Ames test In Vitro

In this Ames test In Vitro post we have briefly explained about Ames test principle, objectives, requirements, the Ames test procedure, uses and limitations.

The Ames test devised by a scientist “Bruce Ames” is used to assess the potential carcinogenic effect of chemicals by using the bacterial strain Salmonella typhimurium.

This strain is mutant for the biosynthesis of histidine amino acid. As a result they are unable to grow and form colonies in a medium lacking histidine.

The Ames test In Vitro

The Ames test is developed by Bruce N. Ames in 1970s to test for determining if the chemical is mutagens. The Ames test is based on the principle of reverse mutation or back mutation. So, the test is also known as bacterial reverse mutation assay.

Test organism: the Ames test uses several strains of bacteria (Salmonella, E.coli) that carry mutation. Eg: A particular strain of Salmonella Typhimurium carry mutation in gene that encodes histidine. So it is an auxotrophic mutant which loss the ability to synthesize histidine (an amino acid) utilizing the ingredients of culture media. Those strains are known as His- and require histidine in growth media.

Culturing His- salmonella is in a media containing certain chemicals, causes mutation in histidine encoding gene, such that they regain the ability to synthesize histidine (His+) This is the reverse mutation. Such chemicals responsible to revert the mutation is actually a mutagen. So, this the Ames test is used to test mutagenic ability of varieties of chemicals.

Requirements

Materials

1. Tips

2. Sterile Petri plates

3. Erlenmeyer flask and beaker

4. Eppendorf tubes

5. Metal loop holder

6. L shaped spreader

Reagents

1. Monosodium Dihydrogen Orthophosphate

2. Sodium Hydrogen Phosphate 

Buffers

1. Add 3.1 g of Monosodium dihydrogen orthophosphate and 10.9 g of sodium hydrogen phosphate (anhydrous) to distilled water to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 month at 4°C.

Ames Test

Image Source: Wikipedia

Procedure

1. For histidine, isolate an auxotrophic strain of Salmonella Typhimurium. (i.e, His-ve) In a simple buffer, make a the Ames test suspension of his-ve Salmonella Typhimurium with test chemical (eg. 2-aminofluorene). Add a pinch of histidine as well.

2. Note: that only a minimal amount of histidine is required for bacteria to begin to proliferate. Only bacteria that have altered to obtain the ability to manufacture histidine establish colonies if histidine is depleted.

3. Prepare a His-ve Salmonella Typhimurium control suspension without the Ames test chemicals. Incubate the suspensions for 20 minutes at 37°C.

4. Prepare two agar plates and spread the suspension between them. Incubate the plates for 48 hours at 37°C. After48 hours count the number of colonies in each plate.

Results

The mutagenicity of chemicals is proportional to number of colonies observed. If large number of colonies on the Ames test plate is observed in comparison to control, then such chemical are said to be mutagens.

Applications

1. The Ames test is used to identify chemical mutagens that cause mutations and are carcinogenic to humans and animals. Some food additives (AF-2), flavouring agents (Safrole), and other substances are both mutagenic and carcinogenic.

2. The Ames test was developed to assess mutagens using eukaryotic cell culture, yeast cells, and animal models. Salmonella isn’t the best organism to use to evaluate mutagens in humans. Certain compounds are not originally mutagens to humans, but when digested, they become mutagens (acted upon by body enzymes). For example, sodium nitrate (NaNO3) is not a mutagen until it is converted to nitrous oxide (HNO2) in the stomach by HCL (a potent mutagen).

3. The Ames test has a high sensitivity for detecting suitable mutations in vast populations of bacteria.

4. The Ames test, not a carcinogenicity test. However, the majority of mutagens discovered by the Ames test (more than 90%) are cancer-causing.

5. It’s a reverse mutation assay for bacteria. As a result, a bacteria’s faulty gene can be transformed into a functional gene.

Limitations

1. The Ames test consists of Salmonella typhimurium strains and so it is not a perfect model for human.

2. Some substances that cause cancer in laboratory animals (dioxin, for example) do not give a positive Ames test (and vice-versa)

Further Reading

Reference