PREPARATION OF CLOSTRIDIUM PARABOTULINUM TOXINS

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Abstract
This paper deals with methods of producing high and consistent yields of Type A and Type B Clostridium parabotulinum toxins. It is shown that highly toxigenic strains readily lose toxin producing activity and that this type of variation may be largely prevented by storing in the cold young cultures to be used as inoculum in toxin production. The best medium of those tested consists of tryptic digest of casein, glucose, and yeast extract or clarified corn steep. Maximum growth occurs in 16 to 18 hr. at 35 °C. Growth is followed by gradual autolysis of the organisms, which is approximately complete in four to six days. Appreciable amounts of toxin are produced during the first 24 hr. of growth and a maximum is reached with the complete lysis of the organisms.Toxin may be completely precipitated from the culture fluid at pH 3.9 and may be partially purified by suspension and reprecipitation at pH 3.9. Acid precipitated toxins may be dried in vacuo, by lyophilization or spray drying. Dry toxins have been stored up to two years without loss of toxicity.Average yields per ml. of culture were:A toxin—1 × 106 LD50's for the mouse (20 gm.)2 × 105 LD50's for the guinea-pig (250 gm.)B toxin—1 × 103 LD50's for the mouse (20 gm.)1 × 105 LD50's for the guinea-pig (250 gm.)Type A toxin has the same order of toxicity for mice, guinea-pigs, chickens, and goldfish per unit of body weight. Type B toxin is 6000 to 8000 times more toxic for guinea-pigs than for mice, chickens, or goldfish on a body weight basis.

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