Graduate Thesis Or Dissertation
 

A comparative study of Clostridium botulinum strains using differential egg yolk media

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  • The purpose of this study was to compare and test the efficiency of the leading kinds of media being used for the isolation and identification of Clostridium botulinum type E strains, and to develop a more reliable method and media for the differentiation of this organism based on the "pearly layer" characteristic. The production of "pearly layer" around the colonies of the different types of C. botulinum on egg yolk enriched media has been used as an aid in differentiation and isolation of this group of bacteria. In this investigation a comparative study was conducted to test the efficiency of the "pearly layer" formation on liver veal agar egg yolk media (LVA) and trypticase agar egg yolk media (TA). Liver veal agar egg yolk media was not entirely satisfactory in that strains of C. botulinum type E and most non-toxic "type E-like" clostridia did not always give a typical reaction. These organisms in many cases produced a faint "pearly layer" or, in some cases, no "pearly layer" on the LVA egg yolk media. When trypticase was substituted as the base in the egg yolk media, more consistant results were obtained in that all C. botulinum types produced the "pearly layer". To separate type E strains from types A and B, sucrose and an indicator, neutral red, were incorporated in the TA media. Since only type E strains ferment sucrose, the acid in the media was detected by the neutral red indicator as formation of a pink halo around the colony. Unfortunately, the fermentation of sucrose in the media suppressed the production of the "pearly Iayer". However, type E's can be differentiated from other types on the sucrose TA egg yolk media by the production of starch-Iike compounds which give an iodine reaction. Types A and B and most non-toxic "type-E-like" clostridia do not produce starch-like granules even on a utilizable sugar such as glucose. Trypticase base, therefore, is much more reliable than liver veal base, not only "pearly layer" formation but also in differentiation of C. botulinum type E's from other types by sucrose fermentation and starch reaction. Throughout this investigation, two morphologically different colony types have been associated with a pure culture of C. botulinum type E. One was the characteristic C. botulinum type E toxic colony which produced "pearly layer" on TA egg yolk plates; the other colony was a non-toxic sporulating isolate which did not produce a "pearly layer". The toxic cultures produced acid and gas from glucose, fructose , maltose, sucrose, and sorbitol. The nontoxic isolates produced acid but no gas in the same sugars. Microscopically, biochemically and morphologically the two types of colonies were very similar to the toxic (TOX) cultures and the nontoxic sporulating (OS) variant described by other investigators.
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