A comparative study was conducted to determine the optimum conditions for sporulation and toxin production by Clostridium botulinum type G, strain 89. One solid and four liquid media were compared for their ability to promote sporulation. After being inoculated, the media were incubated at 35 C for 12 days, at 30 C for 16 days, and at 26 C for 21 days. Spores were harvested by centrifugation, washed 3 times and resuspended to give a 35 × concentration, then counted by the MPN procedure. Spores grown on the solid medium at 35 C for 16 days gave higher counts than those grown at the same temperature in the liquid media. Toxin production was studied in eight media at 35, 30 and 26 C over a 24-day period with samplings every 2 to 3 days. Three of the media contained trypsin and five were trypsinized after growth. Toxin titers were determined by intraperitoneal injection of mice and expressed as MLD/ml of culture. Higher toxin titers were obtained at 26 and 30 C in media containing 0.4% glucose.

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