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

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

P E Georghiou¹, P A Blagden, L Winsor

¹Department of Chemistry, Memorial University of Newfoundland, St. John's, Canada.

Mutation Research|January 1, 1989

View abstract on PubMed

Summary

Increasing bacteria in the Ames mutagenicity assay may increase false positives from pre-existing revertants (PER). PER levels depend on culture handling, potentially masking true mutagenicity. Monitor PER in modified Ames tests.

Area of Science:

Microbiology
Genotoxicity Testing
Molecular Biology

Background:

The Ames S. typhimurium mutagenicity assay is a standard tool for detecting chemical mutagens.
Modifications to increase assay sensitivity, such as using higher bacterial concentrations, have been proposed.
Elevated bacterial counts may inadvertently increase spontaneous revertants (SR) due to pre-existing revertants (PER).

Purpose of the Study:

To investigate the contribution of pre-existing revertants (PER) when using high bacterial concentrations (10^9) in the Ames test.
To determine the impact of different bacterial culture handling and storage methods on PER levels.
To assess the risk of PER interfering with the accurate detection of mutagenicity.

Main Methods:

Bacterial cultures (Salmonella typhimurium TA98) were prepared from various sources: frozen permanent, lyophilized permanent, master plate, and isolated colony.
Cultures were used to inoculate overnight broth.
The number of viable bacteria and pre-existing revertants (PER) per 10^9 viable bacteria were quantified.

Main Results:

The number of PER per 10^9 viable bacteria varied significantly based on the source of the inoculum.
Average PER counts were highest from frozen permanent cultures (267) and lowest from isolated colonies (13).
Lyophilized permanent (188) and master plate (57) cultures also showed substantial PER levels.

Conclusions:

The handling and storage of bacterial cultures significantly influence the number of pre-existing revertants (PER) in the Ames test.
High PER levels can elevate spontaneous revertant counts, potentially leading to false negatives in mutagenicity detection.
Monitoring PER is crucial when modifying the Ames test with increased bacterial concentrations to ensure assay reliability.