2 Sample Test Kit, if tested in 5 strains in triplicates, 5 doses, +/- S9, negative, positive controls, sterility controls for buffer and S9. Strains, media, ampicilline, PB/NF induced rat liver S9 and controls (4-NQO, 2-NF, 2-AA, ) are included in the kit. S9 cofactor solution and plastic ware are not included in the kit.
Strains :
TA98, TA100, TA1535, TA1537, E.coli WP2 uvrA, [pKM101]
Positive Controls :
2-Nitrofluorene for TA98, 4-Nitroquinoline-N-Oxide for TA100, N4-Aminocytidine for TA1535, ICR191 for TA1537, 2-Aminoanthracene for all strains with S9
Scientific Background
Salmonella strains with specific genetic modifications are used in the Ames Test, they are unable to grow without the amino acid histidine (his- genotype). Only Salmonella strains that have undergone a genetic mutation, i.e. a mutagenic reversion to histidine prototrophy can survive and grow in absence of histidine (his+ genotype). Thus, only in the presence of mutagenic compounds, an increase in back-mutations occur to this his+ genotype. The Salmonella colonies which are growing on agar plates or in liquid media, are called “revertants”. An increased number of revertants in relation to the strain-specific spontaneous mutation rates obtained with the solvent control is a clear indication for a mutagenic test substance. See also OECD TG471. Many mutagenic compounds are only mutagenic upon metabolization. The Salmonella strains which do not have a metabolic system are therefore exposed to a test compound in the presence of microsomal fraction S9 of a rat liver homogenate.
Test Principle
Bacteria are exposed to different concentrations of a test sample (11 mg / strain), as well as a positive and a negative control in a medium containing limited quantities of histidine (S. typhimurium) to support approximately two cell divisions. The cultures in each condition (negative control, test samples and positive controls) are poured in 6 well plates. The cells are exposed to the compound in absence and presence of S9. Rat liver post mitochondrial S9 fraction mimics the effect of the chemical after metabolization. Within three days, cells that have undergone a genetic reversion event to amino acid prototrophy will grow and form colonies. The revertant counts will be compared to those grown in the solvent (negative) control wells. A dose-dependent and significant increase in the number of revertant colonies upon exposure to test sample relative to the solvent controls indicates that the sample is mutagenic. A sterility plate for S9 and buffer is used a contamination control.
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