Preparation of Inoculum and Incubation

use strain producing only C2 toxin

C. botulinum type C strain 92-13, which was kindly provided by Prof. S. Nakamura, Kanazawa University, Kanazawa, Japan, is suitable for an inoculum, because it produces only C2 toxin. Prepare the inoculum by incubating the strain overnight at 37 °C in cooked meat medium (Difco Laboratories) supplemented with 1 % ammonium sulfate, 1 % glucose, 1 % yeast extract and 0.2% cysteine hydrochloride (pH 7.5). Transfer a 1-ml portion of the culture into 4 I of the toxin production medium in a flat-bottomed bottle (ca. 23 cm in diameter) and culture for 2 days at 37 °C.

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