Technique for measuring 50% end points in cytotoxicity assays for Clostridium difficile toxins

Serial dilutions of Clostridium difficile culture filtrates were incubated overnight with HeLa cell monolayers. Cells were fixed in formalin, stained with crystal violet, rinsed, and drained. Cell rounding could be observed microscopically in the stained monolayers. Absorbance of the retained dye on monolayers in the drained wells was measured at 595 nm-405 nm. End points could also be estimated visually. The dilution at which dye absorbance was reduced by 50% agreed with that determined by microscopic observations. Five replicate dilution series showed high reproducibility. Specificity was verified by neutralisation with crude rabbit antibody to C difficile toxins. Cytotoxicity in faecal specimens was assayed in the same way, allowing reporting of titres, comparison with standard toxin preparations, and determination of the extent of neutralisation to be made. This novel assay technique has proved effective and reliable in a clinical setting and should allow the gathering of more information on the epidemiology of antibiotic associated colitis.