In situ and suspension protocols for chemically-induced mutation at the tk locus in L5178Y MOLY cells: dose response and colony size distribution

Abstract

We used EMS up to concentrations of 0.25 μl/ml (292 μg/ml) to induce mutations at the tk locus in L5178Y MOLY cells, measured the cellular response by the in situ mutation assay protocol and compared these results to those obtained in a concomitant suspension assay. EMS induced mutagenic responses with both protocols. The mutant fraction for the solvent control was 89 mutants per million viable colonies for the suspension protocol and 426 mutations per million viable cells plated for the in situ protocol. These numbers increase to 447 and 2073 respectively, with 0.25 μl/ml EMS treatment. Sizing curves indicated that the in situ protocol detected a greater proportion of smaller colonies than did the suspension protocol. Not only were the number of small colonies greater than large colonies in the in situ protocol, but their rate of increase was also slightly higher than that of the large colonies. The in situ protocol also reduces the time and cost of experimentally performing the assay compared to the suspension protocol. In this paper we compare the use of the suspension and in situ protocols to measure chemically-induced mutations and demonstrate that the latter method detects a larger fraction of induced mutations at the tk locus in L5178Y MOLY cells.