Bacterial revertant tests

We have had many attempts to develop short term tests aimed at securing the needed Information in a shorter time and less expensively. The bacterial revertant test is clearly outstanding in this regard. This still has defects which may be amenable to correction. Improvement in fields other than mutagenesis (and cancer) has been extremely uneven, and there is no counterpart "success story." An objective of the... 

The Ames salmonella-microsome test is a principal sensitive mutagen screening test. Compounds are tested on the mutants of Salmonella typhimurium for reversion from a histidine requirement back to prototrophy. A positive result is seen by the growth of revertant bacteria (which do not require an external histidine source). A microsomal activation system should be included in this assay. The use of five different bacterial test strains are generally required. 

It induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn. 

Symbols El, antimutagenic effect on IQ (A) and I, that on Trp-p-1 (B). Control means the absence of the test compounds. The abbreviations Meth, Eth, n-Pro, and n-But denote methoxymethylene, ethoxymethylene, n-propoxymethylene, and n-butoxymethylene derivatives of 3-(alkoxy)methylene-2-thioxopyrrolidines shown in Table 5. Salmonella typhimurium TA98 was simultaneously treated with IQ (0.5 pg), Trp-p-1 (0.5 pg), and the test compounds (HMTP 130-520 pg/ml other compounds 500 pg/ml) in the presence of S9-mix for 20 min at 37° C. After 48 h incubation, the bacterial survivors and revert-ants were counted, and ratios of the numbers of revertants against those of the survivors were calculated. The ratio obtained from the control (treated with only IQ or Trp-p-1) is defined as 100% mutagenicity. A statistically significant difference between the control and the treating was determined by t-test ( (p < 0.05) and (p < 0.01). 

A), 3-[4-(methylthio)-3-butenyl]-5-isobutyl-2-thiohydantoin, (B), 3-[4-(methylthio)-3-butenyl]-5-benzyl-2-thiohydantoin and (C), 3-[4-(methylthio)-3-butenyl]-5-[2-(methyl-thio)ethyl]-2-thiohydantoin. Symbols , Salmonella typhimuriumTA98 was simultaneously incubated with IQ (0.5 pg) and the 3,5-disubstituted 2-thiohydantoins (0-500 pg/ml) for 20 min at 37° C and then cultured for 48 h at the same temperature , the bacterial strain was treated with the same amount of IQ for 20 min at 37° C, rinsed with a buffered saline to eliminate the mutagen, and incubated with the test compounds for an additional 20 min. The bacteria were cultured for 48 h at 37° C. Mutagenicity is expressed in terms of the percentage in the revertant number found with or without the test compounds, where the mutagenicity of IQ is defined as 100%. 

As with most bacterial tests, plate-counting techniques are used to determine the number of revertant bacteria. For the Ames test, plate counting requires a 48-hour growth period, plus time to count both the test plates and control plates used to monitor spontaneous reversion. 

It is essential that the bacterial strains be grown in this medium and that the test plates also be of the same composition. Failure to assure these conditions may give unreliable results, since in rich media (i.e., rich nutrient agar), the pol A" strain appears to have a selective advantage and hence the occasional pol A" revertants present in pol Aj" cultures will overgrow (see also Rosenkranz etal ). 

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