Tetanus Toxin Production on a Simplified Medium

The routine preparation of tetanus toxin for the purpose of antitoxin production, or for conversion into toxoid, appears to offer difficulties similar to those formerly encountered in the case of diphtheria toxin, although perhaps in lesser degree. Active immunization against tetanus by means of toxoid, now coming into general use, demands that an attempt be made to place the manufacture of this important biological product on as satisfactory a basis as it has now been possible to reach in the case of diphtheria toxin. 1 , 2 The traditional explanation for failures and irregularities with the latter, namely the types of peptones and meat infusions used in the medium, has been shown to be completely erroneous, although still held to be applicable in dealing with tetanus. In this country, Witte, or the very similar Berna peptone is generally held to be indispensable, together with veal infusion. Elsewhere, enzymatic digests are employed, often with the addition of even more complex materials.

The writers have undertaken a study of the nutritional requirements of the tetanus bacillus and a parallel study of factors influencing the production of its toxin. Since an exact definition of the substances essential to growth may well prove to be a time-consuming matter, it seems worth while to report at this time the production of potent toxin on a simplified, but by no means chemically defined medium, and to indicate that the concentration of iron in the medium, as in the case of diphtheria, is at least one of the controlling factors.

As a basis for experiment we have employed the casein acid hy-drolysate medium described by one of us2 for the production of diphtheria toxin. This medium itself fails to support growth of any of the tetanus strains investigated, as was to be expected, but when it is enriched by the addition of tryptophane, glucose and 1 % of a commercial liver extract, growth occurs with all the strains which have been tried.