Background: The Ames assay is commonly used to evaluate the mutagenic potential of tobacco products for regulatory purposes. A significant increase in the number of His+ revertant colonies observed in agar containing Salmonella bacteria and a test chemical indicates mutagenicity. To assess cytotoxicity in this assay, thinning of the background bacterial lawn is microscopically determined. With the development of new in vitro exposure systems that permit continuous cellular exposure to smoke or aerosol, the Ames assay method required modification by spreading the bacterial solution on top of agar in a 35 mm plate. However, studies detailing the use of bacterial lawn thinning as an indicator of cytotoxicity are needed.

Objective: To determine if microscopic analysis can detect thinning of the bacterial background lawn on top of the agar with or without metabolic activation (S9). We hypothesize that thinning can be generated by limiting the concentration of histidine/biotin in the spread solution.

Methods: Ames assay was performed using a 35 mm agar plate spread technique with a bacteria solution (with or without S9) containing varying amounts of histidine-biotin. Salmonella strains (TA98, TA100, TA102, TA1535, TA1537 and TA97a) were used to determine the number of His+ revertants and lawn thinning after 48/72 hours of incubation.

Results: We observed the reproducible thinning of the background lawn by limiting the amount of histidine/biotin. Lawn thinning was clearly observed without S9. However, the presence of 10 % S9 compromised the lawn assessment but was improved by reducing the S9 to 5 %. Using 5 % S9, the positive and negative control values for revertant colonies were within historical control ranges (similar to 10 % S9) for all strains.

Conclusions: Thinning of the background bacterial lawn was clearly observed in all strains using the Ames assay with spread technique in the presence or absence of 5 % S9 and microscopic analysis.