Each test strain is incubated at 37°C, with shaking, for 8-10 hours to achieve a density of 1-2×109 bacterial cells per ml.
Test chemical is assayed across typically 5 analysable dose levels with solvent and positive controls, all performed in either duplicate, or more commonly in triplicate to provide statistical robustness. The test is operated both with and without S9 exogenous metabolism.
In some cases, for example if the test compound is a volatile liquid, it is pre-incubated in a sealed vessel for 1 hour at 37°C, with shaking.
Either after pre-incubation or directly for solid test compounds, the test compounds and top agar (trace histidine / tryptophan) are mixed and poured onto the agar plate.
Plates are inverted and incubated at 37°C for 3 days.
The numbers of revertant colonies are counted, either manually or using an automatic plate counter.