Ames MPF

Ames featured

Assay principles

The Ames MPF™ assay is an adapted microfluctuation method for performing the Ames bacterial mutagenicity test. Hisˉ or Trpˉ tester strains are incubated with test chemical and scored for colony growth, indicating reversion to His+ or Trp+ phenotype has occurred.

Diagram of Ames plateIn this liquid format of the test, small cultures are scored in 384-well microplates using an indicator medium colour change (purple to yellow).

A test chemical is assayed across 6 dose levels with concurrent zero-dose and positive controls, all performed in triplicate to provide statistical robustness. The test is operated both with and without S9 exogenous metabolism.

Assay Protocol

In 24-well microplates, histidine auxotrophic bacteria are exposed to 6 doses of a test chemical in the absence or presence of S9 (typically 4.5% S9 mix), as well as both a positive and negative control. The 6 test chemical doses are typically prepared in a half-log serial dilution. Test chemical exposure is performed in triplicate and lasts for 90 minutes with shaking. The exposure medium contains sufficient histidine to support ~2 cell divisions during the exposure period. After this time, exposure cultures are diluted in histidine-free medium containing a pH indicator dye. The contents of each well of the 24-well plate are then aliquoted into 48 wells of a 384-well microplate: 1 x 384-well microplate = 1 replicate of a compound test (either with or without S9).

After 48-hours further incubation, 384-well plates are scored by differentially counting coloured wells: spectrophotometric scoring removes operator bias, increasing confidence in the accuracy of the data. Cells that have mutated back to histidine prototrophy, either spontaneously or as a result of test chemical / positive control treatment, are able to grow into colonies. In wells where this occurs, the cells’ metabolism reduces the pH of the medium changing the indicator colour from purple to yellow.  These yellow revertant wells are counted for each dose and compared to the zero-dose.