Ames Test - General Procedure

General Procedure

The Ames test uses several strains of the bacterium Salmonella typhimurium that carry mutations in genes involved in histidine synthesis i.e. it is an auxotrophic mutant, so that they require histidine for growth. The method tests the capability of mutagen in creating mutations that can result in a reversion back 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 (e.g. strains TA-1537 and TA-1538) or point (e.g. strain TA-1531) 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 benzopyrene, are not mutagenic themselves but their metabolic products are.

The bacteria are spread on an agar plate with a 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.