Evaluation of the effect of agar on the results obtained in the L5178Y mouse lymphoma assay.

The L5178Y TK+/- mouse lymphoma assay is widely used in mutagenicity testing. Trifluorothymidine-resistant (TFTr) mutants are quantitated following growth in agar-supplemented cloning medium. In an attempt to evaluate the effect of agar on plating efficiency, we have tested several lots of Difco Noble agar (cat. No. 0142-01-8; normally used in this assay) and compared it with Baltimore Biological Laboratory (BBL) agar (cat No. 11849). We find that BBL agar gives a higher and less variable plating efficiency than any of the Noble lots tested. Colonies plated in BBL agar tend to appear significantly earlier on the plates than those cloned in Noble agar. The absolute mutant number and the induced mutant frequency quantitated from a treated culture is generally higher in BBL compared to Noble agar. To determine if this higher frequency is due to increased mutant recovery rather than "sneak through" of nonmutant cells, we isolated 97 mutants from treated cultures (44 large colonies and 53 small colonies) and 69 mutants from untreated cultures (24 large colonies and 45 small colonies) and tested them for TFT resistance. All but one (a large colony from an untreated culture) were found to be TFTr, indicating that the mutant frequency is due to an increased mutant recovery. The spontaneous mutant frequency was quantitated for 122 untreated cultures. Showing little variation within and between experiments, the spontaneous mutant frequency yielded a mean of 57.7, with a standard deviation of 14.4. Under our laboratory conditions, BBL agar gave reliable results, and we prefer it for use in cloning L5178Y mouse lymphoma cells.