A simple method of detection of toxigenic Clostridium botulinum type B strains

39 spontaneous nontoxigenic (tox −) mutants were isolated from 20 toxigenic (tox +) strains of Clostridium botulinum types B, A and F. The growth of tox + strains different from that of tox − mutants on a medium supplemented with Tween 80. On VL-Tween agar, tox − mutants formed irregular, glisteing, translucent colonies whereas in tox + colonies the centers were covered by an opaque (‘hoarfrosting’) substance. In Tween-liquid medium, tox + cultures were distinguished from tox − cultures by the formation of a turbid and flocculent growth. Of four different Tween preparations tested (Tweens 20, 40, 60 and 80), only Tween 80 proved to be capable of detecting toxigenic strains. The results indicated a correlation between toxigenicity of C. botulinum and its ability to metabolize Tween 80. The metabolites form the characteristic ‘hoarfrosting’ substance on Tween agar. We recommend the use of Tween 80 for the detection of tox + C. botulinum strains in genetic studies.