Fluctuation Method
The Ames test was initially developed using agar plates (the plate incorporation technique), as described above. Since that time, a popular alternative to performing the Ames test has been developed, which is known as the "fluctuation method". The fluctuation method is performed entirely in liquid culture and is scored by counting the number of wells that turn yellow from purple in a 96-well microplate.
This technique is the same in concept as the traditional plate incorporation method, with bacteria being added to a reaction mixture with a small amount of histidine, which allows the bacteria to grow and mutate, reverting back to being able to synthesize their own histidine. By including a pH indicator, the frequency of mutation is counted as the number of wells out of 96 which have changed color (caused by a drop in pH due to metabolic processes of reproducing bacteria). As with the traditional Ames test, the sample is compared to the natural background rate of reverse mutation in order to establish the genotoxicity of a substance. The 96-well plates are incubated for up to five days, with mutated (yellow) colonies being counted each day and compared to the background rate of reverse mutation using established tables of significance to determine the significant differences between the background rate of mutation and that for the tested samples.
The fluctuation method is comparable to the traditional pour plate method in terms of sensitivity and accuracy, however, it does have a number of advantages, namely, allowing for the testing of higher concentrations of sample (up to 75% v/v), increasing the sensitivity and extending its application to low-level environmental mutagens.
The fluctuation method also has a simple colorimetric endpoint; counting the number of positive wells out of a possible 96 wells is much less time consuming than counting individual colonies on an agar plate. Several commercial kits are available. Most kits have consumable components in a ready-to-use state, including lyophilized bacteria, and tests can be performed in a non-specialized laboratory and the only equipment necessary are a 37 °C incubator and a multichannel pipette.
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