Factors Affecting Quantification of Clostridium perfringens Alpha Toxin by the Hemolysin Indicator Plate Test and...

Application of the following methods, electroimmunodiffusion on cellulose acetate, radial immunodiffusion, lecithovitellin agar plate, and hemolysin indicator plate, for quantification of alpha toxin of Clostridium perfringens revealed that the hemolysin indicator plate method was the most sensitive detecting 0.0003 unit/ml. Sensitivity of the hemolysin indicator plate method was enhanced 67-fold when alpha toxin was diluted with Brewer Thioglycollate medium (Difco, 0236) rather than physiological saline solution. The proteose-peptone ingredient of Thioglycollate medium was the major contributor to increased sensitivity. Less refined agar yielded a larger hemolytic zone. This was attributed, in part, to its higher calcium content. The reducing agent, sodium thioglycollate, diminished the hemolytic zone. In terms of clarity of hemolytic zone, the optimum human red blood cell concentration was 2.1 × 10⁶/ml of base agar. Incubation of hemolytic indicator plates at 35 rather than 21 or 45 C yielded greater hemolytic zones. When incubation time was extended, hemolytic zones also increased in size.