Starter cultures (10 ml) of C. perfringens were prepared by
inoculating cook meat stocks into tubes containing 10 ml of fluid
thioglycollate broth (FTG) (Difco) and incubated overnight (18 h) at
37 C. Sporulating cultures of C. perfringens
were prepared by inoculating 0.2 ml of an FTG starter culture into
10 ml of Duncan–Strong (DS) sporulation medium. After incubation for 24 h at 37 C, sporulation was confirmed by phase-contrast microscopy. Spore preparations were
cleaned by repeated centrifugation and washings with sterile
distilled water until the spores were >99% free of sporulating cells,
cell debris and germinated spores. After suspension in distilled
water at a final OD600 of w6, they were stored at 20 C until used