Serum

276 DIFGO MANUAL

Fraction has also been used for the propagation, stabilization and maintenance
of the Treponemes in vitro.

Bacto-PPLO Serum Fraction is the partially purified serum fraction required
for growth by the pleuropneumonia-like organisms described by Smith and
Morton.^ It has the advantage over sera or ascitic fluid in that only 1 per cent
is required for enriching broth or solid media for culturing PPLO and does not
exhibit the inhibitory effect which normal sera has upon some PPLO strains.

Bacto-PPLO Serum Fraction was shown by Rose and Morton^ to replace
effectively the serum or albumin fractions commonly employed in the cultivation
of avirulent Treponemes.

1 J. Bact. J 61:395:1951. 2 Am. J. Syphilis Gonorrh. Venereal Diseases,

36:1:1952.

BAGTO-SUPPLEMENT B (B276)

Bacto-Supplement B is a sterile yeast concentrate for use in supplementing
media for culturing fastidious microorganisms with exacting growth require-
ments. It is particularly recommended for use in Chocolate Agar, prepared from
Bacto-G C Medium Base or Bacto-Proteose No. 3 Agar and Bacto-Hemoglobin,
employed in the cultural diagnosis of gonococcal infections. It is also recom-
mended for use as an enrichment in both solid and liquid media for the isolation
and propagation of Hemophilus influenzae as described by Neter.^'^ This supple-
ment, with crystal violet added (Bacto-Supplement A), may be recommended
for the preparation of selective enriched media for these organisms.

Bacto-Supplement B is processed to preserve both the thermolabile and thermo-
stable growth accessory factors of fresh yeast, including glutamine, coenzyme
(v factor), cocarboxylase and other growth factors for the most exacting strains
of Neisseria gonorrhoeae and H. influenzae. It also contains the hematin or x
factor required by H. influenzae.

Bacto-Supplement B is used in one per cent concentration in either solid or
liquid media. It is added aseptically after the medium has been sterilized in the
autoclave and cooled below 50° C.

The use of this enrichment in media is discussed in detail under Bacto-G C
Medium Base, page 122, Bacto-Proteose No. 3 Agar, page 116 and Bacto-Brain
Heart Infusion, page 77.

Bacto-Supplement B is supplied in packages of six bottles of 5 ml. each.

1 Science, 106:350:1947. 2 j_ Bact., 54:70:1947.

BACTO-SUPPLEMENT A (B246)

Bacto-Supplement A is a sterile yeast concentrate containing crystal violet
as a selective agent for use in supplementing media for culturing fastidious micro-
organisms with exacting growth requirements. It is particularly recommended
for use in Chocolate Agar prepared from Bacto-Proteose No. 3 Agar or Bacto-
G C Medium Base and Bacto-Hemoglobin employed in the cultural diagnosis
of gonococcal infections. It is also suggested for use as a selective enrichment
in solid media containing Bacto-Hemoglobin for the isolation and propagation
of Hernophilus influenzae.

Bacto-Supplement A is processed to preserve both the thermolabile and thermo-
stable growth accessory factors, including glutamine, coenzyme (v factor),
cocarboxylase, and other growth factors for the most exacting strains of Neisseria
gonorrhoeae and H. influenzae. It also contains the hematin or x factor required

INGREDIENTS OF CULTURE MEDIA 277

by H. influenzae. In addition, this enrichment contains crystal violet which sup-
presses growth of many of the common contaminants on the final medium. In
gonococcal isolations, these contaminants, if permitted to develop, may interfere
with the growth and detection of the gonococcus.

Bacto-Supplement A is used in one per cent concentration in either solid or
liquid media containing Bacto-Hemoglobin. It is added aseptically after the
medium has been sterilized in the autoclave and cooled below 50°C.

The use of this enrichment in media is discussed in detail under Bacto-G G
Medium Base, page 122 and Bacto-Proteose No. 3 Agar, page 116.

Bacto-Supplement A is supplied in packages of six bottles of 5 ml. each.

BAGTO-GHAPMAN TELLURITE SOLUTION (B291)

Bacto-Chapman Tellurite Solution is a 1 per cent solution of potassium
tellurite prepared and standardized especially for use with Bacto-Mitis Salivarius
Agar, as discussed on page 154. The final medium is prepared by adding exactly
1.0 ml. to 1000 ml. of sterile melted Mitis Salivarius Agar at 50-55 °G., mixing
thoroughly and pouring into plates at once to obtain the proper selectivity. The
resulting selective medium duplicates that described by Chapman.^'- It permits
the isolation of Streptococcus mitis, S. salivarius and enterococci from grossly
contaminated specimens, other organisms generally being inhibited. Bacto-Chap-
man Tellurite Solution is also used in the preparation of PPLO Enrichment
Broth as discussed on page 82.

Bacto-Chapman Tellurite Solution is supplied in packages of six ampuls of
5 ml. each. The solution must be stored in a glass stoppered container free from
organic matter.

^ Am. J. Digestive Diseases, 13:105:1946. 3 Trans. N. Y. Acad. Sciences, 10:45:1947.

BAGTO-TELLURITE BLOOD SOLUTION (B139)

Bacto-Tellurite Blood Solution is defibrinated beef blood to which one per
cent potassium tellurite has been added. This mixture is prepared expressly for
use in the isolation of Corynebacterium diphtheriae. To 100 ml. sterile medium
prepared from Bacto-Dextrose Proteose No. 3 Agar (as discussed on page 147),
are added 5 ml. Bacto-Tellurite Blood Solution with aseptic precautions. This
mixture is heated to 75-80° G. until it takes on the appearance of Chocolate
Agar, then cooled to 50° C, poured into previously sterilized petri dishes, and
allowed to solidify. The surface of the medium is streaked with the suspected
diphtheritic material and the plates are then incubated at 35-37°G. for 24-48
hours.

Surface colonies of C. diphtheriae on the Tellurite Agar are black in color,
slightly raised, and may have entire or irregular margins. Most contaminating
organisms fail to grow on the complete medium due to its inhibitory action.

Bacto-Tellurite Blood Solution is not sterile, and cannot be recommended for
use except as described above. The tellurite present prevents growth of micro-
organisms and, during the heating at 75-80° C. in preparation of the medium,
contaminating organisms are killed or are inhibited by the potassium tellurite
during the incubation period. It is recommended that plates be inoculated the
same day as prepared.

Bacto-Tellurite Blood Solution is supplied in single bottles or in packages
of six bottles of 25 ml. each.

278 DIFGO MANUAL

BAGTO-MUELLER TELLURITE SERUM (B266)

Bacto-Mueller Tellurite Serum is a sterile selective enrichment used in the
preparation of the tellurite plating medium described by Mueller and Miller^
for the isolation of Corynebacterium diphtheriae. It is prepared according to the
directions given by the authors and contains lactate, pantothenate, beef serum
and potassium tellurite, as a selective agent. To prepare the complete medium,
add 25 ml. of Bacto-Mueller Tellurite Serum to 1000 ml. of sterile Bacto-
Mueller Tellurite Base at 50 °G. under aseptic conditions. Mix thoroughly avoid-
ing formation of air bubbles and distribute 20 ml. into sterile 95 mm. plates.
The resultant medium is transparent. A complete discussion of this medium is
given on page 149 under Bacto-Mueller Tellurite Base.

Bacto-Mueller Tellurite Serum is supplied in single bottles or in packages of
six bottles of 25 ml. each.

1 J. Bact., 51:743:1946.

ENZYMES

In this section are included enzymes generally used for the preparation of
Bacteriological culture media. We are pleased to present several new enzyme
preparations for microbiological and clinical laboratory procedures, including
Bacto-Penase, a potent Penicillinase, Bacto-Chicken Pancreas, for the liberation
of folic acid for microbiological assay, Bacto-Trypsin 1%, for Hemagglutination,
Bacto-Lysozyme and Bacto-Lysozyme Substrate.

PANGESTIN, DIFGO, 1:75 (B150)

{Pancreatic Enzymes)

Pangestin is an active preparation of pancreatic enzymes. It is strongly
amylolytic, proteolytic, and lipolytic. It is also free from starch and sugar. One
part of Pangestin, Dif co, 1 : 75 will digest 75 parts of cooked potato starch under
the conditions of the U.S. P. test for amylase in pancreatin and will also digest
75 parts of casein under conditions of the U.S. P. test for trypsin in pancreatin.
The optimum reaction for pancreatic amylase is pH 7.1, for pancreatic trypsin
it is pH 7.8, and for pancreatic lipase it is pH 8.0.

Pangestin, Difco, 1:75 is available in 500, 100 or 25 gram quantities.

PEPSIN, DIFGO, 1:10,000 (B151)

Pepsin is employed in the preparation of culture media solely because of its
ability to break down protein materials. One part of Pepsin, Difco, 1 : 10,000 will
digest 10,000 parts of freshly coagulated egg albumen under the conditions of
the U.S.P. test for pepsin. Pepsin acts best at a reaction of pH 1 .8.

Pepsin, Difco, 1:10,000 is available in 500, 100 or 25 gram quantities.

PAPAIN, DIFGO (B253)

Papain, Difco, is prepared from the fruit of the papaya (Carica papaya).
It is recommended as a source of proteolytic enzyme of vegetable origin for the
digestion of meat and other protein materials. Papain has also been used in

INGREDIENTS OF CULTURE MEDIA 279

treating red blood cells in blood grouping work by Kuhns and Bailey.^ Papain,
Difco is tested by and meets the National Formulary^ specification for papain
in that 0.1 gram digests 2.0 g. Bacto-Beef suspended in 50 ml. distilled water in
2 hours at 52 °C. Digestion with Papain may be carried out at an acid or alkaline
reaction, but is most active at pH 6.0-6.5 at 52°C.

Asheshov,^ in a description of the preparation of bacteriological culture media
using papain, stated that this enzyme was relatively heat stable, being most active
at 60-70°C. Temperatures above 70°G. diminished the rate of digestion and
activity stopped at 80°C.; best digestion was obtained at pH 7.0 and at a tem-
perature of 60-65 °C.

Papain, Difco is available in 500, 100 or 25 gram quantities.

1 Am. J. Clin. Path., 20:1067:1950. ^ Can. J. Pub. Health, 32:468:1941.

2 National Formulary, Eighth Edition:37i :i946.

RENNIN, DIFCO (B287)
Rennin, Difco is a desiccated enzyme obtained from the glandular layer of
the stomach of the calf. It is used for the enzymatic coagulation of milk. Rennin,
Difco conforms in every respect to the National Formulary standards for this
material.

Rennin, Difco is available in 500, 100 and 25 gram quantities.

TRYPSIN, DIFCO, 1:250 (B152)

Trypsin is used in the preparation of culture media to break down proteins
into simpler compounds which are more readily available for bacterial nutrition.
One part Trypsin, Difco, 1 : 250 will digest 250 parts of casein under the condi-
tions of the U.S. P. test for trypsin in pancreatin. Trypsin acts best at pH 7.8.

Trypsin, Difco, 1 : 250 has been used in the digestion of sputum and other body
fluids in the isolation of Mycobacterium tuberculosis and other acid-fast bacilli.
Haynes,^ using this method in comparison with the Hanks flocculation method,
reported 11 per cent more positive specimens with trypsin concentration. The
technique is easily performed and requires a minimum of time. Trypsin, Difco,
1 : 250 has proven especially useful for treating red blood cells to increase their
specificity in hemagglutinating systems with anti-Rh sera and other blood group
antibodies. A method that has been employed successfully for treating the red
cells with the enzyme is described by Wheeler, Luhby and Scholl.-'^ Wiener and
Katz* also found Trypsin, Difco, 1 : 250 to be well suited for the preparation of
red cells for Rh studies. Such trypsin-modified cells are extremely sensitive indi-
cators for the detection of anti-Rh and other blood group antibodies. Agglutina-
tion reactions with such cells are specific, easily read, immediate and unlikely to
show failure of agglutination or prozones. The complete prepared reagent for
Rh testing is discussed on page 280 under Bacto-Trypsin 1% for Hemagglutina-
tion. Rosenthal, Dameleshak and Brukhardt^ used Trypsin, Difco, 1 : 250 in
treating red blood cells in their study of cells in acquired hemolytic anemia.
Morris^ used Trypsin, Difco, 1 : 250 in his study of hemagglutination by murine
encephalomyelitis virus. He reported that virus which failed to agglutinate human
group O erythrocytes at 20 °C. did so in the presence of trypsin. Additional uses
of Try'psin in the microbiological laboratory are given under Bacto-Trypsin page
280.

Trypsin, Difco, 1 :250 is available in 500, 100 or 25 gram quantities.

1 J. Lab. Clin. Med., 27:806:1942. ^ j, Immunol., 65:39:1950.

2 Paper presented at a Symposium on Serology * j. inununol., 66:51 : 1951.

of Blood Groups, American Association of Im- ^ Am. J. Clin. Path., 21:635:1951.
munologist, 1949. « J. Immunol., 68:97: 1952.

280 DIFCO MANUAL

B AGTO-TR YPSIN ( B 1 53 )

Bacto-Trypsin is a filter sterilized and carefully standardized desiccation of
tryptic enzymes. It is rehydrated by the addition of sterile distilled water. Bacto-
Trypsin is recommended for the neutralization of the antitryptic power of serum
and also for use in blood culture.

Wright first made the observation that, in the treatment of suppurating wounds,
there may be a stage at which the infecting organisms grow luxuriantly and that
this stage was coincident with the lysis of the leucocytes. This destruction of the
white blood cells sets free the tryptic enzymes which neutralize the antitryptic
power of the blood and permits growth of the invading organisms. By utilizing
this principle Douglas and Colebrook^ evolved a method for blood culture work
which has been satisfactory.

Owen^ recommended Trypsin-Beef Tea as an ideal medium for blood culture.
His medium consisted of 10 ml. Bacto-Trypsin added aseptically to 90 ml. sterile
Beef Broth. Typhoid and other organisms tend to show early and luxuriant
growth in this medium. An alternative method consisted of collecting the blood
in sterile tubes containing 0.5 ml. Bacto-Trypsin per milliliter of blood. The
blood and trypsin are mixed, incubated at 35-3 7 °G. for a short period and the
mixture is then smeared directly upon agar media.

For tryptic digests of protein for the preparation of culture media, use Trypsin,
Dif CO, 1 : 250 as discussed above. For Rh Blood Grouping use Bacto-Trypsin 1 %
for Hemagglutination described below or Bacto-Trypsin, Difco, 1 : 250 discussed
above.

Bacto-Trypsin is supplied in packages of one half dozen and one dozen vials
of 10 ml. each.

1 Lancet, 2:181:1916. ^ J. Lab. Clin. Med., 2:198:1916-17.

BACTO-TRYPSIN 1% (B454)
for Hemagglutination

Bacto-Trypsin 1% for Hemagglutination is a carefully standardized water
soluble trypsin preparation designed especially for conditioning erythrocytes for
Rh determinations and other hemagglutination tests.

Hubener^ and Thomsen^ first showed that enzymes present in certain bacterial
filtrates could activate latent red cell agglutinogens. Friendenreich^ referred to
this type of reaction as "T agglutination". Burnet, McCrae and Stone* observed
that filtrates from Vibrio cholerae modified the virus hemagglutination of human
red cells. Pickles^ discovered that such V. cholerae treated cells became specifi-
cally agglutinable in saline dilutions of "incomplete" anti-Rh sera. Morton and
Pickles^ noted that trypsin behaved similarly to culture filtrates in increasing the
sensitivity and specificity of red cells sensitized with "incomplete" antibody and
also that it enhanced the specific agglutination of other hemagglutinins in the
absence of any detectable antibody of the "incomplete" type. The authors used
the beneficial effect of trypsin on erythrocytes as the basis for an improved test
for Rh sensitization.

Wheeler, Luhby and ScholF'^ and Wheeler and Taylor^ studied the effect of
trypsin and other enzymes on cells employed in Rh determinations and found
trypsin to be particularly suitable for conditioning erythrocytes for such tests.
The titers of anti-Rh sera were greater when determined with the trypsin-
modified cell technique than were those obtained with the albumin-tube technique
and in many cases the sensitivity of the titrations equalled that of the indirect

INGREDIENTS OF CULTURE MEDIA 281

Coombs test. The prozones commonly experienced in titrations using untreated
cells in albumin solutions, were completely eliminated when trypsin-modified
cells were employed. Wheeler^o found Trypsin, Dif co, 1 : 250 and Bacto-Trypsin
1% to be eminently satisfactory for use in treating erythrocytes employed in Rh
determinations. The latter preparation was preferable because of its greater
purity, ready solubility and ease of solution. Wiener and Katz^^ in their
studies on the use of enzyme-treated red cells in tests for Rh sensitization found
that the use of trypsinated red cells was a sensitive and specific method for testing
Rh-Hr antibodies and antibodies of other specificities. Enzyme-treated cells gave
more sensitive results in tests for Rh sensitization than other standard tests
hitherto introduced and equalled them in specificity. The titers obtained using
trypsinated erythrocytes were four or five times as high as by the albumin plasma
conglutination technique. In no case of Rh sensitization did the test with enzyme-
treated cells fail to demonstrate the presence of Rh antibodies although in several
of these cases the other tests were negative. Furthermore, the trypsinated cell
technique was observed to be a reliable method for demonstrating autosensitiza-
tion even in the absence of clinical manifestations. These authors used Trypsin,
Dif CO, 1 : 250 and noted no damage to any of the hemagglutinogen loci by this
enzyme.

Unger, Tortora and Mappi^^ jn their isoimmunization studies in rabbits used
Trypsin, Difco, 1:250 for conditioning the erythrocytes and observed that the
trypsinated cell method was more sensitive than either the saline agglutination
or albumin plasma methods for the detection of isoantibodies. Rosenfield and
Vogel^^ studied the effect of trypsinated erythrocytes on the identification of
hemagglutinins. They confirmed Wheeler's observations that the prozone phenom-
enon obtained with certain high titer sera in albumin is not observed with
optimally trypsin-treated erythrocytes. They found trypsinated cells to be sensitive
to agglutination by extremely small amounts of Rh antibody. Like Wiener and
Katz^^ these authors did not encounter an instance of sensitization to Rh-Hr that
was not detectable by this technique. This fact makes the enzyme technique just
as valuable in routine tests for Rh sensitization as the indirect antiglobulin test.
Rosenfield and Vogel also observed that trypsin treatment of the cells influenced
the A and B factors so that agglutination was of better avidity and the titration
values were higher than in saline. The O factor was not changed. The M and N
factors were largely destroyed by enzyme activity. The S factor was enhanced by
trypsin treatment. The Fy^ (Duffy) factor was destroyed by enzyme treatment.
Trypsin improved the Le^ and Le^ agglutination. Trypsin was also found to
accentuate hemolysin activity upon the red cells treated with this enzyme.
Trypsin enhanced agglutination with Anti-P sera. Agglutinogen for cold auto-
agglutinins was altered by enzyme treatment resulting in higher titration values
for cold agglutinins in general and a wider thermal amplitude. They recom-
mended careful standardization of the technique of treating cells with trypsin so
as to avoid false positive reactions. Rosenthal, Damelshak and Brukhardt^* used
Trypsin, Difco 1 : 250 in treating red blood cells in their study of cells in acquired
hemolytic anemia. Morris^^ used Trypsin, Difco 1 : 250 in his study of hemag-
glutination by murine encephalomyelitis virus. He reported that virus which
failed to agglutinate human group O erythrocytes at 20° C. did so in the presence
of trypsin. Bacto-Trypsin 1% is ideally suited for these determinations.

Bacto-Trypsin 1% for Hemagglutination is reconstituted by adding 10 ml.
Bacto-Hemagglutination Buffer to each 10 ml. vial. The resultant solution is
sparklingly clear and equivalent in tryptic activity to a 1 per cent solution of
Trypsin, Difco, 1 :250. The reaction of the final solution will be pH 7.2. The re-
constituted solution should be kept refrigerated at 2-6° G. or colder until used.
Bacto-Trypsin 1% for Hemagglutination when reconstituted is used according to

282 DIFGO MANUAL

the methods described by Wheeler, Luhby and Scholl,'^'^ Wiener and Katz^i or
by Rosenfield and Vogel.^^

Bacto-Trypsin 1% for Hemagglutination is supplied in packages of ten vials
of 10 ml. each.

*Ztschr. Immunitats Exper. Therap., 45:223: ^ j, Jmujunol., 65:39: 1950.

1925- . 0 Am. Assoc. Blood Banks, 1951.

2 Ztschr. Immunitats Exper. Therap., 52:85: i" Personal Communication, 1951.

1927- "^ J. Immunol., 66:51:1951.

8 The Thomsen Hemagglutination Phenomenon, '^ The Laboratory Digest, 15:5:1951.

Levin & Munksgaard, 1930. " New York Academy of Sciences, 13:213:1951.

* Brit. J. Exp. Path., 27:228:1946. 1* Am. J. Clin. Path., 21:635:1951.

6 Nature, 158:880:1946. "J. Immunol., 68:97:1952.
« Nature, 159:779: i947-
' Symposium on Serology of Blood Groups. Am,

Assoc. Immunol., 1949.

BAGTO-HEM AGGLUTINATION BUFFER (B454D)

Bacto-Hemagglutination Buffer, prepared according to the formula of Wheeler,
Luhby and Scholl,^ is a desiccation consisting of sodium chloride, disodium phos-
phate and potassium dihydrogen phosphate. It is recommended for the prepara-
tion of isotonic buffered saline for use in hemagglutination studies. It is especially
recommended for rehydrating Bacto-Trypsin 1 % and for suspending and washing
red cells preparatory for hemagglutination.

Bacto-Hemagglutination Buffer is rehydrated by dissolving 8.6 grams in 1000
ml. distilled water. The resultant solution is isotonic for blood cells and has a pH
of 7.3.

Bacto-Hemagglutination Buffer is conveniently packaged in vials containing
8.6 grams, sufficient for one liter, and supplied in boxes of ten vials.

1 J. Immunol., 65:39:1950.

INVERTASE FOR ANALYTICAL USE (B154)

Invertase for Analytical Use is an enzymatic extract of yeast cells. It is pack-
aged in ampuls of 10 ml. each of a standardized extract, 5 ml. of which will
cause complete inversion of 50 ml. of a 10 per cent sucrose solution in one hour
at room temperature. Invertase for Analytical Use is most active at a reaction
of pH 4.4-4.6. The K value of Invertase Analytical is K (A.O.A.G.) =0.1.

Invertase for Analytical Use is supplied in packages of one-half dozen and one
dozen ampuls of 10 ml. each.

BACTO

CHICKEN PANCREAS (B459)

DEHYDRATED

Bacto-Chicken Pancreas is standardized desiccated chicken pancreas used in
the enzymatic liberation of folic acid from its conjugated state. It is prepared
according to the method described by the Official Agricultural Chemists.^ Bacto-
Chicken Pancreas, is recommended for use in techniques requiring this enzyme,
among which the following may be cited :

Official Methods of Analysis of the Association of Official Agricultural Chem-
ists,2 Methods of Vitamin Assay,^ Sreenivasan, Harper and Elvehjem,* Burk-

INGREDIENTS OF CULTURE MEDIA 283

holder, McVeigh and Wilson,^ Mimms and Laskowski,^ Bird, Bressler, Brown,
Campbell and Emmett'^ and Laskowski, Mimms and Day.®
Bacto-Chicken Pancreas is supplied in 10 gram packages.

1 A.O.A.C, Seventh Edition: 785: 1950. ^ Arch, of Biochem., 7:287:1945.

^A.O.A.C, Seventh Edition: 786: 1950, « J. Biol. Chem., 160:^93:1945.

'The Association of Vitamin Chemists, Inc., ' J. Biol. Chem., 159:631 : 1945.

Second Edition : 231 :i95i. s j, giol. Chem., 157:731:1945.
^ J. Biol. Chem., 177:117:1949.

B AGTO-PEN ASE ( B345 )

Bacto-Penase is a potent sterile thermolabile biological extract capable of
neutralizing the antibiotic properties of all known types of penicillin. It is non-
toxic to microorganisms and, therefore, particularly well suited for use in media
for culturing bacteria from blood and other body fluids containing penicillin
and for estimating penicillin levels in such fluids.^-- "Diagnostic Procedures and
Reagents"^ recommend that a penicillin inactivator, penicillinase, be included
in media for culturing organisms from blood of patients under penicillin therapy.
Bacto-Penase is ideally suited for this purpose. It is also satisfactory for use in the
sterility testing of penicillin and products containing penicillin and for ascertain-
ing the microbial counts on penicillin-containing products. Bacto-Penase or
Bacto-Penase Concentrate is recommended as a penicillinase for use in all official
procedures for the sterility testing of penicillin as specified by the U. S. Govern-
ment.* Most laboratories prefer to use Bacto-Penase Concentrate in such pro-
cedures.

One ( 1 ) ml. of Bacto-Penase will inactivate 50,000 units or more of penicillin
as indicated by the following test procedure:

Prepare dilutions of Bacto-Penase of 1 : 2, 1 : 3, 1 : 4, 1 : 5, 1 : 6, 1 : 7, 1 : 8, 1 : 9 and
1:10 in sterile distilled water. Add 1 ml. of each dilution to a series of test tubes
containing 15 ml. of Bacto-Fluid Thioglycollate Medium. Add 1.0 ml. of a solu-
tion of penicillin containing 5000 units per ml. to each tube and allow to remain
at room temperature for two hours. Inoculate each tube with 1.0 ml. of a 24-hour
broth culture of Micrococcus pyogenes var. aureus P209 diluted 1:1000 and
incubate at 37°C. for 24 hours. Determine the highest dilution permitting growth
in this period and multiply this dilution of Bacto-Penase by 5000. This will give
the actual units of penicillin that 1.0 ml. of Bacto-Penase will inactivate.

M. pyogenes var. aureus P209 will show growth in 18-24 hours in 15 ml. of
Bacto-Fluid Thioglycollate Medium or Bacto-Brain Heart Infusion with PAB
and Agar containing 50,000 units of penicillin and 1 ml. of Bacto-Penase. Suitable
controls show that less than 3 units of penicillin per 15 ml. of medium inhibit
growth completely in the absence of Bacto-Penase, and that Bacto-Penase is not
inhibitory to the growth of microorganisms. The maximum efficiency of Bacto-
Penase against penicillin will be obtained if the reacting substances are contained
in small volumes.

In culturing blood or other body fluids containing penicillin it is recommended
that 1 ml. of Bacto-Penase be added to each 100 ml. or less of sterile Bacto-
Brain Heart Infusion with PAB or Bacto-Brain Heart Infusion with PAB and
Agar. This will assure inactivation of the maximum amount of penicillin that
would be present in 10 ml. of blood or other body fluid. A medium so prepared
will also inactivate sulfonamides and up to 1000 units of streptomycin introduced
with the inoculum.

The sterility of penicillin is determined by adding the test sample to sterile
Bacto-Fluid Thioglycollate Medium containing Bacto-Penase and incubating the
tubes at 35-3 7 °C. for 4 days. The test is performed in tubes containing 15 ml. of

284 DIFGO MANUAL

medium. At least 1 ml. of Bacto-Penase for each 50,000 units of penicillin to be
tested should be incorporated in 15 ml. of sterile Bacto-Fluid Thiogly collate
Medium under aseptic conditions. The medium containing Bacto-Penase must not
be heated. Most laboratories find it more convenient to use Bacto-Penase Con-
centrate for sterility testing. The sterility testing of penicillin products for yeasts
and molds is accomplished by adding the test samples to Bacto-Sabouraud Liquid
Medium and incubating for 5 days at room temperature. The sterility testing of
products for yeasts and molds which have high concentrations of penicillin may
require the addition of Bacto-Penase Concentrate to the medium.

Bacto-Penase is stable when stored at refrigerator temperatures. At normal
room temperature slow deterioration of Bacto-Penase does occur, and for that
reason this product, or media prepared with it, should be stored at 2-6° C.

Bacto-Penase is regularly supplied in packages of one-half dozen tubes of 20
ml. each.

1 Science, loi :365: 1945. * Compilation of Regulations for Tests and

2 Science, 102:355:1945. Methods of Assay and Certification of Anti-

3 Diagnostic Procedures and Reagents, Third biotic Drugs, Federal Security Agency, Food
Edition: 55: 1950. and Drug Administration.

BACTO-PENASE CONCENTRATE (B346)

Bacto-Penase Concentrate is a highly potent sterile thermolabile biological ex-
tract possessing the capacity to neutralize the antibiotic properties of all known
types of penicillin. It is non-toxic to microorganisms and is therefore, particu-
larly satisfactory for use in the sterility testing of penicillin or products containing
penicillin, and in ascertaining the microbial counts on penicillin-containing prod-
ucts. Bacto-Penase Concentrate is recommended as a penicillinase for use in all
official procedures for the sterility testing of penicillin as specified under the
regulations promulgated by the U. S. Government.^ Bacto-Penase Concentrate
may be used in media for culturing organisms from blood and other body fluids
containing penicillin, and for estimation of penicillin levels in such fluids, but
for these purposes Bacto-Penase is recommended.

One ml. of Bacto-Penase Concentrate will inactivate 500,000 units of penicillin,
as indicated by the following test procedures:

Prepare dilutions of Bacto-Penase Concentrate of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,
1:8, 1:9 and 1 : 10 in sterile distilled water. Add 1 ml. of each dilution to a series
of test tubes containing 15 ml. of Bacto-Fluid Thioglycollate Medium. Add 1 ml.
of a solution of penicillin containing 50,000 units per ml. to each tube and allow
to remain at room temperature for two hours. Inoculate each tube with 1 ml. of
a 24-hour broth culture of Micrococcus pyogenes var. aureus P209 diluted 1 to
1000 and incubate at 35-37°C. for 24 hours. Determine the highest dilution
permitting growth in this period, and multiply this dilution of Bacto-Penase
Concentrate by 50,000. This will give the actual units of penicillin that Bacto-
Penase Concentrate will inactivate.

The sterility of penicillin is determined by adding the test sample to sterile
Bacto-Fluid Thioglycollate Medium containing Bacto-Penase Concentrate and
incubating the tubes at 35-37°C. for 4 days. The test is performed in tubes con-
taining 15 ml. of medium. At least 1 ml. of Bacto-Penase Concentrate for each
500,000 units of penicillin to be tested should be incorporated in 15 ml. of sterile
Bacto-Fluid Thioglycollate Medium under aseptic conditions. The medium con-
taining Bacto-Penase Concentrate must not be heated. Bacto-Penase Concentrate
is recommended for testing the sterility of 50,000 units or larger amounts of peni-
cillin, while Bacto-Penase may be used for the sterility testing of smaller amounts
of penicillin.

INGREDIENTS OF CULTURE MEDIA 285

The sterility testing of penicillin products for yeasts and molds is accomplished
by adding the test samples to Bacto-Sabouraud Liquid Medium and incubating
for 5 days at room temperature. The sterility testing of products for yeasts and
molds which have high concentrations of penicillin may require the addition of
Bacto-Penase Concentrate to the medium.

When employed in media such as Bacto-Brain Heart Infusion with PAB and
Agar for culturing blood or other body fluids containing penicillin, it is recom-
mended that Bacto-Penase Concentrate be used in a concentration to give a final
unitage of 100 units or more per ml. of medium.

Bacto-Penase Concentrate is stable when stored at refrigerator temperatures.
At normal room temperature slow deterioration of Bacto-Penase Concentrate
does occur, and for that reason this product, or media prepared with it, should
be stored at 2-6°C.

Bacto-Penase Concentrate is regularly supplied in packages of one-half dozen
tubes of 20 ml. each or packages of one-half dozen bottles of 100 ml. each.

1 Compilation of Regulations for Tests and biotic Drugs, Federal Security Agency, Food

Methods of Assay and Certification of Anti- and Drug Administration.

B AGTO-LYSOZ YME ( B465 )

BAGTO-LYSOZYME SUBSTRATE (B461)

B AGTO-LYSOZ YME BUFFER (B464)

Bacto-Lysozyme is a crystalline enzyme prepared from fresh egg white, con-
taining sufficient Bacto-Lysozyme Buffer to give a solution of pH 6.2 when re-
hydrated with distilled water. Bacto-Lysozyme Substrate is a dried standardized
irradiated culture of Micrococcus lysodeikticus recommended for use in the
turbidimetric assay of Lysozyme activity of body fluids, extracts from animal
tissues and other materials suspected of containing this enzyme. It is supplied
with sufficient Bacto-Lysozyme Buffer to give a suspension of pH 6.2 when re-
hydrated with distilled water. Bacto-Lysozyme Buffer, rehydrated in distilled
water is a phosphate solution at pH 6.2 used in the test. The test employs the
principles of Fleming,^ Goldsworthy and Florey,^ Boasson^ and Hartsell and
Smolelis.* A detailed discussion of the test is available upon request.

1 Proc. Roy. Soc. (London), B, 93:306:1922. ^ J. Immunol., 34:281:1938.

sBrit. J. Exp. Path., 11:192:1930. * J. Bact., 58:731 :»949'

286 DIFCO MANUAL

BILE PRODUCTS

B AGTO-OXG ALL ( B 1 28 )

Bacto-Oxgall is a dehydrated fresh bile, prepared especially for use in making
bile media. It is manufactured from large quantities of fresh bile by rapid
evaporation of the water content. The use of Bacto-Oxgall insures a regular
source of supply independent of the slaughterhouse, and assures a degree of
uniformity impossible to obtain with fresh materials, eliminating the uncertainty
of variable results. The equivalent of fresh bile is attained in a 10 per cent solu-
tion of Bacto-Oxgall.

Bacto-Oxgall is recommended for use in the preparation of media for the
detection and propagation of intestinal organisms. Specifically, a large number
of bile-containing media have been devised for use in the bacteriological exami-
nation of water. Bacto-Oxgall is most frequently used in the preparation of
culture media for water analysis. The Ninth Edition of "Standard Methods for
the Examination of Water and Sewage"^ recommends Brilliant Green Lactose
Bile Broth as discussed on page 37, for confirming positive presumptive tests of
coliform bacteria in water. In this text Brilliant Green Lactose Bile Agar, as
discussed on page 52, is described as a selective agar medium for the direct plate
count of the coliform group. Bacto-Oxgall is recommended as an ingredient of
both these media. Brilliant Green Lactose Peptone Bile 2 per cent is also ap-
proved in "Standard Methods for the Examination of Dairy Products"- for
detecting the presence of coliform bacteria in milk.

Solutions of Bacto-Oxgall, added to broth cultures of pneumococci, produce
lysis of the cells, and can therefore be used in bile solubility tests for differentiat-
ing pneumococci from the bile-insoluble streptococci. The general procedure of
the bile solubility test is to add one part of a sterile 10 per cent solution of Bacto-
Oxgall to nine or ten parts of culture. Evans^ and McKinney* have used Bacto-
Oxgall for this purpose. Greey^ reported excellent results using dry Bacto-Oxgall
sprinkled directly on Blood Agar plates. According to this procedure pneumococ-
cus colonies are dissolved and disappear entirely, but leave evidence of their
presence by means of the fixed blood cells in the clear medium. Colonies of
Streptococcus viridans are not dissolved or otherwise altered. Bacto-Oxgall is also
used in solution for the enrichment of blood cultures.

Bacto-Oxgall has been used in the preparation of a selective medium for fungi.
Littman^ described an agar medium for the isolation of pathogenic fungi, em-
ploying Bacto-Oxgall, crystal violet and streptomycin as inhibiting agents for
bacteria. A complete discussion of this medium is given on page 240.

^Standard Methods for the Examination of ^ J. Bact., 31 1423: 1936.

Water and Sewage, gth Edition, 1946. * J. Bact., 27:373:1934.

"Standard Methods for the Examination of ^ J. Infectious Diseases, 64:206:1939.

Dairy Products, 9th Edition, 1948. ^ Science, 106:109:1947.

BAGTO-BILE SALTS (B129)

Bacto-Bile Salts was originally developed for use in the Lactose Bile Salt Agar
of MacConkey,^ one of the best known of the plating media for the isolation of
organisms of the colon-typhoid group. As originally described by MacConkey,
the medium was a peptone lactose, or glucose, agar containing 0.5 per cent
sodium glycocholate. Bacto-Bile Salts fulfilled the requirements of the original
sodium glycocholate. However, in recent studies, we have developed Bacto-Bile
Salts No. 3 which has been found to be more suitable than the Bacto-Bile Saitb.
previously employed in the MacConkey Agar.

INGREDIENTS OF CULTURE MEDIA 287

Bacto-Bile Salts has proven particularly valuable in the preparation of Bacto-
Tetrathionate Broth. This medium is discussed on page 157.

Bacto-Bile Salts is readily soluble in distilled water and is neutral in reaction.
It may, therefore, be used in preparing media without adjustment of reaction
or filtration.
ij. Hyg., 5:333:1905.

BACTO-BILE SALTS NO. 3 (B130)

Bacto-Bile Salts No. 3 is a modification of Bacto-Bile Salts and is prepared
especially for use in Bacto-MacConkey Agar, Bacto-S S Agar and Bacto- Violet
Red Bile Agar. In our investigations on these media we found that the Bacto-Bile
Salts gave a somewhat heavy precipitate around lactose fermenting colonies,
which, under certain conditions, made it difficult to detect colonies of the patho-
genic lactose non-fermenters. Bacto-Bile Salts No. 3 obviates this fault, giving a
clearer medium and also reducing the precipitation around the coliform colonies,
thus facilitating the detection of lactose non-fermenting colonies. A complete
discussion of these media, using Bacto-Bile Salts No. 3, is given on pages 131, 134,
and 61, respectively.

Bacto-Bile Salts No. 3 is readily soluble in distilled water and is neutral in
reaction. It may, therefore, be used in preparing media without adjustment of
reaction or filtration.

SODIUM TAUROGHOLATE, DIFGO (B278)

Sodium Taurocholate, Difco is the sodium salt of a conjugated bile acid used
generally in bacteriological culture media for the isolation and cultivation of
members of the enteric group. Like whole bile, it has the cultural characteristic
of inhibiting the growth of Gram-positive organisms and spore forming bacteria,
without inhibiting the development of Gram-negative bacilli.

Sodium Taurocholate, Difco contains about 75 per cent sodium taurocholate
along with other naturally occurring salts of bile acids. It is readily soluble in
distilled v/ater forming a clear amber solution, neutral in reaction.

SODIUM DESOXYGHOLATE, DIFGO (B248)

Sodium Desoxycholate, Difco is the sodium salt of desoxycholic acid. It is
used in bacteriological culture media and in the pneumococcus bile solubility
test. Sodium desoxycholate is readily soluble in distilled water, giving a clear
colorless solution neutral in reaction.

Sodium desoxycholate, like bile and other bile salts when used in bacterio-
logical culture media, is inhibitive to Gram-positive cocci and spore forming
organisms but is not inhibitory to Gram-negative enteric bacilli. Since sodium
desoxycholate is a salt of a highly purified bile acid it is used in culture media in
lower concentrations than is naturally occurring bile.

Leifson^ described media for the enumeration of coliform organisms from
milk, water, sewage, etc., and for the isolation of enteric pathogens using sodium
desoxycholate to inhibit Gram-positive organisms. He also prepared a selective
medium using sodium desoxycholate and sodium citrate for the isolation of
intestinal pathogens. Coliform as well as Gram-positive organisms are inhibited
on this Desoxycholate Citrate Agar. Bacto-Desoxycholate Agar and Bacto-Desoxy-
cholate Citrate Agar, prepared with sodium desoxycholate, are discussed on pages
63 and 138, respectively.

Sodium desoxycholate is also used in 10 per cent concentration in the bile
solubility test for pneumococci.

1 J. Path. Bact., 40:581:1935.

288 DIFCO MANUAL

DEHYDRATED MEATS
FOR INFUSIONS

BAGTO-BEEF (B131)

Bacto-Beef is desiccated powdered fresh lean beef. It is prepared especially for
use in making beef infusion media. Large quantities of beef are processed at one
time in order to secure a uniform and homogenous product. The nutritive
qualities of the fresh beef are retained in Bacto-Beef and may be preserved in
the infusions prepared from it. Media prepared from Bacto-Beef are superior to
beef extract media and are equal to infusion media made from market beef.

The equivalent of 500 grams of fresh lean beef is secured from 100 grams of
Bacto-Beef. An excellent infusion, however, can be prepared by using 50 grams
of Bacto-Beef per 1000 ml. of distilled water. This mixture is infused at 50° C.
for one hour. It is then heated to boiling for a few minutes to coagulate some of
the proteins and is filtered. Peptone and the other ingredients of the medium are
then added to the filtrate. After adjustment of the reaction to pH 7.5-7.8 and
subsequent boiling for a few minutes there will be a further coagulation which
should be removed by filtration before the medium is sterilized.

Infusion media prepared from Bacto-Beef have been recommended for use
in the microbial examination of butter.^ Herrold- has also used an infusion from
Bacto-Beef in preparing Blood Agar for primary cultivation of the gonococcus.
Bacto-Beef is used in the National Formulary^ specifications in determining the
proteolytic activity of papain.

1 J. Dairy Science, 16:289: 1933. 3 National Formulary, Eighth Edition:37i : 1946.

2 J. Infectious Diseases, 42:79:1928.

BAGTO-BEEF HEART FOR INFUSIONS (B132)

Bacto-Beef Heart for Infusions is prepared from fresh beef heart tissue and
is particularly recommended for preparing heart infusion media. Bacto-Beef
Heart for Infusions is processed from large quantities of raw material, retaining
all the nutritive and growth-stimulating properties of the fresh tissues.

One hundred grams of Bacto-Beef Heart for Infusions are the equivalent of
500 grams of fresh heart tissue. Generally, excellent infusions can be prepared
using 50 grams of Bacto-Beef Heart for Infusions per 1000 ml. of distilled water.
For best results infuse at 50° C. for 1 hour, then heat to boiling for a few minutes
to coagulate some of the proteins, and filter. Peptone and the other ingredients
of the medium should then be added to the filtrate, the reaction adjusted to
pH 7.5-7.8, and the medium boiled and filtered before sterilizing.

Bacto-Beef Heart for Infusions is recommended for the preparation of St.
John's Medium^ for the cultivation of Endamoeba histolytica by Brown in
'"Diagnostic Procedures and Reagents"- of the American Public Health Associa-
tion. Morton, Smith and Leberman^ used Bacto-Beef Heart for Infusions in a
medium recommended for the cultivation and isolation of Pleuropneumonia-like
organisms.

Bacto-Beef Heart for Infusions is not to be confused with Bacto-Beef Heart
(for antigens) as discussed on page 315. The latter product is prepared for an.

INGREDIENTS OF CULTURE MEDIA 289

entirely different purpose and is not interchangeable with Bacto-Beef Heart for
Infusions.

1 Am. J. Trop. Med., 12:301:1932. s Am. J. Syphilis Gonorrh. Venereal Diseases,

2 Diagnostic Procedures and Reagents, 3rd Edi- 35:361:1951-
1100:417:1950.

BAGTO-LIVER (B133)

Bacto-Liver is prepared from large quantities of carefully trimmed fresh beef
liver and is recommended for use in preparing liver infusion media. The nutritive
factors of fresh liver tissue are retained in infusions prepared from Bacto-Liver.

The equivalent of 500 grams of fresh liver is obtained with 135 grams of
Bacto-Liver. An excellent infusion can be prepared by using 75, or even 50, grams
of Bacto-Liver per 1000 ml. of distilled water. The latter concentration is recom-
mended for media for general culture purposes. The infusion is made by warm-
ing the mixture to 50° C. and holding it at this temperature with frequent agita-
tion for one hour. The mixture is heated to boiling for a few minutes to coagulate
a portion of the proteins, and is filtered. Peptone and other ingredients of the
medium are then added to the filtrate. After adjustment of the reaction to pH
7.5-7.8 and subsequent boiling, there will be a further coagulation which should
be removed by filtration before the medium is sterilized.

BAGTO-VEAL (B134)

Bacto-Veal is desiccated powdered fresh lean veal, retaining the growth-
promoting properties of the fresh tissue, and is recommended for use in the
preparation of veal infusion media.

Eighty-five grams of Bacto-Veal are the equivalent of 500 grams of fresh lean
veal. An excellent infusion can be prepared by using 50 grams of Bacto-Veal per
1000 ml. of distilled water, but larger quantities may be used if desired. Bacto-
Veal should be infused at 50° C. for 1 hour, then heated to boiling for a few
minutes to coagulate some of the proteins, and filtered. Peptone and the other
ingredients may then be added to the filtrate and the reaction adjusted to pH
7.5-7.8. The medium is boiled and filtered before sterilization.

Veal Broth for the preparation of diphtheria toxin can be prepared by infusing
80 grams of Bacto-Veal in 1000 ml. of distilled water at 38°C. for 1 hour. To
remove the muscle sugars the infusion is then inoculated with a vigorously grow-
ing culture of Escherichia coli and incubated overnight at room temperature.
After heating to 85 °C. and filtering, 20 grams of peptone and 5 grams of sodium
chloride are added and the reaction adjusted to pH 8.0. The medium is then
boiled for 5 minutes, filtered and the filtrate distributed in 100 ml. amounts in
300 ml. Erlenmeyer flasks and sterilized in the autoclave for 15 minutes at 15
pounds pressure (121°C.). The sterile medium is inoculated with an 18-hour
culture of Corynebacterium diphtheriae which has been previously acclimated to
the medium. A potent toxin can usually be harvested after incubation at 36°G.
for 4-7 days.

290 DIFCO MANUAL

SOLDIFYING AGENTS

BAGTO-AGAR (B140)

The introduction of agar-agar as a solidifying agent for culture media was an
important step in the advancement of the science of bacteriology. Credit for the
use of agar for this purpose is generally given to Fannie Hesse. ^-^ Agar media
have made possible many of the advances of bacteriology, for the use of these
solid media paved the way for our present day methods of pure culture isolation
and study.

Bacto-Agar is a purified agar from which the extraneous matter, pigmented
portions, and salts are reduced to a minimum. Bacto-Agar is available in the form
of fine, light-colored granules, which are convenient for weighing and handling.
It is usually employed in solid culture media in concentrations of 1-2 per cent.
Recently the use of small quantities of agar (0.05-0.3 per cent) in media has
become quite general for the determination of motility and the growth of
anaerobes and micro-aerophiles. The addition of such amounts of agar to liquid
media, as discussed under Bacto-Brain Heart Infusion on page 77, permits all
degrees of oxygen tension to exist and thus aids in the development of many
fastidious aerobic and anaerobic organisms.

The value of the use of small quantities of agar in media for sterility testing
was pointed out by Falk, Bucca and Simmons^ and has been incorporated in
the thioglycollate medium for sterility testing of biologies and antibiotics by
official procedures.*'^'®''^

^ Hueppe: Die Methoden der Bakterienfor- ^ Compilation of Regulations for Tests and

schung, 1891. Methods of Assay and Certification of Anti-

2 J. Bact., 37:485: 1939. biotic Drugs, Federal Security Agency, Food

5 J. Bact., 37:121 :i939. and Drug Administration.

•♦National Institute of Health Circular: Culture ^Pharmacopeia of the United States, XIV Re-
Media for the Sterility Test, and Revision, vision: 758: 1950.
Feb. 25, 1946. ' National Formulary, 9th Edition: 768: 1950.

SPEGIAL AGAR (NOBLE) (B142)

Special Agar (Noble) is a carefully washed agar, free from all impurities
which would interfere with its efficiency when employed in the preparation of
Noble's^ Cyanide Citrate Agar, for the direct plating and counting of the coli-
aerogenes group. It is prepared according to the procedure suggested by Noble
and each lot is tested for its usefulness in the preparation of his medium. Noble
and Tonney- specify the use of Special Agar (Noble) as an ingredient of their
Brilliant Green Lactose Bile Agar for the direct plate count of the coliform group
in water. The medium, Bacto-Brilliant Green Bile Agar, is discussed on page 52.

^ J. Am. Water Works Assoc, 19:182:1928. ^ j_ Am. Water Works Assoc, 27:108:1935.

B AGTO-GELATIN ( B 1 43 )

Gelatin was first employed as a solidifying agent for bacteriological culture
media by Koch in 1881. This innovation paved the way for the future of the
science although gelatin media were soon replaced by others containing agar as
the solidifying material.

INGREDIENTS OF CULTURE MEDIA 291

Bacto-Gelatin is a high grade gelatin in granular form for convenience in
handling. This granular form of gelatin is readily soluble in warm water. Solu-
tions of Bacto-Gelatin are light in color and sparklingly clear in appearance.
They generally require no adjustment of reaction when used in culture media, as
in the Nutrient Gelatin described in "Standard Methods of Water Analysis" of
the American Public Health Association. In a 12 per cent concentration Bacto-
Gelatin has a melting point between 28 and 30° C. and a reaction of pH 6.8.

The use of Bacto-Gelatin in culture media for studies of gelatinolysis (elabo-
ration of gelatinolytic enzymes) by bacteria is recommended by the Committee
on Bacteriological Technic^ of the Society of American Bacteriologists. Levine
and Carpenter- and Levine and Shaw^ also employed Bacto-Gelatin in the media
used in their studies of gelatin liquefaction. Garner and Tillett* used culture
media prepared with Bacto-Gelatin in their study of the fibrinolytic activity of
hemolytic streptococci.

The formula for Nutrient Gelatin as given in "Standard Methods" specifies

the use of 12 per cent gelatin. Other formulae may call for larger or smaller

quantities of gelatin or for combinations of gelatin and agar.

1 Pure Culture Study of Bacteria, 4:No.3:i936. ^ J. Bact., 9:225:1924.

3 J. Bact., 8:297:1923. * J. Exp. Med., 60:255:1934.

CARBOHYDRATES, POLYHYDRIC
ALCOHOLS, AND GLUCOSIDES

Carbohydrates, polyhydric alcohols, glucosides and salts of organic acids are
extensively employed in culture media as a source of energy for bacteria and,
more particularly, for differentiating genera and identifying species. The ability
of an organism to attack a particular carbohydrate is a definite characteristic of
bacterial species and under controlled conditions remains constant for the organ-
ism throughout generations of cultivation on media.

These carbohydrates, polyhydric alcohols, glucosides and salts of organic acids
are prepared with the utmost care to eliminate admixtures, other sugars or con-
taminating materials. They are standardized to the highest known degree of
purity, having in view the consensus of authority concerning their respective
properties. In addition to the carbohydrates described below we also prepare
filter sterilized solutions of a selected number of carbohydrates. These solutions
are prepared in 10 per cent concentration and are discussed on page 293.

B AGTO-DEXTROSE ( B 1 55 )

Bacto-Dextrose is a specially prepared dextrose (c?-glucose) of unusual purity,
recommended for use as a readily available source of energy for bacteria and
also for fermentation studies. It is free from all other sugars and from starch,
proteins, alcohol and heavy metals. In appearance it is a pure white crystalline
powder. Its specific rotation lies between -f 52.5 and -|-52.76.

Bacto-Dextrose is widely used in studies of the fermentative processes of
bacteria. In fluid media it is usually employed in a concentration of 0.5 per cent,
but in solid media it may be used in higher concentrations. Dextrose media also
have a distinct value in the rejuvenation of cultures because this carbohydrate
is readily assimilated by most bacteria. As small an amount of dextrose as 0.05

292 DIFCO MANUAL

per cent added to a carbohydrate-free medium causes a definite increase in the
rate of growth of many microorganisms.

BACTO-LAGTOSE (B156)

Bacto-Lactose is a highly purified disaccharide, recommended for use in the
study of the fermentation reactions of bacteria. It is free from dextrose as deter-
mined by culture of Salmonella typhosa and controlled by determinations of
changes in hydrogen ion concentration. It contains no trace of casein or other
proteins, starch, alcohol or heavy metals. Its specific rotation is +52.4 with
0.5° tolerance.

Lactose media are of distinct value for the presumptive test for coliform
bacteria in water and milk. Many of the differential media used for identifica-
tion of organisms of the colon-typhoid-dysentery group also contain this carbo-
hydrate either alone or in combination with other fermentable substances. In
fluid media, lactose is usually employed in a concentration of 0.5 per cent, but
in solid media higher concentrations may be used.

ADDITIONAL COMPOUNDS

In addition to the above we also prepare the following purified carbohydrates,
polyhydric alcohols, glucosides and salts of organic acids. These are especially
suited for bacteriological culture media.

Adonitol (Adonite) (B157) Invert Sugar Syrup (B166) Rhamnose (Isodulcitol)

Aesculin (B158) Lactose (see Bacto-Lactose) (B175)

/-Arabinose (B159) (B156) Saccharose (Sucrose) (B176)

Cellobiose (Bi6q) Levulose (J-Fructose, from Salicin (B177)

Dextrin (B161) Inulin) (B167) Soluble Starch (B178)

Dextrose (<f -Glucose, see Maltose (B168) Sodium Hippurate (B330)

Bacto-Dextrose) (B155) Maltose, Technical (B169) ^/-Sorbitol (Sorbite)

Dulcitol (Dulcite) (B132) ^-Mannitol (Mannite) CB170) (B179)

J-Galactose (B163) J-Mannose (B171) Trehalose (UiBoJ

Glycerol (B282) Melezitose (B172) ^/-Xylose (B181)

t-Inositol (B164) Melibiose (B173)

Inulin (B165) Raffinose (B174)

CARBOHYDRATE SOLUTIONS IN AMPULS

Carbohydrate solutions, sterilized by filtration, are prepared for the use of
laboratories desiring fermentation media in which the carbohydrate has not been
heat sterilized. These carbohydrates are ampuled in 10 ml. amounts, each ampul
containing 1 gram of the carbohydrate in sterile solution. Carbohydrate solutions
in ampuls are convenient and economical when small quantities of fermentation
media are required.

For the preparation of carbohydrate media, the basic sugar-free medium is
prepared and sterilized as usual. The desired carbohydrate solution is then added
aseptically and the medium is dispensed into sterile containers with aseptic pre-
cautions. To prepare a medium containing one per cent of the carbohydrate the
contents of one ampul are added to 90 ml. of medium; to prepare a medium
with 0.5 per cent of the carbohydrate, the contents of one ampul are added to
190 ml. of the sterile sugar-free base. The final medium should always be incu-
bated before use to insure its sterility.

In conjunction with these solutions reference should be made to Bacto-

INGREDIENTS OF CULTURE MEDIA 293

Phenol Red Broth Base, Bacto-Phenol Red Agar Base, Bacto-Purple Broth
Base and Bacto-Purple Agar Base as discussed on pages 186-190. The following
carbohydrate solutions are supplied in packages of one-half dozen or one dozen
ampuls of 10 ml. each:

Dextrose Solution 10% (B155A) Maltose Solution 10% (B168A)
Galactose Solution 10% (B163A) Mannitol Solution 10% (B170A)
Glycerol Solution 10% (B282A) Saccharose Solution 10% (B176A)

Lactose Solution 10% (B156A)

DYES AND INDICATORS

Dyes and indicators are essential to the preparation of most differential culture
media. In such media the dyes may act as bacteriostatic agents, as inhibitors of
growth or as indicators of changes in the degree of acidity or alkalinity of the
substrate. It is obvious, therefore, that only dyes of known purity and known
dye content should be used in the preparation of media.

Great care is necessary in the preparation of differential or selective media,
even with dyes standardized by the most modern methods. All ingredients of the
medium must be used in the amounts specified in the formula and the reaction
of the medium must be adjusted with extreme care in order to obtain proper
results. Certain of the dyes listed in this section are identified by their Color
Index number (C.I. No.) according to "Color Index" of the Society of Dyers
and Colourists, 1924. The actual dye content of each dye is shown on each label.

BAGTO-BASIG FUGHSIN (B191)
For Endo Agar and Other Basic Fuchsin Media

Bacto-Basic Fuchsin (C.I. No. 676) is certified for use in culture media and
for the general histological and bacteriological staining by the Commission on
Standardization of Biological Stains.

Bacto-Basic Fuchsin is recommended for the preparation of Endo Agar and
other basic fuchsin media. In Endo Agar this dye is partially decolorized with
sodium sulfite. On this medium lactose fermenting organisms form red colonies
and change the color of the surrounding medium from faint pink to red; typical
Escherichia coli colonies, in addition to being red in color, generally exhibit a
brilliant metallic sheen. Organisms not fermenting lactose form uncolored
colonies which do not alter the appearance of the medium.

In Tryptose Agar, discussed on page 111, Bacto-Basic Fuchsin may be em-
ployed in a concentration of 1:100,000 for differentiation of strains of Brucella.
In Bacto-Fuchsin Lactose Broth, discussed on page 41, the dye is present in a
concentration of 1 : 66,000.

BAGTO-BRILLIANT GREEN (B192)

For Brilliant Green Media

Bacto-Brilliant Green (C.I. No. 662) is certified for use in culture media by
the Commission on Standardization of Biological Stains.

Bacto-Brilliant Green is recommended for use in preparing the Brilliant

294 DIFCO MANUAL

Green Bile Media described in "Standard Methods for the Examination of
Water and Sewage"^ and "Standard Methods for the Examination of Dairy
Products."^ This dye, in the proper concentration in media, exhibits the desired
selectivity, being inhibitive to Gram-positive non-confirming organisms, permit-
ting the unrestricted development of the colon-aerogenes group.

^ Standard Methods for the Examination of 2 Standard Methods for the Examination of
Water and Sewage: gth Edition, 1946. Dairy Products, 8th Edition, 1941.

BAGTO-CRYSTAL VIOLET (B193)

(Gentian Violet)

Bacto-Crystal Violent (C.I. No. 681) is certified for use in bacteriological
culture media by the Commission on Standardization of Biological Stains.

Crystal violet and its impure form, gentian violet, have long been used in
culture media because of their selective inhibitory action toward the Gram-
positive bacteria. Although Bacto-Crystal Violet has a wide range over which
it is not significantly toxic to the Gram-negative bacteria and is still definitely
bacteriostatic toward the Gram-positive organisms, it should be used with ex-
treme care to preserve the proper ratio of dye to medium and to maintain the
correct reaction.

In Tryptose Agar, discussed on page 111, Bacto-Crystal Violet may be used
in a concentration of 1 : 700,000 for isolation of Brucella strains from infected
milk. Bacto-Crystal Violet is employed in Bacto-Supplement A, a selective
enrichment for gonococci and meningococci. A complete discussion of this
supplement is given on page 276 or under Bacto-Proteose No. 3 Agar, page 116^
and Bacto-G C Medium Base, page 122.

BAGTO-EOSIN Y (B194)

Bacto-Eosin Y (C.I. No. 768) is certified for use in bacteriological culture
media by the Commission on Standardization of Biological Stains.

Bacto-Eosin Y is recommended for use in conjunction with Bacto-Methylene
Blue in the preparation of Eosin Methylene Blue Agar according to the Levine
formula^ as specified in "Standard Methods for the Examination of Water and
Sewage"^ or in accordance with the formula of Holt-Harris and Teague.^ These
media are discussed in detail on pages 35 and 133.

1 Bull. 62, Iowa Eng. Exp. Sta., 1921. ^ j. Infectious Diseases, 18:596:1916.

2 Standard Methods for the Examination of
Water and Sewage, 9th Edition: 187: 1946.

BACTO-METHYLENE BLUE (B195)

Bacto-Methylene Blue (C.I. No. 922) is certified for use in bacteriological
culture media and for all other purposes by the Commission on Standardization
of Biological Stains.

Bacto-Methylene Blue is recommended for use in conjunction with Bacto-
Eosin Y in the preparation of Eosin Methylene Blue Agar according to the
Levine formula^ as specified in "Standard Methods for the Examination of
Water and Sewage,"^ or in accordance with the formula of Holt-Harris and
Teague.^ These media are discussed in detail on pages 35 and 133.

1 Bull. 62 Iowa Eng. Exp. Sta., 1921. ^ j_ Infectious Diseases, 18:596:1916.

' Standard Methods for the Examination of
Water and Sewage, gth Edition: 187:1946.

INGREDIENTS OF CULTURE MEDIA 295

B AGTO-THIONIN ( B 1 96 )

Bacto-Thionin (CI. No. 920) is recommended for use in preparing the
thionin medium employed in the differentiation of Brucella types. In Tryptose
Agar, as discussed on page 111, Bacto-Thionin may be used for the differentiation
of Brucella in a concentration of 1 : 200,000. Upon Thionin Tryptose Agar
Brucella melitensis and B. suis grow readily while B. abortus fails to develop.

SULFONPHTHALEIN AND OTHER INDICATORS

In selecting this group of indicators^-- we have chosen those generally employed
in most laboratories. The use of these indicators in the preliminary adjustment
of culture media reactions, or as indicators of bacterial metabolism in the media
themselves, is well-known and needs no further explanation. These indicators
have been subjected to both bacteriological and chemical assay. They are non-
toxic in the concentration usually employed in media, and indicate color changes
at various reactions as shown below.

* Clark: The Determination of Hydrogen Ions, => Pharmacopeia of the United States, XIV 1970:
1928. 1950.

Indicators Showing Preparation, pH Ranges and Color Changes

pH Color

A* Range pK Acid Alkali

Bacto-Cresol Red (acid) (B204) 26.2 0.2-1.8 — Red- Yellow

( o-Gresolsulfonphthalein )
Bacto-Meta Cresol Purple (acid) (B205) . . 26.2 1.2-2.8 1.51 Red-Yellow

( m-Cresolsulf onphthalein )
Bacto-Thymol Blue (acid) (B202) 21.5 1.2-2.8 1.5 Red-Yellow

( Thymolsulfonphthalein )
Bacto-Brom Phenol Blue (B199) 14.9 3.0-4.9 3.98 Yellow-Blue

( Tetrabromophenolsulf onphthalein )
Bacto-Brom Cresol Green (B200) . 14.3 3.8-5.4 4.67 Yellow-Blue

(Tetrabromo-o-cresolsulf onphthalein)
Bacto-Brom Cresol Purple (B201) 18.5 5.2-6.8 6.3 Yellow-Purple

( Dibromo-o-cresolsulf onphthalein )
Bacto-Brom Thymol Blue (B202) 16.0 6.0-7.6 7.0 Yellow-Blue

( Dibromo thymolsulfonphthalein )
Bacto-Phenol Red (B203) 28.2 6.8-8.4 7.9 Yellow-Red

( Phenolsulf onphthalein )
Bacto-Cresol Red (alkaline) (B204) 26.2 7.2-8.8 8.3 Yellow-Red

( o-Cresolsulf onphthalein )
Bacto-Meta Cresol Purple (alkaline) (B205) 26.2 7.4-9.0 8.32 Yellow-Purple

( m-Cresolsulfonphthalein )
Bacto-Thymol Blue (alkaline) (B206) .... 21.5 8.0-9.6 8.9 Yellow-Blue

(Thymolsulfonphthalein )

♦A — ml. 0.0 IN NaOH required per 0.1 g. indicator. Dilute to 250 ml. with distilled
water for 0.04 per cent solution for use as indicator for colorimetric pH determi-
nations.

296

DIFGO MANUAL

Color
pH Range Acid Alkali

Preparation

Methyl Red (B207) . . . . 4.4-6.0 Red-Yellow

Neutral Red (B208)

6.8- 8.0 Red-Yellow

Litmus, Pure (B209) .... 4.5- 8.3 Red-Blue
Phenolphthalein (B210) . 8.3-10.0 Colorless-Red

0.1 g. in 300 ml. ethanol
and dilute to 500 ml.
with distilled water

0.1 g. in 60 ml. ethanol
make up to 100 ml.

LO g. in 100 ml. water

1.0 g. in 60 ml. ethanol
and make up to 100
ml. with ethanol.

BIOCHEMICALS

i>-AMINOBENZOIG ACID, DIFGO (B240)

/?-Aminobenzoic acid is recommended for use in liquid culture media for the
prevention of bacterial stasis due to any sulfonamide drug. It should be added to
all liquid media used for blood cultures of patients under sulfonamide therapy,
and in the culture of exudates or other materials containing sulfonamide com-
pounds. As pointed out by Lockwood^ and McLeod^ most culture media nor-
mally contain some sulfonamide inhibitors which generally are not sufficient
to neutralize completely the sulfonamides likely to be encountered. The addition
of 5 mg. of p-aminobenzoic acid to 100 ml. of medium will more than suffice
to neutralize the bacteriostatic effect of 1.5 mg. per cent sulfonamide drug. This
quantity of p-aminobenzoic acid is not toxic to fastidious pathogens even though
the inocula contain only a few organisms. The report of Janeway^ shows the
value of p-aminobenzoic acid in culture media wherever sulfonamides are en-
countered.

1 J. Immunol., 35- 155:1938.

2 J. Exp. Med., 72:217:1940.

8 J. Am. Med. Assoc, 116:941:1941

i>.AMINODIMETHYLANILINE MONOHYDRO-

CHLORIDE, DIFGO (B249)

/?-Aminodimethylaniline Monohydrochloride, Difco (dimethyl-p-phenylene-
diamine hydrochloride) is recommended for use in the detection of oxidase
production by microorganisms. Its practical application in the bacteriological
laboratory was first described by Gordon and McLeod^ who employed this
reagent as an aid in differentiating colonies of oxidase-producing microorganisms
from those not elaborating this enzyme. These authors found the oxidase test to
be particularly helpful in the detection of Neisseria gonorrhoeae colonies growing
among colonies of extraneous organisms in the primary culture of gonococcal
exudates on Chocolate Agar. Since its introduction for this purpose p-amino-
dimethylaniline monohydrochloride has been widely adopted for use in the
cultural diagnosis of gonorrhoea. ^'^ Oxidase positive colonies flooded or sprayed
with p-aminodimethylaniline monohydrochloride turn pink within a minute or

INGREDIENTS OF CULTURE MEDIA 297

two and darken to become black. Oxidase negative colonies do not change color.

p-Aminodimethylaniline monohydrochloride is used in 1 per cent concentra-
tion in distilled water. The solution should be prepared on the same day it is to
be used. However, if kept longer, it should be refrigerated and then warmed be-
fore use. In no case should the solution be used if more than 5 days old or if a
precipitate is present. A complete discussion of the use of this reagent is given
under Bacto-Proteose No. 3 Agar and Bacto-G C Medium Base, pages 116 and
122, for use of the oxidase test in the cultural diagnosis of gonococcal infections.

Tetramethyl p-phenylenediamine hydrochloride may also be used in exactly
the same way as the dimethyl reagent. The tetramethyl compound has the dis-
advantage of sometimes coloring the surrounding medium and is much more
expensive than the dimethyl.

1 J. Path. Bact., 31:185:1928. 3 Diagnostic Procedures and Reagents, 3rd Edi-

2 Seventh Annual Year Book (1936-37) p. 133. tion: 107: 1950.
Suppl., Am. J. Pub. Heahh, 27:No. 3:1937.

/>-AMINODIMETHYLANILINE OXALATE,

DIFGO (B329)

p-Aminodimethylaniline Oxalate, Difco, is recommended for the detection of
oxidase production by microorganisms. It has the advantage over the mono-
hydrochloride salt in that it is more stable in the powdered form and also in
solution. The oxidase reagent is prepared by dissolving 1 gram of p-Aminodi-
methylaniline Oxalate, Difco in 100 ml. distilled water by heating gently. This
solution is used to flood plates in determining the oxidase reaction. Positive
colonies assume a pink color, finally turning to maroon and black. Carpenter^
reported that the dry crystalline oxalate salt is more stable than the mono-
hydrochloride salt. No change was observed after six months storage. Aqueous
solutions were also more stable. Both salts showed about the same toxicity for
microorganisms. The oxalate salt is slightly less soluble in cold distilled water,
but solution is hastened by warming gently. Carpenter^ further stated the oxalate
salt possesses the additional advantages over the monohydrochloride in that it
does not form the marked black precipitate on Chocolate Agar sometimes ob-
served with the use of the monohydrochloride, especially when freshly prepared
solutions are not employed.

1 Science, 105:649:1948. ^Diagnostic Procedures and Reagents, 3rd Edi-

tion: 107: 1950.

SODIUM THIOGLYGOLLATE, DIFGO (B233)

Sodium thioglycollate or thioglycollic acid is recommended for use in liquid
culture media for testing the sterility of biological and other materials contain-
ing heavy metal compound preservatives, such as the mercurials. The active
sulfhydryl group annuls the toxicity of the metallic preservatives, thus per-
mitting the development of any viable organisms present and, further, behaves
similarly to glutathione, cysteine and the alkali sulfides in being able to lower
the oxidation-reduction potential of the medium. This principle was first de-
scribed by Trenkmann^ who showed that the presence of an alkali sulfide
induced "aerobic growth of anaerobic organisms".

Quastel and Stephenson^ showed likewise that cysteine and thioglycollic acid
made possible the growth of anaerobes through the lowering of the oxidation-
reduction potential of the medium. Brewer^-* combined the principle of lowering

298 DIFCO MANUAL

the oxidation-reduction potential with Kitchen's^ method of adding 0.05 to 0.1
per cent agar to the medium for the growth of anaerobes. Such a combination
was found particularly useful as a sterility test medium for biological containing
mercurial preservatives, since the sodium thioglycollate neutralizes the toxic
effect of any mercurial carried over with the inoculum, while the agar provides
anaerobic conditions necessary for growth.

Since sodium thioglycollate is toxic for some organisms, especially with inocula
containing very few organisms, it is recommended that no more of the sodium
thioglycollate be added to the medium than is required for mercurial neutraliza-
tion. Sodium thioglycollate in 0.05 per cent concentration is specified in the
thioglycollate media of the National Institute of Health^ for sterility testing.
These media are discussed under Bacto-Fluid Thioglycollate Medium and Bacto-
N.I.H. Thioglycollate Broth on pages 195 and 198. Bacto-Brewer Anaerobic
Agar, discussed on page 126, contains sodium thioglycollate, and is employed
as a solid medium for the cultivation of anaerobes.

1 Centr. Bakt. I Abt., 23:1038:1898. ^ J. Infectious Diseases, 29:390:1921.

2 Biochem. J., 20:1125:1926. ^National Institute of Health Circular: Culture
8 J. Bact., 39:10:1940. Media for Sterility Test, 2nd Revision, Feb. 5,

-J. isact., 39:10:1940.

* J. Am. Med. Assoc, 115:598:1940

1946.

THIOGLYGOLLIG ACID, DIFCO (B250)

Thioglycollic Acid, Difco is recommended for use in media designed for the
cultivation of organisms in the presence of mercurial salt preservative. It may
also be used for the preparation of media of a low Eh. In the preparation of
media for the sterility testing of biologicals, thioglycollic acid or sodium thiogly-
collate may be employed. A complete discussion of these media is given under
Bacto-Fluid Thioglycollate Medium page 195 and Bacto-N.I.H. Thioglycollate
Broth page 198.

UREA, DIFCO (B190)

Urea, Difco is purified urea recommended for the preparation of bacterio-
logical culture media. The ability of an organism to hydrolyze urea is often a
salient characteristic in its identification. The use of Urea Broth and Urea Agar
Base for the identification of enteric organisms is discussed in detail on pages
170 and 171.

POTASSIUM TELLURITE, DIFCO (B384)

Potassium Tellurite, Difco is recommended for use as a selective agent in
media for the isolation and differentiation of diphtheria bacilli. It has also
been used as a selective agent in media designed for isolation of streptococci
and the enrichment and isolation of pleuropneumonia-like organisms. Potassium
Tellurite, Difco is readily soluble and well suited for use in media requiring this
chemical, as each batch is checked to determine its inhibitive properties. Bacto-
Chapman Tellurite Solution as discussed on page 277 is prepared with Potassium
Tellurite. A complete description of the media using this selective agent is given
under Bacto-PPLO Enrichment Broth, page 82 and Mitis Salivarius Agar,
page 154.

INGREDIENTS OF CULTURE MEDIA 299

MISCELLANEOUS INGREDIENTS

BAGTO-EGG ALBUMEN, GOAGULATED (B147)

BAGTO-EGG YOLK, GOAGULATED (B148)

BAGTO-WHOLE EGG, GOAGULATED (B149)

At the suggestion of, and in cooperation with, R. S. Spray, we have prepared
Bacto-Egg Albumen, Coagulated; Bacto-Egg Yolk, Coagulated; and Bacto-Whole
Egg, Coagulated. In his extensive studies on the use of egg for the cultivation
and differentiation of anaerobic bacteria Spray^ used these dehydrated egg prod-
ucts with excellent results.

Bacto-Egg Albumen, Coagulated is free from all traces of egg yolk. Bacto-
Egg Yolk, Coagulated is similarly free from traces of egg albumen, while Bacto-
Whole Egg, Coagulated, as the name indicates, contains both of these materials.
All three are prepared for use by making a suspension of 0.5 gram of material
in 10 ml. of distilled water or Nutrient Broth in a test tube. Spray has also
found that a solution of Bacto-Tryptone was distinctly advantageous as a diluent
for these materials. The suspensions are sterilized by autoclaving for 15 minutes
at 15 pounds pressure (121°C.). After sterilization the media will have a reaction
of pH 7.2 and will present a loosely flocculent appearance with a layer of fluid
above the solid material.

The tubes are inoculated deeply by loop or pipette. Characteristic reactions
indicative of proteolysis are produced by most anaerobes. The addition of re-
duced iron or lead acetate to the media greatly intensifies the blackening of the
media produced by the action of proteolytic organisms.
1 J. Lab. Clin. Med., 18:512:1932-33.

BAGTOJSOELEGTRIG GASEIN (B145)

Bacto-Isoelectric Casein is a protein of exceptional purity, prepared by re-
peated precipitation of casein at its isoelectric point. This casein of more than
ordinary purity is recommended for use in routine bacteriological culture media
and also for the most exacting special procedures. It is suitable for the Fuld
Gross method for the determination of tryptic activity. Bacto-Isoelectric Casein
is quickly and completely soluble in a concentration of 1-2 per cent in a slightly
alkaline solution.

Sodium caseinate is readily prepared by suspending Bacto-Isoelectric Casein
in water for a few minutes and then making the suspension slightly alkaline with
sodium hydroxide. This alkaline condition should be maintained until solution
is complete. When prepared in this manner the solution of Bacto-Isoelectric
Casein is light in color and only lightly opalescent.

GASEIN PURIFIED, DIFGO (B336)

Casein Purified is recommended for all procedures requiring a casein of high
purity. Repeated precipitation and washing of casein at the isoelectric point

300 DIFCO MANUAL

produces a purified casein of uniform quality. Casein Purified is soluble in dilute
solutions of sodium hydroxide forming the sodium salt.

CASEIN TECHNICAL, DIFCO (B337)

Casein Technical is a technical grade of this protein recommended for use
in the preparation of culture media, and in other techniques not requiring a
purified product.

SODIUM CASEINATE, DIFCO (B187)

Sodium Caseinate is a valuable ingredient of many culture media. It has
frequently been referred to as "Nutrose". Sodium Caseinate, Difco is a purified
product recommended for use in all media in which "Nutrose" or sodium case-
inate has been employed.

EGG ALBUMEN SOLUBLE, DIFCO (B255)

WHOLE EGG SOLUBLE, DIFCO (B254)

Egg Albumen Soluble and Whole Egg Soluble are desiccated egg albumen
and whole egg, respectively. They are available to bacteriologists who may desire
to use such products for the preparation of culture media or for studies of
bacterial metabolism. Egg Albumen Soluble and Whole Egg Soluble when
rehydrated in 13.5 per cent concentration will give the equivalent of the original
form of these products.

BACTO-RICE POWDER (B146)

Bacto-Rice Powder was prepared at the suggestion of L. R. Cleveland and
is recommended for use in the propagation of Endamoeha histolytica. Cleveland
and his co-workers^ »2'^ have given detailed descriptions of the methods they
employed for the cultivation of this organism on a medium prepared from
Bacto-Liver Infusion Agar with rice powder. Their method is discussed in detail
under Bacto-Endamoeba Medium, page 97. Essentially, this procedure is to
cover each slant of the medium with fresh horse serum-saline solution and to
add sterile rice powder to each tube prior to inoculation with the organism.
Bacto-Rice powder is sterilized in a dry heat oven at 160°F. for one hour.
Scorching must be prevented.

1 Arch. Protistenkunde, 70:323:1930. * Am. J. Hyg., 12:606:1930.

2 Science, 72:149:1930.

Tissue Culture Media
Reagents, Difco

As IN the field of dehydrated culture media for Bacteriology and Mycology,
Difco Laboratories has pioneered in the preparation of dehydrated reagents
for in vitro propagation and maintenance of tissue cells. The task of preparing
and distributing these highly specialized products was undertaken at the request
of the Tissue Culture Association. Each of these reagents, before being released
for distribution, is tested and certified for use in tissue culture by the Central
Laboratory of the Tissue Culture Association. They are applicable to the slide,
roller tube and flask culture techniques commonly employed for propagation and
study of tissue cells in vitro. All desiccated reagents are dried from the frozen
state to preserve, unaltered, the properties of the original material. The reagents
currently available are those commonly employed for culturing tissue cells,
maintaining tissue banks, and for the propagation of Viruses. Additional reagents
will be made available as required for these and other procedures related to
tissue culture.

TG CHICKEN PLASMA (B354)

TG Chicken Plasma is sterile desiccated whole chicken plasma certified for
use in culturing tissue cells in vitro. It is prepared from selected disease-free
cockerels 4-8 months old and is packaged in 5 ml. quantities.

TC Chicken Plasma is reconstituted to its original volume by adding, asep-
tically, 4.5 ml. sterile TC Reconstituting Fluid or TC Triple Distilled Water to
each bottle. The Reconstituting Fluid may be added conveniently with a sterile
5 ml. syringe. Solution of the material is accelerated by gentle end-over-end rota-
tion of the bottle. Do not shake. Allow 30 minutes for complete solution of the
desiccated plasma.

The reconstituted plasma is alkaline in reaction due to loss of carbon dioxide
in the desiccation process and requires adjustment with carbon dioxide to pH
7.2-7.4 if a normal clotting time is desired. Adjustment of the reaction with
carbon dioxide to pH 7.2-7.4 accelerates the clotting time when Embryo Extract
and tissue explants are added. The adjustment of the reaction of the recon-
stituted plasma is best accomplished by first adding TC Phenol Red Solution
1% to the plasma to give a final Phenol Red concentration of 0.002 per cent,
(add 1 ml. TC Phenol Red Solution 1% to 9 ml. TC Triple Distilled Water, then
add 0.1 ml. of resultant Phenol Red Solution to 5 ml. of reconstituted Chicken
Plasma) and then introducing carbon dioxide to bring the reaction to pH
7.2-7.4. Three methods of introducing carbon dioxide that have given good
results are outlined:

1. Transfer the reconstituted TC Chicken Plasma containing 0.002 per cent
Phenol Red to a clean, dry, sterile cotton stoppered or loosely capped test tube.
Place the tube in a beaker or container of sufficient depth so that the walls ex-
tend one or more inches above the mouth of the tube. Introduce carbon dioxide
into the beaker to displace the air and then cover the beaker with paper or
suitable cover. Gently agitate the plasma in the tube occasionally to bring all
parts of the solution in contact with the carbon dioxide. Observe color change in

301

302 DIFGO MANUAL

the indicator from cerise alkaline reaction to reddish orange almost neutral
reaction. Reasonable care should be exercised to prevent overacidification as this
will tend to coagulate the plasma spontaneously. When the indicator color
change begins to turn from cerise to reddish orange, double check the pH by
removing a drop of the plasma aseptically and transferring it to a few drops of
distilled water to which indicator has been added on a spot plate or in a test tube.
The recheck of the reaction by the test tube or spot plate method is essential
because indicators do not always give true color changes in undiluted protein
solutions. Failure to adjust the reaction of Chicken Plasma may give prolonged
clotting times. For example, undiluted Chicken Plasma containing 0.002 per cent
Phenol Red may give a color reaction indicative of pH 7.2 whereas the true
reaction as determined by dilution in a test tube or spot plate may be pH 8.0 or
greater. Also, color reactions with Phenol Red in undiluted plasma indicative of
pH 7.2 reaction may actually be more acid by the test tube dilution and spot
plate methods. When the reaction has been adjusted to pH 7.2-7.4, stopper
tightly to prevent escape of carbon dioxide. Store in refrigerator at 2-6°C. if not
used immediately. Storage for periods longer than one week is not recommended.

2. Transfer the reconstituted plasma containing Phenol Red to a clean, dry
sterile tube. Bubble sterile carbon dioxide or a mixture of sterile carbon dioxide
and nitrogen through the solution until the cerise color changes to reddish
orange. Recheck pH by test tube or spot plate as outlined above. Adjust final
reaction to pH 7.2-7.4.

3. Same as method ^2 except blow sterile alveolar air through the plasma solu-
tion to give pH 7.2-7.4 using precautions stated above.

It has been our observation that Embryo Extracts diluted with Balanced Salt
Solutions containing calcium in levels comparable with TC Balanced Salt Solu-
tion (Earle's BSS) give a faster clotting rate and a firmer coagulum with TG
Chicken Plasma than do extracts prepared with a lower calcium content.

TG CHICK EMBRYO EXTRACT EEioo (B355)

TC Chick Embryo Extract EE^qq is sterile desiccated whole, undiluted Chick
Embryo Extract certified for use in tissue culture procedures requiring embryo
extract. It is prepared from selected eleven-day old chick embryos, and is
packaged in 2 ml. quantities.

TC Chick Embryo Extract EE^qq is reconstituted by adding, aseptically, 2.0
ml. of TC Reconstituting Fluid or TC Triple Distilled Water to the 2 ml. bottle.
When the contents of the bottle have uniformly combined with the water, they
are diluted further as desired with TC Balanced Salt Solution. A 20 per cent
Embryo Extract, EE20, has been found to be useful in tissue culture procedures.
This concentration is obtained by adding 4 volumes (8 ml.) of TC Balanced
Salt Solution to the reconstituted Embryo Extract EEjoq- The Reconstituting
Fluid and the Balanced Salt Solution may be added conveniently by means of
a sterile syringe and needle.

The diluted Embryo Extract EE20, or other desired concentration, is allowed to
stand at 35-3 7 °C. for 30-60 minutes to obtain maximum solution of the nutrients.
Solution of the desiccated material is accelerated by gentle end-over-end rota-
tion of the bottle. Do not shake. A small portion of the desiccated Embryo Extract
may remain in suspension. This insoluble residue should be removed by centrifu-
gation of the bottle for 10 minutes at 2500-3000 rpm. The supernatant liquid is
now ready for use. Unused portions of the diluted Embryo Extract should be
stored in tightly stoppered containers in the refrigerator (2-6°C.). Storage of the
reconstituted TC Chick Embryo Extract for periods longer than one week, even
though refrigerated, is not recommended.

It has been our observation that Embryo Extracts diluted with Balanced Salt

TISSUE CULTURE REAGENTS 303

Solutions containing calcium in levels comparable with TG Balanced Salt Solu-
tion (Earle's BSS) give a faster clotting rate and a firmer coagulum w^ith TG
Ghicken Plasma than do extracts prepared with a lower calcium content.

TG CHICK EMBRYO (B470)

TC Chick Embryo is sterile whole minced Chick Embryo dried from the
frozen state and certified for use in tissue culture procedures requiring chick
embryo extract. It is prepared from selected eleven-day old chick embryos and
is packaged in 2 ml. quantities.

TC Chick Embryo is prepared for use by adding 2 ml. TC Reconstituting
Fluid or TC Triple Distilled Water to each vial to wet the desiccated embryo
tissue thoroughly. Four volumes (8 ml.) TC Balanced Salt Solution are then
added to each bottle. The solids are suspended by rotating the bottles gently in
an end-over-end motion. Do not shake. The suspension is warmed to 35-3 7 °C.
for 30-60 minutes with occasional rotation to resuspend the solids. The bottles
are then centrifuged at 2500-3000 rpm for 5 minutes to sediment the particulate
matter following which, the supernatant is drawn off aseptically and trans-
ferred to a clean sterile container. The resultant extract is a 20 per cent Chick
Embryo Extract, EEgo, and is ready for use. Unused portions of the extract should
be stored at 2-6° C. Storage of the extract for periods longer than one week are
not recommended.

It has been our observation that Embryo Extracts diluted with Balanced Salt
Solutions containing calcium in levels comparable with TG Balanced Salt Solu-
tion (Earle's BSS) give a faster clotting rate and a firmer coagulum with TG
Ghicken Plasma than do extracts prepared with a lower calcium content.

TG BEEF EMBRYO EXTRACT EEioo (B396)
TC Beef Embryo Extract EE^qq is sterile desiccated undiluted Beef Embryo
Extract certified for use in tissue culture procedures requiring mammalian
embryo extract. It is prepared from fresh bovine embryos 60-90 days of age
and is packaged in 2 ml. quantities.

TC Beef Embryo Extract EEj^qq is reconstituted to its original volume by
adding, aseptically, 2.0 ml. TC Reconstituting Fluid or TC Triple Distilled
Water to each bottle. When the contents of the botde has uniformly combined
with the liquid it is then diluted further as desired with TC Balanced Salt
Solution. A 20 per cent Embryo Extract, EE20, has been found useful in tissue
culture procedures. This concentration is obtained by adding four volumes
(8 ml.) of TC Balanced Salt Solution to the reconstituted Embryo Extract EEiqo-
The Reconstituting Fluid and Balanced Salt Solution may be added conveniently
by means of a sterile syringe and needle.

The diluted Embryo Extract EE20, or other desired concentration, is allowed
to stand at 35-3 7 °C. for 30-60 minutes to obtain maximum solution of the
nutrients. Solution of the desiccated material is accelerated by gentle end-over-
end rotation of the bottle. Do not shake. A small portion of the desiccated
Embryo Extract may remain in suspension. This insoluble residue should be
removed by centrifugation of the bottle for 10 minutes at 2500-3000 rpm. The
supernatant liquid is now ready for use. Unused portions of the diluted Embryo
Extract should be stored in tightly stoppered containers in the refrigerator at
2-6°C. Storage of the reconstituted TC Beef Embryo Extract for periods longer
than one week, even though refrigerated, is not recommended.

304 DIFCO MANUAL

TG BEEF EMBRYO (B471)

TC Beef Embryo is sterile whole minced Beef Embryo dried from the frozen
state and certified for use in tissue culture procedures requiring beef embryo
extract. It is prepared from beef embryos 60-90 days of age and is packaged in
2 ml. quantities.

TC Beef Embryo is prepared for use by adding 2 ml. TC Reconstituting
Fluid or TC Triple Distilled Water to each vial to wet the desiccated embryo
tissue thoroughly. Four volumes (8 ml.) TC Balanced Salt. Solution are then
added to each bottle. The solids are suspended by rotating the bottles gently in
an end-over-end motion. Do not shake. The suspension is warmed to 35-37'^C.
for 30-60 minutes with occasional rotation to resuspend the solids. The bottles
are then centrifuged at 2500-3000 rpm for 5 minutes to sediment the particulate
matter following which, the supernatant liquid is drawn off aseptically and trans-
ferred to a clean sterile container. The resultant extract is a 20 per cent Beef
Embryo Extract, EE20, and is ready for use. Unused portions of the extract
should be stored at 2-6°C. Storage of the extract for periods longer than one
week are not recommended.

It has been our observation that Embryo Extracts diluted v>^ith Balanced Salt
Solutions containing calcium in levels comparable with TG Balanced Salt Solu-
tion (Earle's BSS) give a faster clotting rate and a firmer coagulum with TG
Chicken Plasma than do extracts prepared with a lower calcium content.

TG GORD SERUM, HUMAN (B356)

TC Cord Serum, Human is sterile desiccated whole human cord serum
certified for use in culturing tissue cells in vitro. It is obtained from blood from
the placenta at childbirth. Each preparation is from the pooled sera from 25
or more placentae. It is packaged in 5 ml. quantities.

TC Cord Serum, Human is reconstituted to its original volume by adding,
aseptically, 4.5 ml. TC Reconstituting Fluid or TC Triple Distilled Water to
each bottle. Solution of the material is accelerated by gentle end-over-end rota-
tion of the bottle. Do not shake. When solution is complete, it is ready for use.
Unused portions of the reconstituted TC Cord Serum, Human should be kept in
tightly stoppered containers in the refrigerator at 2-6° C. Storage of the recon-
stituted TC Cord Serum, Human for periods longer than one week, even though
refrigerated, is not recommended.

The Reconstituted TC Cord Serum, Human is alkaline in reaction due to
loss of carbon dioxide in the desiccation process and if desired may be adjusted
to pH 7.2-7.4 in an atmosphere of carbon dioxide as described for the adjust-
ment of Chicken Plasma page 301.

TG HORSE SERUM (B357)

TC Horse Serum is sterile desiccated whole, pooled horse serum certified for
use in culturing tissue cells in vitro. It is obtained from blood from fasted,
healthy, lean geldings, and is packaged in 5 ml. quantities.

TISSUE CULTURE REAGENTS 305

TC Horse Serum is reconstituted for use by adding, aseptically, 4.5 ml. TC
Reconstituting Fluid or TC Triple Distilled Water to each bottle. The Reconsti-
tuting Fluid may be added conveniently with a sterile syringe and needle. Solu-
tion oi the material is accelerated by a gentle end-over-end rotation of the bottle.
Do not shake. When solution is complete, the serum is ready for use. Unused
portioas of the reconstituted TC Horse Serum should be kept in tightly stoppered
contaiiers in the refrigerator at 2-6°C. Storage of the reconstituted TC Horse
Serum for periods longer than one week, even though refrigerated, is not recom-
mended.

• The reconstituted TC Horse Serum is alkaline in reaction due to loss of carbon
dioxide in the desiccation process and if desired may be adjusted to pH 7.2-7.4
in an atmosphere of carbon dioxide as described for the adjustment of Chicken
Plasma page 301.

TC ASCITIC FLUID (B472)

TC Ascitic Fluid is sterile, pooled, whole human ascitic fluid certified for use
in tissue culture procedures. It is obtained from cirrhotic patients, free from bile
salts, and is packaged in 10 ml. ampuls. TC Ascitic Fluid has a reaction of
pH 6.9-/.0 and requires adjustment to 7.2-7.4 before being used as a nutrient
for tissue cells. Adjustment is made by adding sterile sodium bicarbonate solu-
tion or by diluting the Ascitic Fluid with TC Balanced Salt Solution and agitating
the mixture gently to drive off some of the carbon dioxide. TC Phenol Red Solu-
tion 1% may be added to the Ascitic Fluid in a concentration of 0.002 per cent
as an indicator of reaction. (Add, aseptically, 0.02 ml. TC Phenol Red Solution
1% to 10 ml. Ascitic Fluid.)

TC BEEF SERUM ULTRAFILTRATE (B473)

TC Beef Serum Ultrafiltrate is a protein-free solution containing 0.001 per
cent Phenol Red indicator. It is prepared by the ultrafiltration of beef serum
through a colloidion membrane filter to remove all protein and preserve filterable
growth accessory factors. It is certified for use in tissue culture and is recom-
mended for all procedures requiring serum ultrafiltrate. TC Beef Serum ultra-
filtrate is especially suited as a basal substrate for culturing tissue cells, maintain-
ing tissue banks, propagating viruses and for maintaining the viability and
motility of Treponema pallida for the Treponema Immobilization test. It is
packaged in 50 ml. quantities in glass sealed ampuls.

TC HORSE SERUM ULTRAFILTRATE (B474)

TC Horse Serum Ultrafiltrate is a protein-free solution containing 0.001 per
cent Phenol Red indicator. It is prepared by the ultrafiltration of horse serum
through a colloidion membrane filter to remove all protein and preserve filterable
growth accessory factors. It is certified for use in tissue culture and is recom-
mended for all procedures requiring serum ultrafiltrate. TC Horse Serum ultra-
filtrate is especially suited as a basal substrate for culturing tissue cells, maintain-
ing tissue banks, propagating viruses and for maintaining the viability and
motility of Treponema pallida for the Treponema Immobilization test. It is
packaged in 50 ml. quantities in glass sealed ampuls.

306 DIFGO MANUAL

TC BALANCED SALT SOLUTION (B351)

TC Balanced Salt Solution is a sterile balanced salt solution certified br use
in tissue culture procedures. It is prepared according to the formula of larle,^
and has the following composition:

Sodium Chloride 6.8 g.

Potassium Chloride 0.4 g.

Calcium Chloride 0.2 g.

Magnesium Sulfate 0.2 g.

Sodium Phosphate Monobasic ... 0.125 g.

Bacto-Dextrose 1. g.

Sodium Bicarbonate 2.2 g.

Triple Distilled Water 1,000 ml.

TG Balanced Salt Solution is stabilized by adjustment of the reactior to pH
7.0 with carbon dioxide during preparation. It can readily be adjustec to pH
7.2-7.4 by permitting carbon dioxide to escape after removal from the bottle.
TC Phenol Red Solution 1% may be added to the TG Balanced Salt Solution
in 0.002 per cent concentration as an indicator of reaction. (Add, aseptically,
0.2 ml. TG Phenol Red Solution 1% to 100 ml. Balanced Salt Solution). TG
Balanced Salt Solution is packaged in 100 ml. bottles.

1 Nat. Cancer Inst., 4:167:1943.

TG RECONSTITUTING FLUID (B352)

TG Reconstituting Fluid is sterile, triple distilled water saturated with carbon
dioxide and is certified for use in tissue culture procedures. It is packaged in
100 ml. quantities. It is used for reconstituting desiccated tissue culture reagents
where a lower pH is desired than obtainable through use of TG Triple Distilled
Water.

TG TRIPLE DISTILLED WATER (B353)

TC Triple Distilled Water is sterile and certified for use in tissue culture
procedures. It is the distillate from triple distillation of water in an all-Pyrex
still and is packaged in 100 ml. bottles.

TG Triple Distilled Water is recommended as a reconstituting fluid for tissue
culture reagents and for the preparation of Balanced Salt Solution and other
media.

TG PHENOL RED SOLUTION 1% (B358)

TG Phenol Red Solution 1% is sterile and certified for use as an indicator of
pH in tissue culture procedures. It is employed in tissue culture media in con-
centrations of 0.001-0.002 per cent. For judging the pH of very small volumes
of media, (1-2 ml.), the concentration of phenol red should be 3-5 times that
given above, as suggested under the discussion of TG Chicken Plasma as given
on page 301. TG Phenol Red Solution is packaged in 1 ml. ampuls and supplied
in boxes of 6 ampuls.

TISSUE CULTURE REAGENTS 307

METHODS OF TISSUE CULTURE

Brief outlines for the use of Difco tissue culture media reagents by the common
methods of tissue culture are given below. These directions apply generally for
any type of animal tissue; however, special investigation may be required to
determine the optimal conditions for culturing a given tissue. Proportions of the
various reagents given in the discussion may be varied to meet individual re-
quirements.

Slide Culture

The sterile tissue to be cultured should be placed on a clean sterile glass
surface and cut into appropriate sized explants while immersed in balanced salt
solution i^pH 7.4). The following sizes are usually suitable for the specified
tissue :

minced tissues 0.5—1,0 mm.

embryonic tissues 1.0 mm.

adult tissues 1.5 mm.

adult skin 2.0 mm.

Further rinsing of the explants in balanced salt solution may be done if desired.
Add one drop of TC Chicken Plasma to a cover-glass, followed by one drop of
TC Embryo Extract EEgo- Larger amounts may be used in the same ratio if
desired. Mix the two with a spatula and spread over an area having a diameter
of 1 cm. Add two or three explants and separate them by about equal distances.
Cover with a well slide and set aside to clot. Later, seal the coverglass to the well
slide with melted paraffin or a mixture of 5 parts paraffin and 1 part yellow
vaseline.

More diffuse and extensive cell migration may be encouraged by diluting the
plasma before using with an equal or double volume of TC Balanced Salt
Solution. Incubate the slide culture at 37°C., observe and continue culture in
accordance with standard procedures.

Roller Tube Culture

Prepare explants as for slide cultures and place them in a small volume of
nutrient solution in a well-slide or petri dish. The nutrient may consist of various
proportions of TC Balanced Salt Solution, TC Horse Serum, or TC Cord Serum,
Human and TC Embryo Extract EE20 (5 parts of Balanced Salt Solution, 3 parts
serum and 2 parts embryo extract is satisfactory for general purposes). Place 2
drops of whole TC Chicken Plasma (50 per cent plasma is also satisfactory) in
a 16x150 mm. tube and rotate until bottom third or half of tube is coated. With
a pipette place 4 or 5 explants on the uncoated area near the top of the tube and
remove excess nutrient from around explants with a Pasteur pipette. Then with
pipette transfer individual explants to plasma coated surface. After about 10
minutes, stopper tubes and place them in the roller drum in the 37°C. incubator
for 2 hours. The nutrient medium (mentioned above) should now be added in
0.5 ml. volumes to each tube and the tubes returned to roller drum in the
incubator. Incubate at 37°C., observe and continue culture in the usual manner.

Flask Culture

Prepare explants in TC Balanced Salt Solution. Explants may be made con-
siderably larger for culture in flasks than for slide or roller tube cultures. While

308 DIFCO MANUAL

the conventional size of the explants has approximated 1x1x1 mm., Earle has
used explants ranging from 0.5x1x2 mm. to 0.5x3x2 mm. In other cases he has
substituted cell suspensions for the explants.

To a 35 mm. flask (Carrel type) add 0.3 ml. of TG Chicken Plasma, either
whole or diluted to 50 or even 25 per cent with TC Balanced Salt Solution,
and spread over bottom of flask. This is then increased in volume to 1 ml. by
introducing 0.6 ml. of TC Balanced Salt Solution and 0.1 ml. of TC Embryo
Extract EEgQ. Add explants and orient them. Stopper flasks and set aside to clot.
Incubate for 1-2 hours to give firm clot, then add 0.5 ml. of nutritional mixture
as described under Roller Tube Culture.

Detailed methods of tissue culture may be found in texts such as those listed
below :

Parker, Methods of Tissue Culture
Paul B. Hoeber, Inc., 1938, 1950.

White, Handbook of Plant Tissue Culture
Ronald Press Company, 1943.

Cameron Tissue Culture Technique
Academic Press, Inc., 1950.

Pomerat, Methods in Medical Research Section IV, 1951
Year Book Publishers, 1951.

Serological Reagents
for Diagnosis of Syphilis

The exacting technique of serological diagnostic methods requires carefully
prepared and standardized materials. The reagents listed in this section are pre-
pared for the diagnosis of syphilis by means of the complement-fixation and
precipitation tests. Each preparation of Bacto-Beef Heart, or Bacto-Kahn
Beef Heart, is tested and approved for the preparation of antigens, before re-
lease. The complete antigens for the diagnosis of syphilis are prepared in strict
accordance with the published methods. Before distribution each lot of antigen
is tested and approved for its sensitivity and specificity by the author serologist
or by a laboratory designated by him for that purpose. Thus, users of Bacto-
Antigens are assured of the most satisfactory reagent for the performance of the
various tests.

Complete antigens prepared with purified cardiolipin and lecithin are de-
scribed in this section. These antigens are in exact agreement with the formula
given by the author serologists using cardiolipin and lecithin prepared according
to the directions given by Pangborn. Additional cardiolipin-lecithin reagents will
be prepared as required. The cardiolipin is prepared under license from the
New York State Health Department.

Additional antigens and reagents for syphilology are available and descriptions
will be given upon request.

REAGENTS FOR
COMPLEMENT-FIXATION TESTS

BAGTO-KOLMER IMPROVED ANTIGEN (B304)

Bacto-Kolmer Improved Antigen is prepared according to the directions
given by Kolmer and Lynch.^ It consists of an alcoholic extract of acetone and
ether extracted Bacto-Beef Heart, fortified with 0.4 per cent Bacto-Gholesterol.
In the Kolmer simplified and quantitative complement-fixation tests for syphilis,
these authors^ report this antigen to give the same sensitivity as cardiolipin anti-
gen (Kolmer); the specificity of the antigens with sera and spinal fluids of pre-
sumably normal nonsyphilitic individuals was found to be equal.

lAm. J. Clin. Path., 18:731:1948.
* Texas State J. Med., 44:312:1948.

BAGTO-KOLMER IMPROVED ANTIGEN (B305)
(REENFORGED)

Bacto-Kolmer Improved Antigen (Reenforced) is prepared according to the
formula described by Kolmer and Lynch. ^ In the preparation of this antigen
Bacto-Beef Heart is extracted with acetone followed by ether and then with

309

310 DIFCO MANUAL

alcohol. The alcoholic extract of the tissue is fortified with 0.4 per cent Bacto-
Cholesterol and the ether-soluble, acetone-insoluble lipoids of the Bacto-Beef
Heart. The antigenic sensitivity of this antigen is similar to that of an antigen
prepared with 0.03 per cent cardiolipin, 0.05 per cent lecithin and 0.6 per cent
cholesterol when used in the Kolmer simplified and quantitative complement-
fixation tests for syphilis;^ the specificity of the antigens with sera and spinal
fluids of presumably normal nonsyphilitic individuals was reported to be equal.
The sensitivity of Bacto-Kolmer Improved Antigen (Reenforced) is slightly in-
creased over that of Bacto-Kolmer Improved Antigen.

lAm. J. Clin. Path. 18:731:1948.
2 Texas State J. Mea., 44:312:1948,

BACTO-EAGLE WASSERMANN (B214)
ANTIGEN

Bacto-Eagle Wassermann Antigen was developed by Eagle^-- for use in his
modification of the Wassermann reaction. It is an alcoholic extract of Bacto-Beef
Heart reenforced with Bacto-Cholesterol. This antigen, when tightly stoppered,
retains its reactivity indefinitely at room temperature.

1 J. Lab. Clin. Med., 22:300:1936.
3 Eagle: Laboratory Diagnosis of Syphilis,
144:1938.

BACTO-B.J.L. WASSERMANN ANTIGEN (B234)

Bacto-B.J.L. Wassermann Antigen is prepared according to the procedure
described by Boemer, Jones and Lukens^. The antigen is an alcoholic ex-
tract of Bacto-Beef Heart to which is added Bacto-Cholesterol. In the authors'
technique, the time and cost of performing the test is greatly reduced without
loss of specificity.2'3 It is suitable for quantitative as well as qualitative tests. The
antigen is stable when stored in tightly stoppered bottles at room temperature.

1 Am. J. Clin. Path., 9:i3:i939-

2 Arch. Derm. Syph., 41:32:1940.

3 Am. J. Clin. Path., 10:282:1940.

BACTO-ANTISHEEP HEMOLYSIN (B217)

(Amboceptor)

The epochal discovery of Bordet concerning the lysis of erythrocytes by
immunized serum is the foundation upon which the indicator system of the
complement-fixation test is built.

Bacto-Antisheep Hemolysin (Amboceptor) is prepared by the method of
Kilduffe.^ It is a highly potent antisheep rabbit serum preserved with glycerol.
It is exceptionally stable and may be stored in the refrigerator for periods of
1 2 months or more without loss of activity if kept properly stoppered.

^ J. Lab. Clin. Med., 10:582:1924-25.

SEROLOGICAL REAGENTS 311

BAGTO-GOMPLEMENT (B383)

Bacto-Complement is fresh guinea pig serum dried from the frozen state. It
is prepared by pooling sera of a large number of mature, well-nourished guinea
pigs. It will retain its potency for a minimum of one year if kept at — 5°C. (the
freezing chamber of the refrigerator). The pooling of the large numbers of sera
as used in the preparation of Bacto-Complement eliminates the possibility of
non-specific reactions with antigens used in the complement-fixation test.

Bacto-Complement is reconstituted by adding 5 ml. of Bacto-Complement
Reconstituting Fluid, using a clean sterile syringe to make the transfer. Rotate
the bottle to effect complete solution of the complement. This solution will
retain its potency for two to three weeks if stored in the freezing compartment
of the refrigerator. After thawing the frozen solution, avoid unnecessary exposure
to room temperature.

Bacto-Complement is recommended for use in all complement-fixation tests.
Variables encountered in performing these tests necessitate that the complement
be titered just prior to use with the same reagents employed in the test.

It has been shown that the use of a satisfactory saline is of utmost importance
in the performance of complement-fixation tests. In many localities the use of
tap water for the preparation of saline is preferred over distilled water. The titer
of complement is often improved by incorporating 0.1 gram of magnesium
chloride or magnesium sulfate per liter of tap or distilled water saline, resulting
in more clear-cut complement-fixation reactions.

Bacto-Complement is supplied in 5 ml. amounts in vials. Each vial is accom-
panied with 5 ml. of Bacto-Complement Reconstituting Fluid.

REAGENTS FOR PRECIPITATION

TESTS

BAGTO-KAHN STANDARD ANTIGEN (B218)

Precipitation tests during the past thirty years have gained in interest and use
as diagnostic agents in syphilis. The Kahn test, described in 1923, was the first
practical precipitation test to gain wide usage. It is readily performed, rapid and
accurate.

Bacto-Kahn Standard Antigen is prepared according to Kahn's directions^ '^-^
and is a highly sensitive and specific reagent. Each lot is standardized to the same
standard degree of sensitivity and specificity. No lot is released without approval
from Dr. Kahn's laboratory. The antigen is stable and should be kept tightly
stoppered in the dark at room temperature. Bacto-Kahn Standard Antigen is
used in the standard and quantitative Kahn procedures with serum and spinal
fluid and is also used in the Universal Reaction of Kahn.^'^

iKahn: The Kahn Test, A Practical Guide, *Kahn: Serology with Lipid Antigen, i and

76:1928. 24:1950.

2 Kahn : Technique of the Standard Kahn 6 Kahn : An Introduction to Universal Sero-
Test, Univ. Mich. Press, 39- '944- logic Reaction in Health and Disease, 1951.

3 Kahn : Serology with Lipid Antigen,
271:1950.

312 DIFCO MANUAL

BAGTO-KAHN SENSITIZED ANTIGEN (B219)

Bacto-Kahn Sensitized Antigen is prepared in strict accordance with the pro-
cedure outlined by Kahn.^' 2,3,4 j^ |g ^j^ especially sensitive and stable antigen.
Each lot is carefully standardized and is rechecked under the personal super-
vision of Dr. Kahn. This antigen is used in the Kahn presumptive procedures
with serum and spinal fluid. To prevent fluctuations in the titer, this antigen
should be kept tightly stoppered, in the dark, at about 21 °C. (not below room
temperature).

iRahn: The Kahn Test, A Practical Guide, 3 Am. J. Clin. Path., 17:130:1947.

143:1928. * Am. J. Clin. Path., 17:770:1947.

2 Kahn : Technique of the Standard Kahn

Test, Univ. Mich. Press, 39:i944-

BAGTO-EAGLE FLOGGULATION (B220)
ANTIGEN

The flocculation technique developed and modified by Eagle^'2,3,4 {§ ^ Ye\2i'
tively simple and highly accurate diagnostic test for syphilis. Eagle's research on
the sterols led him to use a combination of corn germ sterol and cholesterol in
the antigen for his flocculation test. With these sensitizing agents he secured large
crystalline aggregates in positive sera, which made the reaction easier to read.

Bacto-Eagle Flocculation Antigen is a sensitive and satisfactory reagent for use
in the Eagle flocculation technique. It is diluted for use according to the titer
shown on the label. The antigen is stable when stored at room temperature. As
described by Eagle^'* this same antigen is used in both the "macro" and "micro"
modification of his flocculation technique.

1 J. Lab. Clin. Med., 17:787:1932. * Eagle: Laboratory Diagnosis of Syphilis,

2 J. Lab. Clin. Med., 22:300:1936. 215:1937.
^ Am. J. Syphilis, 22:22:1938.

(

BACTO-HINTON INDICATOR (B221)

The Hinton Glycerol Cholesterol Reaction for the detection of syphilis is
recommended for its accuracy and simplicity. For the actual reading of the test
only one tube need be considered. Like all other serological diagnostic tests, how-
ever, the success of this test depends upon the sensitivity of the reagents used as
well as rigid adherence to the details of the technique.

Bacto-Hinton Indicator is the cholesterolized heart extract used in Hinton's^
Glycerol Cholesterol Reaction for syphilis, and also in "A Modification of the
Hinton Test applied to Spinal Fluid" by Davies.^ This extract has been prepared
according to the directions given by Hinton and is also recommended for use in
the "micro" and "capillary" serological tests as described by Davies.^

This indicator is stable when it is stored, tightly stoppered, at room tempera-
ture. In the event that the cholesterol precipitates, the indicator is warmed to
redissolve the cholesterol before diluting it with salt solution for use in the per-
formance of the test.

Each lot of Bacto-Hinton Indicator is tested in Dr. Hinton's laboratory and is
not released for distribution until his approval is received.

ij. Lab, Clin. Med., 18:198:1932-33.

a Am. J. Clin. Path. 7:240:1937.

•J. Lab. Clin. Med., 22:959:1936-37.

SEROLOGICAL REAGENTS 313

BACTO-BJ.L. FLOGGULATION (B235)
ANTIGEN

Bacto-B.J.L. Flocculation Antigen is prepared according to the directions given
by Boerner, Jones and Lukens.^ It is an alcoholic extract of Bacto-Beef Heart.
It is stored in the dark in a tightly stoppered bottle at room temperature. Bacto-
B.J.L. Flocculation Antigen is satisfactory for the performance of the micro-
scopic and macroscopic flocculation tests described by these authors. Their tech-
nique has the advantage of simplicity, reproducibility and a desirable degree of
sensitivity and specificity.

1 Am. J. Clin. Path., Tech. Sup., 4:141:1940.

GARDIOLIPIN ANTIGENS

BAGTO-V D R L ANTIGEN (B388)

Bacto-V D R L Antigen is a cardiolipin-lecithin antigen for the V D R L Slide
and Tube Test Procedures used in the serological diagnosis of syphilis.

Bacto-V D R L Antigen is prepared according to the directions given by
Harris, Rosenberg, and Riedel,^ and consists of cardiolipin, cholesterol and
lecithin dissolved in absolute ethyl alcohol. Cardiolipin and lecithin are prepared
according to directions given by Pangborn.2'3.4.5

Buffered saline used in dilution of the Bacto-V D R L Antigen is prepared
according to the directions given by Harris, Rosenberg, and Riedel.^

Bacto-V D R L Antigen is used in the techniques as given in detail in the
Manual of Serologic Tests for Syphilis, Federal Security Agency, Public Health
Service, Division of Venereal Disease.^

Bacto-V D R L Antigen is supplied in 5 ml. units. Each unit consists of 5 ml.
antigen and 50 ml. Buffered Saline for diluting the antigen in preparing the
emulsion for the VDRL Slide and Tube Test Procedures for the serological diag-
nosis of syphilis.

1 J. Venereal Disease Inform., 27:169:1946. ^ j, giol. Chem., 161:71:1945.

2 Proc. Soc. Exp. Biol. Med., 48:484:1941. « J. Venereal Disease Inform., Supplement

3 J. Biol. Chem., 143:484:1942. No. 22, 1949.
*J. Biol. Chem., 153:343: i944-

BAGTO-KAHN GARDIOLIPIN ANTIGEN (B404)

Bacto-Kahn Cardiolipin Antigen is prepared according to the directions given
by Kahn and McDermott^ and Kahn^ and consists of cardiolipin, lecithin and
cholesterol in absolute ethyl alcohol. The cardiolipin and lecithin are prepared
according to the directions given by Pangborn.^'*'^-^ The amount of lecithin
present may vary slightly to give a suitable antigen; however, generally the anti-
gen consists essentially of 0.1 per cent cardiolipin, LO per cent lecithin, and
0.025 per cent cholesterol.

Bacto-Kahn Cardiolipin Antigen is recommended for use in conjunction with
standard Kahn antigen in the performance of the Kahn Standard Test with
serum and spinal fluids as well as quantitative procedures in accordance with the
directions given by Kahn.^ This antigen may also be used in a microflocculation
procedure as described by Kahn and McDermott.^ Each batch of Bacto-Kahn
Cardiolipin Antigen is tested and approved in Dr. Kahn's laboratory to assure
an antigen of the correct specificity and sensitivity.

314 DIFGO MANUAL

A complete discussion of the Kahn Cardiolipin Antigen and its use in the
serological diagnosis of syphilis is given in "Serology with Lipid Antigen with
Special Reference to Kahn and Universal Reactions" by Kahn.^

Bacto-Kahn Cardiolipin Antigen is supplied in 5 ml. bottles.

1 Am. J. Clin. Path., 18:364:1948. *J, Biol. Chem., 143:484:1942.

2 Kahn, Serology with Lipid Antigen, p. 159, ^ j giol. Chem., i53:343:i944-
164, 259, 263, and 283, 1950. ^J Biol. Chem., 161:71:1945.

sProc. See. Biol. Med., 48:484:1941.

BACTO-KOLMER CARDIOLIPIN (B438)
ANTIGEN

Bacto-Kolmer Cardiolipin Antigen is used in the Kolmer Complement Fixa-
tion Test for Syphilis as described by Kolmer and Lynch. ^ It consists of a solu-
tion of purified cardiolipin, lecithin and cholesterol (0.03 per cent cardiolipin,
0.05 per cent lecithin and 0.6 per cent cholesterol) in absolute ethyl alcohol in
the optimum combination to give maximum sensitivity consistent with specificity
in tests with sera and spinal fluids. Cardiolipin and lecithin are prepared accord-
ing to directions given by Pangborn.-'^*-^

Bacto-Kolmer Cardiolipin Antigen is tested and approved by Dr. Kolmer's
laboratory before release, assuring an antigen of the proper sensitivity and spec-
ificity. The dose to employ as given on the label was determined by a method
of antigenic titration described by Kolmer arid Boerner.^

This method determines the "optimum dose" instead of "antigenic units," and
the dose as given on the label is the optimum amount to use. It corresponds to
10 to 20 antigenic units by the older method of titration. The titration should be
checked before use. If employed for any other method, the titrations should be
conducted, and the dose determined, in accordance with the method employed.
The dilution should be prepared in quantities sufficient for one day's use, and a
new dilution made daily.

Bacto-Kolmer Cardiolipin Antigen is supplied in 5 ml. bottles.

1 J. Venereal Disease Inform., 29:166:1948. ^ J. Biol. Chem., 161:71:1945.

2 Proc. Soc. Exp. Biol. Med., 48:484:1941. ^ j^olmer and Boerner, Approved Laborator

3 J. Biol. Chem., 143:484:1942. Technic, 4th Edition: 679: 1945.
*J. Biol. Chem., I53:343;i944-

BACTO-HINTON CARDIOLIPIN (B440)
INDICATOR

Bacto-Hinton Cardiolipin Indicator is prepared according to the directions
given by Hinton, Stuart and Grant^ consisting of purified cardiolipin, lecithin
and cholesterol in absolute ethyl alcohol. The cardiolipin and lecithin are pre-
pared according to the directions given by Pangborn.2'3.4.5

Bacto-Hinton Cardiolipin Indicator is recommended for the regular Hinton
qualitative and quantitative tests. Each batch of Bacto-Hinton Cardiolipin Anti-
gen is tested and approved by Dr. Hinton's laboratory before being released for
distribution.

Bacto-Hinton Cardiolipin Indicator is supplied in 5 ml. bottles.

1 Am. J. Syphilis Gonorrh. Venereal Dis- » j. gjol. Chem., 143:484:1942.

eases, 33:587:1949. * J. Biol. Chem., 153 = 343:1944

" Proc. Soc. Biol. Med., 48:484:1941. ^ j, gjol. Chem., 161:71:1945.

SEROLOGICAL REAGENTS 315

REAGENTS FOR PREPARATION
OF ANTIGENS

BAGTO-BEEF HEART (B222)

The discovery that alcoholic extracts of normal organs would act as antigens
for tests for syphilis and that the antigenic principles were associated with
lipoids, has resulted in much experimentation and research. The first description
of the preparation of desiccated heart muscle and its use for the extraction of
phosphatides belongs to Rubow.^ This method for the preparation of dried tissue
was improved by Erlandsen.^ Dried heart muscle was employed by Neymann and
Gager^ in 1917 for the preparation of Wassermann test antigen. In 1922, Kol-
mer* described the technique of desiccating beef heart for use in the antigen
employed in his modification of the complement-fixation test for syphilis. Many
extracts of desiccated beef heart tissues are now employed as the source of the
necessary antigenic lipoidal substances for use in both complement-fixation and
flocculation tests.

Bacto-Beef Heart is a desiccated and powdered fresh beef heart tissue, trimmed
free from fat, possessing highly polytropic properties as each lot represents the
heart muscle of not less than several hundred animals. It has not been treated
with any solvent other than water. It contains a minimum of fat and moisture.
The production at one time of large quantities of Bacto-Beef Heart assures a
polytropic material, yielding extracts which are only slightly hemolytic and anti-
complementary and at the same time highly antigenic. Each lot is tested and
approved for its sensitivity and specificity by the laboratory designated for that
purpose by the author-serologists for use in the preparation of the following anti-
gens which are listed alphabetically:

B. J. L. Flocculation Antigen Hinton Indicator

B. J. L. Wassermann Antigen Kline Antigen

Eagle Flocculation Antigen Kolmer Improved Antigen

Eagle Wassermann Antigen Kolmer Improved Antigen (Reenforced)

Bacto-Beef Heart has also been recommended for use in the preparation of the
antigens used in such other tests for the diagnosis of syphilis as those of Mazzini,^
Rosenthal^ and Rytz.'^-^ It is also used in alcoholic^ and cholesterolized antigen, ^^
Bacto-Beef Heart is not recommended for the preparation of the antigens used
for the Kahn tests. Bacto-Kahn Beef Heart as discussed on page 316, is recom-
mended for the preparation of Kahn Standard Antigen and Kahn Sensitized
Antigen.

Bacto-Beef Heart should be stored in the refrigerator at 2-6°G. to secure the
most satisfactory antigens.

A description of the preparation of the more widely used antigens employing
Bacto-Beef Heart is available upon request.

1 Arch, exp. Path. Pharmakol., 52:173:1905. ^ J. Lab. Clin. Med., 21:934:1936.

2 Zeit. physiol. Chem., 51:71:1907. 8 J. Lab. Clin. Med., 22:1186:1937.

'J. Immunol., 2:573:1917. » Kolmer: Infection, Immunity, and Biologic

* Am. J. Syphilis, 6:76:1922. Therapy, 455:1924.

^ Am. J. Clin. Path., 9:163:1939. 10 Kolmer: Infection, Immunity, and Biologic

^ Proc. Soc. Exp. Biol. Med., 27:61:1929, Therapy, 456:1924.

316 DIFCO MANUAL

BAGTO-KAHN BEEF HEART (B223)

Bacto-Kahn Beef Heart^-^'^**'^ is recommended particularly for the preparation
of antigens for use in the Kahn tests for the diagnosis of syphilis and for use in
the Kahn Universal Reaction. Dried beef heart, even when stored in the refrig-
erator, may undergo changes which render it unsatisfactory for use in preparing
antigens for the Kahn procedures, although it continues to be satisfactory for use
in the preparation of other antigens. Bacto-Kahn Beef Heart is prepared spe-
cifically for use in the preparation of Kahn antigens. No lot is released for dis-
tribution until it has been found satisfactory for the preparation of Kahn Stand-
ard Antigen and Kahn Sensitized Antigen in Dr. Kahn's laboratory. Bacto-Kahn
Beef Heart should be stored in the refrigerator at all times.

The antigens employed in the Kahn tests are stable. For that reason Bacto-
Kahn Beef Heart should be converted into antigen as soon as the material has
been received from our laboratories. A description of the method of preparation
of Kahn Standard Antigen and Kahn Sensitized Antigen is available upon
request.

iKahn: The Kahn Test, A Practical Guide, SKahn: The Kahn Test, A Practical Guide,

Chapter 3:1928. Chapter 4:1928.

3 Kahn: Technique of the Standard Kahn * Am. J. Clin. Path., 17:117:1947.

Test, Univ. Mich. Press, p. 39, I944- ^ Kahn, Serology with Lipid Antigen, 271:1950.

BAGTO-GHOLESTEROL ( B224)

Bacto-Cholesterol is prepared especially for cholesterolizing antigens. It is of
the highest purity and is ash free.

The addition of pure cholesterol to reenforce antigens was first recommended
by Sachs. ^ By this means, antigens are rendered more sensitive without increasing
their anticomplementary or hemolytic powers.

Bacto-Cholesterol is also recommended for use as a standard in blood cho-
lesterol and other cholesterol determinations.

^ Klin. Wochschr., Berlin, 48:2066:1911.

BAGTO-GORN GERM STEROL (B225)

Bacto-Com Germ Sterol is a highly purified reagent which was developed in
cooperation with Dr. Eagle for use as a sensitizing agent in the antigen for his
tests.

This sterol is used in conjunction with Bacto-Cholesterol by Eagle^ in the
antigen for his flocculation technique for syphilis. In positive sera the crystalline
aggregates are large and readily lend themselves to detection.

ij. Lab. Clin. Med., 17:787:1932.

KAHN OVERSENSITIVE GORREGTION SOL'N. (B448)
KAHN UNDERSENSITIVE GORREGTION SOL'N. ( B449 )

Bacto-Kahn Oversensitive Correction Solution and Bacto-Kahn Undersensitive
Correction Solution are used for the correction of oversensitive and under-
sensitive alcoholic extracts of Bacto-Kahn Beef Heart in the preparation of Kahn
Standard Antigen and Kahn Sensitized Antigen as described by Wheeler,
Brandon and Kahn^'- and Wheeler and Brandon.^

1 Am. J. Clin. Path., 17:117:1947.
^ Am. J. Clin. Path., 17:130:1947.

2 Am. J. Clin. Path., 17:770:1947.

Diagnostic Reagents

BAGTO-THROMBOPLASTIN (B226)

Bacto-Thromboplastin is stabilized, desiccated rabbit brain substance for use
in determining the prothrombin time of bloods from patients with obstructive
jaundice, hepatitis, cirrhosis, hepatic neoplasia, biliary fistula, colitis, cholangitis,
hemorrhagic disease of the newborn, and other conditions wherein hypopro-
thrombinemia is suspected. It is particularly recommended for use in following
prothrombin activity in patients on dicoumarol, tromexan or other anticoagulant
therapy. It is applicable to the procedures described by Quick,i'2'3 Smith,
Warner and Brinkhouse,^ the bedside test of Smith et al.^, the micro-prothrombin
test of Kato,® and Kato and Poncher,'' the dilute plasma technique of Campbell,
Smith, Roberts and Link,^ Shapiro, Sherwin, Redish and Campbell,^ Shapiro^",
and its extensions^i'^^-^^'^* and is recommended for all tests requiring a potent
thromboplastin. Bacto-Thromboplastin has given exceptionally good results in
the preparation of the Two-Stage Reagent for the quantitative determination of
prothrombin concentration as described by Ware and Seegers.^^

Quick Prothrombin Time Test^
Reagents:

1. Bacto-Thromboplastin

Empty the contents of one ampul Bacto-Thromboplastin into a clean dry
test tube. Add 4.0 ml. of 0.85 per cent sodium chloride solution. Mix lightly
to suspend and wet all particles. Incubate the mixture in a water bath at
45-48°C., for 10 minutes, during which time gently twirl the tube momen-
tarily at three-minute intervals to resuspend the solids. Avoid excessive agi-
tation as this will yield an unsatisfactory heavy milky extract.

Place a thin layer of absorbent cotton over the wide end of a clean dry
pipette. Insert the covered end into the tube containing the incubated
thromboplastin mixture and carefully lower it through the suspension to the
bottom of the tube keeping the end of the pipette pressed firmly against
the cotton. Withdraw as much extract as possible through the cotton by
gentle suction and transfer the filtered extract to a clean container. The
filtered extract should be opalescent without particles. Some laboratories
may prefer to use the extract without filtration. In such cases it is best to
employ a pipette with a wide tip orifice.

The Thromboplastin Extract should be used within six hours following
preparation. Unused portions of the Thromboplastin Extract should be
frozen solidly in the freezing compartment of the refrigerator, thawed and
reincubated for 5 minutes at 37°C. before being used in the test. Extract
properly frozen will retain full potency for at least three months. Repeated
thawing and freezing of the extract is not recommended. Accordingly, if the
extract is prepared for stock use it should be distributed in amounts of
approximately one day's usage or single test dose (0.1 ml.) and then frozen
and stored as recommended above. In such cases the amount required for
a day's use or one test can then be withdrawn from the freezer, thawed and
reincubated as specified.

2. Bacto-Sodium Oxalate 0.1 Molar or dissolve 1.34 grams of anhydrous re-
agent grade sodium oxalate in 100 ml. of distilled water.

317

318 DIFCO MANUAL

3. Bacto-Calcium Chloride 0.02 Molar or dissolve 0.222 gram of anhydrous
reagent grade calcium chloride in 100 ml. distilled water.

4. Bacto-Sodium Chloride 0.85 per cent or dissolve 0.85 gram reagent grade
sodium chloride in 100 ml. distilled water.

Procedure:

Obtain 4.5 ml. of blood by venipuncture and immediately mix it with 0.5 ml.
of sodium oxalate solution. Within one-half hour centrifuge the oxalated blood
to separate the plasma from the formed elements.

Place the tubes containing thromboplastin extract and the tubes containing
calcium chloride solution in the 37 °C. water bath to bring them to this working
temperature.

Transfer 0.1 ml. of plasma to a small dry serologic tube supported by a rack
in the 37 °C. water bath. Add 0.1 ml. thromboplastin extract and twirl the tube
to mix the contents. After the tube has been in the water bath a few seconds, add
0.1 ml. calcium chloride solution by blowing it forcibly into the mixture and
simultaneously start the stop watch. Keep the tube in the water bath and shake
it lightly until within a few seconds of the expected clotting time; then by gently
tilting the tube in a horizontal position obseive the formation of the clot or
fibrin web which is the end point.

Many laboratories may prefer to use the following loop technique. Imme-
diately upon the addition of the calcium chloride solution and starting of the
stop watch, insert a clean iron, nichrome or platinum wire with a terminal loop
about ^" in diameter into the tube and stir thoroughly. Draw the loop from the
back of the tube to the front through the plasma-thromboplastin-calcium chloride
mixture at the rate of 2 sweeps per second, and observe the time at which the
clot forms. When the clot forms it will usually adhere to the loop and is easily
recognizable. Stop the watch at the first evidence of the clot and record the time
required for the plasma to clot after adding the calcium chloride to the plasma-
thromboplastin mixture. This is the prothrombin time.

The time elapsing between the addition of calcium chloride and the end point
is the prothrombin time. The test should be run in duplicate or triplicate to
obtain the minimum clotting time.

Some laboratories find it helpful to check the prothrombin time of the test
plasma diluted with an equal volume of 0.85 per cent sodium chloride solution
as well as the whole plasma. Where such a dilution of the test plasma is made,
this must be taken into consideration when interpolating the prothrombin
activity.

For determining the prothrombin activity of a patient's plasma by the Quick
method it is desirable to construct a prothrombin activity curve for a given
thromboplastin using several normal human plasmas. The following is a typical
example :

This is a typical prothrombin activity curve constructed with Bacto-Thrombo-
plastin using normal human plasmas. It was constructed by running prothrombin
time tests on 10, 20, 30, 40, 60 and 100 per cent plasma of normal individuals,
and plotting the prothrombin time against per cent prothrombin employed. The
indicated concentration of plasmas used were obtained by diluting the normal
plasmas with 0.85 per cent sodium chloride solution.

The prothrombin activity of the blood to be tested is obtained by determining
the prothrombin time of the whole plasma, locating this time on the prothrom-
bin activity curve and, by means of the curve, converting the prothrombin time
to the per cent of prothrombin activity.

A curve is submitted with each lot of Bacto-Thromboplastin to serve as a guide
for laboratories using Bacto-Thromboplastin according to the method of Quick.

DIAGNOSTIC REAGENTS

319

70]

(A(

CT<

D--

rHROMBOPLAStIN

|.„

PROTHROMBIN ACTIVITY CURVE

J T 1 I 1 I I r T r , .

*s.

^**

■*■

—

0 10 20 30 40 50 60 70 80 90 100
Per cent Prothrombin

However, variability in prothrombin times obtained by different technicians
makes it imperative that the technician running the test construct a curve with
two or more normal plasmas using a given thromboplastin to make sure his or
her values correspond with the guide. Otherwise, the technician must construct
his own prothrombin activity curve using three or more normal human plasmas.
Interpretation :

When Bacto-Thromboplastin is used in the Quick prothrombin time test, clot
formation is obtained in 12-14 seconds with normal plasma. The quantitative
relationship between the prothrombin content of plasma and its clotting time as
obtained by the Quick test is discussed in detail by Quick^^-^^-^^ and in the
"Manual for Prothrombin and Blood Clotting Determinations for Laboratory
Technicians" published by the Newton Health Department, Newton, Massa-
chusetts.

Safe and effective limits of prothrombin activity in controlled dicoumarol
therapy are discussed by: Allen, Barker and Waugh,^^ Wright and Prandonij^o
Butsch and Stewart,^! Echstam,22 Evans,^^ Barker, Cramer, Hurn and Waugh,^*
Peters, Guyther and Brambel,-^ Barker, Hines, Kvale and Allen,^^ Allen^^ and
in leaflets descriptive of dicoumarol and its clinical application published by the
licensees of dicoumarol, Eli Lilly and Company,^^ Abbott Laboratories^^ and E. R.
Squibb and Company.^^

The necessity of repeated prothrombin determinations in following and deter-
mining the dosage in anticoagulant therapy has been emphasized by Wellman
and Allen.31 Bacto-Thromboplastin is eminently suited for this control procedure.

Link-Shapiro Dilute Plasma Technique^'^'^^

Reagents:

1. Thromboplastin Extract. Prepare thromboplastin extract as described for
the Quick technique and dilute with an equal volume of sodium chloride
0.85 per cent.

320 DIFCO MANUAL

2. Sodium Oxalate 0.1 Molar. Dissolve 1.34 grams of anhydrous reagent grade
sodium oxalate in 100 ml. of distilled water.

3. Calcium Chloride 0.02 Molar. Dissolve 0.222 gram of anhydrous reagent
calcium chloride in 100 ml. distilled water.

4. Sodium Chloride 0.85 per cent. Dissolve 0.85 gram reagent grade sodium
chloride in 100 ml. distilled water.

Procedure:

Obtain 4.5 ml. of blood by venipuncture and immediately mix it with 0.5 ml.
sodium oxalate solution. Centrifuge the oxalated blood at 1700-2000 r.p.m. to
separate the plasma from the formed elements. Pipette the plasma into a clean,
dry test tube.

Prepare a 12.5 per cent plasma by adding 1 ml. of plasma to 7 ml. 0.85 per
cent sodium chloride solution in a test tube.

Add 1 ml. of calcium chloride solution to 1 ml. of thromboplastin extract in
a second test tube.

Place all reagents and plasma in a water bath at 37°C. to bring them to this
working temperature.

Add 0.1 ml. diluted plasma to a small serological tube firmly secured in a
vertical position in a glass walled water bath at 37°G.

Add 0.2 ml. calcium chloride-thromboplastin mixture to the tube by blowing
it directly into the plasma in the tube from the tip of a graduated pipette and
simultaneously start the stop watch. Prothrombin time may be determined either
by the tilting tube method or the wire loop technique described in the Quick
Prothrombin Time Technique discussed above.

Stop the watch at the first evidence of the clot and record the time required
for the plasma to clot after adding the thromboplastin-calcium chloride mixture.
This is the prothrombin time of the dilute plasma. Standard values for diluted
normal human plasma lie between 35-42 seconds. Two or more known normal
plasma diluted similarly to the plasma under test should be run as controls. The
prothrombin time of whole plasma may be performed by using this same tech-
nique, but omitting the dilution of the plasma.

Complete details for prothrombin time determinations by the Smith Bedside*
and Kato Micro Prothrombin Test^''^ are available upon request.

Precautions — factors influencing prothrombin time:

1. Storage temperature of desiccated Bacto-Thromboplastin in ampuls.

Bacto-Thromboplastin, kept properly refrigerated below 6°C., will retain
its potency for years. At room temperature, 22-23 °C., there may be a slight
reduction in potency after one or two months. Storage temperature higher
than 25 °C., will accelerate destruction of the active thromboplastin prin-
ciple.

2. Reagents.

The calcium chloride, sodium chloride and sodium oxalate used in the
various prothrombin time tests should be anhydrous and of reagent grade.
Prolonged clotting times obtained with Bacto-Thromboplastin have been
traced most frequently to a faulty calcium chloride reagent. A calcium
chloride solution either more or less concentrated than that indicated for
use in the Quick technique may prolong the clotting time. Anhydrous
calcium chloride is hygroscopic, and absorbs moisture from the atmos-
phere. Compensation for the moisture content must be taken into con-
sideration when preparing the 0.02M solution.

3. Thromboplastin Extract.

Thromboplastin extracts that are deeply opaque or milky may give pro-

DIAGNOSTIC REAGENTS 321

longed clotting times. Deeply opaque or dense thromboplastin extracts
result through excessive agitation of the suspension during the extraction
period and may give unsatisfactory results. Unfiltered extracts which are
not deeply opaque even though they contain gross particles of brain tissue
generally give satisfactory results.

4. Temperature for Extraction of the Thromboplastin.

A temperature of 45-48° C, for 10 minutes is most satisfactory. Tempera-
tures in excess of 50° C, will cause deterioration of the thromboplastin
principle. The optimum temperature for performing the prothrombin time
test is 36-38°C.

5. Aging of Thromboplastin Extract.

Thromboplastin extract is subject to deterioration. There should be no
loss of potency of the extract if used within 6 hours after its preparation.
Prolonged aging results in a gradual loss in potency. Extracts held over
from one day to another should be frozen solid to retard deterioration.
These frozen extracts should be thawed and incubated for 5 minutes at
37°C., before being used. Extracts properly frozen will retain full potency
for at least two weeks. Repeated thawing and freezing of the extract is
not recommended.

6. Age of Plasma at Time of Test.

Fresh plasma should be used in running the test. Plasmas allowed to stand
at room temperature or ice box temperature for more than 2-3 hours may
give clotting times slightly higher than does fresh plasma.

7. Calcium Chloride Addition.

Usually if the thromboplastin extract is allowed to remain in contact with
the plasma for 30 seconds before the calcium chloride is added, the clot-
ting time is shortened in comparison with that obtained if the calcium
chloride is added immediately following the addition of the extract. Either
procedure is satisfactory if adhered to consistently. Our curve is based
upon the addition of calcium chloride a few seconds after the addition
of the thromboplastin extract.

8. Cleanliness of Glassware.

Soaps and other detergents used in cleaning glassware must be completely
removed by rinsing. Frequently glassware appears optically clean but re-
mains chemically contaminated with adsorbed soap or detergent. These
materials interfere with prothrombin procedures as reported by Leh-
mann.32 He also stated that results will still be irregular if the container
in which the blood has been collected is not chemically clean.

9. Ionic Contamination.

Metallic ions present in excess in the distilled water used in making the
saline for extraction of the thromboplastin may lead to prolonged clotting
times. Water from copper stills or water which has been in contact with
copper vessels may contain enough copper to be detrimental.

10. Agitation of Plasma-Thromboplastin-Calcium Chloride Mixture.

Failure to mix thoroughly the reagents with the plasma at the time of
addition may result in a prolonged clotting time. Also, excessive agitation
of the plasma-thromboplastin-calcium chloride mixture will often prolong
the clotting time.

11. Silicone coated apparatus.

Some laboratories prefer to use syringes, needles and glassware that have
been coated with silicone for handling blood for prothrombin time deter-
minations. Where silicone coated glassware is used, the prothrombin time
will be slightly prolonged over that normally obtained with uncoated glass-
ware. According to Quick,^^ one should use 0.01 Molar Calcium Chloride

322

DIFGO MANUAL

solution for running prothrombin times of plasma collected and handled
in silicone coated glassware.

12. Time of drawing blood.

Blood drawn for prothrombin determinations should be taken previous to
meal time and not directly after meals. High lipid content of plasma
following ingestion of high fat food materials may prolong the clotting
time.

13. Effect of Salicylates and other drugs.

Aspirin and other salicylates, as well as caffeine as shown by Link,^* when
ingested may influence the clotting mechanism by prolonging the pro-
thrombin time.

1 J. Biol. Chem., logrlxxiii: 1935.

2 J. Am. Med. Assoc, 110:1658:1938.

3 J. Hem., 4:1281:1949.

* J. Exp. Med., 66:801:1937.

s J. Am. Med. Assoc, 113:380:1939.

* Am. J. Clin. Path., 10:147:1940.
'J. Am. Med. Assoc, 114:749:1940.
8 J. Biol. Chem., 138:1:1941-

» Proc Soc Exp. Biol. Med., 50:85:1942.
^0 Exp. Med. Surgery, 2:103:1944.

11 Proc Soc. Exp. Biol. Med., 52:12:1943.

12 Proc Soc. Exp. Biol. Med., 53:218:1943.

13 Arch. Surgery, 50: 137:1 945-
i*Am. J. Clin. Path., 17:405:1947.
15 Am. J. Clin. Path., 19:471:1949.

i« J. Am. Med. Assoc, 114:1342:1940.
"Am. J. Clin. Path., 19:1016:1949.

942-
942.

1944-

18 Quick, The Physiology and Pathology of

Hemostasis, 1951.
""^ J. Am. Med. Assoc, 120:1009:1942.
Am. Med. Assoc, 120:1015
Am. Med. Assoc, 120:1025
==> Minnesota Med., 27:455:1944.
23 New England J. Med., 230:13
^ Surgery, 17:207:1945.
25 J. Am. Med. Assoc, 130:398:1946.
20 Minnesota Med., 29:250:1946.
f. Am. Med. Assoc, 134:323:1947.
icumarol Leaflet, Eli Lilly & Go.
28 Dicumarol Leaflet, Abbott Laboratories.

30 Dicumarol Leaflet, E. R. Squibb & Sons.

31 Proc Mayo Clinic, 26:257:1951.
62 Lancet, Vol. I, (XXIV) : 1133:1950.

33 Personal Communication, 1950.

34 Chicago Med. Bull., 51:53:1948.

20 7

28 Die

BAGTO-SODIUM CHLORIDE 0.85 per cent (B379)

Bacto-Sodium Chloride 0.85 per cent is recommended for use in all prothrom-
bin techniques requiring 0.85 per cent sodium chloride solutions. It is prepared
with reagent grade sodium chloride and triple distilled water.

Bacto-Sodium Chloride 0.85 per cent is supplied in bottles of 100 ml. in
packages of 6.

BAGTO-SODIUM OXALATE 0.1 MOLAR (B377)

Bacto-Sodium Oxalate 0.1 Molar is recommended for use in all prothrombin
techniques requiring 0.1 Molar sodium oxalate solutions. It is prepared with
anhydrous, reagent grade sodium oxalate in triple distilled water.

Bacto-Sodium Oxalate 0.1 Molar is supplied in 25 ml. bottles in packages of 6.

BAGTO-GALGIUM GHLORIDE (B378)
0.02 MOLAR

Bacto-Calcium Chloride 0.02 Molar is recommended for use in all prothrom-
bin techniques requiring 0.02 Molar solutions of calcium chloride. It is prepared
with anhydrous, reagent grade calcium chloride and triple distilled water.

Bacto-Calcium Chloride 0.02 Molar is supplied in 5 ml. and 25 ml. bottles in
packages of 6.

DIAGNOSTIC REAGENTS 323

BAGTO-PROTHROMBIN 2 STAGE (B407)
REAGENT

Bacto-Prothrombin 2 Stage Reagent is a desiccated reaction mixture dried
from the frozen state, prepared according to the specifications of Ware and
Seegers/ for use in the two-stage procedure for the quantitative determination
of prothrombin concentration and Ac globulin. The two-stage prothrombin con-
centration determination, as the name implies, is carried out in two steps. The
fibrinogen is first removed from the plasma under test by adding thrombin. The
clot which forms is defibrinated and the resulting fluid allowed to stand for a
short time for destruction of the added thrombin by the antithrombin present in
the serum. The second step consists of measuring quantitatively the unaltered
prothrombin in the fibrinogen-free plasma. This is accomplished by adding
Bacto-Prothrombin 2 Stage Reagent and Bacto-Ac Globulin to the fibrinogen-
free plasma to convert the prothrombin to thrombin and subsequently measuring
the thrombin activity on a standardized solution of fibrinogen. A thrombin unit
is that amount of thrombin which will clot 1 ml. of standardized fibrinogen
solution in 15 seconds under conditions imposed by the two-stage analysis. One
unit of thrombin is derived from each unit of prothrombin. Therefore, the pro-
thrombin unitage is the same as that of the thrombin. Complete details of the
two-stage procedure for measuring prothrombin concentration are outlined by
Ware and Seegers.^

Bacto-Prothrombin 2 Stage Reagent is supplied in vials of 10 ml. which are
rehydrated by adding 10 ml. 0.6 per cent sodium chloride solution. Bacto-
Prothrombin 2 Stage Reagent as well as the resulting solution should be refrig-
e: ated when not in use.

Bacto-Ac Globulin is also available for use in conjunction with Bacto-Pro-
thrombin 2 Stage Reagent in the two-stage procedure for the quantitative deter-
mination of prothrombin concentration.
lAm. J. Clin. Path., 19:471:1949.

BAGTO-Ac GLOBULIN (B447)

Bacto-Ac Globulin is desiccated beef serum dried from the frozen state pre-
pared according to the specifications of Ware and Seegers,^ for use as a source of
accelerator globulin in the two stage procedure for the quantitative determina-
tion of prothrombin. It is recommended for use in conjunction with Bacto-Pro-
thrombin 2 Stage Reagent for the total conversion of prothrombin to thrombin.

Bacto-Ac Globulin is rehydrated by adding 2 ml. sterile distilled water to each
vial to give a solution comparable with fresh beef serum. The reconstituted Ac
Globulin is further diluted with 0.85 per cent sodium chloride solution to give a
final concentration of 1 : 600 as used in the two stage prothrombin concentration
test of Ware and Seegers. Bacto-Ac Globulin as well as the solution should be
stored in the refrigerator at 2-6° C.

Bacto-Ac Globulin is supplied in 2 ml. vials in packages of 6.

lAm. J. Clin. Path., 19:471:1949.

324 DIFCO MANUAL

BAGTO-PROTHROMBIN FREE (B445)
BEEF PLASMA

Bacto-Prothrombin Free Beef Plasma is prepared according to the method of
Quick^'2.3 for use in the prothrombin consumption tests and for diluting human
plasma for prothrombin time determinations according to the method of Rosen-
field and Tuft.-i

To rehydrate Bacto-Prothrombin Free Beef Plasma, add 4.5 ml. distilled water
and rotate gently in an end over end motion to effect solution without generating
excessive air bubbles. The reconstituted plasma is equivalent to freshly prepared
deprothrombinized beef plasma. Bacto-Prothrombin Free Beef Plasma as well as
the reconstituted plasma should be stored in the refrigerator at 2-6° C.

Bacto-Prothrombin Free Beef Plasma is supplied in 5 ml. vials in packages
of 6.

1 Blood, 4:1281:1949. 3 Quick, The Physiology and Pathology of

2 Am. J. Clin. Path., 19:1016:1949. Hemostasis, 142:1951.

* Am. J. Clin. Path., 17:405:1947.

BAGTO-PROTHROMBIN FREE (B446)
RABBIT PLASMA

Bacto-Prothrombin Free Rabbit Plasma is prepared according to the method
of Quicki'2.3 for use in the prothrombin consumption test. It may also be used
for diluting plasmas for prothrombin time determinations by the method de-
scribed by Rosenfield and Tuft.*

To rehydrate Bacto-Prothrombin Free Rabbit Plasma, add 4.5 ml. distilled
water to each 5 ml. vial. Rotate gently in an end over end motion to effect solu^
tion without generating excessive air bubbles. The reconstituted plasma is equiv-
alent to freshly prepared deprothrombinized rabbit plasma. Bacto-Prothrombin
Free Rabbit Plasma as well as the reconstituted plasma should be stored in the
refrigerator at 2-6° G.

Bacto-Prothrombin Free Rabbit Plasma is supplied in 5 ml. vials in packages
of 6.

^ Bloody 4j^i 281 M 949. _ 8 Quick, The Physiology and Pathology of

: 405: 1947-

2 Am. J. Clin. Path., 19:1016:1949. Hemostasis, 142:1951.

*Am. J. Clin. Path., 17:

BAGTO-GEPHALIN GHOLESTEROL (B238)
ANTIGEN

Bacto-Cephalin Cholesterol Antigen is recommended for use in the Hanger
flocculation test^'^ for determining the index of disturbance of the liver paren-
chyma. It is a stable desiccation consisting of 1 part especially prepared sheep
brain cephalin and 3 parts Bacto-Cholesterol.

The Hanger flocculation test is regarded as an index of inflammatory and
certain other forms of degenerative disturbances of the liver parenchyma, and
does not necessarily parallel hepatic function tests. Cephalin cholesterol emul-
sions, properly prepared, are not flocculated by sera from normal individuals but
are flocculated by sera from patients with active disturbance of the liver paren-
chyma. The degree of flocculation parallels the severity of active liver disease
and may, therefore, be employed prognostically in estimating the degree and

(

DIAGNOSTIC REAGENTS 325

persistence of the active process. Jaundice due to biliary obstruction usually may
be distinguished from hepatogenous jaundice by this flocculation test.

Since its introduction in 1938 the Hanger flocculation test has won the
unanimous approval and acceptance of its many investigators. The publications
of Rosenberg,^ Rosenberg and Soskin,* Phole and Stewart,^ Mateer, Baltz,
Marion, Hollands and Yagle,^ Nadler and Butler/ Lawson and Englehardt,^
Yardumian and Weisband,^ and Mateer, Baltz, Marion and MacMillin^o express
the high regard which the authors have for the test and characterize it as a
valuable adjunct to the other tests in the study of hepatic dysfunction.

Kopp and Solomon,^^ Mirshy and Von Brecht^^ ^nd Lippencott, EUerbrook,
Hesselbrock, Gordon, Gottlieb and Marbel^^ obtained positive cephalin choles-
terol flocculation reactions with sera from malarial patients. Guttman, Potter,
Hanger, Moore, Pierson and Moore^* confirmed these observations with sera
from malarial infected patients and found the changes in the blood to simulate
those in acute hepatitis — hypoalbumenemia, decreased capacity of the serum
albumin fraction to inhibit the flocculating action of gamma globulin, and an
increase in the globulin content of serum in this disease. In malaria infection, the
formation of serum protein was more profoundly deranged than many other
functions ascribed to the liver and these changes were well demonstrated by a
series of cephalin cholesterol flocculation tests.

Mateer, Baltz, Steele, Brouwer and Colvert^^ in their studies on chronic sub-
clinical impairment of the liver, regarded the cephalin cholesterol flocculation
test as the most satisfactory single screening test available to detect cases of early
hepatic impairment. Mateer, Baltz, Comanduras, Steele and Brouwer^^ recom-
mended the cephalin cholestrol flocculation test as (a) the best of all screening
tests to detect early hepatic impairment in subclinical cases and preoperative
cholelithiasis cases; (b) valuable in chronic hepatitis and cirrhosis cases, and (c)
extremely helpful in detecting residual impairment in post-icteric repair stage of
acute hepatitis, and helpful in early stages.

The Hanger flocculation test when properly executed is extremely simple
and reliable. It is a more sensitive index of hepatic disturbance than many of the
functional studies and gives by far the best correlation with clinical observations.
Because of its simplicity, as well as its reliability and efficiency, the flocculation
test is advocated as an ideal routine test for determining active disease of the
liver.

Preparation of the Test Antigen

The test antigen is prepared from the desiccated material in the following
manner:

1. Add 5 ml. of anesthetic ether per unit bottle to effect solution of the con-
tents. If turbidity persists, add one drop of distilled water to obtain a clear
solution. This solution constitutes the stock ether antigen of Hanger and is stable
for months if kept tightly stoppered to prevent evaporation.

2. The final test antigen is prepared by adding (slowly and with stirring)
1 ml. of the stock ether antigen solution to 35 ml. of distilled water warmed to
65-70°C. and then heating slowly to boiling. The mixture is allowed to simmer
until the final volume is reduced to 30 ml. During the heating, all coarse granular
clumps should be dispersed, resulting in a stable, milky, translucent emulsion
from which all traces of ether are driven off. The antigen is cooled to room tem-
perature and then is ready for use. The liquid emulsion when properly prepared
and stored in the refrigerator at 2-6° G. is stable.

Excessive bacterial contamination of the test antigen emulsion or of the serum-
saline-antigen test mixture may give rise to falsely positive flocculation. This may

326 DIFGO MANUAL

be overcome by using clean, dry, sterile glassware in which to prepare and store
the emulsion and in which to perform the test. The addition of sodium ethyl
mercuri-thiosalicylate (Merthiolate), in a concentration of 1:10,000 to the test
antigen emulsion and to the saline used for diluting the serum, has given good
results. The antigen emulsion should be stored at refrigerator temperature,
2-6° C., when not in use.

Performance of the Hanger Flocculation Test

The Hanger flocculation test is performed by adding 1 ml. of the aqueous
lipoid emulsion to a test tube (preferably a centrifuge tube) containing 0.2 ml.
of the patient's serum diluted with 4 ml. of 0.85 per cent sodium chloride. After
thorough shaking and stoppering with cotton, the tube is allowed to stand undis-
turbed in the dark at room temperature ( 20-25 °C.) and a notation is made at
the end of 24 and 48 hours as to the amount of flocculation and precipitation
that has taken place. With normal human sera the emulsion remains as a stable
homogeneous suspension, but with sera from patients with diffuse hepatitis the
lipoid material tends to flocculate or precipitate to the bottom of the tube.
A + + + + reaction indicates a complete precipitation leaving the supernatant
liquid water-clear. Gradations of the reaction between negative and -f ++H-
are designated in terms of +, ++ and +++• No test should be regarded as
negative until 48 hours have elapsed without flocculation.

Neefe and Reinhold^^ discovered that the cephalin cholesterol flocculation
reaction was influenced by light and that this photosensitivity gave rise to falsely
positive reactions with normal sera. Hanger^^ and Mateer^^-^^ confirmed Neefe's
and Reinhold's observations. Their findings suggested that the test antigen
emulsion and the serum should be protected from light and that the serum-
saline-antigen test mixture should be incubated in the dark at room temperature
( 20-25 °C.). Higher or lower incubation temperatures are not indicated. The
patient's serum should not be diluted until the test is to be performed.

A rapid Cephalin Cholesterol Flocculation Test using centrifugation has been
described by Moloney, Donovan and Whoriskey.-*^ While less sensitive than the
regular Hanger test, the centrifugation technique is rapid, permitting the reading
of the test within a few minutes. Saifer and Zymaris-^ have described an easily
performed accurate photometric microprocedure for the quantitative determina-
tion of electrophoretically pure gamma globulin (Cohn's Fraction H) in the
range of 100 to 1,000 micrograms of protein using Bacto-Cephalin Cholesterol
Antigen in the presence of Hayem's solution.

Bacto-Cephalin Cholesterol Antigen is supplied in 5 ml. bottles in packages
of 6. Each bottle or unit is sufficient for the preparation of 5 ml. of stock ether
antigen and 150 ml. of the final test antigen.

1 Trans. Assoc. Am. Physicians, 53:148:1938. ^Science, 99:20:1944.

3 J. Clin. Invest., 18:261:1939. i3 j. Qjn. Invest., 24:616:1945.

3 Arch. Surgerv-, 43:231:1941. -* J. Clin. In\est., 24:296:1945.

* Am. J. Digestive Diseases, 8:421:1941. " j. Am. Med. Assoc, 133:909: i947>

^ J. Clin. Invest., 20:241:1941. i^ Gastroenterology, 8:52:1947.

^ Am. J. Digestive Diseases, 9:13:1942. "Science, 100:83:1944.

^Surgery, 11:732:1942. 1* Personal Communication, 1944.

8 New Orleans Med. Surgical J., 95:60:1942. ^^ Personal Communication, 1944.

9 Am. J. Clin. Path., 13:383:1943- 20 Am. J. Clin. Path., Tech. Supp., 18:568:
1° J. Am Med. Assoc, 121:723:1943. 1948.

^ Am. J. Med. Sci., 205:90:1943. 21 j, Clin. Investigation, 31:1:1952.

DIAGNOSTIC REAGENTS 327

BAGTO-THYMOL TURBIDITY REAGENT (B328)

Bacto-Thymol Turbidity Reagent is recommended for use in the Maclagan
thymol turbidity test and its modifications for indicating hepatic parenchymal
impairment. It is a barbital buffered saturated thymol solution with a reaction of
pH 7.55, prepared according to the method described by Maclagan,^ and the
modification of Mateer et al.^-^

It was observed by Maclagan,^ that a turbidity or precipitate formed in a
buffered saturated thymol solution when serum from patients with certain types
of liver disease was added to it. The degree of turbidity paralleled closely the
severity of parenchymatous hepatic impairment and provided a quantitative
measure of the extent of the damage done. Clinical tests indicated the thymol
turbidity reaction to be a valuable indicator of liver dysfunction particularly in
infective hepatitis and cirrhosis. Findings were negative or only weakly positive in
obstructive jaundice.

Clinical studies employing the thymol turbidity test and other tests used lor
detecting hepatic impairment have been reported by Watson and Rappaport,*
Neefe,^ Neefe and Reinhold^ and Mateer et al.-'^-'^ These authors have all as-
signed the thymol turbidity test a significant place as an adjunct in studying liver
disease. Their results parallel, to a degree, those obtained with the cephalin
cholesterol flocculation reaction; however, significant differences in positivity and
sensitivity displayed by the two tests on the same sera indicate that the chemical
mechanisms of the tests are different.

In a study of the sensitivity and value of the thymol turbidity and thymol
flocculation tests Mateer et al.^-^ showed that the buffer solution with a reaction
of pH 7.55, gave more satisfactory results than at pH 7.8 as originally suggested
by Maclagan, and could be used for acute as well as chronic cases. With this
slightly more acid thymol buffer solution they reported that the sensitivity of
the test was greatly increased and that no false positives were obtained. In
addition, the modified solution was of equal value in chronic cases and for
following the progress of acute impairment. Mateer's observation of the greater
sensitivity of the thymol turbidity reagent at pH 7.55 has been confirmed by
Neefe^ et al. Bacto-Thymol Turbidity Reagent is prepared according to the
modification suggested by Mateer et al.--^ with a reaction of pH 7.55.

Procedure:

Measure 0.05 ml. of unheated serum into a dry tube and add 3 ml. of Bacto-
Thymol Turbidity Reagent. Allow to stand for one-half to one hour, and
examine in a comparator with a black line on a white background against the
turbidity standards of Kingsbury et al.^ Bacto-Kingsbury Turbidity Standards
are now available. If the turbidity exceeds the 100 mg. per cent standard, dilute
with a further measured volume of buffer as required. The result is expressed
in arbitrary units equal to the appropriate standard divided by ten with allow-
ance for dilution. The standard dilution is 1-60, so that if the final dilution is
1-120 and the mixture then matches the 70 mg. per cent tube, the result is 14
units.

-, , f , TT • Standard tube reading X final dilution of serum

Expressed as a formula : Units = -rrr^

bUU

Normal limits are 0 to 4 units. Tubes showing positive results, as evidenced by
turbidity within one hour, if held overnight usually show flocculation. This

328 DIFCO MANUAL

flocculation may also be used as a measure of hepatic damage.^'^ The reaction
is inhibited by heating to 56°C. for 30 minutes, so that inactivated sera cannot
be used. The turbidity standards referred to are those in common use for urine
protein estimation. They should be checked every few months against diluted
serum of known protein content as recommended by Kingsbury^. Also, a photo-
electric method for measuring the turbidity quantitatively in this test has been
described by Shank & Hoagland.^o

Occasionally a slight turbidity or a light flocculent precipitate forms in the
reagent upon long standing. This slight turbidity in no way interferes with the
result obtained; however, if any flocculent be present, it should be removed by
filtration through ash-free filter paper into chemically clean receptacles. The re-
agent should be stored in the refrigerator at 2-6° C.

Bacto-Thymol Turbidity Reagent is supplied in 25 ml. bottles in packages of 6.

iBrit. J. Exp. Path., 25:234:1944- « Gastroenterology, 7:393:1946.

2 Gastroenterology, 8:52:1947- 'Cyclopedia of Med., 1947.

3 J. Am. Med. Assoc, 133:909:1947- ^ Am. J. Med., 8:60:1950.

4 J. Lab. Clin. Med., 30:983:1945. ® J- Lab. Clm. Med., 11:981:1926.
6 Gastroenterology, 7:1:1946. ^° J- Biol. Chem., 162:133:1946-

BAGTO-KINGSBURY STANDARDS (B349)

Bacto-Kingsbury Standards are prepared according to the specifications of
Kingsbury, Clark, Williams and Post^ for use in the Maclagan Thymol Turbidity
Test and in the estimation of urinary albumin. The set consists of seven stand-
ards, corresponding to 5, 10, 20, 30, 40, 50 and 75 per cent albumin as published
by Kingsbury et al.^ These standards are especially recommended for use with
Bacto-Thymol Turbidity Reagent (see page 327) in the Maclagan test^ and
modifications^'* indicating hepatic parenchymal impairment.

1 J. Lab. Clin. Med., 11:981:1926. a Gastroenterology, 8:52:1947.

a Brit. J. Expl. Path., 25:234:1944. * J. Am. Med. Assoc, i33:909:i947'

PHENOLSULFONPHTHALEIN AMPULS, DIFCO (B221)
FOR RENAL FUNCTION TEST

Phenolsulfonphthalein Ampuls, Difco contain a carefully prepared solution
for determining renal functional activity. Each ml. of the solution contains 0.006
grams (Ho grain) of the dye in the form of its monosodium salt. The ampuls
contain sufficient solution to allow the withdrawal of one ml. Phenolsulfon-
phthalein Ampuls, Difco conform to the specifications for Phenolsulfonphthalein
Injection as given in the U. S. Pharmacopeia.

When phenolsulfonphthalein, in the form of its sodium salt, is injected either
intramuscularly or intraveneously, a large percentage is normally excreted in the
urine within a comparatively short time. This observation by Abel and Rowntree^
has been used by Rowntree and Geraghty^ as the basis of a most accurate test for
the functional activity of the kidney. This test is universally known as a renal
function test.

After intramuscular injection the first traces of dye begin to appear in the
urine in 5-10 minutes and the normal kidney will excrete from 60-80 per cent
of the total amount injected within 2 hours.

After intravenous administration the excretion of the phenolsulfonphthalein is
normally more rapid; 35-45 per cent being eliminated in 15 minutes, 50-65 per
cent in 30 minutes, and 65-80 per cent during the first hour.

If the initial appearance of the phenolsulfonphthalein in the urine is delayed,

DIAGNOSTIC REAGENTS 329

or the amount excreted is below normal, an impairment of renal function is indi-
cated. The degree of dysfunction may be estimated by the proportionate amount
excreted. The functional efficiency of each kidney may be determined by cathe-
terizing each ureter and collecting the excretions separately.

For the performance of the test, exactly 1 ml. of the solution containing 6 mg.
of phenolsulfonphthalein is carefully withdrawn from a Phenolsulfonphthalein
Ampul, Difco, by means of an accurately graduated syringe. This solution is in-
jected either intramuscularly or intravenously, aseptic technique being observed
throughout. {Note: More than 1 ml. has been placed in each ampul in order that
exactly 1 ml. may be easily withdrawn) . To insure copious excretion of urine, the
patient is given 200-400 ml. of water 20-30 minutes before the beginning of the
test. If this precaution is not taken, a late appearance of phenolsulfonphthalein
in the urine may be due to lack of excretion. The patient's bladder must be
emptied, preferably by catherization, the time noted, and 1 ml. of phenolsul-
fonphthalein solution injected. When the injection is made intramuscularly, the
most suitable site is in the lumbar region. The area surrounding the point of in-
jection must be free from edema. If the patient is catheterized the urine may be
allowed to drain into a test tube containing a drop of 25 per cent sodium hydrox-
ide solution and the time noted for the appearance of phenolsulfonphthalein
denoted by the first faintly pink tinge. In patients having no obstruction to the
free passage of urine, the catheter may now be withdrawn. If there is no urinary
obstruction and the passing of the catheter be disagreeable or inconvenient, it
may be omitted, in which case the first appearance of the dye in the urine is not
noted. When the phenolsulfonphthalein is administered intramuscularly, the
patient is instructed to void into a suitable receptacle at the end of 70 minutes
and again into another receptacle at the end of the second hour. If the injection
is made intravenously, it is customary for the urine to be collected at the end of
15 minutes, 30 minutes or 1 hour.

The amount of phenolsulfonphthalein in the urine may be estimated by making
the sample alkaline with a sufficient quantity of 25 per cent sodium hydroxide
solution, diluting to 1 liter, and after filtering, if necessary, comparing the depth
of color with that of a standard solution. When the color is faint, it is advisable
to dilute to only 500 ml., in which case the result should be divided by 2.

If the comparison is made by means of the ordinary biological colorimeter, it
is customary to use a 50 per cent standard, prepared by diluting 0.5 ml. of the
same solution used for injection, to 1 liter, after the addition of a few drops of
25 per cent sodium hydroxide solution to insure the development of the full
alkaline color. If this standard solution is kept in a cool place and not exposed
to strong light, it will keep for several months.

The depth of color may be also conveniently, but less accurately, estimated by
placing a small quantity of the diluted alkaline sample in a tube or vial and com-
paring the depth of color with similar tubes or vials containing amounts of alka-
line phenolsulfonphthalein corresponding to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
60, 80 and 100 per cent. The 100 per cent standard is prepared by diluting 1 ml.
of solution from a Phenolsulfonphthalein Ampul, Difco, and a few drops of 25
per cent sodium hydroxide solution to 1 liter. The other standards are made by
appropriate dilution of the 100 per cent standard.

Complete details of the phenolsulfonphthalein renal function test will be found
in the references of the original articles as given below and also in such texts as
"Manual of Clinical Laboratory Methods" by Cummer, "Diagnostic Methods"
by Webster, "Approved Laboratory Technic" by Kolmer, Spaulding and Robin-
son and other similar texts.

=}:

Pharmacol ., 1:231:1909.
Phsurmacol., i: 579:1910.

330 DIFGO MANUAL

BAGTO-GOAGULASE PLASMA (B286)

Bacto-Coagulase Plasma is a desiccated plasma recommended for the deter-
mination of coagulase production by staphylococci. The coagulase test is gen-
erally accepted as affording the best single criterion of potential pathogenicity of
staphylococci. The use of plasma dried on filter paper was first suggested by
Chapman.^ Foley^ also described a method for the desiccation of rabbit plasma
for use in the coagulase test.

The ability of staphylococci to produce coagulase, an enzyme capable of clot-
ting plasma, was first reported by Loeb^ in 1903. Daranyi* indicated the practical
significance of this test, and since then numerous investigators have studied this
reaction to determine its relationship to the pathogenicity of staphylococci.
Chapman, Berens, Peters and Curcio^ in a study of coagulase and hemolysin pro-
duction by staphylococci, showed that strains producing coagulase were usually
pathogenic regardless of their hemolytic or chromogenic properties. Hallman^
used coagulase production as a means of differentiating pathogenic from non-
pathogenic staphylococci isolated from the nares of a large number of individuals.
Cruickshank'' recommended that coagulase production be used as a test for the
pathogenicity of the staphylococci. Chapman, Berens, Nilson and Curcio^ stated
that coagulase production was the most reliable single test for the differentiation
of pathogenic from nonpathogenic staphylococci. Fairbrother^ concluded that
coagulase is formed only by pathogenic staphylococci and constitutes an impor-
tant criterion for the classification of these organisms into pathogenic and non-
pathogenic strains. Moss, Squires and Pitts^^ also showed the importance of
coagulase production in the determination of the pathogenicity of staphylococci.
Many other reports have been made showing the value of this test for the differ-
entiation of pathogenic from nonpathogenic staphylococci.

The coagulase test may be performed using the tube method or the slide tech-
nique. Chapman, Berens and Stiles^^ described a tube coagulase test using a 1-3
dilution of fresh plasma. Incubation of the plasma and emulsified culture was at
37°C. and readings were made at one hour intervals for four hours. Generally,
rabbit plasma has been used in the various tests. Human plasma and human
whole blood have also been employed. Chapman^^ compared the value of human
plasma and human whole blood with rabbit plasma for testing the coagulating
power of staphylococci. He reported that rabbit plasma clotted more rapidly and
more firmly than did the human plasma or whole blood. Whole human blood,
however, clotted more easily than did the corresponding plasma. Chapman^^
noted that not all human bloods were satisfactory and that other factors also
enter into a satisfactory coagulase test. There must be sufficient culture, grown
under satisfactory conditions, to react with a satisfactory plasma or whole blood.
Conditions that give a satisfactory coagulase test for the determination of patho-
genicity are discussed by Chapman.i-^-i^ Evans^^ used Bacto-Coagulase Plasma
and the tube technique in his studies of coagulase positive staphylococci. He re-
ported the 19 coagulase positive staphylococci comprised a rather homogenous
group. They fermented mannitol under anaerobic conditions and were able to
grow in a synthetic medium devoid of biotin.

Bacto-Coagulase Plasma is well suited for the coagulase test. An outline for
the performance of the test follows :

Dissolve the contents of one ampul (100 mgs.) in 3.0 ml. of distilled water.
Place 0.5 ml. of the plasma solution in a Wassermann tube supported in a rack.
Add two drops of a 16-24 hour Bacto-Brain Heart Infusion culture of the organ-
ism under test, or two drops of a Bacto-Brain Heart Infusion suspension of a 16-
24 hour culture on a slant of Heart Infusion Agar. Incubate at 37°C. Most

DIAGNOSTIC REAGENTS 331

coagulase positive staphylococci will clot Bacto-Coagulase Plasma within one
hour. A second reading should be made, however, after three hours incubation
at 37°C., before the organism can be regarded as coagulase negative. Any degree
of clotting, however slight, is considered positive.

Bacto-Coagulase Plasma is satisfactory for the slide technique as reported by
Chapman!^ ^j^^j Moss.^^ It is also satisfactory for the slide test as described by
Cadness-Graves, Williams, Harper and Miles,^^ discussed by Needham, Ferris
and Spink, -1 This technique is particularly useful for presumptive identification
or screen test for coagulase positive staphylococci in large scale bacteriological
surveys. This test is performed by preparing a homogeneous suspension of a
suspected colony in a drop of water on a slide with a minimum of spreading. Mix
a large loop of rehydrated Bacto-Coagulase Plasma with the suspension. Coagu-
lase positive staphylococci will produce macroscopic clumping within 5-15
seconds. Delayed clumping is not indicative of a positive test. Morphological con-
firmation by the Gram stain is made simultaneously.

Bacto-Coagulase Plasma is stable when stored in the refrigerator. The plasma
solution prepared from Bacto-Coagulase Plasma may be kept in the refrigerator
for several days without loss of potency. It is not satisfactory for use, however,
if gross contamination occurs. After being kept in the refrigerator the plasma
solution is cold enough to delay clotting for 10-15 minutes. This delay can be
prevented by warming the plasma solution to 37°C. before use. Caution: Bacto-
Coagulase Plasma must be kept in the refrigerator in order to preserve its sta-
bility.

ij. Bact., 41:431:1941. 12 J. Bact., 47:211:1944.

2 Science, 95:416:1942. ^ J. Bact., 50:119:1945.

8 J, Med. Res., 10:407:1903. "J. Bact., 50:234:1945.

*Zentr. Bakt. I Abt. Orig., 99:74:1926. is Trans. N. Y. Academy Sciences, 9:52:1946.

6 J. Bact., 28:343:1934. i^J. Bact., 52:151:1946.

^Proc. Sec. Exp. Biol. Med., 36:789:1937. "J. Bact., 53:367:1947.

Path. Bact., 45:295:1937- ^^ J- Bact., 55:793:1948.

Bact., 35:311:1938. 1^ Personal Communication.

J. Path. Bact., 50:83:1940. 20 Lancet, 1:736:1945.

"Am. J. Clin. Path., 11:857:1941. 21 Am. J. Clin. Path., Tech. Supp., 9:83:1945.
^J. Bact., 41:431:1941.

ij;

BAGTO-SENSITIVITY DISKS (B403)

Bacto-Sensitivity Disks are sterile paper disks containing known amounts of the
more commonly employed antibiotics and other therapeutic agents. They are
recommended as a rapid, practical, clinically accurate and inexpensive means of
determining the relative sensitivity of microorganisms to these therapeutic agents.
Bacto-Sensitivity Disks are especially valuable in selecting the antibiotics effec-
tive against chronic or persistent infections refractory to primary antibiotic
therapy. Three concentrations of each antibiotic are available, indicating if the
microorganism is very sensitive, moderately sensitive, slightly sensitive or resist-
ant. With proper interpretation the bacterial susceptibilities as determined with
these Disks give the clinician a qualitative in vitro measure of sensitivity equal to
that of any other procedure with less effort and often without the added time
necessary for pure culture isolation as in the test tube method. Bacto-Sensitivity
Disks are available containing three concentrations of Aureomycin, Bacitracin,
Chloromycetin®*, Dihydrostreptomycin, Penicillin, Polymyxin B (Aero-
sporin®**). Streptomycin and Terramycin.

The use of paper disks in testing the potency of penicillin and other bacterio-

* Chloramphenical: Parke, Davis and ** Polymyxin B: Burroughs Wellcome and

Company. Company.

332 DIFCO MANUAL

static or bactericidal materials has been described by numerous investigators.
Likewise, many investigators have used this technique to determine the resistance
or susceptibility of microorganisms to antibiotics or other inhibitory substances.
Morley^ dried sulfathiazole and penicillin on paper disks for determining the
sensitivity of wound bacteria to these materials. O'Toole^ reported that this
method proved to be simple and economical. She suggested modifications making
the test suitable for determining the sensitivity of various species of micro-
organisms in the routine laboratory and gave tables showing the stability of the
dried disks, and also the size of zones produced by various concentrations of
penicillin, streptomycin and sulfathiazole. Bondi, Spaulding, Smith and Dietz^
described a method for the rapid determination of the susceptibility of micro-
organisms to penicillin and other antibiotics stressing that this method can be
used at the time of primary isolation, thereby eliminating the time required for
pure culture isolation and identification prior to selecting the antibiotic for treat-
ment. They saturated sterile disks with solutions of the antibiotic and placed
them on the surface of Blood Agar plates immediately after inoculation with
clinical specimens. Kolmer, Spaulding and Robinson* described the paper disk
technique of determining susceptibility of bacteria to antibiotics. They pointed
out that the method is a practical, simple and a rapid means of estimating sensi-
tivity. Many modifications of these and similar tests are in current use to deter-
mine the resistance or sensitivity of microorganisms to antibiotics and other
therapeutic agents.

The use of antibiotics in the treatment of many infectious diseases is an estab-
lished procedure. The sensitivity of groups of many pathogenic bacteria to the
various antibiotics and other therapeutic agents is known, and infections caused
by these microorganisms usually respond to treatment with the designated anti-
biotic. However, the clinician frequently finds that an infection is not responding
as expected to a specific treatment. In such cases he must accurately and rapidly
determine the most effective antibiotic to use to overcome the infection. If treat-
ment is not effective against the organism, there is danger of the possible sensitiza-
tion of a patient to an antibiotic or other therapeutic agent which may be re-
quired at some future date. It is also possible to increase the resistance of micro-
organisms to these materials if ineffective dosages are used in treatment.

Determination of susceptibility of microorganisms to various antibiotics has
shown a wide variation in individual strains. The publications of Finland, Frank
and Wilcox^ '^''^•^ clearly show a wide range of sensitivity between different
species or groups of organisms. Barber and Rozwadowska-Dowzenko^ reported
that the proportion of staphylococci isolated from hospital patients resistant to
penicillin increased from 14 per cent in 1946 to about 60 per cent in 1948. Davi-
son^o suggested that penicillin blood levels in 1949 had to be 15 times higher than
in 1946 in order to inhibit the growth of 80 per cent of the Gram-positive cocci
found in nasal exudates. The more sensitive organisms are killed by inadequate
dosage of penicillin while the more resistant strains survive, becoming progres-
sively more resistant. Miller and Sherlock-Hubbard^^ reported that over the
seven year period, 1945-1951, the resistance of pathogenic staphylococci to peni-
cillin had increased markedly. In addition during the last two years comparative
resistance to the broad spectrum antibiotics on the part of the usually sensitive
organisms has increased. Increased resistance of the tubercle bacillus to strepto-
mycin was also shown. The clinician must rely on the laboratory for the deter-
mination of the sensitivity of microorganisms in cases where prompt response is
not obtained with the usual antibiotic therapy. The use of Bacto-Sensitivity Disks
supplies this information for all the more commonly employed antibiotics with
a minimum of laboratory time.

Sweeney, Davis and Barnes^^ ^^^^ Bacto-Sensitivity Disks to determine the

DIAGNOSTIC REAGENTS 333

susceptibility of 10 Shigella strains and one Proteus strain to bacitracin, dihydro-
streptomycin, penicillin, Chloromycetin and terramycin. They reported this
method could be used to determine if an organism is sensitive, moderately sensi-
tive or resistant and concluded that the test was adaptable to a field laboratory
or mobile unit. Christensen and Lipsett^^ reported that Bacto-Sensitivity Disks
could be satisfactorily used to show the relative sensitivity of a variety of micro-
organisms to six antibiotics, using Bacto-Heart Infusion Agar, with or without
blood as a test medium. Mitchell, Arnold and Lett^* used Bacto-Sensitivity Disks
in their study of the sensitivity of Pseudomonas aeruginosa isolated from external
otitis. They reported this method as a quick aid to the physician in his choice of
therapy, since the test could be run directly, using the specimen as inoculum.
Spaulding and Anderson^^ called attention to the necessity for the determination
of the susceptibility of microorganisms to various antibiotics in treating certain
types of infections. They described and evaluated a disk technique which is
readily performed and within the scope of any laboratory capable of culturing
clinical material. The test gives information which is a valuable and often an in-
dispensable guide to successful treatment of severe, refractory or relapsing infec-
tions. They state that disks are presently available that may be transferred
directly to the inoculated plate and are admirably suited to the needs of the
clinical laboratory. Bacto-Sensitivity Disks are of this type, containing known
amounts of the various antibiotics, thus eliminating the need of making standard-
ized solutions of the antibiotics.

Pike, Schulze and McCullough^^ using Bacto-Tryptose Agar and Bacto-Sensi-
tivity Disks determined the sensitivity of Mima polymorpha, isolated from a
patient, and reported prompt recovery upon administration of the selected anti-
biotic. Lind and Swanton^^ reported that the Disk method of determining
whether an organism is sensitive to one or more antibiotics is reliable, relatively
inexpensive, rapid and when used as a guide to therapy is of definite value to the
physician and patient. Trafton and Lind^^ in a study of urinary infections pointed
out that sensitivity testing by the Disk method permitted a rapid, accurate and
inexpensive determination of the susceptibility of microorganisms to the various
antibiotics. They suggested that physicians should be encouraged to use these
tests routinely and reported clinical data showing that the Disk method gives a
fairly reliable guide to prognosis when selecting an antibiotic. Fusillo and
Kuhns,^^ using Bacto-Sensitivity Disks described a method for antibiotic sensi-
tivity testing using one concentration of each antibiotic with a single plate of
medium to give the entire antibiotic sensitivity spectrum thereby saving time,
media and equipment. Broom, Martineau and Young^o used Bacto-Sensitivity
Disks in determining the sensitivity of organisms in their evaluation study of the
Disk Method in treating 100 clinical cases. Their results showed the indispensa-
bility of sensitivity testing from the clinical standpoint and demonstrated the
practicability of the disk technique in routine diagnosis. Koch and Bourgeoises-
reported increased resistance of staphylococci to penicillin as well as aureomycin
and terramycin. They used Bacto-Sensitivity Disks in determining the resistance
of staphylococci and showed that the Disk Method correlated in every instance
with results obtained by the two fold tube dilution technique. Gould and Bowlegs
in a study of the determination of bacterial sensitivity to antibiotics reported the
Disk technique to be more accurate than "ditch and cylinder" methods, and less
tedious. They pointed out that the serial dilution tube method is much more
laborious than the Disk method, and subject to more errors. Closely similar re-
sults were obtained when the Disk method was carried out in parallel with the
serial dilution tube method and reported that the Disk technique is as accurate
as the more complicated tube dilution method for all purposes. They expressed
their results as "sensitive, relatively resistant and resistant," and stated this suffices

334 DIFCO MANUAL

for clinical purposes. Since in vivo sensitivities cannot be accurately gauged from
in vitro levels of sensitivity, mathematical expressions of these might even be
misleading.

There have been some interpretations in the literature to indicate that the tube
dilution method of determining sensitivity is the only accurate procedure and
that the Disk method does not present the desired information. Continued wide-
spread use of the Disk Technique and proper interpretation of results have dem-
onstrated the clinical accuracy, efficiency and reliability of this procedure. The
tube dilution method is applicable in research procedures and in cases where
quantitative determinations are required and where proper consideration can be
made of resistant variants and other factors. The tube dilution method because
of the time and materials involved is not suitable for routine diagnostic work,
and has not offered any advantages over the Disk method in clinical procedures.

The results obtained with Bacto-Sensitivity Disks, as with other sensitivity tests
similarily conducted, are of a qualitative rather than a quantitative nature. The
many variables that enter into a test of this type such as the size of the inoculum,
type of growth of the organism, amount of inoculum on the plate, thickness of
the medium, incubation conditions, diffusion rate of the antibiotic, rate of dete-
rioration of the antibiotic during incubation, preclude its quantitative nature.
Sensitivities are to be determined by the presence of and not entirely by the
diameter of the zone of inhibition around the disks. Organisms showing a zone
of inhibition around the disk with the smallest concentration of the antibiotic
would be considered very sensitive to that antibiotic; an organism showing no
zone around the lowest, but a zone around the intermediate disk would be classed
as sensitive or moderately sensitive, while a zone around only the highest concen-
tration disk would indicate that the organism is but slightly sensitive to the
antibiotic. Resistant organisms produce no zone of inhibition even around the
disk containing the highest concentration. As was pointed out by Jawetz in a
personal communication, the information obtained by the use of Sensitivity Disks
is not only important as an aid in determining which antibiotic may be effective
against a specific infection, but also which antibiotics are ineffective. He stressed
that knowledge of the resistance and sensitivity of an organism is equally im-
portant in saving time and expense in treatment.

Information of this type has proven to be invaluable to the clinician under
practical conditions in selecting the most appropriate antibiotic for treating in-
fections. It is stressed that the differences in the diameters of the zones of inhibi-
tion obtained with like concentrations of the various antibiotics do not necessarily
indicate their relative therapeutic efficiency. The rate of diffusion of the various
therapeutic agents from the disks and through the medium influence the diameter
of the zone of inhibition. Howe^s in a study of sensitivity testing using paper disks
considered only the presence or absence of bacterial growth and gave no attention
to the diameter of the zone of inhibition.

The importance of culture media in sensitivity testing has been shown by
many investigators. Repeated comparative tests in our laboratory have shown
that Bacto-Heart Infusion Agar, with or without added blood, is well suited for
the plating medium, producing excellent growth of a large variety of pathogenic
microorganisms. Hemolytic reactions on this medium containing blood are typi-
cal, being an added advantage in diagnostic work. Tryptic soy media have been
employed in sensitivity testing but we have found no advantage in their use.
Waisbren, Carr and Dunnett-* reported inhibition of activity with certain anti-
biotics using media of this type. Bacto-Brain Heart Infusion is recommended as
a liquid medium for obtaining a rapid profuse growth of the test organism to be
used for inoculating the Heart Infusion Agar employed in the disk plate tech-
nique of sensitivity testing. This medium also was employed in a turbidimetric

DIAGNOSTIC REAGENTS 335

assay method suitable for an antibiotic screen test using a large variety of test
organisms by Joslyn and Galbraith.^^

Performance of the Test

Bacto-Sensitivity Disks are sterile and should be removed from the vials using
aseptic technique. Antibiotics are differentiated by the color of the disk and
concentrations are identified by the markings on each disk. The vials should be
tightly stoppered when not in use, and stored in the refrigerator at 2-6° C.

The sensitivity test may be performed on plates of medium inoculated with
either the isolated organism or the pathological material containing these
organisms as suggested by Bondi, Spaulding, Smith and Dietz.^ Wood-^ suggested
the inoculation of duplicate plates with the specimens, placing the sensitivity
disks on one and using the second plate for the primary isolation of the etiological
agent. With either of these procedures, often the antibiotic of choice can be
determined prior to the isolation and identification of the organism.

Plates of Bacto-Heart Infusion Agar, with or without added blood, are pre-
pared. These are inoculated either by smearing or streaking with a heavy sus-
pension of the test organism or clinic specimen containing the causative organism,
to obtain confluent or solid growth. Excessive moisture is to be avoided. Im-
mediately following the inoculation of the medium, Bacto-Sensitivity Disks are
placed on the surface spaced about 3-4 cm. apart. Use a flamed tweezer in re-
moving the disks from the vial so as not to contaminate the remaining disks.
Plates are incubated at 35-3 7 °C. for 12 hours or until growth develops, and
then examined for zones of inhibition of growth. It is recommended that in any
case the etiological agent be isolated since mixed cultures may not always give
dependable sensitivities.

Heavy inoculation of plates permits more rapid sensitivity determinations
with the possibility of earlier treatment. Experience has demonstrated that with
some organisms plates so inoculated have shown definite zones of inhibition
within 5-6 hours after inoculation. Generally 5-6 sensitivity disks can be placed
on a regular 95 mm. petri plate. Some laboratories may prefer to use large size
petri plates (140 mm.) permitting the testing of the sensitivity of an organism
to a larger number of antibiotics or therapeutic agents. Crowding of disks is to be
avoided.

If desired, a base and "seed layer" technique may be employed. For this pur^
pose, pour 15 to 20 ml. of sterile Bacto-Heart Infusion Agar in sterile 95 mm.
plates and allow to solidify (use 60 ml. for 140 mm. plates). The inoculum is
incorporated in sterile melted Bacto-Heart Infusion Agar with or without blood
at 45 °C. This inoculated seed agar is poured on the base layer and distributed
evenly over the surface by tilting the plate to give a thin "seed layer" inoculation.
The disks are placed on the surface of this layer after it has solidified.

The sensitivity of tubercle bacilli can be determined using Bacto-Sensitivity
Disks in combination with Bacto-Peizer TB Medium Base and Bacto-Peizer TB
Medium Enrichment. The medium used in this test is prepared according to the
formula described by Peizer and Schecter.-^ This procedure has been compared
with other methods and found to be as accurate and less time consuming than
other techniques presently in use.

The method for determining the sensitivity of the tubercle bacillus to thera-
peutic agents using Bacto-Sensitivity Disks is essentially similar to the determina-
tion of sensitivities of other organisms. Either the pure culture or the clinic
specimen, such as treated sputum, spinal fluid, urine, etc., provided these contain
a moderate number of acid-fast organisms, Gaffky III or IV, may be used for
the inoculation. The complete medium is prepared from Bacto-Peizer TB

336 DIFCO MANUAL

Medium Base and Bacto-Pelzer TB Medium Enrichment. The medium is poured
into sterile plates or flat wide-mouth bottles and allowed to solidify. A heavy
suspension of the organism or the clinic specimen is smeared on the surface of
the medium and the Bacto-Sensitivity Disks, dihydrostreptomycin or streptomycin
1, 10 and 100 meg., are placed on the surface about 3 cm. apart. The plates are
incubated for 48 hours and then sealed with either cellophane tape or melted
paraflin and incubation continued at 35-3 7 °C. Tubercle bacilli are frequently
isolated and their sensitivities determined in 10-14 days; some strains may re-
quire up to 6-8 weeks incubation for suitable growth. Sensitivity of the organisms
to the antibiotic is indicated by the presence of zones of inhibition surrounding
the disks.

Interpretation

A moderate zone of inhibition around the lowest concentration of one or more
antibiotics would classify the organism as being sensitive to any or all of these
antibiotics even though there may be a difference in zone size. Resistant organisms
are not inhibited in the maximum concentration of the antibiotic while very
sensitive strains show marked zones of inhibition even in the lowest concentra-
tions. In the case of very sensitive strains, one may obtain zones of inhibition
around the disk containing the lowest concentration nearly as large as those
obtained around the higher concentrations, due to the diffusion characteristics of
the test.

Results of this technique cannot necessarily be expressed in terms of the tube
dilution method. The presence of zones of inhibition of growth around the disks
indicates that the organism is sensitive to the antibiotic. The diameter of the zone
is dependent not only upon the sensitivity of the organism but also upon the
diffusability of the antibiotic and other factors which may limit the availability
of the antibiotic to the organism in the medium.

Hemolytic properties of microorganisms must be considered when using plates
of Blood Agar for the test. Growth of hemolytic organisms is characterized by a
lysed, cleared or discolored background with the presence of colonies, while
zones of inhibition leave the medium unchanged (no hemolysis). Upon longer
incubation the hemolysin produced by the organism may diffuse toward the disk,
giving a cleared zone, without growth, around the periphery of the zone of inhibi-
tion. The true zone of inhibition on Blood Agar includes both the unchanged
non-hemolytic as well as the hemolytic areas showing no bacterial growth.

Frequently one will observe isolated colonies growing within the zone of
inhibition. These represent resistant variants of the strain or culture, under test.
The appearance of several such resistant colonies in the zone of inhibition does
not indicate the true resistance of the culture which is demonstrated by the zone
of inhibition of growth. Bacto-Sensitivity Disks thus permit the isolation of re-
sistant variants from a culture.

Bacto-Sensitivity Disks are stable for one year when stored in the refrigerator
at 2-6°C.

Bacto-Sensitivity Disks are available in vials of 25 disks each. They are sup-
plied in complete sets and as replacement packages. A complete set (B403) con-
sists of 1 vial each of three concentrations of the more commonly used antibiotics.

DIAGNOSTIC REAGENTS

337

Antibiotic

Aureomycii
Bacitracin

joacitracin

Chloromycetin

Dihydrostrepto-

mycin
Penicillin
Polymyxin B

(Aerosporin)
Terramycin

Color

Yellow
Brown
Grey
Pink

Plain

10 meg.

2 units

10 meg.

1 meg.

Marking

Green 0.5 unit
Blue 5 meg. (50

units)
Terra-cotta 10 meg.

30 meg.

60 meg.

10 units

20 units

30 meg.

60 meg.

10 meg.

100 meg.

1 unit

10 units

10 meg.

(100

30 meg. (300

units]

units)

30 meg.

60 meg.

Sets are also available consisting of one vial each of the lowest (B403A), inter-
mediate (B403B) or highest (B403C) concentration of each therapeutic agent.

Replacement packages consist of six vials of any one concentration of any
antibiotic.

Streptomycin (white disks) in concentrations of 1.0, 10 and 100 meg. are avail-
able also in replacement packages.

1 J. Path. Bact., 57 = 379: i945-

a Am. J. Med. Tech., 12:251:1946.

^ Am. J. Med. Sciences, 213:221:1947.

* Approved Laboratory Technique, 5th Edi-
tion: 557:1 951.

BJ. Lab. Clin. Med., 35:188:1950.

* J. Lab. Clin. Med., 35:205:1950.
'Am. J. Clin. Path., 20:325:1950.
8 Am. J. Clin. Path., 20:335:1950.
8 Lancet, 2:641:1948.

10 Laryngoscope, 60: 131 : 1950.

"Can. Jf. Pub. Health, 43:35:1952.

^ Navy Med. Research Inst. Research Report,

Project NM 005 048, 04.13.
"News Letter S.A.B., 17:15:1951. Paper

read at Mich. Branch S.A.B. Mtg. April,

1951-

1* Laryngoscope, 6 1 : 649 : 1 95 1 .

"J. Am. Med. Assoc, 147:1336:1951.

^8 Am. J. Clin. Path., 21:1094:1951.

1' J. Antibiotics and Chemotherapy, 2:30:1952.

18 J. Urol. In Press.

18 Med. Technicians Bull., 3:7:1952.

* Bact. Proceedings, Boston, p. 120:1952.

21 Antibiotics and Chemotherapy, 2:229:1952.

22 Edinburgh Med. J., 59:178:1952.

23 Surg. Gynecol. Obstet., 9:669:1950.
2* Am. J. Clin. Path., 21:884:1951.

35 J. Bact., 59:711:1951.

^ Personal Communication. 1950.

^Am. J. Clin. Path., 20:682:1950.

BAGTO-GONGENTRATION DISKS, PENICILLIN (B452)
BAGTO-SUBTILIS SPORE SUSPENSION (B453)

Bacto-Concentration Disks, Penicillin are used to determine the penicillin level
of milk, serum, plasma, urine and other body fluids. Concentrations of from
0.05 to 5 units penicillin per ml. may be determined by this method. The Con-
centration Disks contain known measured amounts of penicillin to correspond to
0.05, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 units per ml. Disks containing Bacto-Penase
are supplied to identify the bactericidal agent as penicillin. The test employs a
standardized medium (Bacto-Whey Agar), a standardized inoculum (Bacto-
Subtilis Spore Suspension), and when the conditions of the test are adhered to,
accurate determinations can be made.

Churchill and Frank^'^ used the Concentration Disks, Penicillin to detect
penicillin in milk. They compared the phosphatase method, growth coagula-
tion method and the Disk method and reported the superiority of the Disk
method. Kosikowsky,^ in a systematic testing of inhibitory substances in milk re-
ported that under optimum conditions concentrations of penicillin as low as
0.05 unit per ml. were detected. Detailed discussion of Bacto-Concentration
Disks is available upon request.
1 Paper read at Mich. Branch S A.B. Mtg. April 1951. 3 In Press, a J. Dairy Science, 35:533:1952-

Miscellaneous Products

DEHYDRATED MEDIA IN
SPECIAL PACKAGES

For the convenience of the user, we have prepared a series of dehydrated
media in small packages. Each bottle contains sufficient dehydrated medium for
1 liter or 100 ml. "Standard Methods Media" are packaged in amounts sufficient
for 1 liter; other media for 100 ml.

These packages of dehydrated media are recommended for use in laboratories
where humidity is high or in laboratories where there is occasional need for a
small quantity of medium.

The special packages of dehydrated media are securely sealed to prevent the
absorption of moisture. Unopened vials will keep for long periods of time with-
out lumping or deterioration.

The following media are packaged in amounts sufficient for 1 liter in boxes
of six :

Bacto-Brilliant Green Bile 2% (B7) Bacto-Lactose Broth (B4)

Bacto-Endo Agar (B6) Bacto-Nutrient Agar (Bl)

Bacto-Levine E.M.B. Agar (B5) Bacto-Nutrient Broth (B3)

Bacto-Formate Ricinoleate Broth (B9) Bacto-Tryptone Glucose Extract Agar

(B2)

The following media are packaged in amounts sufficient for 100 ml. in boxes
of six:

Bacto-Brain Heart Infusion (B32) Bacto-Phenol Red Lactose Broth (B94)

Bacto-Heart Infusion Agar (B44) Bacto-Phenol Red Maltose Broth

(B96)
Bacto-Heart Infusion Broth (B38) Bacto-Phenol Red Mannitol Broth

(B97)
Bacto-Phenol Red Agar Base (B98) Bacto-Phenol Red Saccharose Broth

(B95)
Bacto-Phenol Red Broth Base (B92) Bacto-Russell Double Sugar Agar

(B84)
Bacto-Phenol Red Dextrose Broth
(B93)

PREPARED MEDIA IN TUBES
AND BOTTLES

For the bacteriologist who requires only a few tubes or bottles of media or
who does not have readily available facilities for the preparation of media, we
prepare a series of media in tubes, ready for use. This group of prepared media
in tubes offers still another convenience to bacteriologists since some of the media
do not lend themselves to dehydration.

Upon request media of any desired formula will be prepared and supplied in
tubes or bottles.

338

MISCELLANEOUS PRODUCTS

339

These media are put up in regular sized tubes fitted with a tightly sealed
screw cap. No cotton plugs are used. The media are freshly prepared, and are
examined for sterility before being released. Culture media in tubes should be
stored in sealed cans to prevent evaporation, and under these conditions should
give good results indefinitely. Agar media should be melted and allowed to
resolidify in a slanted position or placed in a warm water bath for several minutes
to provide a fresh moist surface prior to inoculation. Media containing blood,
ascitic fluid, antibiotics or other thermolabile materials, should not be heated
prior to use.

We supply the following media in tubes, packed in boxes of one dozen or one-
half dozen tubes each:

Lead Acetate Agar
Levine E.M.B. Agar
Littman Oxgall Agar
Loeffler Blood Serum
Lowenstein-Jensen Medium
North Gelatin Agar
Nutrient Agar
Nutrient Broth
Nutrient Gelatin
Peizer TB Medium
Penase Ascites Medium
Petragnani Medium
Petroff Egg Medium
Potato Dextrose Agar
Root Canal Medium
Russell Double Sugar Agar
Sabouraud Dextrose Agar
Sabouraud Maltose Agar
Snyder Test Agar
TB Serum Broth
Tryptose Agar

BIOOOT

Ascitic Agar

B88T

BIOOIT

Blood Agar

B5T

B1013T

Bovine TB Medium

B294T

B37T

Brain Heart Infusion

B70T

B37AT

Brain Heart Infusion with

B1017T

PAB

B50T

B37BT

Brain Heart Infusion with

BIT

PAB and Agar

B3T

B1002T

Chocolate Agar

BUT

B1012T

Chocolate Tellurite Agar

B400T

B1003T

Cystine Hemoglobin Agar

B1015T

B67T

Dextrose Agar

BIOIOT

B63T

Dextrose Broth

BlOllT

B1006T

Dorset Egg Medium

B13T

B385TA

Dubos Albumin Broth

B1014T

B373T

Dubos Oleic Agar

B84T

B385T

Dubos Serum Broth

B109T

B256T

Fluid Thioglycollate Medium

BllOT

B1007T

Glucose Ascites Medium

B247T

B1008T

Glycerol Agar

B291T

B44T

Heart Infusion Agar

B64T

B1016T

Lash Serum Medium

BAGTO-DIPHTHERIA CULTURE TUBES (B1004T)
AND OUTFITS (B1005T)

The advantages of early diagnosis in suspected cases of diphtheria require no
enumeration. As an emergency aid at times when delay may be of serious conse-
quence, we have prepared a Bacto-Diphtheria Culture Outfit which consists of
a Bacto-Diphtheria Culture Tube (an hermetically sealed and sterilized Loeffler
Blood Serum slant) and a sterile applicator with a data sheet. The outfit is com-
plete, neat and convenient. The hermetically sealed diphtheria culture tube pre-
serves the sterile medium indefinitely, keeping it ready for instant use. This tube is
etched for convenience in breaking.

STERILE APPLICATORS IN TUBES (B1009T)

Sterile applicators in tubes have been prepared for use in conjunction with
Bacto-Diphtheria Culture Outfits, as discussed above or for use in the collection
of other specimens.

340 DIFCO MANUAL

These media are packaged in 100 ml. amounts in screw capped bottles. They
are especially recommended for blood culture work, each bottle containing
sufficient medium for up to 10 ml. of blood or other body fluid as inoculum.

BACTO

PENASE BROTH (B347B)

Bacto-Penase Broth is prepared especially for use in culturing organisms from
blood and other body fluids from patients under penicillin, streptomycin, or
sulfonamide treatment. It is prepared from Bacto-Brain Heart Infusion and
contains sufficient Bacto-Penase and para-Aminobenzoic Acid to inactivate any
penicillin or sulfonamide that might be carried over in a body fluid inoculum.
One hundred ml. of this medium will inactivate 10,000 units of penicillin and up
to 1000 units of streptomycin.

Each bottle of Bacto-Penase Broth contains 100 ml. of medium so as to
permit the use of as much as 10 ml. of blood or other body fluid as an inoculum.
Inoculation must be made under aseptic conditions. Contamination can best be
avoided if the cap is held in the hand, bottom down, to avoid its contact with
contaminated objects. After replacing the cap the bottle is gendy agitated to
distribute the inoculum uniformly throughout the medium. Incubate at 37 °C.
Observations for growth are made at desired intervals, preferably after 24 hours
and on each succeeding day for 14 days. Material for examination may be re-
moved with either a sterile pipette or loop using aseptic precautions.

Bacto-Penase Broth is stable when stored at refrigerator temperatures. At
normal room temperatures, the Bacto-Penase in the medium deteriorates slowly
and for that reason the medium must be placed in the refrigerator immediately
upon receipt and kept there until used. Stability tests to date indicate that the
Bacto-Penase in the medium is stable for at least a year when stored in the
refrigerator. This medium must not be heated before inoculation as the Bacto-
Penase is thermolabile.

Bacto-Penase Broth is supplied in packages of one-half dozen bottles of 100 ml.
each.

BACTO-PENASE MEDIUM (B348B)

Bacto-Penase Medium is prepared especially for use in culturing organisms
from blood and other body fluids from patients under penicillin, streptomycin,
or sulfonamide treatment. It is prepared from Bacto-Brain Heart Infusion with
PAB and Agar, and contains sufficient Bacto-Penase and para-Aminobenzoic
Acid to inactivate any penicillin or sulfonamide that might be carried over in a
body fluid inoculum. One hundred ml. of this medium will inactivate 10,000
units of penicillin and up to 1000 units of streptomycin. The presence of the
small amount of agar in the medium provides a range of oxidation-reduction
potentials, permitting the development of anaerobes and microaerophiles, as well
as aerobes.

Each bottle of Bacto-Penase Medium contains 100 ml. of medium so as to
permit the use of as much as 10 ml. of blood or other body fluid as an inoculum.
Inoculation must be made under aseptic conditions. Contamination can best be
avoided if the cap is held in the hand, bottom down, to avoid its contact with
contaminated objects. After replacing the cap the bottle is gently agitated, to
distribute the inoculum uniformly throughout the medium. Incubate at 37°C.

MISCELLANEOUS PRODUCTS 341

Observations for growth are made at desired intervals, preferably after 24 hours
and on each succeeding day for 14 days.

Upon storage, and especially upon the addition of the inoculum, there may
be a partial flocculation of the agar in the medium. This in no way interferes
with the nutritional value of the medium.

Bacto-Penase Medium is stable when stored at refrigerator temperatures. At
normal room temperatures, the Bacto-Penase in the medium deteriorates slowly
and for that reason the medium must be placed in the refrigerator immediately
upon receipt and kept there until used. Stability tests to date indicate that the
Bacto-Penase in the medium is stable for at least a year when stored in the re-
frigerator. This medium must not be heated before inoculation as the Bacto-
Penase is thermolabile.

Bacto-Penase Medium is supplied in packages of one-half dozen bottles of
100 ml. each.

BAGTO-FLUID THIOGLYGOLLATE (B256B)
MEDIUM

Bacto-Fluid Thioglycollate Medium conforms to the formula specified by the
National Institute of Health for the sterility testing of biologicals, and for the
sterility testing of antibiotics according to the method of the Food and Drug
Administration, as well as the specifications given in the U. S. Pharmacopeia
and the National Formulary. Bacto-Fluid Thioglycollate Medium is especially
recommended for culturing organisms from inocula containing mercurial pre-
servatives. This medium has been used for the cultivation of anaerobes from
blood and other body fluids since the small concentration of agar present in the
medium prevents reaeration of the medium after sterilization by convection cur-
rents and thus maintains a low oxidation-reduction potential. Brain Heart In-
fusion with PAB and Agar or Bacto-Penase Medium not containing thioglycollate,
have proven more satisfactory for the cultivation of some anaerobes and aerobes
from materials not containing mercurials.

Each bottle of Bacto-Fluid Thioglycollate Medium contains 100 ml. of medium
so as to permit the use of as much as 10 ml. of blood or other body fluid
as an inoculum. To test sterility use 1-2 ml. of the solution under assay. Inocula-
tion must be made under aseptic conditions. Contamination can best be avoided
if the cap is held in the hand, bottom down, to avoid its contact with con-
taminated objects. After replacing the cap the bottle is gently agitated to distrib-
ute the inoculum uniformly throughout the medium and then incubated at
37 °C. Observations for growth are made at desired intervals, preferably after
24 hours and on each succeeding day for 14 days.

Upon storage, and especially upon the addition of the inoculum, there may be
a partial flocculation of the agar in the medium. This in no way interferes with
the nutritional value of the medium.

Just prior to inoculation the bottles of the medium should be heated in a
boiling water bath or flowing steam at 100°C. for 10 minutes to drive out dis-
solved gases, and then cooled to below 40° C. without excessive agitation.

Bacto-Fluid Thioglycollate Medium is supplied in packages of one-half dozen
bottles of 100 ml. each.

Index

A C Broth, 202

A G Globulin, 323

A G Medium, 201

Acetyl-methyl-Carbinol Test, 54

Acidogenic Microorganisms, 190

Adonitol (Adonite), 292

Aerosporin Disks, 331

Aesculin, 292

Agar, 290

rf/- Alanine, 268

Amboceptor, 310

Amino Acid Assay Media, 230-236

Amino Acids, 268

/j-Aminobenzoic Acid, 296

/>-Aminodimethylaniline Monohydro-

chloride, 296
/>-Aminodimethylaniline Oxalate, 297
Anaerobe Cultivation, 79, 81, 84, 85,

104, 126, 127, 201
Anaerobic Incubation, 17, 112, 118
Analysis Peptones, 265
Antibiotic Disks, 331
Antibiotics Assay, 203-211
Antigens, Reagents for Preparation, 315,

316
Antisheep Hemolysin, 310
Applicators in Tubes, 339
/-Arabinose, 292
Arginine Assay Medium, 235
Ascitic Agar, Tubes, 339
Ascitic Fluid, 273
Ascitic Fluid, TG, 305
Asparagine, 268

Aureomycin Assay, 203, 204, 206
Aureomycin Disks, 331
Autoclave Sterilization, 18
Azide Blood Agar Base, 155
Azide Dextrose Broth, 48

Bi2 Assay Medium USP, 221
Bacitracin Assay, 203, 204, 206

Bacitracin Disks, 331

B A G G Broth, 47

Balanced Salt Solution, TG, 306

Basic Fuchsin, 293

Bean Pod Agar, 247

Beef, 288

Beef Blood, 272

Beef Blood Serum, 273

Beef Embryo, TG, 304

Beef Embryo Extract, TG, 303

Beef Extract, 269

Beef Heart for Antigens, 315, 316

Beef Heart for Infusion, 288

Beef Lactose Agar, 69

Beef Serum, 273

Beef Serum Ultrafiltrate, TG, 305

Bile Products, 286, 287

Bile Salts, 286

Bile Salts No. 3, 287

Bile Solubility Test, 287

Biochemicals, 296-298

Biotin Assay Medium, 220

Bismuth Sulfite Agar, 139

B J L Flocculation Antigen, 313

B J L Wassermann Antigen, 310

Blood Agar Base, 87, 88, 115, 125

Blood Agar, Tubes, 339

Blood Gulture, 77, 79, 83, 87, 88, 100,

115, 340
Bordet Gengou Agar Base, 95
Boric Acid Broth, 49
Bottles, Complete Media, 338, 340, 341
Bouillon, 80

Bovine TB Medium, Tubes, 339
Brain Heart Infusion, 77 , 338
Brain Heart Infusion Agar, 90, 242
Brain Heart Infusion, Tubes, 339
Brain Heart Infusion with P A B, 79
Brain Heart Infusion with P A B and

Agar, 79
Brain Heart Infusion with P A B and

Agar, Tubes, 339
Brain Liver Heart (Semisolid), 129
Brain Veal Agar, 94
Brewer Anaerobic Agar, 126

343

344

INDEX

Brewer Anaerobic Agar without Dex-
trose or Eh Indicator, 127
Brewer Thioglycollate Medium, 197
Brilliant Green, 293
Brilliant Green Agar, 144
BriUiant Green Bile 2%, 57, 59, 338
Brilliant Green Bile 5%, 76
Brilliant Green Bile Agar, 52
Brom Cresol Green, 295
Brom Cresol Purple, 295
Brom Phenol Blue, 295
Brom Thymol Blue, 295
Brucella,26,102yll 1,262
B T B Lactose Agar, 194

Cabbage Infusion Agar, 129
Cabbage Infusion Broth, 129
Calcium Chloride 0.02 Molar, 322
Carbohydrate Free Base, 186, 187, 189
Carbohydrate Solutions in Ampuls, 292
Carbohydrates, 291, 292
Carbohydrates, Filter Sterilized, 292
Carbon Dioxide Incubation, 112, 118
Cardiolipin Antigens, 313
Caries Detection, 190
Casamino Acids, 265
Casamino Acids, Technical, 267
Casein, 299, 300
Casitone, 264

Castaneda Technique, 102, 113
Cellobiose, 292

Cephalin Cholesterol Antigen, 324
C F Assay Medium, 229
Chapman Stone Medium, 153
Chapman Tellurite Solution, 277
Chick Embryo Extract, TC, 302
Chick Embryo, TC, 303
Chicken Pancreas, 282
Chicken Plasma, TC, 301
Chlamydospore Production, 246
Chloramphenicol Assay, 203, 206
Chloramphenicol Disks, 331
Chlorine Inactivation, 71
Chloromycetin Disks, 331
Chocolate Agar, 116
Chocolate Agar, Tubes, 339
Chocolate Tellurite Agar, Tubes, 339
Cholesterol, 316
Choline Assay Medium, 227
Citrate Utilization, 55, 182
Clostridium, 27, 84, 85, 104, 126, 127
Coagulase Plasma, 330

Coagulase Test, 78, 330
Coagulation — Sterilization, 128
Coli-Aerogenes Differentiation, 35, 52,

54-55, 175, 182, 183
Coliform Count, 52, 61, 63, 64, 175
Coliform Detection, 30, 34, 35, 37, 39,

41-46, 49, 60, 61, 63, 64
Coliform Group, 28
Complement, 311
Complement Fixation Test Reagents,

309, 311, 314
Concentration Disks, Penicillin, 337
Confirmed Test (Coliform), 34, 35, 37,

39, 46, 60
Conradi Drigalski Agar, 146
Cooked Meat Medium, 85
Cooledge Broth, 76
Cord Serum, Human, TC, 304
Corn Germ Sterol, 316
Corn Meal Agar, 246
Corn Meal Agar with Dextrose, 246
Corynebacterium diphtheriae, 26, 128,

147, 149
Cresol Red, 295
m-Cresolsulfonphthalein, 295
o-Cresolsulfonphthalein, 295
Crystal Violet, 294
Crystal Violet Agar, 194
Crystal Violet Lactose Broth, 43
C S Vitamin B12 Agar, 223
/-Cystine, 268
Cystine Assay Medium, 235
Cystine Heart Agar, 9 1
Cystine Hemoglobin Agar, Tubes, 339
Czapek Dox Broth, 245
Czapek Solution Agar, 245

Dairy Products Analysis, 57-76
Dental Media, 73, 190, 339
Desoxycholate Agar, 63, 133
Desoxycholate Citrate Agar, 138
Desoxycholate Lactose Agar, 64
Dextrin, 292
Dextrose, 291, 292
Dextrose Agar, 125
Dextrose Agar, Tubes, 339
Dextrose Broth, 101
Dextrose Broth, Tubes, 339
Dextrose Heart Agar, 129
Dextrose Infusion Broth, 129
Dextrose Proteose No. 3 Agar, 147
Dextrose Solution 10%, 293

INDEX

345

Dextrose Starch Agar, 124
Dextrose Tryptone Agar, 67
Diagnostic Reagents, 317-337
Dibromo-o-cresolsulfonphthalein, 295
Dibromothymolsulfonphthalein, 295
Differential Media, 130-194
Differential Plate Media, 130, 131-156
Differential Tube Media, 159-194
Dihydrostreptomycin Disks, 331
Dimethyl-/?-phenylenediamine hydro-
chloride, 296
Diphtheria Culture Tubes and Outfits,

339
Diphtheria Differentiation, 147, 149
Diphtheria Isolation, 128, 147, 149
Dorset Egg Medium, Tubes, 339
Double Sugar Agar, 162
Dubos Albumin Broth, Tubes, 339
Dubos Broth Base, 105
Dubos Broth Base without Tween 80,

107
Dubos Medium Albumin, 274
Dubos Medium Serum, 274
Dubos Oleic Agar, Tubes, 339
Dubos Oleic Agar Base, 107
Dubos Oleic Albumin Complex, 275
Dubos Oleic Serum Complex, 274
Dubos Serum Broth, Tubes, 339
Dulcitol (Dulcite), 292
Dyes, 293-295

Eagle Flocculation Antigen, 312

Eagle Wassermann Antigen, 310

E G Medium, 45

Egg Albumen, 299, 300

Egg Meat Medium, 84

Egg, Whole, 299, 300

Egg Yolk, 299

Eijkman Lactose Medium, 44

Eijkman Test, 44, 45

E M B Agar, 133

Embryo, Beef, TC, 303, 304

Embryo, Chick, TC, 303

Embr>'o Extract, Chick, TC, 302

Embryo Extract, Beef, TC, 303

Endamoeba histolytica, 27, 97, 273, 300

Endamoeba Medium, 97

Endo Agar, 34, 134, 338

Enrichments, 271-278

Enrichments, Enteric, 156—158

Enteric Cultural Reactions, 160, 161

Enteric DifferentialTube Media, 162-1 94

Enteric Isolation, 130
Enteric Microorganisms, 28
Enterococci, 46-52, 154, 155
Enterococci Confirmatory Agar, 51
Enterococci Confirmatory Broth, 52
Enterococci Presumptive Broth, 50
Enzymatic Hydrolysates, 255-264
Enzymes, 278-285
Eosin Y, 294
Extracts. 269-271

Fecal Streptococci, 46-52
Fermentation Reactions, 186-190
Flocculation Tests, Syphilis, 311-314
Fluid Thioglycollate Medium, 195, 211
Fluid Thioglycollate Medium, Bottles,

341
Fluid Thioglycollate Medium, Tubes,

339
Folic Acid Assay Medium, 225
Formate Ricinoleate Broth, 39, 60, 338
Friewer Shaughnessy Medium, 178
rf-Fructose, 292
Fuchsin Basic, 293
Fuchsin Lactose Broth, 41
Fungi, 27, 90, 237-254

//-Galactose, 292

Galactose Peptonized Milk, 76

Galactose Solution 10%, 293

Galactose Whey Agar, 76

G C Medium Base, 122

Gelatin, 290

Gelatin Liquefaction, 32, 151, 153

Gentian Violet, 294

Gentian Violet Lactose Bile, 76

<f -Glucose, 291

Glucose Ascites Medium, Tubes, 339

Glycerol, 292

Glycerol Agar, Tubes, 339

Glycerol Solution 10%, 293

Gonococcus, 116, 122, 124, 271, 276

Guides for Selection of Media, 25-28

H Antigen Production, 177

H Broth, 177

H D Broth, 46

Hanger Flocculation Test, 324

Heart Infusion Agar, 69, 87, 338

346

INDEX

Heart Infusion Agar, Tubes, 339

Heart Infusion Broth, 80, 338

Hemagglutination Buffer, 282

Hemagglutination, Trypsin 1%, 280

Hemoglobin, 271

Hemolysin, Antisheep, 310

Hemophilus, 27, 78, 95, 271, 276

Hinton Cardiolipin Indicator, 314

Hinton Indicator, 312

Hormone Broth, 80

Horse Serum, 273

Horse Serum Saline 1-6, 273

Horse Serum, TC, 304

Horse Serum Ultrafiltrate, TC, 305

Human Cord Serum, TC, 304

Huntoon Hormone Broth, 80

Hydrogen Sulfide Production, 165, 166,

169, 173, 176, 178, 181
Hydrolysates, 255-268
Hydroly sates, Acid, 265-268

Indicators, 293, 295, 296

Indole Production, 53, 173, 260

Infusion Media, 76-98

Mnositol (Inosite), 292

Inspissation, 128

Inulin, 292

Invert Sugar Syrup, 292

Invertase, 282

Isodulcitol, 292

Isoelectric Casein, 299

Isoleucine Assay Medium, 234

Kahn Beef Heart, 316

Kahn Cardiolipin Antigen, 313

Kahn Oversensitive Correction Solution,

316
Kahn Sensitized Antigen, 312
Kahn Standard Antigen, 311
Kahn Undersensitive Correction Solu-
tion, 316
Kingsbury Standards, 328
Kligler Iron Agar, 165
Kolmer Cardiolipin Antigen, 314
Kolmer Improved Antigen, 309
Kolmer Improved Antigen (Reenforced ) ,

309
Koser Citrate Medium, 55
Kracke Blood Culture Medium, 83
Krumwiede Triple Sugar Agar, 164

Lactobacilli, 72-76, 190, 192, 193, 212,

213
Lactose, 292
Lactose Broth, 30, 338
Lactose Peptone Bile, 76
Lactose Solution 10%, 293
Lash Serum Medium, Tubes, 339
Lauryl Tryptose Broth, 39
Lead Acetate Agar, 169
Lead Acetate Agar, Tubes, 339
Legumin Trypagar, 129
Leucine Assay Medium, 230
Levine E M B Agar, 35, 134, 338
Levine E M B Agar, Tubes, 339
Levulose, 292
Lima Bean Agar, 247
Linden Thioglycollate Medium, 197
Link-Shapiro Technique, 319
Listeria, 114
Litmus, 296

Litmus Lactose Agar, 145
Litmus Milk, 192
Littman Oxgall Agar, 240
Littman Oxgall Agar, Tubes, 339
Liver, 289

Liver Function Test, 324, 327
Liver Infusion Agar, 129
Liver Veal Agar, 98
Loeffler Blood Serum, 128
Loeffler Blood Serum, Tubes, 339
Lowenstein- Jensen Medium, Tubes, 339
Lysine Assay Medium, 233
Lysodeikticus, 285
Lyso2yme, 285
Lysozyme Buffer, 285
Lysozyme Substrate, 285

MacConkey Agar, 130, 131
MacConkey Broth, 56
Malaria Diagnosis, 324
Malonate Broth, 183
Malt Agar, 65, 243
Malt Extract, 271
Malt Extract Agar, 244
Malt Extract Broth, 202, 242
Maltose, 292

Maltose Solution 10%, 293
Maltose, Technical, 292
<f-Mannitol (Mannite), 292
Mannitol Salt Agar, 150

INDEX

347

Mannitol Solution 10%, 293

^-Mannose, 292

M B-B C P Medium, 42

Meats, Dehydrated, 288, 289

Melezitose, 292

Melibiose, 292

Meningococci, 26, 77, 79, 87, 90, 100,

115
Mercurial Inactivation, 195, 198, 199,

200
Meta Gresol Purple, 295
Methionine Assay Medium, 232
Methyl Red, 296
Methyl Red Test, 54
Methylene Blue, 294
Micro Assay Culture Agar, 212
Microbiological Assay, 203-236, 267
Micro Inoculum Broth, 213
Mildew Test Medium, 246
Milk Analysis, 57-72
Milk, Skim, 74
Mitis Salivarius Agar, 154
Motility, 173, 176, 178, 184
Motility Sulfide Medium, 176
Motility Test Medium, 184
M R-V P Medium, 54
Mueller Hinton Medium, 93
Mueller Tellurite Base, 149
Mueller Tellurite Serum, 278
Mycin Assay Agar, 208
Mycin Assay Broth, 207
Mycobacterium tuberculosis, 27, 105-

110,274,275
Mycology Media, 237-254
Mycological Agar, 243
Mycological Broth, 243

Neisseria DiEerentiation^ 119

Neisseria gonorrhoeae, 26, 116, 119, 122,

124
Neomycin Assay, 203
Neopeptone, 263

Neurospora Culture Agar, 214, 242
Neutral Red, 296
Neutral Red Medium, 76
Neutralizing Buffer, 71
Niacin Assay Medium, 216
N I H Agar Medium, 198
N I H Thioglycollate Broth, 198
Nitrate Agar, 185
Nitrate Broth, 184
Nitrate Reduction, 184, 185

Noble, Special Agar, 290
North Gelatin Agar, 129
North Gelatin Agar, Tubes, 339
Nutrient Agar, 32, 338
Nutrient Agar, Tubes, 339
Nutrient Agar 1.5%, 127
Nutrient Broth, 29, 338
Nutrient Broth, Tubes, 339
Nutrient Gelatin, 32
Nutrient Gelatin, Tubes, 339
Nutrient Phosphate Agar, 129
Nutritive Caseinate Agar, 69
Nutrose, 300

Oxgall, 286

Oxydase Reagent, 296, 297

Pancreas, Chicken, 282

Pancreatic Enzyme, 278

Pangestin, 278

Pantothenate Assay Medium, 219

Papain, 278

Pasteur ella tularensis, 27, 91

Pathogenic Microorganisms, 76-194

Peizer TB Medium, Tubes, 339

Peizer TB Medium Base, 110

Peizer TB Medium Enrichment, 275

Penase, 203, 211,2S5

Penase Ascites Medium, Tubes, 339

Penase Broth, Bottles, 340

Penase Concentrate, 284

Penase Disks, 337

Penase Medium, Bottles, 340

Penassay Base Agar, 204

Penassay Broth, 203

Penassay Seed Agar, 205

Penicillinase, 283, 284

Penicillin Assay, 203-206

Penicillin Concentration Disks, 337

Penicillin Disks, 331

Penicillin Inactivation, 82, 83, 283, 284

Pepsin 1:10,000, 278

Peptone, Bacto, 256

Peptone Analysis, 265

Peptone Colloid Medium, 104

Peptone Iron Agar, 181

Peptone Media, 99-129

Peptones, 255-264

Peptonized Milk, 75, 264

Peptonized Milk Agar, 76

348

INDEX

Petragnani Medium, Tubes, 339
Petroff Medium, Tubes, 339
Phenolphthalein, 296
Phenol Red, 295
Phenol Red Agar Base, 187, 338
Phenol Red Broth Base, 186, 338
Phenol Red Carbohydrate Agars, 189
Phenol Red Carbohydrate Broths, 187
Phenol Red Dextrose Agar, 189
Phenol Red Dextrose Broth, 187, 338
Phenol Red Lactose Agar, 189
Phenol Red Lactose Broth, 187, 338
Phenol Red Maltose Agar, 189
Phenol Red Maltose Broth, 187, 338
Phenol Red Mannitol Agar, 189
Phenol Red Mannitol Broth, 187, 338
Phenol Red Saccharose Agar, 189
Phenol Red Saccharose Broth, 187, 338
Phenol Red Solution, TC, 306
Phenol Red Tartrate Agar, 183
Phenolsulfonphthalein, 295
Phenolsulfonphthalein Ampuls, 328
Phenylalanine Assay Medium, 235
Phytopathology Media, 246-248
Plasma, Chicken, TC, 301
Plate Count, Butter, 69
Plate Count, Milk, 57, 68, 69
Plate Count, Water, 32, 33
Pleuropneumonia-Like Organisms

(PPLO), 82, 89, 275
Pneumococci, 26, 77, 79, 87, 88, 90,

100, 101, 115
Polymyxin Assay, 210, 211
Polymyxin B Disks, 331
Polymyxin Base Agar, 210
Polymyxin Seed Agar, 211
Potassium Tellurite, 298
Potato Dextrose Agar, 64, 243
Potato Dextrose Agar, Tubes, 339
Potato Infusion Agar, 95
PPLO Agar, 89
PPLO Enrichment Broth, 82
PPLO Serum Fraction, 275
Precipitation Test Reagents, 311-314
Precipitation Tests, Syphilis, 311-314
Preparation Dehydrated Media, 21
Pressure-Temperature, 18
Presumptive Test (Coliform) 30, 37, 39,

46, 49, 60
Proteose No. 3 Agar, 116
Proteose Peptone, 257
Proteose Peptone No. 2, 259
Proteose Peptone No. 3, 259
Proteose Tryptone Agar, 68

Proteus identification, 170-173

Prothrombin Activity Curve, 319

Prothrombin Determinations, 317, 323

Prothrombin Free Beef Plasma, 324

Prothrombin Free Rabbit Plasma, 324

Prothrombin 2 Stage Reagent, 323

Protone, 264

Prune Agar, 248

Purple Agar Base, 190, 338

Purple Bile Salt Agar, 76

Purple Broth Base, 189, 338

Purple Lactose Agar, 146

Purple Milk, 76, 194

Pyridoxine Assay Medium, 226

Quaternary Inactivation, 71
Quick Test, 317

RafEnose, 292

Reconstituting Fluid, TC, 306

Renal Function Test, 328

Rennin, 279

Rhamnose (Isodulictol), 292

Riboflavin Assay Medium, 214

Rice Powder, 300

Root Canal Medium, Tubes, 339

Rosenow Brain Broth, 77

Russell Double Sugar Agar, 162, 338

Russell Double Sugar Agar, Tubes, 339

Sabouraud Dextrose Agar, 238
Sabouraud Dextrose Agar, Tubes, 339
Sabouraud Liquid Medium, 200, 240
Sabouraud Maltose Agar, 239
Sabouraud Maltose Agar, Tubes, 339
Sabouraud Maltose Broth, 240
Saccharose (Sucrose), 292
Saccharose Mannitol Agar, 163
Saccharose, Solution 10%, 293
Salicin, 292
Salmonella, 28, 130, 131, 133, 134, 138,

139, 144, 157, 158
Sanitation Control, 71
Selenite Broth, 158
Sensitivity Disks, 331
Serological Reagents, 309-316
Serum, Horse, 273
Serum, Horse, TC, 304
Serum, Human Cord, TC, 304

INDEX

349

S F Medium, 46

Shigella, 28, 130, 131, 133, 134, 138

SI M Medium, 173

Simmons Citrate Agar, 182

Skim Milk, 74

Snyder Test Agar, 190

Snyder Test Agar, Tubes, 339

Sodium Caseinate, 300

Sodium Chloride 0.85 per cent, 322

Sodium Desoxycholate, 287

Sodium Hippurate, 292

Sodium Oxalate 0.1 Molar, 322

Sodium Taurocholate, 287

Sodium Thioglycollate, 297

Solidifying Agents, 290

c?-Sorbitol (Sorbite), 292

Soy tone, 264

Special Agar (Noble), 290

SS Agar, 134

Standard Methods Dairy and Food

Products, 25, 57-67
Standard Methods Water and Sewage,

25, 29-56
Staphylococcus, 26, 150, 151, 153, 155,330
Staphylococcus Medium No. 110, 151
Starch Agar, 129
Starch, Soluble, 292
Sterile Applicators in Tubes, 339
Sterility Test Media, 195-202
Sterilization, 18
Stock Culture Agar, 98
Stock Cultures, 85, 98
Stone Gelatin Agar, 194
Streptococci, 26, 77, 79, 87, 88, 90, 100,

101, 115, 154, 155
Streptococci Detection, 46-52, 154, 155
Streptomycin Assay, 203, 206-208
Streptomycin Assay Agar, 208
Streptomycin Disks, 331
Streptomycin Inactivation, 82, 83
Subtilis Spore Suspension, 337
Sucrose, 292

Sulfonamide Resistance, 93, 120
Sulfonphthalein Indicator, 295
Supplement A, 276
Supplement B, 276

TB Broth Base, 108
TB Broth Base without Tween 80, 109
TB Serum Broth, Tubes, 339
Tellurite Blood Solution, 277
Tellurite Solution, 277

Tergitol-7 Agar, 175

Terramycin Disks, 331

Testicular Agar, 129

Tetrabromo-o-Cresolsulfonphthalein,
295

Tetrabromophenolsulfonphthalein, 295

Tetrathionate Broth Base, 157

Thermoacidurans Agar, 70

Thermophilic Spore Count, 67, 70

Thiamine Assay Medium, 217

Thioglycollate Medium without Dex-
trose, 199

Thioglycollate Medium without Dex-
trose or Indicator, 200

Thioglycollate Medium without Indi-
cator, 200

Thioglycollic Acid, 298

Thiol Broth, 83

Thiol Medium, 82

Thionin, 295

Thromboplastin, 317

Thymol Blue, 295

Thymolsulfonphthalein, 295

Thymol Turbidity Reagent, 327

Tissue Culture Media Reagents, 301-
308

Tissue Culture Methods, 307, 308

Tomato Juice Agar, 73

Tomato Juice Agar, Special, 73

Toxin Production, 257, 265, 267

Trehalose, 292

Trichomonas Culture, Lash Serum Me-
dium Tubes, 339

Triple Distilled Water, TC, 306

Triple Sugar Agar, 164-166

Triple Sugar Iron Agar, 166

Trypsin, 280

Trypsin 1%, 280

Trypsin 1:250, 279

Trypsin Digest Agar, 74

Tryptone, 53, 260

Tryptone Glucose Extract Agar, 33, 57,
338

/-Tryptophane, 269

Tryptophane Assay Medium, 235

Tryptose, 262

Try'ptose Agar, 65, 111

Tryptose Agar, Tubes, 339

Tryptose Blood Agar Base, 115

Tryptose Broth, 102, 113

Tryptose Phosphate Broth, 100

Tube Media, 338

Tubercle Bacillus, Media Tubes, 339

Tubes, Complete Media, 338-339

350

INDEX

Turbidity Standards, 328
/-Tyrosine, 269
Tyrosine Assay Medium, 235
Tyrothrycin Assay, 209-210
Tyrothrycin Assay Agar, 210
Tyrothrycin Assay Broth, 209

Ulrich Milk, 193

Ultrafiltrate, Beef Serum, TC, 305

Ultrafiltrate, Horse Serum, TC, 305

Urea, 298

Urea Agar Base, 171

Urea Agar Base Concentrate 173

Urea Broth, 170

Urea Broth Concentrate, 171

Urease Production, 170-173

Vitamin Assay, 212-230
Vitamin Free Casamino Acids, 267
Vitamin Free Yeast Base, 251, 253
Voges-Proskauer Test, 54

Wassermann Test Reagents, 309-310

Water Analysis, 29-56

Whey Agar, 76, 337

Whey Broth, 76

W L Differential Medium, 249

W L Nutrient Medium, 248

Wort Agar, 244

rf-Xylose, 292

VDRL Antigen, 313

Veal, 289

Veal Infusion Agar, 89

Veal Infusion Broth, 81

Veal Infusion Medium, 81

Vibrio fetus, 82

Violet Red Bile Agar, 61

Visual Sanitation Test, 71

Yeast Beef Agar, 206

Yeast Carbon Base, 251, 252

Yeast Classification, 250

Yeast Dextrose Agar, 76

Yeast Extract, 270

Yeast Mold Count, 64, 65

Yeast Morphology Agar, 250, 251

Yeast Nitrogen Base, 251, 252

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