Ames test devised by a scientist “Bruce Ames” and colleagues at the University of California in Berkeley, is a method for evaluating mutagenic effects of chemicals, drugs or implant device. This test is used to assess the potential carcinogenic effect of chemicals by using histidine auxotrophs of Salmonella typhimurium (which lacks the ability to biosynthesis histidine because of a mutation in an essential gene) or tryptophan auxotrophs of Escherichia coli.
Not every mutagen is also a carcinogen BUT many mutagenic chemicals are also carcinogenic (capable of causing cancer in humans or other animals), knowing that a compound is mutagenic to bacteria is a warning of possible danger.
Mutant Salmonella strains are unable to grow and form colonies in a medium lacking histidine. When these mutant bacterial cells are treated with chemicals having mutagenic effect, those chemicals may cause a reversal of mutation in bacterial cells enabling them to grow on a media lacking in histidine. Chemicals found to be mutagenic in Ames test can be tested for their potential to induce carcinogenic effect in animals.
Histidine auxotrophs of Salmonella typhimurium are spread on the surface of a culture media lacking histidine in the presence of the suspected mutagen. As the medium does not contain an essential nutrient, histidine in this case, auxotrophic/mutant strains cannot grow, even if a very large population of cells is spread on the medium. However, if a particular bacterial strain became able to revert back to wild type (known as back mutants or revertants), they will grow and form colonies. If the reversion rate is increased in the presence of suspected mutagen (with the evidence of the appearance of more number of colonies in the presence of suspected mutagen) presence of mutagenicity in the test chemical is confirmed. Higher the number of such colonies, higher is the mutagenic potential of the suspected mutagen.
Compounds identified as mutagenic in the Ames test are further tested for their potential carcinogenic properties by using animal models.
Ames test can be carried out using tryptophan auxotrophs of Escherichia coli but in that case, medium lacking amino acid, Tryptophan should be used.
- Inoculate a single fresh colony of standard strains of Salmonella typhimurium in nutrient broth and incubate for 10-12 hour at 37°C.
- Prepare different concentrations of the test compound and activate using enzymes from rat liver*.
- Prepare minimal glucose agar (MGA) plates freshly before use and label it.
- Spread the nutrient broth (from 1) across the minimal glucose agar plates using an L-shaped glass spreader.
- Dispense the activated complex in a paper disc placed on the centre of the minimal glucose agar plates. For negative control instead of activated complex, distilled water is dispensed over the paper disc.
- Cover the Petri plates with sterile aluminium foil to protect the testing sample from photoreactive substances.
- Incubate the plate at 37°C for 24/48 hours.
- Observe the development of bacterial colonies around the paper disc in both test and control agar plate and compare them.
*Note: Many carcinogens in their natural/inactive forms are not carcinogenic or mutagenic, but undergo modifications in the liver by the liver enzymes (called mixed function oxygenases) that convert them into active substances. Activated forms of these compounds are highly reactive and thus mutagenic toward DNA. So, liver enzyme preparation is added in the Ames test to mimic liver metabolism.
- Detection and screening of potentially hazardous (mutagenic/carcinogenic) chemicals present in the environment, food or drugs.
- Commonly used by the pharmaceutical industry to test various drugs and chemicals before using them for clinical trials.
References and further reading
- Bio-protocol: Microbial Mutagenicity Assay: Ames Test