Quantifying Clostridial Alpha Toxin Activity

To determine the alpha toxin activity in the supernatants of strains JIR12058, EHE-NE18, CP56, JIR4866 and JIR4860, the lecithinase activity was assayed in an egg yolk agar well diffusion assay [30 (link),31 (link),32 (link)]. Therefore, small holes were pierced into Columbia agar supplemented with 2% egg yolk (v/v) with the rear end of a 20–200 µL pipette tip (diameter 7 mm), which were filled with 20 µL of supernatant per tested strain. A standard was included on each plate by preparing a two-fold dilution series of Phospholipase C (Sigma Aldrich) ranging from 1 to 0.0313 U/mL. Plates were incubated anaerobically overnight at 37 °C and scanned with a GS-800 calibrated densitometer (Bio-Rad Laboratories, Hercules, CA, USA). Alpha toxin activity was indicated by the development of turbidity. The area of the opaque zones was measured using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA).