The Ames test is an assay that measures bacterial revertant colony formation; it was named to honor Bruce Ames, who first identified and reported the utility of this assay in detecting mutations in 1974 (Ames, 1974). The Ames assay is the most commonly used assay to predict mutagenic potential of a test compound across several scientific disciplines and industries.
The Ames test is designed to evaluate the potential of a test compound to induce reverse mutations in bacteria.
The Ames test is a bacterial short-term test for identification of carcinogens using mutagenicity in bacteria as an endpoint. It includes mammalian metabolism to activate promutagens. A high but not complete correlation has been found between carcinogenicity in animals and mutagenicity in the Ames test. The latter detects mutations in a gene of a histidine-requiring bacterial strain that produces a histidine-independent strain. The Ames test is one of the most frequently applied tests in toxicology. Almost all new pharmaceutical substances and chemicals used in industry are tested by this assay.
The use of the Ames test is based on the assumption that any substance that is mutagenic for the bacteria used in his test may also turn out to be a carcinogen; that is, to cause cancer. Although, in fact, some substances that cause cancer in laboratory animals do not give a positive Ames test, the ease and low cost of the test make it invaluable for screening substances in our environment for possible carcinogenicity.
General Procedure
The Ames test uses several strains of the bacterium Salmonella typhimurium that carry mutations in genes involved in histidine synthesis. These strains are auxotrophic mutants, i.e. they require histidine for growth, but cannot produce it. The method tests the capability of the tested substance in creating mutations that result in a return to a “prototrophic” state, so that the cells can grow on a histidine-free medium.
The tester strains are specially constructed to detect either frameshift or point mutations in the genes required to synthesize histidine, so that mutagens acting via different mechanisms may be identified. Some compounds are quite specific, causing reversions in just one or two strains.The tester strains also carry mutations in the genes responsible for lipopolysaccharide synthesis, making the cell wall of the bacteria more permeable,and in the excision repair system to make the test more sensitive. Rat liver extract is optionally added to simulate the effect of metabolism, as some compounds, like benzo pyrene, are not mutagenic themselves but their metabolic products are.
The bacteria are spread on an agar plate with small amount of histidine. This small amount of histidine in the growth medium allows the bacteria to grow for an initial time and have the opportunity to mutate. When the histidine is depleted only bacteria that have mutated to gain the ability to produce its own histidine will survive. The plate is incubated for 48 hours. The mutagenicity of a substance is proportional to the number of colonies observed.
