The influence of culture conditions on the susceptibility of Salmonella typhimurium in the Salmonella mutagenicity test.

To determine the variability of tester strain susceptibility in the Salmonella mutagenicity assay and to optimize the culturing procedure we examined the influences of the overnight culture period (12-16 h), the use of an additional short-term culturing procedure (1-4 h) and the Salmonella density (cfu/plate) on the rate of revertants per plate. As tester strains we used Salmonella typhimurium TA97, TA98, TA100 and TA102. As shown previously for other microbial genotoxicity short-term tests (i.e. with Escherichia coli PQ37), we observed the highest susceptibility of the Salmonella strains, i.e. the highest amounts of revertants per plate, when using a 12 h overnight culture followed by a 2 h short-term culturing procedure. We calibrated the bacterial count to 100 x 10(6) cfu per assay by photometric measurement (600 nm). Initially, we used 0-1 nmole 2,4,7-trinitro-9-fluorenone with S. typhimurium TA97, 0-15 nmole daunomycin with strain TA98, 0-25 nmole sodium azide with strain TA100 and 0-15 nmole methylmethanesulfonate with strain TA102 as reference compounds in the standard plate incorporation test. Subsequently, to evaluate the results of the culturing procedure variations we examined 22 well-known mutagenic and direct-acting (-S9-mix) compounds out of different chemical classes using both the standard and a modified culturing procedure. The comparison of these results showed a 64% (for 4-nitroquinoline-N-oxide) to 421% (for sodium azide) increased amount of revertants for the modified test protocol.