Assessment of experimental conditions affecting spontaneous mutation level of Salmonella strains used in the Ames test

Abstract

Introduction. The bacterial reverse gene mutations test (the Ames test) is widely used to assess chemicals’ mutagenic activity. The spontaneous mutation level of test strains is a mandatory characteristic that has to be monitored in a laboratory performing mutagenicity studies using the Ames test. In this regard, it is important to assess the factors affecting the spontaneous mutation level in the experiment and, therefore, on the general conclusion on the test item mutagenicity. Material and methods. A plate incorporation test version was used both in the presence and absence of a metabolic activation system. Results. We summarized the historical control data obtained in the laboratory in 2016-2020, determine the fluctuation limits in the number of revertant colonies for each strain, and identify the factors affecting the negative control variability. No significant differences were found in the spontaneous background of test strains when using DMSO or water as solvents, polypropylene or polystyrene tubes, as well as Petri dishes of different types. In the case of the TA1535, TA102 and TA100 cultures, no influence of the presence of the S9 mixture on the spontaneous reversion range was revealed (p≤0.05). Statistically significant differences in the number of spontaneous revertants (at + S9 or -S9) were found for the strains that allow detecting frameshift mutations, TA97 and TA98. It has been shown that the volume of the selective medium and the brand of gelling agent in its composition are important factors leading to the variability of the historical negative control. Conclusion. To ensure the quality of experiments according to the principles of good laboratory practice and the reliability of the data obtained using the bacterial reverse mutation method, it is necessary to standardize the operations in advance of experiments.