Reversion Assay

Figure 10.14 is a simplified diagram of the test procedure. A suitable organic solvent, e.g., dichloromethane, or solvent mixture (see Nielsen, 1992) is used to extract the POM from the environmental sample, the solvent is evaporated, and the residual POM is redissolved in dimethyl sulfoxide (DMSO). Serial dilutions of this DMSO solution are then added to a series of tubes, each containing agar and one of the several his ~ test strains of bacteria, e.g., TA98, which is widely used for frameshift mutagens.

The resulting solution is then overlaid onto Petri dishes containing a minimal glucose agar medium (with a trace of histidine to allow cell division) and incubated in the dark for 48-72 h at 37°C. The plates are removed from the incubator and any colonies present are counted.

A significant increase in the number of his + colonies above the background count (the number of colonies on Control Plate C when no sample is added) on Test Plate A indicates that the POM sample contains a chemical(s) that is (are) a direct mutagen with the Ames strain employed. If no direct activity is observed on Test Plate A, it does not necessarily mean there are no bacterial mutagens in the sample. As noted earlier, carcinogenic PAHs such as BaP and benz[a]anthracene are promutagens and must first be metabolized to reactive intermediates (metabolites), which then attack the bacterial DNA and cause his —> his+ reversions. Therefore, in practice, a small amount (e.g., 1-2 mg per plate) of an enzyme system is added to another portion of the original test sample, Test Plate B (+ S9), and the assay conducted in parallel with Test Plate A ( — S9).

After incubation, the spontaneous his~^>his + reversions found on background Plate C are subtracted from the colony count on Test Plate B. If a statistically significant number of colonies remain (i.e., "net revertants"), the POM sample contains one or more chemicals that are promutagens. Sam-

Add: 0.05 ml of test Sample in DMSO

Medium on plate supplemented with just enough histidine and biotin to allow a few cell divisions

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