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Ames Test

The Ames test is used to test the mutagenicity of compounds. It involves exposing different strains of Salmonella typhimurium bacteria that are sensitive to mutagens to the test compounds. If the compounds cause more bacterial colonies to grow on a histidine-deficient plate compared to a control plate without the compound, then the compound is considered mutagenic and possibly carcinogenic. The Ames test is useful for rapidly and inexpensively screening many chemicals for potential carcinogenicity.

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639 views16 pages

Ames Test

The Ames test is used to test the mutagenicity of compounds. It involves exposing different strains of Salmonella typhimurium bacteria that are sensitive to mutagens to the test compounds. If the compounds cause more bacterial colonies to grow on a histidine-deficient plate compared to a control plate without the compound, then the compound is considered mutagenic and possibly carcinogenic. The Ames test is useful for rapidly and inexpensively screening many chemicals for potential carcinogenicity.

Uploaded by

Anirudh Acharya
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© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Ames Test

Developed by Bruce Ames and his colleagues in the 1970s. Tests the mutagenicity of different compounds Is used to test many chemical rapidly and inexpensively. Uses special bacteria that are very sensitive to many mutagenic agents

Ames Test
The picture (courtesy of Bruce Ames) shows a qualitative version of the Ames test. A suspension of a histidinerequiring (His) strain of Salmonella typhimurium has been plated with a mixture of rat liver enzymes on agar lacking histidine. The disk of filter paper has been impregnated with 10g of 2-aminofluorene, a known carcinogen. The mutagenic effect of the chemical has caused many bacteria to regain the ability to grow without histidine, forming the colonies seen around the disk. The scattered colonies near the margin of the disk represent spontaneous revertants.

Characteristics of mutants strains of S.typhimurium used for Ames Test cannot synthesize histidine. are very susceptible to additional mutations because they lack the normal repair mechanisms found in bacteria. more permeable than wild-type bacteria to external chemicals, including potential mutagens.

Different mutant strains of S.typhimurium with different mutations in their DNA

TA 1535 has a base substitution that produces a missense mutation in the gene coding for the first enzyme of histidine synthesis. The mutant enzyme has a proline where a leucine is in the wild-type enzyme. TA 100 is very similar to 1535, but is also supposed to detect a different range of mutagens. TA 1537 has a frameshift mutation (deletion of one nucleotide) in a different gene than is mutated in 1535. TA 1538 has a different frameshift mutation (insertion of one nucleotide) in the same gene that is mutated in TA 1537. TA 98 is similar to 1538 but is supposed to detect more mutagens than 1538 does. TA 102 is significantly different from the others. It has an ochre mutation which means that it has a nonsense mutation. This mutation occurs in the same gene as is mutated in the strain TA 1535.

Mutations
A mutation is any change in a DNA sequence from the original sequence of nucleic acids

Spontaneous mutation are just errors that occur during DNA replication when
cells divide, there is no obvious cause on which to blame the mutation. - An average of nearly one mutation (error) in DNA every time one cell divides. - Cells have ways to repair the mutated DNA, and they usually do, but if the mistake is overlooked, the change in the DNA is carried on in future replications in the cell.

Induced Mutations
- New mutations created by treating an organism with a mutagenizing agent. - Mutations may be induced by exposure to ultraviolet rays and alpha, beta, gamma, and X radiation, by extreme changes in temperature, and by certain mutagenic chemicals such as nitrous acid, nitrogen mustard, and chemical substitutes for portions of the nucleotide subunits of genes.

- H. J. Muller, an American geneticist, pioneered in inducing mutations by


X-ray radiation (using the fruit fly, Drosophila).

Types of point mutations


Missense mutations: transitions, transversions.
Nonsense mutations: amber, ochre, opal. Frameshift mutations: deletion, insertion. .

Mutagenic Agents
These are substances, conditions and forms of energy that significantly increase the frequency of mutations. Examples of a forms of energy are ultraviolet light, x-rays, cosmic energy, gamma radiation, alpha particles, beta particles and neutrons. Examples of substances that are mutagens are nitrous acid, hydroxylamine, ethyl methanesulfonate, 5-bromouracil, celery, benzo (a) pyrene, acridine dyes, fungally contaminated peanuts or peanut butter, and many, many more.

Mode of action of chemical mutagenic agents

Alkylating agents are chemicals that donate alkly groups to other molecules. Ethyl methanesulfonate (EMS) is an example.

Intercalation agents

Compounds that can slide between the nitrogenous bases in a DNA molecule. This tends to cause a greater likelihood for slippage during replication, resulting in an increase in frameshift mutations.

Principle of Ames test


In order for any of the mutant cells to survive on a plate that lacks histidine, they must regain the ability to synthesize histidine by undergoing another mutation that corrects the original mutation that prevented the production of missing enzyme .

This type of mutation is -- back mutation or reversion.second mutation returns the mutation to wild type(his+).

Procedure
Preparing bacteria plates
Obtain pre-poured minimal agar Petri plates which contain no histidine at all. The salmonella stain will be kept at room temperature. Agar contains glucose and trace amounts of histidine already; some histidine is necessary to allow a couple of cell divisions to take place. This agar will be kept on heating blocks until it liquefies. Aseptically mix agar and bacteria and prepare the plates with bacteria stains. Work fast so that agar doesnt solidify while ur doing this and move to the next step quickly. Pour the agar-bacteria mix in a plate and spread evenly across the entire surface of the plate.

Let the plate flat on the counter for ~10-15 min facing upwards until agar hardens. The mutagens to be tested is then carefully added to the tester stains. Invert plates and place it in a 37 C incubator for 48 hours. Consequently a controlled plate must me carried out along side the test plate in same condition but without the addition of any test chemicals. After 48 hours the number of mutant bacterial colonies that appear on the test plate is compared with the number that appear on the control plate with no chemical. Safely dispose of the materials and wash your hands and forearms with soapy water immediately after taking off your gloves.

Report
If the number of colonies appearing on a treated plate is more than that of control plate, it is said to be mutagenic and the chemical used is probably carcinogenic. The mutagenicity of a substance is proportional to the number of colonies observed.

Limitations
Salmonella typhimurium is a prokaryote, -its not a perfect model organism for humans. Rat liver( S9 fraction) is used to mimic the mammalian metabolic conditions. Some compounds are not active carcinogens but can be converted into cancer-causing compounds in the body. To make the Ames test sensitive for such potential carcinogens a compound to be tested is first incubated in S9fraction that contains metabolic enzymes. However there are difference in metabolism and mutagenicity of chemicals between human and rat.

Conclusion
Ames test has been applied to thousands of chemicals and commercial products. An early demonstration of its usefulness is tested by this method. Eg:- in 1975 many hair dyes sold in United states contained compounds that were mutagenic to bacteria. These compounds were then removed from most hair dyes.

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Presented By Kushal.N

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