Formation of tetanus toxin within the bacterial cell.

The ability of a strain of Clostridium tetani to produce toxin is conventionally determined by measuring the Lf titer of the culture supernatant fluid after lysis is completed sometime after the third or fourth day of incubation. Although the flocculation procedure has been repeatedly found useful in the development of a suitable nutritional background for toxin production (Mueller and 1liller, 1954, 1955), it is not appropriate for studying the dynamics of tetanus toxin synthesis. The work of Raynaud (1951) and Raynaud et al. (1955) established the existence of significant quantities of extractable toxin within the bacillary bodies. In order to study the early phases of tetanus toxin synthesis it is necessary to measure the toxin in whole bacilli taken from young cultures before the onset of autolysis. The method reported here involves the injection of washed bacilli into mice under conditions where multiplication in vivo and continued toxin formation are believed to be minimal. When bacilli from a relatively nontoxigenic medium are transferred to a more suitable medium, application of this technique reveals that there is a marked increase in the amount of toxin per cell unit during the early phases of growth. This finding provides an experimental basis for evaluating the factors required for toxin formation without the added complication of cell lysis.