BD Diagnostic Systems (publ.). Difco Manual (Manual of Microbiological Culture)
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11th Edition
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Table of Contents
Foreword................................................................................................... vii
Introduction.................................................................................................ix
Monographs .................................................................................................1
Culture Media and Ingredients, Dehydrated ..............................................19
Culture Media, Prepared ..........................................................................585
Stains and Indicators................................................................................595
Serology and Immunology.......................................................................607
Reference Guides.....................................................................................811
Indices......................................................................................................843
Alphabetical Index .............................................................................845
Numerical Index.................................................................................855
vi The Difco Manual
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
Reprinted 1956
Reprinted 1958
Reprinted 1960
Reprinted 1962
Reprinted 1963
Reprinted 1964
Reprinted 1965
Reprinted 1966
Reprinted 1967
Reprinted 1969
Reprinted 1971
Reprinted 1972
Reprinted 1974
Reprinted 1977
Tenth Edition 1984
Reprinted 1985
Reprinted 1994
Reprinted 1996
Eleventh Edition 1998
Copyright 1998 by
Difco Laboratories,
Division of Becton Dickinson and Company
Sparks, Maryland 21152 USA
The Difco Manual vii
Foreword
This edition of the DIFCO MANUAL, the eleventh published since 1927, has been extensively revised and
rewritten. The purpose of the Manual is to provide information about products used in microbiology. The
Manual has never been intended to replace any official compendium or the many excellent standard text
books of scientific organizations or individual authors.
Difco is perhaps best known as the pioneer in bacteriological culture media. Numerous times one will find
the trademarks Difco
®
or Bacto
®
preceding the names of materials used by scientists in their published
papers. Because Difco products have been readily available worldwide longer than any others, Difco
products have become the common-language reagents of the microbiological community. Standardized
products readily available worldwide are essential for corroborative studies demanded by rigorous science.
Recommendation and approval have been extended to our products by the authors of many standard text
books and by the committees on methods and procedures of scientific societies throughout the world. Difco
products continue to be prepared according to applicable standards or accepted formulae. It is expected that
they will be used only by or under the supervision of microbiologists or other professionals qualified by
training and experience to handle pathogenic microorganisms. Further, it is expected that the user will be
throughly familiar with the intended uses of the formulations and will follow the test procedures outlined in
the applicable official compendia and standard text books or procedures manual of the using laboratory.
Grateful acknowledgment is made of the support we have received from microbiologists throughout the
world. It is our desire to continue and extend our services to the advancement of microbiology and related
sciences.
Difco Laboratories
Division of Becton Dickinson and Company
Foreword
The Difco Manual ix
Introduction
Introduction
Microbiology, through the study of bacteria, emerged as a defined
branch of modern science as the result of the monumental and immortal
research of Pasteur and Koch. In 1876, Robert Koch, for the first time
in history, propagated a pathogenic bacterium in pure culture outside
the host’s body. He not only established Bacillus anthracis as the
etiological agent for anthrax in cattle, but he inaugurated a method of
investigating disease which ushered in the golden age of medical
bacteriology.
Early mycologists, A. de Bary and O. Brefeld, and bacteriologist,
R. Koch and J. Schroeter, pioneered investigations of pure culture
techniques for the colonial isolation of fungi and bacteria on solid
media. Koch, utilizing state-of-the-art clear liquid media which he
solidified with gelatin, developed both streak and pour plate methods
for isolating bacteria. Gelatin was soon replaced with agar, a solidifying
agent from red algae. It was far superior to gelatin in that it was
resistant to microbial digestion and liquefaction.
The capability of Koch to isolate disease-producing bacteria on solidi-
fied culture media was further advanced by manipulating the cultural
environment using meat extracts and infusions so as to reproduce, as
closely as possible, the infected host’s tissue. The decade immediately
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 cultivation of bacteria.
The fundamental principles of pure culture isolation and propagation
still constitute the foundation of microbiological practice and research.
Nevertheless, it has become more and more apparent that a successful
attack upon problems unsolved is closely related to, if not dependent
upon, a thorough understanding of the subtle factors influencing
bacterial metabolism. With a suitable culture medium, properly
used, advances in microbiology are more readily made than when
either the medium or method of use is inadequate. The microbiologist
of today is, therefore, largely concerned with the evolution of methods
for the development and maintenance of microbial growth upon which
an understanding of their unique and diversified biological and
biochemical characteristics can be investigated. To this end, microbi-
ologists have developed innumerable enrichment culture techniques
for the isolation and cloning of microorganisms with specific nutri-
tional requirements. These organisms and their unique characteristics
have been essential to progress in basic biological research and modern
applied microbiology.
The study of microorganisms is not easy using microscopic single cells.
It is general practice to study pure cultures of a single cell type. In the
laboratory, microbiological culture media are utilized which contain
various nutrients that favor the growth of particular microorganisms in
pure cultures. These media may be of simple and defined chemical
composition or may contain complex ingredients such as digests of
plant and animal tissue. In particular, the cultivation of bacteria is
dependent upon nutritional requirements which are known to vary
widely. Autotrophic bacteria are cultivated on chemically defined or
synthetic media while heterotrophic bacteria, for optimal growth, may
require more complex nutrients such as peptones, meat or yeast
extracts. These complex mixtures of nutrients readily supply fastidious
heterotrophic bacteria with vitamins and other growth-promoting
substances necessary for desired cultivation. The scientific literature
abounds with descriptions of enriched, selective and differential
culture media necessary for the proper isolation, recognition and
enumeration of various bacterial types.
Almost without exception whenever bacteria occur in nature, and this
is particularly true of the pathogenic forms, nitrogenous compounds
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 dissimilated into simpler
compounds then assimilated into cellular material. Such metabolic
alterations are affected by enzymatic 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
activity of bacterial enzymes which are both numerous and varied. The
processes 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. The study of bacterial
metabolism, which defines the organized chemical activities of a cell,
has led to the understanding of both catabolic or degradative activities
and anabolic or synthetic activities. From these studies has come a
better understanding of the nutritional requirements of bacteria, and in
turn, the development of culture media capable of producing rapid and
luxuriant growth, both essential requisites for the isolation and study
of specific organisms.
Studies to determine the forms of carbon, hydrogen, and nitrogen which
could most easily be utilized by bacteria for their development were
originally carried on by Naegeli
1
between 1868 and 1880, and were
published by him in the latter year. Naegeli’s report covered the use of
a large variety of substances including carbohydrates, alcohols, amino
acids, organic nitrogen compounds, and inorganic nitrogen salts.
The first reference to the use of peptone for the cultivation of microor-
ganisms is that made by Naegeli in the report referred to above, when
in 1879, he compared peptone and ammonium tartrate. Because of its
content amino acids and other nitrogenous compounds which are
readily utilized by bacteria, peptone soon became one of the most
important constituents of culture media, as it still remains. In the light
of our present knowledge, proteins are known to be complex compounds
composed of amino acids joined together by means of the covalent
peptide bond linkage. When subjected to hydrolysis, proteins yield
polypeptides of various molecular sizes, metapeptones, proteoses,
peptones and peptides, down to the level of simple amino acids. The
intermediate products should be considered as classes of compounds,
rather than individual substances, for there exists 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 hydrolysis. Not all the
x The Difco Manual
products of protein decomposition are equally utilizable by all
bacteria. In their relation to proteins, bacteria may be divided into two
classes; those which decompose naturally occurring proteins, and those
which require 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.
Duval,
2,3
for example, reports that cysteine and leucine are essential in
the cultivation of Mycobacterium leprae. Kendall, Walker and Day
4
and Long
5
reported 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
6
to the meningococcus and Streptococcus;
Cole and Lloyd
7
and Cole and Onslow
8
to the gonococcus; and Jacoby
and Frankenthal
9
to the influenza bacillus. More recently Feeley, et
al.
34
demonstrated that the nonsporeforming aerobe, Legionella
pneumophila requires L-cysteine
.
HCI for growth on laboratory media.
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 such
as those with Legionella pneumophila 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
requirements. Bacteriologists are therefore continuing to expend much
energy on the elucidation of the variations in 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 realized, 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,
10
acid,
11,12,13
enzymatic digestion,
8,14,15,16,17,18
and partial digestion of plasma
10
have been described for the preparation
of protein hydrolysates.
The use of protein hydrolysates, particularly gelatln and casein, has
led to especially important studies related to bacterial toxins by
Mueller, et al.
20-25
on the production of diphtheria toxin; that of Tamura,
et al.
25
of toxin of Clostridium welchii; that of Bunney and Loerber
27,28
on scarlet fever toxin, and of Favorite and Hammon
29
on Staphylococcus
enterotoxin. In addition, the work of Snell and Wright
30
on the
microbiological assay of vitamins and amino acids was shown to
be dependent upon the type of protein hydrolysate utilized. Closely
associated with research on this nature are such studies as those of
Mueller
31,32
on pimelic acid as a growth factor for Corynebacterium
diphtheriae, and those of O’Kane
33
on synthesis of riboflavin by
staphylococci. More recently, the standardization of antibiotic suscep-
tibility testing has been shown to be influenced by peptones of culture
media. Bushby and Hitchings
35
have shown that the antimicrobial ac-
tivities of trimethoprim and sulfamethoxazole are influenced consider-
ably by the thymine and thymidine found in peptones of culture media.
In this brief discussion of certain phases of bacterial nutrition, we have
attempted to indicate the complexity of the subject and to emphasize
the importance of continued study of bacterial nutrition. Difco Labo-
ratories has been engaged 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 dependable laboratory adjuncts
in all fields of microbiology.
References
1. Sitz’ber, math-physik. Klasse Akad. Wiss. Muenchen, 10:277,
1880.
2. J. Exp. Med., 12:46, 1910.
3. J. Exp. Med., 13:365, 1911.
4. J. Infectious Diseases, 15:455, 1914.
5. Am. Rev. Tuberculosis, 3:86, 1919.
6. Brit. Med. J., 2:398, 1918.
7. J. Path. Bact., 21:267, 1917.
8. Lancet, II:9, 1916.
9. Biochem, Zelt, 122:100, 1921.
10. Centr. Bakt., 1:29:617, 1901.
11. Indian J. Med. Research, 5:408, 1917-18.
12. Compt. rend. soc. biol., 78:261, 1915.
13. J. Bact., 25:209, 1933.
14. Ann. de L’Inst., Pasteur, 12:26, 1898.
15. Indian J. Med. Research, 7:536, 1920.
16. Sperimentale, 72:291, 1918.
17. J. Med. Research, 43:61, 1922.
18. Can. J. Pub. Health, 32:468, 1941.
19. Centr. Bakt., 1:77:108, 1916.
20. 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., 37:103, 1939.
25. J. Immunol., 40:21, 1941.
26. 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.
33. J. Bact., 41:441, 1941.
34. J. Clin. Microbiol., 8:320, 1978.
35. Brit. J. Pharmacol., 33:742, 1968.
Introduction
The Difco Manual 3
Section I Monographs
Original Difco Laboratories
Manufacturing facility.
Section ! Monographs
Difco Laboratories, originally
known as Ray Chemical, was
founded in 1895. This company
produced high quality enzymes,
dehydrated tissues and glandular
products to aid in the digestion
process. Ray Chemical acquired
Digestive Ferments Company,
a company that specialized in
producing digestive enzymes
for use as bacterial culture media
ingredients. The experience of
processing animal tissues, puri-
fying enzymes and performing
dehydration procedures created
a smooth transition to the
preparation of dehydrated
culture media. In 1913, the Digestive Ferments Company moved to
Detroit, Michigan, and dropped the name, Ray Chemical.
After 1895, meat and other protein digests were developed to stimulate
growth of bacteria and fungi. The extensive research performed on
the analysis of pepsin, pancreatin and trypsin (and their digestive
processes) led to the development of Bacto
®
Peptone. Bacto Peptone,
first introduced in 1914, was used in the bacteriological examination
of water and milk as a readily available nitrogen source. Bacto Peptone
has long been recognized as the standard peptone for the preparation
of bacteriological culture media.
The development of Proteose Peptone, Proteose Peptone No. 2 and
Proteose Peptone No. 3 was the result of accumulated information that
no single peptone is the most suitable nitrogen source for growing
fastidious bacteria. Proteose Peptone was developed for use in the
preparation of diphtheria toxin of high and uniform potency. Bacto
Tryptose was originally formulated to provide the growth requirements
of Brucella. Bacto Tryptose was also the first peptone prepared that
did not require the addition of infusions or other enrichments for the
isolation and cultivation of fastidious bacteria.
The Digestive Ferments Company began the preparation of diagnostic
reagents in 1923. Throughout the development of products used in the
diagnosis of syphilis and other diseases, Difco worked closely with
and relied on the direct involvement of expert scientists in the field.
Bacto Thromboplastin, the first manufactured reagent used in
coagulation studies, was developed in the early 1930s. This product
was another in a long line of many “firsts” for Difco Laboratories.
In 1934, the Digestive Ferments Company chose an acronym, “Difco,”
to rename the company. The focus of Difco Laboratories was to
develop new and improved culture media formulations.
After World War II, the microbiology and health care fields expanded
rapidly. Difco focused on the development of microbiological and
immunological products to meet this growing demand. In the 1940s,
Difco pursued the challenging task of producing bacterial antisera
and antigens. Lee Laboratories, a subsidiary, remains one of the
largest manufacturers of bacterial antisera. Additional “firsts” for Difco
Laboratories came in the 1950s with the development of C Reactive
Protein Antiserum, Treponemal Antigen and Antistreptolysin Reagents.
Throughout the 1950s and 1960s, Difco continued to add products
for clinical applications. Bacto Blood Cultures Bottles were developed
to aid in the diagnosis and treatment of sepsis. Difco Laboratories
pioneered in the preparation of reagents for in vitro propagation and
maintenance of tissue cells and viruses.
With the discovery of penicillin, a brand new branch of microbiology
was born. Difco initiated developmental research by preparing
antibiotic disks for use in a “theorized” disk diffusion procedure.
The result was Bacto Sensitivity Disks in 1946, followed by Dispens-
O-Discs
™
in 1965.
In the 1960s, Difco Laboratories became the largest manufacturer of
microbiological culture media by acquiring the ability to produce agar.
Difco offers the same premier “gold standard,” Bacto Agar, today.
Bactrol
™
Disks were introduced by Difco Laboratories in 1972. Bactrol
Disks are water-soluble disks containing viable microorganisms of
known cultural, biochemical and serological characteristics used for
quality control testing. Bactrol Disks became the first of many
products manufactured by Difco for use in quality control.
In 1983, Difco purchased the Paul A. Smith Company, later to be known
as Pasco
®
. A semi-automated instrument, the Pasco MIC/ID System, is
used for bacterial identification and sensitivity testing. The Pasco Data
Management System can be used in industrial and clinical laboratories,
either alone or as a back up to automated systems.
In 1992, ESP
®
, an automated continuous monitoring blood culture
system, was introduced. ESP was the first blood culture system to
detect both gas production and consumption by organism growth. The
technology continued with ESP Myco, an adaptation to the system that
allowed for growth, detection and susceptibility testing of mycobacteria
species. The ESP clinical system was sold to AccuMed International
in 1997.
In 1995, Difco Laboratories celebrated 100 years in business. In 1995,
Difco was the first U.S. microbiology company to receive ISO 9001
certification. The International Organization for Standardization (ISO)
verifies that Difco Laboratories maintains quality standards for the
worldwide microbiology industry.
In 1997, Difco Laboratories, the “industrial microbiology leader,” was
purchased by the “clinical microbiology leader,” Becton Dickinson
Microbiology Systems, to form the largest microbiology company in
the world. Together, Becton Dickinson Microbiology Systems and
Difco Laboratories look forward to an even stronger future with our
combined commitment to serving microbiologists worldwide.
History of Difco Laboratories
4 The Difco Manual
Monographs Section I
The science of microbiology evolved from a series of significant
discoveries. The Dutch microscopist, Anton van Leeuwenhoek, was
the first to observe bacteria while examining different water sources.
This observation was published in 1676 by the Royal Society in
London. Anton van Leeuwenhoek was also the first to describe the
parasite known today as Giardia lamblia. In 1667, the discovery of
filamentous fungi was described by Robert Hooke.
After microorganisms were visually observed, their growth or
reproduction created a major controversy. The conflict was over the
spontaneous generation theory, the idea that microorganisms will grow
spontaneously. This controversy continued for years until Louis
Pasteur’s renowned research. Pasteur realized that the theory
of spontaneous generation must be refuted for the science of
microbiology to advance. The controversy remained even after
Pasteur’s successful experiment using heat-sterilized infusions.
Two important developments were required for the science of
microbiology to evolve. The first was a sophisticated microscope; the
second was a method for culturing microorganisms. Compound
microscopes were developed in Germany at the end of the sixteenth
century but it was not until the early nineteenth century that
achromatic lenses were developed, allowing the light in the microscope
to be focused.
In 1719, Leeuwenhoek was the first to attempt differentiation of
bacteria by using naturally colored agents such as beet juice. In 1877,
Robert Koch used methylene blue to stain bacteria. By 1882,
Robert Koch succeeded in staining the tubercle bacillus with
methylene blue. This landmark discovery was performed by using
heat to penetrate the stain into the organism. Two years later Hans
Christian Gram, a Danish pathologist, developed the Gram stain. The
Gram stain is still widely used in the differentiation of gram-positive
and gram-negative bacteria.
In 1860, Pasteur was the first to use a culture medium for growing
bacteria in the laboratory. This medium consisted of yeast ash, sugar
and ammonium salts. In 1881, W. Hesse used his wife’s agar
(considered an exotic food) as a solidifying agent for bacterial growth.
The study of fungi and parasites lagged behind other microorganisms.
In 1839, ringworm was the first human disease found to be caused by
fungi, followed closely by the recognition of Candida albicans as
the cause of thrush. It was not until 1910 that Sabouraud introduced
a medium that would support the growth of pathogenic fungi. The
interest of scientists in studying fungi was often related to crop
protection. There continues to be a close connection between
mycology and botany today.
By 1887, a simple device called the Petri dish revolutionized
microbiology. With the invention of the Petri dish, the focus turned to
culture media formulations. With all the research being performed,
scientists began to replace gelatin with agar because it was resistant to
microbial digestion and liquefaction.
The study of immunity began after the discovery of the tubercle
bacillus by Robert Koch. With this acclaimed discovery, the
involvement of bacteria as agents of disease became evident. The first
rational attempts to produce artificial active immunity were by
Pasteur in 1880 during his work with cholera.
Antibiotics had a dramatic beginning with the famous discovery of
penicillin by Alexander Fleming in 1928. Fleming found a mold spore
that accidentally landed on a culture of staphylococci. It was not until
the late 1930s that scientists could purify penicillin and demonstrate
its antibacterial effects. Commercial production of penicillin began
as a combined wartime project between the United States and
England. This project was the beginning of the fermentation industry
and biotechnology.
Around 1930, certain growth factors, including factor X and V, were
shown to be important in bacterial nutrition. In the early 1950s, most
of the vitamins were also characterized as co-enzymes. This detailed
information lead scientists to develop an understanding of
biochemical pathways.
A “booming” development of microbiology began after World War II.
Molecular biology, biotechnology and the study of genetics were fields
of extraordinary growth. By 1941, the study of microbiology and
genetics came together when Neurospora crassa, a red bread mold,
was used to study microbial physiology. The study of bacterial
genetics moved dramatically forward during the 1940s following the
discovery of antibiotic resistance. The birth of molecular biology
began in 1953 after the publication by Watson and Crick of the
structure of DNA.
In 1953, viruses were defined by Luria as “submicroscopic entities,
capable of being introduced into specific living cells and of
reproducing inside such cells only”. The work of John Enders on
culturing viruses lead to the development of vaccines. Enders
Early years at Difco Laboratories.
History of Microbiology and Culture Media