difco manual

11th Edition Important Notice ● Product labels/package inserts take precedence over the formula or instructions listed in the manual. ● Generic names may be substituted for trade names in the ingredient list, providing more information regarding animal origin, i.e. “pancreatic digest of casein” instead of Casitone. ● During 1999, catalog numbers will be changed to meet new UCC/EAN128 labeling requirements. Please visit us at www.bd.com/microbiology for more information. 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 [...]... contaminants to be The Difco Manual Agar Granulated is qualified to grow recombinant strains of Escherichia coli (HB101) and Saccharomyces cerevisiae Agar Granulated may be used for general bacteriological purposes where clarity is not a strict requirement This agar was developed to address the special needs of the Biotechnology Industry for large scale applications Noble Agar is the purest form of Difco agar... during the manufacturing process and on the final product Certificates of Analysis supply information from the manufacturer on lot specific final testing of a product 8 The Difco Manual Section I A typical analysis was performed on Difco peptones and hydrolysates to aid in the selection of products for research or production needs when specific nutritional characteristics are required The specifications... (“heat loving”) organisms grow only at temperatures greater than 45°C; psychrophilic (“cold loving”) organisms require temperatures below 20°C Human pathogenic organisms are generally mesophiles The Difco Manual Common Media Constituents Media formulations are developed on the ability of bacteria to use media components CONSTITUENTS SOURCE Amino-Nitrogen Peptone, protein hydrolysate, infusions and extracts... that no single peptone is the most suitable nitrogen source for culturing fastidious bacteria Extensive investigations were undertaken at Difco Laboratories using peptic digests of animal tissue prepared under varying digestion parameters Bacto Tryptone was developed by Difco Laboratories while investigating a peptone particularly suitable for the elaboration of indole by bacteria Meat (fresh, frozen or... group of red-purple marine algae (Class Rhodophyceae) including Gelidium, Pterocladia and Gracilaria These red-purple marine algae are widely distributed throughout the world in temperate zones The Difco Manual Section I Monographs trapped in the frozen water The ice is then washed from the agar, eliminating the contaminants The Ice Agar process results in greater consistency and freedom from interposing... used in a 1 for 1 substitution Bile Salts Bile Salts No 3 Bile Salts and Bile Salts No 3 are used as selective agents for the isolation of gram-negative microorganisms, inhibiting gram-positive The Difco Manual Monographs cocci Bile is derived from the liver The liver detoxifies bile salts by conjugating them to glycine or taurine A bile salt is the sodium salt of a conjugated bile acid Bile Salts and... are used as selective agents in microbiological culture media They are used to aid in the isolation of gram- negative microorganisms, inhibiting gram-positive organisms and spore forming bacteria The Difco Manual Section I Soytone Soytone is an enzymatic digest of soybean meal The nitrogen source in Soytone contains the naturally occurring high concentrations of vitamins and carbohydrates of soybean TC... demonstrate acceptable clarity and growth promoting characteristics Yeast Extract and Yeast Extract, Technical also provide vitamins, nitrogen, amino acids and carbon in microbiological culture media The Difco Manual 11 Monographs Section I Media Preparation The preparation of culture media from dehydrated media requires accuracy and attention to preparation The following points are included to aid the user... Organisms appropriately • Maintain appropriate records • Report deficiencies to the manufacturer The following table is a troubleshooting guide to assist in the preparation of reliable culture media The Difco Manual Section I PROBLEM Monographs A B C D E F G Abnormal color of medium Incorrect pH • • • • • • • • • • Nontypical precipitate Incomplete solubility • • • • • • • Darkening or carmelization Toxicity... requiring lower temperatures than dry heat Moist heat is the most popular method of culture media sterilization When used correctly, it is the most economical, safe and reliable sterilization method The Difco Manual Moist Heat Sterilization Water boils at 100°C, but a higher temperature is required to kill resistant bacterial spores in a reasonable length of time A temperature range of 121-124°C for 15 minutes . 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. 1968. Introduction