Spontaneous revertants in modified S. typhimurium mutagenicity tests employing elevated numbers of the tester strain

It has been proposed that increases in the number of bacteria applied to each plate can enhance the sensitivity of the Ames S. typhimurium mutagenicity assay. These procedures have the potential to elevate the number of spontaneous revertants (SR) by increasing the contribution of pre-existing revertants (PER) present before application of the bacteria to the limited histidine test plates. We have investigated the contribution of PER when 10(9) bacteria are applied to the plates and found that the number of PER is dependent on the handling and storage of the cultures used to inoculate the overnight broth. The average number of PER/10(9) viable bacteria after overnight growth in broths inoculated from a frozen permanent, lyophilized permanent, master plate, and an isolated colony, of TA98 were 267, 188, 57 and 13 respectively. The resultant elevation of the number of SR for a strain may result in a failure to identify a mutagenic response. It is recommended that the number of PER be monitored in any modification of the Ames test that makes use of elevated numbers of bacteria.