The Ames Test
The Ames test is used to determine mutagenic activity in water extracts are component of AS/NZS 4020. Water extracts are mixed with specific bacteria and any measured change in genetic nature of the bacteria is regarded as evidence of mutagenic activity. The Ames is a reverse mutation assay.
The Ames test was developed to determine whether a chemical is a mutagen. The test assumes that if a substance is mutagenic it may also be carcinogenic. A number of mutagenic compounds first detected by the Ames test have shown to be carcinogenic in animal tests, including some hair dyes and several protein pyrolysis products produced when cooking foods.
The test is also used to determine whether mutagenic compounds are present in potable water as a result of water treatment processes. Water extracts are mixed with a test strain of Salmonella typhimurium that carries a defective (mutant) gene making it unable to synthesise the amino acid histidine from the ingredients in the culture medium. However, some types of mutations can be reversed, a back mutation, with the gene regaining its function. These revertants are able to grow on a medium lacking histidine.
Histidine requiring strains are used for the Ames test. Each tester strain contains a different type of mutation in the histidine operon (eg frameshift mutagens and base-pair substitutions). The standard tester strains contain other mutations that greatly increase their ability to detect mutagens. The tester strains are relatively non-virulent because they lack the galactose operon which is essential for making the lipopolysaccharide coating required for virulence.
Salmonella typhimurium is a prokaryote is and consequently not the ideal model of the human body, hence liver enzymes are added to the test. Liver enzymes are used as some chemicals are not mutagenic in themselves but are converted into mutagens when they are metabolised in the body.
Tester strains are grown in a specialised nutrient broth as some nutrient broths that contain proteins extracted from beef at high temperature are possibly mutagenic.
The cells are combined with water extract samples, positive controls and controls with and without liver enzymes.
Spontaneous reversion of the tester strains to histidine independence is measured in each Ames test and is expressed as the number of spontaneous revertants per plate. Spontaneous reversion for each strain is at a frequency that is characteristic of the strain.
A positive mutagenic response is measured when the mean number of revertants between the control and test extract exceeds the sum of two standard deviations. A mutgenic reponse does not necessarily mean the test material is harmful to humans, but indicates that there are substances that require further investigation.