Isolation of nontoxinogenic mutants of Vibrio cholerae in a colorimetric assay for cholera toxin using the S49 mouse lymphosarcoma cell line.

A mouse lymphosarcoma (S49) cell line that is growth-inhibited by agents that elevate intracellular concentrations of adenosine 3':5'-cyclic phosphate was used in a sensitive and convenient colorimetric assay for cholera toxin. S49 cells suspended in Dulbecco's modified Eagle's minimal essential medium containing 10(-5)--10(-6) M RO 20-1724, an analogue of 4-(3,4-demethoxybenzyl)-2-imidazolidinone and a phosphodiesterase inhibitor, were growth-inhibited by subnanogram concentrations of cholera toxin. Effects of toxin were detected by the absence of a yellow pH change (phenol red indicator) which normally accompanies the production of acid metabolites by lymphoma cells. An assay using S49 cells grown in microtiter plates, which is capable of detecting 10 pg of cholera toxin or 0.01 units of cholera antitoxin, was used in screening for nontoxinogenic mutants of Vibrio cholerae strain 569B. The properties of two mutants of the Tox--phenotype, which lacked biologically and immunologically detectable toxin products, are described.