Production of Types A and B Spores of Clostridium botulinum by the Biphasic Method: Effect on Spore Population, Radiation Resistance, and Toxigenicity

Spores of three strains each of type A and type B Clostridium botulinum were produced both by a biphasic (solid medium overlaid with an aqueous phase) and by a "conventional" (deep broth culture) procedure. Sporogenesis by the biphasic system was more rapid, convenient, and economical, and yielded as many or more heat-resistant (80 C, 10 min) spores per milliliter as by the conventional technique. Of several aqueous phases [thiamine-hydrochloride, yeast extract, (NH(4))(2)SO(4)] tested with strain 62A, the highest spore colony counts were obtained with 2.0% (NH(4))(2)SO(4). The six strains formed maximum spore numbers in 5 to 6 days of incubation. Spores produced by the two methods had essentially equal radiation resistances (D and lag values), and their subcultures gave similar toxin titers (LD(50) values).