Development and standardization of a cytotoxic micro-assay for detection of enterotoxins: survey of enterotoxins from Escherichia coli of infant origin.

The development of a practical cytotoxic micro-assay for detection of enterotoxins in crude bacterial lysates of E. coli and other Gram-negative bacteria, is described. This quantitative assay is based on growth inhibition of mouse-fibroblasts, maintained in suspension or by inhibition of uptake of DNA precursors. Guidelines for performing the assay and evaluating the results by statistical considerations, are described. The choice of a relatively cheap medium and a suitable number of target cells to achieve cell doubling in 24 h is given. The concentrations of proteins in the crude lysates from strains of different origin, are not of equal potency; a predetermined but different protein concentration for strains from infants, adults or porcine origin, are recommended for detection of the toxins and for achieving reproducible results. Production of toxic proteins is enhanced by mitomycin C in toxigenic strains and not in non-toxigenic strains. Screening of a limited number of lysates from E. coli strains originating from infants and a comparison of the cytotoxicity of several known toxigenic and non-toxigenic strains from human adults and porcine origin, are presented.