Media for identification and differentiation of Clostridium perfringens and Clostridium botulinum types a, b, and e Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/z316q4180

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  • During the development of the media for the identification and differentiation of Clostridium perfringens and C. botulinum types A, B and E, 16 different double layer media were prepared and tested with known cultures of 28 C. botulinum strains, 20 C. perfringens strains and 11 other clostridial species. The semisolid media incorporate a hydrogen sulfide indicator system, a carbohydrate and a pH indicator in the bottom layer, nitrate source and trypticase in the top layer, thus providing five different biochemical and physiological tests in a single screw-cap tube (H₂S production, nitrate reduction, carbohydrate fermentation, indole formation and motility). The media are used for the confirmation of Q. perfringens colonies from selective agar plates and for the differentiation and presumptive identification of C. botulinum types A, B and E and other clostridial species found on differential egg yolk agar media. The "jelly-like" consistency of Media Nos. 14 A/B and 15 A/B allows the inoculation of the test tube with a 1 ml plastic pipette without breaking the column. These media, when inoculated with active cultures grown in fluid thioglycolate medium for 4-6 hours or overnight at 35°C give results within 6-8 hours at 35°C or within 14-16 hours at 30°C. The media do not require an anaerobic jar for their incubation. The test tubes, with their caps tightly closed, are incubated in a 35°C or 30°C incubator. Both Medium No. 14 A/B and Medium No. 15 A/B, with the exception of the carbohydrate, have the same base composition. Medium No. 14 A/B uses lactose while Medium No. 15 A/B uses sucrose. During this study 101 anaerobic organisms were isolated from 41 different food samples. Forty-eight were identified as C. perfringens, one as C. botulinum type A, two as C. acetobutylicum, one as C. tertiuxn, and two as C. tetani. The proposed names for Media Nos. 14 A/B and 15 A/B are trypticase nitrate lactose; iron agar (TNLI) and trypticase nitrate sucrose iron agar (TNSI) respectively.
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