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Ames Test – Introduction, Principle, Procedure
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The Ames test is a widely employed method that uses bacteria to test whether a given chemical can cause mutations in the DNA of the test organism. More formally, it is a biological assay to assess the mutagenic potential of chemical compounds.
The test was developed by Bruce N. Ames in 1970s to determine if a chemical at hand is a mutagen.
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.
A sample’s mutagenic potential is assessed by exposing amino
acid-requiring organisms to varying concentrations of chemical and
selecting for the reversion event. Media lacking the specific amino acid
are used for this selection which allow only those cells that have
undergone the reversion to histidine / tryptophan prototrophy to survive
and grow. If the test sample causes this reversion, it is a mutagen.
What Is The Objective Of Ames Test?
It is used to determine the mutagenic activity of chemicals by observing whether they cause mutations in sample bacteria.
What Is the Uses of ames test?
Ames test is used to identify the revert mutations which are present in strains
it can also be used to detect the mutagenicity of environmental
samples such as drugs, dyes, reagents, cosmetics, waste water,
pesticides
What Is the Advantage of ames test?
It is a Simple, rapid processes
Ames test can detects suitable mutants in large population of bacteria with high sensitivity.
Low cost
What Is the Disadvantage of ames test?
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.
What Is the Method of ames test?
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 (let’s say 2-aminofluorene). Also add small amount of histidine.
Ps: small amount of histidine is required for initial growth of
bacteria. Once histidine is depleted only those bacteria mutated to gain
the ability to synthesize histidine form colony. 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. The mutagenicity of chemicals is proportional to number of
colonies observed. If large number of colonies on test plate is observed
in comparison to control, then such chemical are said to be mutagens.
*Very few number of colonies can be seen on control plate also. This
may be due to spontaneous point mutation on hisidine encoding gene
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i ) Isolate an auxotrophic strain of Salmonella Typhimurium for histidine. (ie. His-ve)
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