A medium consisting of a complete acid hydrolysate of casein supplemented with cystine, tryptophan, growth accessory factors, glucose, and inorganic salts, has been developed for the cultivation of Clostridium septicum. Toxin equivalent to 400 to 700 L.D50 per ml. has been obtained regularly in this medium. The principal factors found to affect the yield of toxin are:—

(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.

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