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Methodology for Staphylococcus Areus and Clostridium Perfringens

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Methodology for Staphylococcus Areus and Clostridium Perfringens 342 MFTHODOLQGY FOR STAPHYLOCOCCUS AU€WJS AND CUIZX'RIDIUM PERF'RINGE:NSK H. W. WALKER AND C. R. EU3Y Iowa State University The staphylococci and micrococcihave been-placed in the family Micrococcaceae in "Bergey ' s Manual of Determinative Bacteriology" (Breed et g., 1957). Classification of these gram-positive, catalase-positive cocci in this family is still somewhat controversial. In 1965, however, the International Subcommittee on Staphylococci and Micrococci proposed a standard test for separating these organisms into the twcJ genera :,f Staphylococcus and Micrococcus. According to this cormnittee, the genus Staphylococcus should contain the parasitic, facultative-anaerobic cocci which produce acid from glucose under anaerobic conditions, and the genus Micrococcus should contain the saprophytic, aerobic cocci which produce acid from glucose aerobically, but not anaerobically. Our interest is mainly with the genus Sta h lococcus which consists of two well accepted species, S. aureus and S. -$=-7-e idermidis Breed et al., 1957). These tw:, species are separgtted as follows: S. aureus ferments mannitol and is coagulase positive, while S. epidermidis does not ferment mannitol and is coagulase negative. S . aureug produces a variety of extracellular products, among which is enterotoxin, a heat-stable exotoxin that causes a severe type of foQd poisoning in man and ~omeother animal species. Animal assays are not convenient for testing for strains that produce this toxin; therefore, other simplified means of detecting pathogenic strains have been used or suggested. Coagulase production is probably the most extensively used test for correlation with enterotoxigenicity (Chapman, 1944; Morrison, 1962); this enzyme causes the formation of a clot or coagulum in citrated or oxalated blood plasma. According to Ivler (1970), approxbnately 97$ of the staphylococci associated with pathological symptoms in man are capable of elaborating this enzyme. Other characteristics that have been correlated with ability to produce enterotoxin have been production of hemolysin and a gDlden pigment (Feldman, 1946), mannitol fermentation under anaerobic cmditions (Joshi and Dale, 1963); gelatin liquefaction (Stone, 1935): prductim of deoxyribonuclease (Iechica -et a1,*7 1969, 1971; Brandish and \'illis, i970; Jay, 1962) and production of protein A, an extracellular substance formed by staphylococci (Forsgren, 1970; Jay, 1971). * Journal Paper No. J-7292 of the Iowa Agriculture and Home Economics 2xpcriment Station, Ames, Iowa. Project No. 1896. Presented at the 25th Annual Reciprocal Meat Conference :,f the American Keat Science Association, 1972. 343 Exceptions to all these indicators of enterotoxin production have been observed. For example, Bergdoll --et al. (1967) and Thatcher and Simon (1956) have observed staphylococcal isolates that were coagulase-negative but prcAuced enterotoxin. All coagulase-positive staphylococci are not necessarily pigmented (Muth, 1971). Production of gelatinase is a poor index of fo& poisoning potential (Chinn, 1936; Clark et al., 1961). These and additional examples have been extensively reviewed by MFnor and Marth (1972). Evidence indicates a relatively high correlation between enterotoxigenicity and coagulase production, deoxyribonuclease production, and production of protein A. No one characteristic, however, is infallible as an index of enterotoxi- genicity. Franklin --et al. (1966) evaluated coagulase-mannitol agar base, deoxyribonuclease test medium, the phosmtase test, the fluorescein amine reaction and the fibrinogen test as substitutes for the tube coagulase test; they found none of these diagnostic tests suitable substitutes for the tube coagulase test in the identification of S. aureus. In the absence of a simple undisputed methd of identifying Fnterotoxigenic strains, the coagulation of plasma is the most widely accepted index of this characteristic and is used as a confirmatorytest for typical colonies occurring on various selective media for staphylococci. The numerous media developed for isolation of staphylococci from other organisms have been classified into three groups on the basis of the selective agents used (Jay, 1962). The first group includes those that use sodium chloride as the selective agent; levels used may vary from 5.5% to l@. Several examples of this type of medium are: Chapman-Stone medium, Mannitol- Salt agar, Staphylococcus Medium No. 110, and Staphylococcus Medium So. 110 fortified with egg yolk. The second group consists of media that depend on various chemicals for their selectivity. Media included in this category are: Tellurite-Glycine agar, Azide agar; and Vogel and Johnson agar. The third group includes media that incorporate antibiotics, such as neomycin and polymyxin, as selective agents. Practically all these media and others not listed have been used successfully for the isolation of coagulase- positlve strains of s. aureus from clinical specimens and nasal and skin swabs, but they have-not always been adapted successfully for use with food analyses. Zebovitz --et al. (1955) developed Tellurite-Glycine medium especially for the detection of staphylococci in cured meats. Black colonies are formed on an agar medium containing tellurite because staphylococci can aerobically reduce the tellurite salt to elemental tellurium. This medium,, however, proved excessively inhibitory when used for other fmd products (Mowe and Nelson, 1962; Donnelly -et ,*, a1 1964; Marshall -et a1,*, 1965a; Chou and Msrth, 1969). Vogel and Johnson (1$1) mQdified the Tellurite- Glycine agar of Zebovitz and co-workers by increasing the mannitol content and by adding phenol red for a pH indicator. Another modification of the Tellurite-Glycine type medium is the 3gg Yolk-Tellurite-Glycine-Qruvate Agar formulated by Baird-Parker (1962). "his medium and Vogel-Johnson medium are two commonly used for enumerating staphylococci. The Food and Drug Administration in its Bacteriological Analytical Manual (1969) recommended Vogel-Johnson Medium in the direct 344 plating method for the detection and enumeration of S. aureus occurring in food; however, this recommendation was changed to BaTrd-Parker medium in an update In 1971. The USDA also uses this medium. This usage gives a certain ethtus to the medium, but does not constitute endorsement as a standard method. Comparisons made on recovery of staphylococci by using various selective media have shown that Vogel-Johnson medium does not always yield the greatest percentage recovery (Sessams and Mercuri, 1969; Marshall et al., 1%5a; Chou and Marth, 1969). The Food Research Institute at the University of Wiscmsin uses the procedure of Foster --et al. (1970) for enumerating staphylococci. This technique consists of surface-plating samples on Egg Yolk-Pyruvate- Tellurite-Glycine Agar (Baird-Parker medium) and incubating for 24-30 hr at 35C. Black colonies surrounded by a clear zone are inoculated into Brain Heart Infusion or Trypticase Soy broths and incubated overnight at 35C. Coagulase activity of these cultures is then determined. In their book, "MicroorganJ.sms in foods; their significance and methods of enumeration," Thatcher and Clark (1968) describe the methods used by the Food and Drug Administration and the Food Research Institute but also recommended Baird-Parker, Egg Yolk-Azide, Milk-Salt, or Tellurite-Polyxayxin- Egg Yolk agars. In several comparative studies, Tellurite-Polymyxin-Egg Yolk agar gave the best overall recoveries (Sessoms and Mercuri, 1969; Marshall et al., l965a; Crisley & &., 1965). Chou and Marth (1%9), however, found that direct plating on Mannitol Salt agar yielded better recovery of coagulase positive staphylococci from naturally contaminated feed-grade meat products than did Tellurite-Polymyxin-Egg Yolk agar. Chapman (1946) developed a medium containing mannitol, gelatin, tryptone, lactose, yeast extract and 7.54 NaCl for isolation of coagulase positive staphylococci, which is known as Staphylococcus Medium No. 110. A modification of this medium, in which the amount of NaCl WBS reduced to 5.58 and smmonium sulfate was added to aid in the detection of gelatbase activity, is known as Chapman-Stone Medium. Yellow or orange colonies surrounded by a clear zone on this medium are considered suspect. These media or modif'ications to which egg yolk has been added also have been used extensively for isolation and enumeration of staphylococci. Many coagulase positive strains produce opacity as a result of lipase activitywhen grown Fn media containing egg ;yolk (Gillespie and Alder, 1952). A medium called Polymyxin-Mannitol-Phenolphthalein Diphosphate agar has been reported as fielding good recoveries of coagulase-positive staphylococci in the analysis of foods without the presence of false p?sitive reactions (Williams, 1972). In this method, poured agar plates +".-e surface inoculated by streaking and incubated at 3°C for 40 hr. When the plates are exposed to the vapor from strong ammonia solution, coagulase- positive staphylococci appear pink, surrounded by a clear zone. The theory is that only coagulase-positive staphylococci produce recognizable acid from mannitol and menolphthalein from phenolphthalein diphosphate within 40 hr at 37C. 34 5 A 24-hour method for the detection of coagulase-positive staphylococci in fish and shrimp has been developed by Insalata --et al. (1972) in cooperation with the Food and Drug Administration. These authors set out t3 devehp a rapid screening method that would yield results within 24 hr. Requirements to be
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