Analysis of toxin A and toxin B production by M7404 <i>spo0A</i> mutant.

<p>The wild type, mutant and complemented derivatives were examined for toxin production by Western immunoblotting using (A) TcdA-specific and (B) TcdB-specific antibodies and precipitated supernatant proteins from the strains indicated. Toxin production by these strains was also quantified. Serial doubling dilutions of culture supernatants were made in MEM alpha medium supplemented with 1% HI FCS and used in Vero cell cytotoxicity assays (C). Morphological changes of the Vero cells were observed and scored by microscopy after 24 hrs. The toxin titre is the reciprocal of the endpoint dilution. Vero cell viability (D) was also measured using MTT reagent. •, M7404 (vector control); ▪, M7404 <i>spo0A</i> mutant (vector control); ▴, complemented derivative. All assays were performed in duplicate on at least three independent culture supernatants; the mean values of these assays are shown together with the standard errors of the means. ***, p ≤ 0.001. For Western immunoblot analysis, three independent culture supernatants were tested; the image shown is representative.