The differentiation of clostridial species by gas chromatography and deep agar colony morphology

An investigation was initiated to determine the feasibility of using gas chromagography and deep agar colony morphology to aid in the identification of clostridial species. Five organisms were studied by gas chromatographic analysis (Clostridium chauvoei, Clostridium hemolyticum, Clostridium novyi type B, Clostridium perfringens type C and Clostridium septicum). The organisms were grown in trypticase soy broth for 24 hours in a special anaerobic system. Cells and spent medium were collected by centrifugation then subjected to fatty acid extraction procedures (chloroform-methanol extraction and saponification). Comparison of the two methods of fatty acid extraction revealed that the saponification method was best for differentiation of the five species tested. Gas chromatographic analysis of the saponified samples yielded fifteen cellular fatty acids. Analysis of variance at the 1% level of significance indicated that eight of the fifteen fatty acids had no significant mean values. A fatty acid profile of each organism was constructed form the statistical results which allowed for differentiation of the five organisms at the species level. Differentiation of clostridial species by comparison of mature deep agar colony forms was demonstrated. ..