The Ames test is a bacterial mutagenicity test that uses strains of Salmonella typhimurium bacteria to determine if a chemical is mutagenic. It is based on the principle of reverse mutation, where auxotrophic mutant bacteria that cannot synthesize the amino acid histidine are exposed to a test chemical. If the chemical is mutagenic, it can induce a reverse mutation in the histidine gene, allowing the bacteria to grow in a histidine-deficient medium. The test involves mixing the chemical with rat liver enzymes before incubating it with the bacterial strains, and counting the number of colonies formed, with more colonies indicating higher mutagenicity. The Ames test is a fast, inexpensive way to screen

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AMES TEST
AMITY UNIVERSITY UTTAR PRADESH
AMITY INSTITUTE OF PHARMACY
LUCKNOW, INDIA
2018-20

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Introduction:
Ames test was brought by Bruce N. Ames in 1970 to test for determining
if the chemical is mutagens.
Because the assay doesn’t use a live model, it is inexpensive easy and fast.
Animal test for all compound is expensive and time expensive thus ames
test is used.
The Ames test is a bacterial short-term test to determine whether a
chemical is mutagenic or not. And one of the most important part about
this test is that it involves mixing of the test compound with rat liver
enzymes.
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AMES TEST

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Principle:
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.
It carries a mutation in a gene that contains histidine. It is an auxotrophic
mutant(nutritional-requirement mutant).
It loses its ability to mix histidine; a particular type of amino acid, by
using the ingredients of the culture media. They are his strain and they
need histidine in growth media.
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Once we put His-salmonella in a medium that has certain chemicals, it
will react with the medium there by causing histidine encoding gene
mutation.
Hence, it regains the ability to blend histidine causing a reverse mutation.
Reverse mutation is made possible because of the chemical mutagen.
Hence, it is safe to say that the ames test determines the mutagenic
property of a variety of chemicals.
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Figure: Colonies are formed in the plate
indicating that the substance being tested for has
mutagenic and carcinogenic properties.

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Procedure:
i ) Isolate an auxotrophic strain of Salmonella Typhimurium for
histidine. (ie. His-ve)
ii) Prepare a test suspension of his- 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.

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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.
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Application:
1) The practical application of Ames test is to screen chemical
mutagens that causes mutation and are carcinogenic to human and
animals.
2) Isoniazid; an anti TB drug is also mutagens.
3) Ames test adopted to use eukaryotic cell culture, yeast cell, as well as
animal model to test mutagens. Since, Salmonella is not a best test
organism to test mutagens for Human.
For example; sodium nitrate (NaNO3) is not mutagens until it is acted
upon by HCL in stomach to form Nitrous oxide HNO2 (a potent
mutagen).
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4) Ames test can detects Suitable mutants in large population of bacteria
with high sensitivity.
5) It is test for mutagenicity not carcinogenicity. However, most of the
mutagens (more than 90%) detected by Ames test are responsible to
cause cancer.
6) It is a bacterial reverse mutation assay. So the defective gene of
bacteria can be mutated into functional gene.
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