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11th Edition

Important Notice ●

Product labels/package inserts take precedence over the formula or instructions listed in the manual.

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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.

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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

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 Reprinted Reprinted Reprinted Reprinted Reprinted Reprinted Reprinted Reprinted Reprinted Reprinted Reprinted Reprinted Reprinted Reprinted Reprinted Tenth Edition Reprinted Reprinted Reprinted Eleventh Edition

1953 1953 1956 1958 1960 1962 1963 1964 1965 1966 1967 1969 1971 1972 1974 1977 1984 1985 1994 1996 1998

Copyright 1998 by

Difco Laboratories, Division of Becton Dickinson and Company Sparks, Maryland 21152 USA vi

The Difco Manual

Foreword

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

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vii

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 solidified 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, microbiologists have developed innumerable enrichment culture techniques for the isolation and cloning of microorganisms with specific nutritional 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

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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 Naegeli1 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 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 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

ix

Introduction

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 Day4 and Long5 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 Hiles6 to the meningococcus and Streptococcus; Cole and Lloyd7 and Cole and Onslow8 to the gonococcus; and Jacoby and Frankenthal9 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 plasma10 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 Loerber27,28 on scarlet fever toxin, and of Favorite and Hammon29 on Staphylococcus enterotoxin. In addition, the work of Snell and Wright30 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 Mueller31,32 on pimelic acid as a growth factor for Corynebacterium diphtheriae, and those of O’Kane33 on synthesis of riboflavin by staphylococci. More recently, the standardization of antibiotic susceptibility testing has been shown to be influenced by peptones of culture

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media. Bushby and Hitchings35 have shown that the antimicrobial activities of trimethoprim and sulfamethoxazole are influenced considerably 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 Laboratories 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.

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Monographs

History of Difco Laboratories 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, purifying enzymes and performing dehydration procedures created Original Difco Laboratories a smooth transition to the Manufacturing facility. preparation of dehydrated culture media. In 1913, the Digestive Ferments Company moved to Detroit, Michigan, and dropped the name, Ray Chemical.

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.

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.

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.

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,

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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 DispensO-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.

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.

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Monographs

Section I

History of Microbiology and Culture Media 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.

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Early years at Difco Laboratories.

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

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demonstrated that a virus could be grown in chick embryos and would lose its ability to cause disease after successive generations. Using this technique, Salk developed the polio vaccine.

With rapid advances in technologies and instrumentation, the basic culture media and ingredients listed in this Manual remain some of the most reliable and cost effective tools in microbiology today.

One organism that has made a great contribution to molecular biology is Escherichia coli. In 1973, Herbert Boyer and Stanley Cohen produced recombinant DNA through plasmid transformation. The researchers found that the foreign gene not only survived, but copied the genetic material. This study and similar others started a biotechnology revolution that has gained momentum over the years.

References

In the 1980s, instrumentation entered the microbiology laboratory. Manual procedures could be replaced by fully automated instruments for bacterial identification, susceptibility testing and blood culture procedures. Immunoassays and probe technologies are broadening the capabilities of the microbiologist.

1. Marti-Ibanez, F. 1962. Baroque medicine, p. 185-195. In F. Marti-Ibanez (ed.). The epic of medicine. Clarkson N. Potter, Inc., New York, N.Y. 2. Wainwright, M., and J. Lederberg. 1992. History of microbiology, p. 419-437. In J. Lederberg (ed.), Encyclopedia of microbiology, vol 2. Academic Press Inc., New York, N.Y.

Microorganism Growth Requirements Microorganism growth on culture media depends on a number of important factors: • Proper nutrients must be available. • Oxygen or other gases must be available, as required. • Moisture is necessary. • The medium must have an appropriate pH. • Proper temperature relations must prevail. • The medium must be free of interfering bioburden. • Contamination must be prevented. A satisfactory microbiological culture medium must contain available sources of: • Carbon, • Nitrogen, • Inorganic phosphate and sulfur, • Trace metals, • Water, • Vitamins. These were originally supplied in the form of meat infusion. Beef or yeast extracts frequently replace meat infusion in culture media. The addition of peptones, which are digests of proteins, provides readily available sources of nitrogen and carbon. The pH of the culture medium is important for microorganism growth. Temperature is another important parameter: mesophilic bacteria and fungi have optimal growth at temperatures of 25-40°C; thermophilic (“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.

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Common M edia Constituents Media formulations are developed on the ability of bacteria to use media components. CO N STITU EN TS

SO U RCE

Amino-Nitrogen

Peptone, protein hydrolysate, infusions and extracts

Growth Factors

Blood, serum, yeast extract or vitamins, NAD

Energy Sources

Sugar, alcohols and carbohydrates

Buffer Salts

Phosphates, acetates and citrates

Mineral Salts and Metals

Phosphate, sulfate, magnesium, calcium, iron

Selective Agents

Chemicals, antimicrobials and dyes

Indicator Dyes

Phenol red, neutral red

Gelling agents

Agar, gelatin, alginate, silica gel

M edia Ingredients Peptone, protein hydrolysates, infusions and extracts are the major sources of nitrogen and vitamins in culture media. Peptones are water-soluble ingredients derived from proteins by hydrolysis or digestion of the source material, e.g. meat, milk. Carbohydrates are employed in culture media as energy sources and may be used for differentiating genera and identifying species. Buffers maintain the pH of culture media.

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Monographs

Selective Agents include Bile Salts, dyes and antimicrobial agents. Bile Salts and desoxycholate are selective for the isolation of gram-negative microorganisms, inhibiting gram-positive cocci. Dyes and indicators are essential in the preparation of differential and selective culture media. In these formulations, dyes act as bacteriostatic agents, inhibitors of growth or indicators of changes in acidity or alkalinity of the substrate. Antimicrobial agents are used in media to inhibit the growth of bacteria, yeasts and fungi. Solidifying agents, including agar, gelatin and albumin, can be added to a liquid medium in order to change the consistency to a solid or semisolid state.

Environmental Factors in Culture M edia Atmosphere Most bacteria are capable of growth under ordinary conditions of oxygen tension. Obligate aerobes require the free admission of oxygen, while anaerobes grow only in the absence of atmospheric oxygen. Between these two groups are the microaerophiles, which develop best under partial anaerobic conditions, and the facultative anaerobes, which are capable of growing in the presence or absence of oxygen. Anaerobic conditions for growth of microorganisms are obtained in a number of ways: • Addition of small amounts of agar to liquid media; • Addition of fresh tissue to the medium; • Addition of a reducing substance to the medium; e.g., sodium thioglycollate, thioglycollic acid and L-cystine; • Displacement of the air by carbon dioxide;

Section I

• Absorption of the oxygen by chemicals; • Inoculation into the deep layers of solid media or under a layer of oil in liquid media. Many microorganisms require an environment of 5-10% CO2. Levels greater than 10% are often inhibitory due to a decrease in pH as carbonic acid forms. Culture media vary in their susceptibility to form toxic oxidation products if exposed to light and air.

Water Activity Proper moisture conditions are necessary for continued luxuriant growth of microorganisms. Organisms require an aqueous environment and must have “free” water. “Free” water is not bound in complex structure and is necessary for transfer of nutrients and toxic waste products. Evaporation during incubation or storage results in loss of “free” water and reduction of colony size or total inhibition of organism growth.

Protective Agents and Growth Factors Calcium carbonate, soluble starch and charcoal are examples of protective agents used in culture media to neutralize and absorb toxic metabolites produced by bacterial growth. NAD (V factor) and hemin (X factor) are growth factors required by certain bacteria; e.g., Haemophilus species, and for enhanced growth of Neisseria species. Surfactants, including Tween® 80, lower the interfacial tension around bacteria suspended in the medium. This activity permits more rapid entry of desired compounds into the bacterial cell and can increase bacterial growth.

Culture Media Ingredients – Agars History Agar was discovered in 1658 by Minora Tarazaemon in Japan. 1 According to legend, this Japanese innkeeper threw surplus seaweed soup into the winter night and noticed it later transformed into a gel by the night’s freezing and the day’s warmth.2 In 1882, Koch was the first to use agar in microbiology.3,4 Walter Hesse, a country doctor from Saxony, introduced Koch to this powerful gelling agent.5 Hesse had learned about agar from his wife, Fanny Hesse, whose family had contact with the Dutch East Indies where agar was being used for jellies and jams.3,5,6 The term ‘agar-agar’ is a Malaysian word that initially referred to extracts from Eucheuma, which yields carrageenan, not agar.5 By the early 1900s, agar became the gelling agent of choice instead of gelatin. Agar was found more suitable because it remained solid at

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the temperatures required for growth of human pathogens and was resistant to breakdown by bacterial enzymes. Production of agar in the United States was started just before the beginning of World War II as a strategic material.5 In the 1940s, bacteriological-grade agar manufactured by the American Agar Company of San Diego, California, served as reference agar for the evaluation of the characteristics of other culture media components, such as peptones.5

Characteristics Agar is a phycocolloid, a water-soluble polysaccharide, extracted from a 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.

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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 contaminants when used in microbiological procedures.

Product Applications Bacto Agar is optimized for beneficial calcium and magnesium content. Detrimental ions such as iron and copper are reduced. Bacto Agar is recommended for clinical applications, auxotrophic studies, bacterial and yeast transformation studies and bacterial molecular genetics applications.7,8 Agar Flake is recommended for use in general bacteriological purposes. The quality is similar to Bacto Agar. The flakes are more wettable than the granules found in Bacto Agar. Agar is derived from a group of red-purple marine algae as pictured above.

For Difco Agars, Gelidium is the preferred source of agar. The most important properties of agar are:5 • Good transparency in solid and gel forms to allow identification of colony type; • Consistent lot-to-lot gel strength that is sufficient to withstand the rigors of streaking but not so stiff that it affects diffusion characteristics; • Consistent gelling (32-40°C) and melting (approximately 85°C) temperatures, a property known as hysteresis; • Essential freedom from metabolically useful chemicals such as peptides, proteins and fermentable hydrocarbons; • Low and regular content of electronegative groups that could cause differences in diffusion of electropositive molecules (e.g., antibiotics, nutrients); • Freedom from toxic substances (bacterial inhibitors); • Freedom from hemolytic substances that might interfere with normal hemolytic reactions in culture media; • Freedom from contamination by thermophilic spores. Agars are normally used in final concentrations of 1-2% for solidifying culture media. Smaller quantities of agar (0.05-0.5%) are used in culture media for motility studies (0.5% w/v) and growth of anaerobes (0.1%) and microaerophiles.2

The M anufacturing Process Difco Laboratories selects the finest Gelidium marine algae from world sources and requires algae harvested from water where the temperature is both constant and temperate. Bacto Agar and Agar Granulated are produced from an Ice Agar purification process. Agar is insoluble in cold water but is colloidally dispersible in water above 90°C.2 When an agar gel is frozen, the agar skeleton contracts toward the center of the mass as a membrane, leaving ice as a separate phase.2 Through a variety of processes, the agar is extracted from the Gelidium, resulting in a liquid agar that is purified. The liquid agar is first gelled and then frozen, causing the soluble and suspended contaminants to be

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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. It is washed extensively and bleached to remove extraneous material. The result is a white powder in dry form, clear and colorless in solution and when solidified in plates. This agar is suitable for immunodiffusion studies, for use in some electrophoretic applications and as a substrate for mammalian and plant tissue culture. Agar Technical is suitable for many general bacteriological applications. This agar is not as highly processed as other Difco agars and has lower technical specifications. This agar is not recommended for growth of fastidious organisms.

References 1. C. K. Tsend. 1946. In J. Alexander (ed.). 6:630. Colloid Chemistry. Reinhold Publishing Corp., New York, N. Y. 2. Selby, H. H., and T. A. Selby. 1959. Agar. In Whister (ed.)., Industrial gums. Academic Press Inc., New York, NY. 3. Hitchens, A. P., and M. C. Leikind. 1939. The introduction of agar-agar into bacteriology. J. Bacteriol. 37:485-493. 4. Koch, R. 1882. Die Atiologie der Turberkulose. Berl. Klin. Wochenschr. 19:221- 230. 5. Armisen, R. 1991. Agar and agarose biotechnological applications. Hydrobiol. 221:157-166. 6. Hesse, W. 1894. Uber die quantitative Bestimmung der in der Luft enthaltenen Mikroorganismen. Mitt. a. d. Kaiserl. Gesh. Berlin 2:182-207. 7. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning, a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, New York, N.Y. 8. Schiestl, R. H., and R. D. Geitz. 1989. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Current Genetics 16:339-346.

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Culture Media Ingredients – Peptones and Hydrolysates Preparation of Peptones The composition of peptones varies with the origin and the method of preparation. Some common sources of peptone include:

Typical fermentation process.

History Peptones were originally described by Naegeli in 1879.3 In this report, Naegeli compared peptone and ammonium tartrate. With the rich amino acid and nitrogen compounds readily utilized by bacteria, peptone soon became one of the most important constituents of culture media. The importance of peptone as a nutritive source was demonstrated by Klinger.4 Bacto Peptone was introduced commercially in 1914, and became the standard peptone for the preparation of bacteriological culture media. The development of Bacto Proteose Peptone, Bacto Proteose Peptone No. 2 and Bacto Proteose Peptone No. 3 resulted from accumulated information 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 dried) Fish (fresh, dried) Casein Gelatin Keratin (horn, hair, feathers) Ground Nuts Soybean Meal Cotton Seed Sunflower Seeds Microorganisms (yeasts, algae, bacteria) Guar Protein Blood Corn Gluten Egg Albumin Demineralized water is added to these protein sources to form a thick suspension. The digestion process follows with an acid or enzyme. Acid and alkaline hydrolyses are performed by boiling the protein with mineral acids or strong alkalis at increased pressure to raise the temperature of the reaction. This procedure can decrease the vitamin content of the protein and a portion of the amino acid content. Digestion with proteolytic enzymes is performed at lower temperatures and normal atmospheric pressure. This process is often less harmful to the protein and amino acids. Microbial Proteoses, Papain, Pancreatin and Pepsin are used most often by Difco Laboratories in the manufacture of peptones. The peptone suspension is then centrifuged and filtered. The suspension is concentrated to approximately 67% total solids and the product now appears as a syrup. This peptone syrup is spray dried and packaged.

Infusions and Extracts

Other non-chemically defined ingredients, including Bacto Liver, Bacto Beef Heart for Infusion and Bacto Yeast Extract can serve as nitrogen or carbon sources. Infusions of meat were first employed as nutrients in culture media. It was discovered that for many routine procedures in the preparation of culture media, extracts have the advantage of greater ease in preparation, uniformity and economy than infusions.

The water-soluble fractions of materials such as muscle, liver, yeast cells and malt are usually low in peptides but contain valuable extractives such as vitamins, trace metals and complex carbohydrates.5 It is common practice to combine infusions and peptones to obtain the best of both products.5 Bacto Yeast Extract, Bacto Malt Extract, Bacto Beef Heart for Infusion and Bacto Beef Extract are examples of extracts and infusions manufactured by Difco Laboratories for use in the preparation of culture media.

Protein Biochemistry

Peptone Performance

Proteins consist of amino acids joined together by means of the covalent peptide bond linkage. When the bonds are hydrolyzed, proteins yield polypeptides of various molecular sizes, proteoses, peptones and peptides down to the level of simple amino acids. Bacteriological peptones are mixtures of various products of protein hydrolysis, organic nitrogen bases, inorganic salts and trace elements.

The quality and performance of peptones, infusions and extracts are very dependent on the freshness or preservation of the raw materials.5 Extensive quality control testing is performed on all peptones and other culture media ingredients 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 for the typical analysis include: • Physical characteristics • Nitrogen content • Amino acids • Inorganics • Vitamins • Biological testing The quality of peptones and culture media ingredients is truly assessed by their ability to support adequate growth of various microorganisms when incorporated into the medium. 6 The nature of peptones, infusions and extracts will then play a major role in the growth performance properties of the medium and, in turn, advance the science of microbiology.6

M edia Ingredients Autolyzed Yeast Autolyzed Yeast is a desiccated product containing both the soluble and insoluble portions of autolyzed bakers’ yeast. Autolyzed Yeast is recommended for the preparation of yeast supplements used in the microbiological assay of riboflavin and pantothenic acid.7,8, Autolyzed Yeast provides vitamins, nitrogen, amino acids and carbon in microbiological culture media. Beef Beef Heart for Infusion Beef and Beef Heart for Infusion provide nitrogen, amino acids and vitamins in microbiological culture media. Beef is desiccated, powdered, fresh lean beef, prepared especially for use in beef infusion media. Large quantities of beef are processed at one time to secure a uniform and homogenous product. Beef Heart for Infusion is prepared from fresh beef heart tissue and is recommended for preparing heart infusion media. Beef Heart for Infusion is processed from large volumes of raw material, retaining all the nutritive and growth stimulating properties of fresh tissues. Beef Extract Beef Extract, Desiccated Beef Extract and Beef Extract, Desiccated are replacements for infusion of meat. Beef Extract and Beef Extract, Desiccated provide nitrogen, vitamins, amino acids and carbon in microbiological culture media. Beef Extract is standard in composition and reaction and generally used to replace infusion of meat. In culture media, Beef Extract is usually employed in concentration of 0.3%. Beef Extract, Desiccated, the dried form of Beef Extract, was developed to provide a product for ease of use in handling. Beef Extract is in the paste form. The products are to be 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 Bile Salts No. 3 contain bile extract standardized to provide inhibitory properties for selective media. Bile Salts No. 3 is a modified fraction of bile acid salts, providing a refined bile salt. Bile Salts No. 3 is effective at less than one-third concentration of Bile Salts. Casamino Acids Casamino Acids, Technical Casamino Acids, Vitamin Assay Casamino Acids, Casamino Acids, Technical and Casamino Acids, Vitamin Assay are derived from acid hydrolyzed casein. Casein is a milk protein and a rich source of amino acid nitrogen. Casamino Acids, Casamino Acids, Technical and Casamino Acids, Vitamin Assay are added to media primarily because of their organic nitrogen and growth factor components; their inorganic components also play a vital role.9 Casamino Acids is recommended for use with microbiological cultures that require a completely hydrolyzed protein as a nitrogen source. In Casamino Acids, hydrolysis is carried on until all the nitrogen in the casein is converted to amino acids or other compounds of relative chemical simplicity. The hydrolysis of Casamino Acids, Technical is carried out as in the preparation of Casamino Acids, but the sodium chloride and iron content have not been decreased to the same extent. Casamino Acids, Vitamin Assay is an acid digest of casein specially treated to markedly reduce or eliminate certain vitamins. It is recommended for use in microbiological assay media and in growth promotion studies. Casein Digest Casein Digest is an enzymatic digest of casein, providing a distinct source of amino acids for molecular genetics media. Casein Digest is used as a nitrogen and amino acid source for microbiological culture media. Casein Digest is similar to N-Z Amine A. This product is digested under conditions different from other enzymatic digests of casein, including Tryptone and Casitone. Casitone Casitone is a pancreatic digest of casein. Casitone is recommended for preparing media where an enzymatic hydrolyzed casein is desired. Casein is a rich source of amino acid nitrogen. This product is used to support the growth of fastidious microorganisms and its high tryptophan content makes it valuable for detecting indole production. Fish Peptone No. 1 Fish Peptone No. 1 is a non-mammalian, non-animal peptone used as a nitrogen source in microbiological culture media. Fish Peptone No. 1 is a non-bovine origin peptone, to reduce Bovine Spongiform Encephalopathy (BSE) risk. This peptone was developed by Difco Laboratories for pharmaceutical and vaccine production and can replace any peptone, depending on the organism and production application.

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Gelatin Gelatin is a protein of uniform molecular constitution derived chiefly by the hydrolysis of collagen.10 Collagens are a class of albuminoids found abundantly in bones, skin, tendon, cartilage and similar tissues of animals. 10 Gelatin is used in culture media to detect gelatin liquifaction by bacteria and as a nitrogen and amino acid source.

Peptamin Peptamin, referred to as Peptic Digest of Animal Tissue, complies with the US Pharmacopeia XXIII (USP). 11 Peptamin provides nitrogen, amino acids, vitamins and carbon in microbiological culture media. Diluting and rinsing solutions, Fluid A and Fluid D, contain 0.1% Peptamin.

Gelatone Gelatone is a pancreatic digest of gelatin, deficient in carbohydrates. Gelatone is used as a media ingredient for fermentation studies and, alone, to support the growth of non-fastidious microorganisms. Gelatone is in granular form for convenience in handling and is distinguished by a low cystine and tryptophan content.

Peptone, Bacto Peptone Bacteriological, Technical Bacto Peptone and Peptone Bacteriological, Technical are enzymatic digests of protein and rich nitrogen sources. Bacto Peptone was introduced in 1914 and became the standard peptone for the preparation of culture media. Peptone Bacteriological, Technical can be used as the nitrogen source in microbiological culture media when a standardized peptone is not essential. Both peptones have a high peptone and amino acid content and only a negligible quantity of proteoses and more complex nitrogenous constituents.

Liver Liver is prepared from large quantities of carefully trimmed fresh beef liver. Liver is a desiccated powder of beef liver. The nutritive factors of fresh liver tissue are retained in infusion prepared from Liver. Liver is used as a source of nitrogen, amino acids and vitamins in microbiological culture media. The reducing substances contained in liver create an anaerobic environment, necessary to support the growth of anaerobes. One hundred thirty-five (135) grams of desiccated Liver are equivalent to 500 grams of fresh liver. Malt Extract Malt Extract is obtained from barley, designed for the propagation of yeasts and molds. Malt Extract is particularly suitable for yeasts and molds because it contains a high concentration of carbohydrates, particularly maltose. This product is generally employed in concentrations of 1-10%. Malt Extract provides carbon, protein and nutrients for the isolation and cultivation of yeasts and molds in bacterial culture media. Neopeptone, Difco Neopeptone is an enzymatic digest of protein. Neopeptone contains many peptide sizes in combination with vitamins, nucleotides, minerals and other carbon sources. Neopeptone is particularly well suited in supplying the growth requirements of fastidious bacteria. This peptone is extremely valuable in media for the cultivation of pathogenic fungi. Growth of these microorganisms is rapid and colony formation is uniform and typical. Oxgall Oxgall is manufactured from large quantities of fresh bile by rapid evaporation of the water content. Bile is composed of fatty acids, bile acids, inorganic salts, sulphates, bile pigments, cholesterol, mucin, lecithin, glycuronic acids, porphyrins and urea. The use of Oxgall ensures a regular supply of bile and assures a degree of uniformity impossible to obtain with fresh materials. It is prepared for use in selective media for differentiating groups of bile tolerant bacteria. Oxgall is used as a selective agent for the isolation of gram-negative microorganisms, inhibiting gram-positive bacteria. The major components of Oxgall are taurocholic and glycocholic acids.

10

Proteose Peptone Proteose Peptone No. 2 Proteose Peptone No. 3 The development of Proteose Peptone, Proteose Peptone No. 2 and Proteose Peptone No. 3 is the result of accumulated information demonstrating that no single peptone is the most suitable nitrogen source for culturing fastidious bacteria. Proteose Peptone is an enzymatic digest of protein high in proteoses. Many factors account for the suitability of Proteose Peptone for the culture of fastidious pathogens, including the nitrogen components, buffering range and the high content of proteoses. Proteose Peptone No. 2 and Proteose Peptone No. 3 are enzymatic digests of protein. Proteose Peptone No. 2 is used for producing bacterial toxins and is suitable for media of nutritionally less-demanding bacteria. Proteose Peptone No. 3 is a modification of Proteose Peptone, adapted for use in the preparation of chocolate agar for propagation of Neisseria species and chocolate tellurite agar for Corynebacterium diphtheriae. Sodium Deoxycholate Sodium Taurocholate Sodium Desoxycholate is the sodium salt of desoxycholic acid. Since Sodium Desoxycholate is a salt of a highly purified bile acid, it is used in culture media in lower concentrations than in naturally occurring bile. Sodium Taurocholate is the sodium salt of a conjugated bile acid. Sodium Taurocholate contains about 75% sodium taurocholate in addition to other naturally occurring salts of bile acids. Sodium Desoxycholate and Sodium Taurocholate, like other bile salts, 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.

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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 Lactalbumin Hydrolysate TC Yeastolate TC Lactalbumin Hydrolysate is an enzymatic digest of lactalbumin for use as an enrichment in tissue culture media. Lactalbumin is a protein derived after removal of casein from milk. TC Yeastolate is a desiccated, clarified, water soluble portion of autolyzed fresh yeast prepared and certified for use in tissue culture procedures. TC Yeastolate is a source of vitamin B complex. Tryptone Peptone Tryptone Peptone is a pancreatic digest of casein used as a nitrogen source in culture media. Casein is the main protein of milk and is a rich source of amino acid nitrogen. Tryptone Peptone is rich in tryptophan, making it valuable for use in detecting indole production.12 The absence of detectable levels of carbohydrates in Tryptone Peptone makes it a suitable peptone in differentiating bacteria on the basis of their ability to ferment various carbohydrates. Tryptose Tryptose is a mixed enzymatic hydrolysate with distinctive nutritional properties. The digestive process of Tryptose results in assorted peptides, including those of higher molecular weight. Tryptose was originally developed as a peptone particularly adapted to the growth requirements of Brucella.

Monographs

References 1. Nash, P., and M. M. Krenz. 1991. Culture Media, p. 1226-1288. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 2. De Feo, J. 1986. Properties and applications of hydrolyzed proteins. ABL. July/August, 44-47. 3. Naegeli. 1880. Sitz’ber, math-physik. Klasse Akad. Wiss. Muenchen. 10:277. 4. Klinger, I. J. 1917. The effect of hydrogen ion concentration on the production of precipitates in a solution of peptone and its relation to the nutritive value of media. J. Bacteriol. 2:351-353. 5. Bridson, E. Y. 1990. Media in microbiology. Rev. Med. Microbiol. 1:1-9. 6. Alvarez, R. J., and M. Nichols. 1982. Formulating microbiological culture media-a careful balance between science and art. Dairy Food Sanitation 2:356- 359. 7. J. Ind. Eng. Chem., Anal. Ed. 1941. 13:567. 8. J. Ind. Eng. Chem., Anal. Ed. 1942. 14:909. 9. Nolan, R. A., and W. G. Nolan. 1972. Elemental analysis of vitamin-free casamino acids. Appl. Microbiol. 24:290-291. 10. Gershenfeld, L., and L. F. Tice. 1941. Gelatin for bacteriological use. J. Bacteriol. 41:645-652. 11. United States Pharmacopeial Convention. 1995. The United States pharmacopeia, 23rd ed. The United States Pharmacopeial Convention. Rockville, MD. 12. J. Bacteriol. 1933. 25:623.

Yeast Extract Yeast Extract, Technical Yeast Extract and Yeast Extract, Technical are water soluble portions of autolyzed yeast containing vitamin B complex. Yeast Extract is an excellent stimulator of bacterial growth and used in culture media. The autolysis is carefully controlled to preserve the naturally occurring B-complex vitamins. Yeast Extract is generally employed in the concentration of 0.3-0.5%, with improved filterability at 20%. Yeast Extract, Technical is used in bacterial culture media when a standardized yeast extract is not essential. Yeast Extract, Technical was developed to 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.

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M edia Preparation The preparation of culture media from dehydrated media requires accuracy and attention to preparation. The following points are included to aid the user in successful and reproducible preparation of culture media. Dehydrated Media and Ingredients • Store in a cool (15-30°C), dark and dry area unless otherwise specified. • Note date opened. • Check expiry (applied to intact container). • Verify that the physical characteristics of the powder are typical. Glassware / Plasticware • Use high quality, low alkali borosilicate glass. • Avoid detergent residue. • Check for alkali or acid residue with a few drops of brom thymol blue pH indicator (yellow is acidic; blue is alkaline).

• Quantities of media in excess of two liters may require an extended autoclave time to achieve sterilization. Longer sterilization cycles can cause nutrient concentration changes and generation of inhibitory substances. Adding Enrichments and Supplements • Enrichments and supplements tend to be heat sensitive. • Cool medium to 45-55°C in a waterbath prior to adding enrichments or supplements. • Ensure adequate mixing of the basal medium with enrichments or supplements by swirling to mix thoroughly. • Sterile broths may be cooled to room temperature before adding enrichment. pH • Commercial dehydrated media are designed to fall within the specified pH range after steam sterilization. The pH tends to fall approximately 0.2 units during steam sterilization.

• Use vessels at least 2-3 times the volume of medium.

• For filter sterilization, adjust the pH, if necessary, prior to filtering.

• Discard (recycle) etched or chipped glassware.

• Avoid excessive pH adjustments.

• Do not used etched glassware. Equipment • Use measuring devices, scales, pH meters, autoclaves and other equipment that are frequently and accurately calibrated. Water • Use distilled or deionized water. • pH 5.5-7.5. Dissolving the Medium • Accurately weigh the appropriate amount of dehydrated medium. • Dissolve the medium completely. • Agitate the medium while dissolving. • Take care to not overheat. Note media that are very sensitive to overheating. Overheated media will frequently appear darker. Do not heat in a microwave. Sterilization • The autoclave set-temperature should be 121°C. • Routine autoclave maintenance is important. Ask manufacturer to check for “hot” and “cold” spots. • The recommended 15 minute sterilization assumes a volume of 1 liter or less. Larger volumes may require longer cycles. Check with your autoclave manufacturer for recommended load configurations.

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Dispensing Media • Ensure gentle mixing during dispensing. • Cool the medium to 50-55°C prior to dispensing to reduce water evaporation. • Dispense quickly. • If using an automatic plate dispenser, dispense general purpose media before dispensing selective media. • Immediately recover or recap tubes to reduce the chance of contamination. Leave Petri dish covers slightly open for 1-2 hours to obtain a dry surface. Storage and Expiry • In general, store steam-sterilized plated media inverted in a plastic bag or other container in a dark refrigerator for up to 1-2 weeks. Quality Control • For media prepared in-house, each lot of every medium must be tested. • Maintain Quality Control 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.

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PRO BLEM

Monographs

A

B

C

D

E

F

G

Abnormal color of medium Incorrect pH

• •

• •

• •

•

•

•

•

Nontypical precipitate Incomplete solubility

•

•

•

•

• •

•

Darkening or carmelization Toxicity Tract substances (Vitamins)

•

•

•

• • •

•

•

O TH ER CAU SES

Storage at high temperature Hydrolysis of ingredients pH determined at wrong temperature Inadequate heating Inadequate convection in a too small flask

•

Loss of gelation property Loss of nutritive value or selective or differential properties

H

•

•

•

•

•

•

• •

Contamination

•

Burning or scorching Airborne or environmental sources of vitamins Hydrolysis of agar due to pH shift Not boiling medium Burning or scorching Presence of strong electrolytes, sugar solutions, detergents, antiseptics, metallic poisons, protein materials or other substances that may inhibit the inoculum Improper sterilization Poor technique in adding enrichments and pouring plates Not boiling agar containing medium

Key A

Deteriorated Dehydrated Medium

D

Incorrect Weighing

G

Repeated Remelting

B

Improperly Washed Glassware

E

Incomplete Mixing

H

Dilution by a Too Large Inoculum

C

Impure Water

F

Overheating

Media Sterilization Sterilization is any process or procedure designed to entirely eliminate viable microorganisms from a material or medium. Sterilization should not be confused with disinfection, sanitization, pasteurization or antisepsis which are intended to inactivate microorganisms, but may not kill all microorganisms present. Sterilization can be accomplished by the use of heat, chemicals, radiation or filtration.1

Sterilization with Heat1 The principal methods of thermal sterilization include 1) moist heat (saturated steam) and 2) dry heat (hot air) sterilization. Heat kills microorganisms by protein denaturation and coagulation. Moist heat has the advantage of being more rapid and 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.

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M oist 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 is an accepted standard condition for sterilizing up to one liter of culture medium. The definition of “autoclave at 121°C for 15 minutes” refers to the temperature of the contents of the container being held at 121°C for 15 minutes, not to the temperature and time at which the autoclave has been set.2 The steam pressure of 15 pounds per square inch at this temperature aids in the penetration of the heat into the material being sterilized. If a larger volume is to be sterilized in one container, a longer period should be employed. Many factors can affect sterility assurance, including size and contents of the load and the drying and cooling time. Certain products may decompose at higher temperature and longer cycles. For this reason, it is important that all loads be properly validated. The basic principles for validation and certification of a sterilizing process are enumerated as follows:3 1. Establish that the processing equipment has the capability of operating within the required parameters.

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2. Demonstrate that the critical control equipment and instrumentation are capable of operating within the prescribed parameters for the process equipment. 3. Perform replicate cycles representing the required operational range of the equipment and employing actual or simulated product. Demonstrate that the processes have been carried out within the prescribed protocol limits and, finally, that the probability of microbial survival in the replicate processes completed is not greater than the prescribed limits. 4. Monitor the validated process during routine operation. Periodically as needed, requalify and recertify the equipment. 5. Complete the protocols and document steps 1-4, above. For a complete discussion of process validation, refer to appropriate references. Ensuring that the temperature is recorded correctly is vital. The temperature must reach all parts of the load and be maintained for the desired length of time. Recording thermometers are employed for the chamber and thermocouples may be buried inside the load.

• Carmelization or darkening of the medium; • Loss of nutritive value; • Loss of selective or differential properties. There are certain media (e.g., Hektoen Enteric Agar and Violet Red Bile Agar) that should not be autoclaved. To dissolve these media formulation, heat to boiling to dissolve completely. It is important to follow all label directions for each medium. Media supplements should be sterile and added aseptically to the sterilized medium, usually at 45-55°C.

Dry Heat Sterilization1 Dry heat is employed for materials such as metal instruments that could be corroded by moist heat, powders, ointments and dense materials that are not readily penetrated by steam. Because dry heat is effective only at considerably higher temperatures and longer times than moist heat, dry heat sterilization is restricted to those items that will withstand higher temperatures. The dry heat time for sterilization is 120 minutes at 160°C.

For best results when sterilizing culture media, plug tubes or flasks of liquids with nonabsorbent cotton or cap loosely. Tubes should be placed in racks or packed loosely in baskets. Flasks should never be more than two-thirds full. It is important to not overload the autoclave chamber and to place contents so that there is a free flow of steam around the contents. After sterilizing liquids, the chamber pressure must be reduced slowly to atmospheric pressure. This allows the liquid to cool below the boiling point at atmospheric pressure before opening the door to prevent the solution from boiling over.

Chemical Sterilization1

In autoclave operation, all of the air in the chamber must be expelled and replaced by steam; otherwise, “hot spots” and “cold spots” will occur. Pressure-temperature relations of a properly operated autoclave are shown in the table below.

Radiation Sterilization1

4

Pressure-Temperature Relations in Autoclave (Figures based on complete replacement of air by steam) PRESSURE IN PO UNDS

5 10 15 20 25 30

TEM PERATURE (°C)

TEM PERATURE (° F)

109 115 121 126 130 135

228 240 250 259 267 275

Over-sterilization or prolonged heating will change the composition of the medium. For example, carbohydrates are known to break down in composition upon overheating. Over-sterilizing media can cause a number of problems, including: • Incorrect pH; • A decrease in the gelling properties of agar; • The development of a nontypical precipitate;

14

Chemical sterilization employs gaseous and liquid sterilants for certain medical and industrial instruments. The gases include ethylene oxide, formaldehyde and beta-propiolactone. The liquid sterilants include glutaraldehyde, hydrogen peroxide, peracetic acid, chlorine dioxide and formaldehyde. Chemical sterilization is not employed in the preparation of culture media. For a complete discussion of this topic, consult appropriate references.

Radiation sterilization is an optional treatment for heat-sensitive materials. This includes ultraviolet light and ionizing radiation. Ultraviolet light is chemically active and causes excitation of atoms within the microbial cell, particularly the nucleic acids, producing lethal mutations. This action stops the organism from reproducing. The range of the ultraviolet spectrum that is microbiocidal is 240-280 nm. There is a great difference in the susceptibility of organisms to ultraviolet radiation; Aspergillus niger spores are 10 times more resistant than Bacillus subtilis spores, 50 times more resistant than Staphylococcus aureus and Escherichia coli, and 150 times more resistant than influenza virus. Because most materials strongly absorb ultraviolet light, it lacks penetrating power and its applications are limited to surface treatments. Much higher energy, 100 to millions of times greater, is generated by ionizing radiations. These include gamma-rays, high energy X-rays and high energy electrons. Ionizing radiation, unlike ultraviolet rays, penetrates deeply into atoms, causing ionization of the electrons. Ionizing radiation may directly target the DNA in cells or produce active ions and free radicals that react indirectly with DNA. Gamma radiation is used more often than x-rays or high-energy electrons for purposes of sterilization. Gamma rays are generated by

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Monographs

radioactive isotopes, cobalt-60 being the usual source. Gamma radiation requires many hours of exposure for sterilization. Validation of a gamma irradiation procedure includes:4 • Establishment of article materials compatibility; • Establishment of product loading pattern and completion of dose mapping in the sterilization container; • Establishment of timer setting; • Demonstration of the delivery of the required sterilization dose. The advantages of sterilization by irradiation include low chemical reactivity, low measurable residues, and few variables to control.3 Gamma irradiation is used for treating many heat-sensitive products that can also be treated by gaseous sterilization, including medical materials and equipment, pharmaceuticals, biologicals, certain prepared media and laboratory equipment.

Sterilization by Filtration1,3 Filtration is a useful method for sterilizing liquids and gases. Filtration excludes microorganisms rather than destroying them. Two major types of filters may be used, depth filters and membrane filters. The membrane filter screens out particles, while the depth filter entraps them. Membrane filters depend largely on the size of the pores to determine their screening effectiveness. Electrostatic forces are also important. A membrane filter with an average pore size of 0.8 µm will retain particulate matter as small as 0.05 µm. For removing bacteria, a pore size of 0.2 µm is commonly used. For retention of viruses and mycoplasmas, pore sizes of 0.01-0.1 µm are recommended. Cocci and bacilli range in size from about 0.3 to 1 µm in diameter. Most viruses are 0.02-0.1 µm, with some as large as 0.25 µm. Rating the pore size of filter membranes is by a nominal rating that reflects the capability of the filter membrane to retain microorganisms of size represented by specified strains. Sterilizing filter membranes are membranes capable of retaining 100% of a culture of 10 7 microorganisms of a strain of Pseudomonas diminuta (ATCC® 19146) per square centimeter of membrane surface under a pressure of not less than 30 psi. These filter membranes are nominally rated 0.22 µm or 0.2 µm. Bacterial filter membranes (also known as analytical filter membranes), which are capable of retaining only larger microorganisms, are labeled with a nominal rating of 0.45 µm. Membrane filters are used for the commercial production of a number of pharmaceutical solutions and heat-sensitive injectables. Serum for use in bacterial and viral culture media are often sterilized by filtration, as well as some sugars that are unstable when heated. Membrane filtration is useful in testing pharmaceutical and medical products for sterility. 1

Sterility Assurance

Sterility Assurance is the calculated probability that a microorganism will survive sterilization. It is measured as the SAL, Sterility Assurance Level, or “degree of sterility”. For sterility assurance, Bacillus stearothermophilus which contains steam heat-resistant spores is employed with steam sterilization at 121°C.

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Testing Sterilizing Agents1,5 Sterilization by moist heat (steam), dry heat, ethylene oxide and ionizing radiation is validated using biological indicators. The methods of sterilization and their corresponding indicators are listed below:

STERILIZATION METHOD

BIOLOGICAL INDICATOR

Steam Dry heat Ethylene oxide Filtration

Bacillus stearothermophilus Bacillus subtilis var. niger Bacillus subtilis var. globigii Pseudomonas diminuta

For moist heat sterilization, paper strips treated with chemicals that change color at the required temperature may be used. The heat-resistant spores of B. stearothermophilus are dried on paper treated with nutrient medium and chemicals. After sterilization, the strips are incubated for germination and growth, and a color change indicates whether they have or have not been activated. Spore strips should be used in every sterilization cycle.

Glossary1,6 Bioburden is the initial population of living microorganisms in the product or system being considered. Biocide is a chemical or physical agent intended to produce the death of microorganisms. Calibration is the demonstration that a measuring device produces results within specified limits of those produced by a reference standard device over an appropriate range of measurements. Death rate is the rate at which a biocidal agent reduces the number of cells in a microbial population that are capable of reproduction. This is determined by sampling the population initially, during and following the treatment, followed by plate counts of the surviving microorganisms on growth media. D value stands for decimal reduction time and is the time required in minutes at a specified temperature to produce a 90% reduction in the number of organisms. Microbial death is the inability of microbial cells to metabolize and reproduce when given favorable conditions for reproduction. Process validation is establishing documented evidence that a process does what it purports to do. Sterility Assurance Level is generally accepted when materials are processed in the autoclave and attain a 10-6 microbial survivor probability; i.e., assurance of less than one chance in one million that viable microorganisms are present in the sterilized article.3 Sterilization process is a treatment process from which the probability of microorganism survival is less than 10-6, or one in a million.

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Monographs

Thermal Death Time and Thermal-Chemical Death Time are terms referring to the time required to kill a specified microbial population upon exposure to a thermal or thermal-chemical sterilizing agent under specified conditions. A typical thermal death time value with highly resistant spores is 15 minutes at 121°C for steam sterilization.

References 1. Block, S. 1992. Sterilization, p. 87-103. Encyclopedia of microbiology, vol. 4. Academic Press, Inc., San Diego, CA. 2. Cote, R. J., and R. L. Gherna. 1994. Nutrition and media, p. 155-178. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R. Krieg (ed.), Methods for general and molecular bacteriology. American Society for Microbiology, Washington, D.C.

Section I

3. The United States Pharmacopeia (USP XXIII) and The National Formulary (NF 18). 1995. Sterilization and sterility assurance of compendial articles, p. 1976-1980. United States Pharmacopeial Convention Inc., Rockville, MD. 4. Perkins, J. J. 1969. Principles and methods of sterilization in health sciences, 2nd ed. Charles C. Thomas, Springfield, IL. 5. Leahy, T. J. 1986. Microbiology of sterilization processes. In F. J. Carleton and J. P. Agalloco (ed.), Validation of aseptic pharmaceutical processes. Marcel Dekker, Inc. New York, N.Y. 6. Simko, R. J. 1986. Organizing for validation. In F. J. Carleton and J. P. Agalloco (ed.), Validation of aseptic pharmaceutical processes. Marcel Dekker, Inc., New York, N.Y.

Q uality Control O rganisms Bacteria Control Strain Source An integral part of quality control testing includes quality control organisms. Microorganisms should be obtained from reputable sources, for example, the American Type Culture Collection (ATCC®) or other commercial sources.

9. Store vials at or below -50°C (freezer) for one year. Organisms will keep longer (indefinitely) if stored in an ultra low temperature freezer or in a liquid nitrogen tank.

To use a frozen culture: 1. Thaw the vial quickly.

M aintenance / Frozen Stock Cultures

2. Use the culture directly or subculture.

If using commercial stock cultures, follow the manufacturer’s recommendations for growth and maintenance.

3. Discard any unused cell suspension.

To prepare frozen stock cultures of Staphylococcus species, Streptococcus species, Enterobacteriaceae and Pseudomonas aeruginosa: 1. Reconstitute the stock culture, if necessary. 2. Inoculate multiple plates of a general purpose medium (e.g., TSA or blood agar). 3. Incubate plates for 18-24 hours in an appropriate atmosphere and at the recommended temperature. 4. Check for purity and correct colony morphology. 5. If necessary, verify biochemical tests. 6. Remove sufficient growth from a confluent area to prepare a 0.5 McFarland standard (1-2 x 108 CFU/ml). For fastidious organisms, adjust to a 1 McFarland. 7. Suspend the growth in 50-100 ml of cryoprotective medium, e.g., Tryptic Soy Broth with 10-15% Glycerol, Skim Milk or sterile defibrinated sheep blood. 8. Dispense 0.5-1.0 ml into sterile glass or plastic freezing vials. Prepare enough vials for one year of storage. Assume only one freeze/thaw cycle per vial. Assume at least one fresh culture every four weeks.

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Working Cultures Prepare no more than three serial subcultures from a frozen stock culture. 1. Inoculate an agar slant or plate with the frozen stock culture and incubate overnight. 2. Store the working culture at 2-8°C or at room temperature for up to four weeks. 3. Check for purity and appropriate colony morphology. OR 1. Use the frozen stock culture directly as a working culture. Maintain anaerobic cultures in Cooked Meat Medium or another suitable anaerobic medium. Alternatively, use frozen anaerobic cultures.

Test Procedure 1. Inoculate an agar plate from the “working culture”. 2. Incubate overnight. 3. Suspend 3-5 isolated colonies with typical appearance in a small volume (0.5-1.0 ml) of TSB. Incubate 4-5 hours in an appropriate atmosphere and temperature. 4. Adjust the turbidity to 0.5 McFarland and 0.08-0.1 absorbance units at 625 nm.

The Difco Manual

Section I

OR 1. Adjust an overnight culture to a 0.5 McFarland. 2. Plate 0.01 ml of the specimen to confirm a colony count of 1-2 x 108 CFU/ml. If using a frozen culture, confirm the appropriate density.

To Test Cultural Response Non-Selective Media Dilute the cell suspension 1:100 in normal saline or purified water. Inoculate each plate with 0.01 ml to give 1-2 x 104 CFU/plate. Reduce the inoculum ten fold, if necessary, to obtain isolated colonies.

Monographs

Results For general-purpose media, sufficient, characteristic growth and typical colony morphology should be obtained with all test strains. For selective media, growth of designated organisms is inhibited and adequate growth of desired organisms is obtained. Color and hemolytic reaction criteria must be met.

Reference National Committee for Clinical Laboratory Standards. 1996. Quality assurance for commercially prepared microbiological culture media, 2nd ed. Approved standard. M22-A2, vol. 16, no. 16. National Committee for Clinical Laboratory Standards, Wayne, PA.

Selective Media and Tubed Media Dilute the cell suspension 1:10 in normal saline or purified water. Streak each plate with 10.01 ml of the suspension to provide 1-2 x 105 CFU/ plate. Reduce the inoculum ten fold, if necessary, to avoid overwhelming some selective media.

Typical Analysis “Typical” chemical compositions have been determined on media ingredients. The typical analysis is used to select products for research or production needs when specific nutritional characteristics are required. The specifications for the typical analysis include: • • • • • •

Physical characteristics, Nitrogen content, Amino acids, Inorganics, Vitamins, and Biological testing.

Nitrogen Total Nitrogen: Total nitrogen is usually measured by the Kjeldhal digestion or titration method. Not all organic nitrogen is nutritive. Percent (%) nitrogen x 6.25 ≈ % proteins, peptides or amino acids present. Amino Nitrogen: The amino nitrogen value shows the extent of protein hydrolysis by measuring the increase in free amino groups. This is a nutritionally meaningful value.

pH

Glossary

Changes in pH from specified values, either after storage or processing, indicate deterioration. These changes are usually accompanied by darkening of the end product. Hydrolysates vary in their pH resistance according to their inherent buffering (phosphate) capacity.

Ash

Phosphates

The higher the ash content, the lower the clarity of the prepared ingredient. The ash content includes sodium chloride, sulfate, phosphates, silicates and metal oxides. Acid-insoluble ash is typically from silicates found in animal fodder.

High-phosphate ingredients may be unsuitable for pH indicator media due to the inherent buffering of phosphates. However, phosphates do aid in gas production, which can be enhanced by deliberate addition of sodium phosphate.

Moisture

Sodium Chloride

Lower moisture levels (