First, filter sterilized bacterial supernatants were incubated on a Vero cell (a kind gift of Dr. E.J. Snijder [27] ) monolayer and cytotoxic effects were quantified after 24 hours by determining the end-point titer ( Figure 3A ) [26] . In the exponential growth phase (5 and 8 hours post inoculation) no cytotoxic effects were detectable (data not shown). In the stationary growth phase (12, 24 and 48 hours post inoculation) we observed increasing cytotoxic effects, indicative of the presence of toxin. Importantly, the observed cytotoxic effects were specific for C. difficile toxin A and B, as a pre-incubation of the filter sterilized bacterial supernatants with anti-toxin, a polyclonal antibody against toxin A and toxin B (Techlab), resulted in complete neutralization of cytotoxic effects on the Vero cells at all time points (data not shown). The tcdC mutant strains showed no significant differences in toxin levels compared to the wild type strain (Fig. 3A) . Next, we used an enzyme immunoassay (Ridascreen, Biopharma) for the direct detection and relative quantification of the secreted toxins. In the exponential growth phase (5 and 8 hours post inoculation) no toxins were detectable (data not shown), consistent with the lack of toxicity towards Vero cells described above. In the stationary growth phase (12, 24 and 48 hours post inoculation) increasing toxin levels were detectable. When we compared the toxin levels at various time points, there were equal amounts of toxins in the wild type and tcdC mutant strains.