From the time of Rous and Yersin, who first reported the toxicity of sterile filtrates from cultures of the diphtheria bacillus some 70 years ago, the means by which diphtheria toxin exerts its lethal effect has not been discovered. Following the injection of large doses of diphtheria toxin into susceptible animals there is a prolonged latent period of many hours during which no ill effect of any kind, either morphological or biochemical, can be detected. It is now generally agreed (1, 2) that all of the familiar signs of diphtheria intoxication including gross and microscopic morphological damage to tissues, increased resistance of the animal to insulin (Corkill (3)), reduced capacity to synthesize carbohydrates (Cross and Holmes (4)), or to metabolize lactic acid (Dawson and Holmes (5)) and decreased stores of muscle phosphocreatine (Pinchot and Bloom, (6)) are all secondary effects which follow by many hours a primary injury the nature of which is unknown. Peters and Cunningham (7) examined various oxidative enzyme systems which had been treated with toxin in vitro, but could find no untoward effects. Pappenheimer and Williams (8), using the metamorphosing silkworm Platysamia cecropia as the susceptible animal, found that only those tissues characterized by an active succinoxidase system, or undergoing rapid growth and development dependent on the succinoxidase system, were sensitive to the action of toxin; whereas tissues which were deficient in the succinoxidase system were insensitive.With the advent of improved tissue culture methods and consequent stabilization of certain mammalian cell lines in vitro, more homogeneous cell populations became available for use in studying the effects of toxin. Lennox and Kaplan (9) and Placido Sousa and Evans (10) found various cell lines to be susceptible to diphtheria toxin.

Various simplified media have been described in which Clostridiura welchii grows well but produces no alpha toxin (1-3). Macfarlane and Knight (4) showed that the addition of autolyzed muscle to a medium which is satisfactory for growth enhanced the production of alpha toxin. Rogers and Knight (5) presented evidence indicating that the effect of autolyzed muscle was in part due to glucosamine. Logan, TyteU, Danielson, and Griner (6) used a pancreatic digest of beef heart as a source of "toxin factor" and furthermore demonstrated the importance of dextrin as a source of carbohydrate in obtaining maximum fields of alpha toxin with certain strains of Cl. welchii. Adams and Hendee (7) used a pancreatic digest of casein for "toxin-promoting factor" and confirmed the finding of Logan et al. that dextrin was an important factor in obtaining high yields of toxin.In the course of the present investigation it has been found that for the production of maximum yields of Cl. welchii alpha toxin, at least two factors are required in addition to dextrin and the basic growth requirements. One of these factors is present in enzymatic digests of certain proteins such as casein and gelatin. The second factor is present in extracts of pancreas and gastric mucosa. Glycerylphosphorylcholine is responsible, at least in part, for the toxin-promoting activity of pancreas and gastric mucosa.

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Summary The production of the alpha and theta toxins of Cl. welchii on a simplified medium based on a pancreatic digest of casein is described. Methods are given for the concentration and purification of the alpha toxin.