The production of a hemolytic substance in young liquid cultures of Cl. botulinum.

A clear zone of hemolysis is usually noted around each colony of Cl. botulinum when the organism is grown anaerobically on blood agar medium at 35° C. for 3 to 4 days. The blood cells within the hemolyzed area are entirely dissolved. Preliminary experiments to demonstrate the production of a soluble hemolytic substance in 24 hour sheep's serum veal broth cultures were unsuccessful. Subsequent tests indicate that the hemolytic substance is formed during the first stages of growth; it is in all probability destroyed by the 24th hour.

The demonstration of the lytic substance depends on (1) the composition of the culture medium, (2) the age of the culture, and (3) the strain. Plain veal infusion broth is unfavorable for production of the lytic agent. The addition of small amounts of glucose or of 20 percent heated (56° C.—30°) sterile sheep's serum stimulates its formation. The first visible signs of growth in a 1 percent glucose veal broth medium, inoculated with spore forms (3-4 day beef heart cultures heated at 80° C. for 10 minutes) are usually noted after 14 to 16 hours incubation at 35° C. Hemolysin can be demonstrated in such a culture after 16 to 18 hours growth and usually persists until the 22nd or the 26th hour, depending on the composition of the medium. The dytic substance invariably disappears from the culture by the 38th hour of growth. After the inoculation of young actively growing vegetative forms (12 to 14 hour cultures), the first signs of turbidity usually appear after 4 to 6 hours incubation. About 2 hours later hemslysin can be demonstrated. The peak of hemolysin production is reached after 8 to 10 hours growth; by the twelfth hour the concentration of the lytic agent has either decreased or entirely disappeared.