α-Toxin of Clostridium perfringens was highly sensitive to formalin; its antigenicity was destroyed rapidly during toxoiding. It was succeeded in toxoiding α-toxin under a strict regulation of protein concentration, formalin concentration, constituents of the medium, temperature and incubation period.
The ratio of egg unit to mouse toxicity changed during toxoiding and a long incubation was required when the egg yolk reaction was used as a measure for the toxicity. The customary usage of the in vitro test may have been one of the reasons for the inconsistent antigenicities of α-toxoid in the past.