(a) Phase of strain employed: Cultures of Cl. septicum may contain a number of variants. Some of these may be characterized by their colonial morphology on blood agar as: (1) rough, non-hemolytic colonies; (2) smooth, non-hemolytic colonies; (3) smooth, hemolytic colonies. Of these three variants, only the last produces toxin.

(b) Quantity of growth and length of time cultures were incubated: The toxin and hemolysin content of cultures increases as the bacterial population increases, reaching a maximum value when the number of bacteria is at a maximum, or shortly thereafter. Upon further incubation, the toxin and hemolysin content decreases.

(c) Presence of a hemolysin-inactivating factor: A substance, possibly lipid, and present in cooked meat, is capable of inactivating hemolysin produced in casein hydrolysate medium and can account for the absence of hemolysin from cultures grown in cooked-meat broth.