Agar diffusion procedures for the assay of lecithinase from Clostridium perfringens.

Lecithinase can be determined by the measurement of the opalescence developing in serum (Nagler, 1939; Seifert, 1939), the precipitation of lecithovitellin (van Heyningen, 1941a; Weed et al., 1942; Theriault, 1945), the release of hemoglobin from erythrocytes (van Heyningen, 1941b; Burrows, 1951), the determination of the amount of acid soluble phosphorus released from lecithin (MacFarlane and Knight, 1941), and the release of CO2 from carbonate buffer in the Warburg apparatus (Zamecnik et al., 1947). A technique for the assay of lecithinase using an agar diffusion procedure has been developed in this laboratory and is described in this report. With the exception of the turbidimetric determination of lecithovitellin precipitation, this technique is as sensitive and accurate as any of the above and much simpler to carry out. Agar diffusion techniques have long been used for the assay of antibiotics, growth factors, etc. In the field of enzymology, the general utility of these methods and the parameters for the assay of trypsin and horse liver esterase have been discussed (Stansley and Ramsay, 1956), and the technique has been used for the assay of amylase (Stark et al., 1953). In this study, human erythrocytes and egg yolk suspensions were used as substrates for the lecithin-lecithinase reaction. With both substrates it was found that the size of the reaction zone was a function of the enzyme concentration, and that this function can be expressed as a linear relationship between the size of the reaction zone and the logarithm of the lecithinase concentration.