Ames test was developed by Bruce N. Ames in 1970s to test for determining if the chemical is mutagens. Ames test it is a biological assay to assess the mutagenic potential of chemical compounds. It utilizes bacteria to test whether a given chemical can cause mutations in the DNA of the test organism.
This 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: 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 Ames test is used to test mutagenic ability of varieties of chemicals.
Principle
Ames test uses several strains of bacteria (Salmonella, E.coli) that carry a particular mutation.
Point mutations are made in the histidine (Salmonella typhimurium) or the tryptophan (Escherichia coli) operon, rendering the bacteria incapable of producing the corresponding amino acid.
These mutations result in his- or trp- organisms that cannot grow unless histidine or tryptophan is supplied.
But 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 to say that when a mutagenic event occurs, base substitutions or frameshifts within the gene can cause a reversion to amino acid prototrophy. This is the reverse mutation.
These reverted bacteria will then grow in histidine- or tryptophan-deficient media, respectively.
Method
I ) Isolate an auxotrophic strain of Salmonella Typhimurium for histidine. (ie. His-ve)
II) Prepare a test suspension of his-ve Salmonella Typhimurium in a plain buffer with test chemical (eg. 2-aminofluorene). Also add a small amount of histidine
Note: small amount of histidine is required so bacteria starts growing. Once histidine is depleted only those bacteria mutated to gain the ability to synthesize histidine form colonies.
III) Also prepare a control suspension of His-ve Salmonella Typhimurium but without test chemicals.
IV) Incubate the suspensions at 37°C for 20 minutes
V) Prepare the two agar plate and spread the suspension on agar plate.
VI) Incubate the plates at 37°C for 48 hours.
VII) After48 hours count the number of colonies in each plate.
Result Interpretation
The mutagenicity of chemicals is proportional to number of colonies observed.
If there is a large number of colonies on the test plate in comparison to control, then such chemical are said to be mutagens.
Very few numbers of colonies can be seen on control plate also. This may be due to spontaneous point mutation on hisidine encoding gene.
Application:
The practical application of Ames test is to screen chemical mutagens that causes mutation and are carcinogenic to human and animals.
It can also be used to detect the mutagenicity of environmental samples such as drugs, dyes, reagents, cosmetics, waste water, pesticides and other substances which are easily solubilized in a liquid suspension.
Merits
Simple, rapid and robust bacterial assay.
Ease and low cost of the test make it invaluable for screening substances in our environment for possible carcinogenicity.
Ames test can detects suitable mutants in large population of bacteria with high sensitivity.
Limitations
Some substances that cause cancer in laboratory animals (dioxin, for example) do not give a positive Ames test (and vice-versa)
Ames assay consists of Salmonella typhimurium strains and so it is not a perfect model for human.
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