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Introduction to Bacterial Mutation Test - Ames Test This test makes use of special strains engineered by Bruce Ames and collaborators. A typical assay will use four strains of Salmonella typhimurium that are incapable of synthesizing histidine, and one strain of Escherichia coli incapable of synthesizing tryptophan. Each of these strains contains a slightly different mutation which deactivates a gene coding for an enzyme required to synthesize a vital amino acid. These strains cannot grow in normal culture medium and have an absolute requirement for supplementation with the required amino acid. Should the gene be caused to revert to an active version, the bacterium will be able to grow in the absence of the amino acid, as would a wild type bacterium. The resulting colonies are referred to as revertants.Certain mutagens attack specific bases. For instance, a high proportion of cytosine to thymine transitions may occur. The strains in this test have different mutations are some are more sensitive to basepair substitutions, or frameshift mutations. Another important feature of these strains are genetic defects that increase membrane permeability. A positive result is claimed in the compound causes a substantial does-related increase in colony counts (usually twice the spontaneous rate) on two occasions. Pour Plate Method If possible, the test compound is dissolved and diluted in an inert solvent. Aqueous solvents are preferred, but when this is not possible DMSO and ethanol are frequently used. The solution is mixed with an aliquot of the bacterial culture and with culture medium containing agar, called top agar. The mixture is poured into a Petri dish containing a layer of pre-set medium called bottom agar. The Petri dishes are incubated at 37°C for up to 72 hours.S9 Mix Many chemicals are not mutagenic or carcinogenic, but their metabolites are. These metabolites are usually generated by liver enzymes. In order to test the metabolites of the chemical under investigation, a crude preparation of enzymes is obtained from the homogenized liver of rats previoiusly treated with Aroclor to enhance liver enzyme levels and activity. This preparation is known as the S9 fraction and contains a wide range of enzymes, to which enzyme cofactors are added. Typically, S9 mix is added to the bacteria just prior to addition of the test substance solution. Two sets of plates are prepared, one containing S9 mix and another with buffer.Pre-Incubation Method This method differs from the pour plate in that the S9 and the test substance are pre-incubated for 30 minutes at 37°C prior to addition of the culture medium.In both the pour plate and pre-incubation methods, the top agar culture medium contains just enough of the essential amino acid for the bacteria to divide a few times resulting in a background lawn of minute colonies. Any bacteria that mutates, either spontaneously or as a result of treatment with the test substance will carry on dividing and form a visible colony. Colonies are counted using a digital image analyzer. The spontaneous mutation rate depends primarily on the bacterial strain but is re-assessed in each experiment using a concurrent control culture which has been treated with the solvent only. The test substance is dosed at a range of concentrations but is only assessed at the five highest levels below the toxic level or at five levels up to the standard limit of 5000 µg/plate if nontoxic. As with all genotoxicity tests, concurrent solvent/vehicle and positive controls are included in each experiment. Toxic effects result in poor growth of the background lawn or a substantial reduction in the spontaneous mutation rate. Triplicate plates are used at each experimental point and the test is normally performed twice. If the first test is negative, the second test normally employs slightly different conditions, perhaps using a different metabolizing system, pout pour plate int he first test, and pre-incubation in the second. 
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