METHODS

Toxin. Type A toxin was obtained from Clostridium botulinum (strain 4587) grown in a medium consisting of 25% tryptic digest of casein, 10% yeast extract, and 1% glucose. The ingredients were prepared as in CCY medium (Gladstone & Fildes, 1940). The toxin was purified by acid precipitation with HCl at pH 3 9, then rendered soluble by elution with 0-2M-Na2HPO4 to pH 6-0. The solution was freeze-dried and stored as a powder in vacuo over phosphorus pentoxide. As required, the toxin was dissolved in a buffer consisting of 1% Na2HPO4 and 0-2% gelatin at pH 6-6 to give a concentration of 2 x 105 or 2 x 106 mouse LD50/ml.(20 g. mice, intraperitoneally). The toxin contained 1* 3 x 107 LD50/mg. N.(Crystalline type A toxin contains 2-4 x 108 LD60/mg..(Lamanna, McElroy & Eklund, 1946), ie this material was c. 5% pure toxin.) In the text the amount of toxin used is expressed in terms of mouse LDr0/ml. of bath fluid which is for convenience called a unit.