Immunodiffusion Detection of Clostridium botulinum Colonies

Toxic colonies of Clostridium botulinum types A, B, E and F were detected by precipitating toxin around the colonies on agar containing antitoxin. Incorporating antitoxin in a gel diffusion agar overlay after colonies had developed was unsatisfactory for detection of type E, but worked well for all types when added directly to the plating medium. Addition of 0.6 IU of antitoxin per ml of agar gave satisfactory results with all types except type E. Zones of precipitation were produced by proteolytic strains of types A, B and F incubated at 35°C for 3 d and by nonproteolytic strains of types B and F incubated at 28°C for 4 d; type E required 1.2 IU of antitoxin per ml of agar and 5 d of incubation at 35°C. Nontoxigenic putrefactive anaerobes produced no zones of precipitation with any of the antitoxins, and toxic colonies of C. botulinum mixed among them were easily distinguished. This method was used successfully for selecting type B colonies from plates containing toxic enrichment cultures of tomato juice.