The Spot-Overlay Ames Test was used in the lab to find the mutagenesis of Diet Coke and ThermaFlu. Along with these substances three mutant strains of salmonella were also tested. TA 1535, TA 1537, TA 1538 all lacked the ability to grow the amino acid histidine unless reverted back by the potential mutagens. After the first week of testing, results showed that both of the potential mutagens, Diet Coke and ThermaFlu were in fact mutagenic. This was established if colonial growth was at least twice as much as that of the negative control. For the continuation of the experiment, Diet Coke and the TA 1535 strain of S. thyphimurium were chosen to continue experimentation for the second week. Testing commenced once both substances had been mixed into six new DMA plates. The purpose of this experiment was to see if different amounts of Diet Coke would increase colony growth. Results for this week showed that at 100ul of Diet Coke, colony growth was at its peak, but as the concentration of Diet Coke kept increasing, colony growth stopped. In conclusion, the potential mutagens tested in the lab proved to be mutagenic, and as the concentration of the mutagen was increased, colony growth would follow until it leveled off. INTRODUCTION
The use of the Ames test is based on the assumption that any substance that is mutagenic may also turn out to be a carcinogen, which causes cancer. “Salmonella / microsome test is the most popular of the bacterial test system. It detects mutagenic substances via their ability to revert histidine auxotrophs of S. typhimurium to wild-type.” (Ames et al., 1973 ; Maron and Ames, 1983; Hofnung and Qullardet, 1986) The trials that will be held in this lab will be tested under the Spot-Overlay Ames Test. It is a widely used technique for screening potential carcinogens by testing for mutagenesis of bacteria. It relies on the observation that the most common cause of cancer is somatic mutations brought about by DNA damage. It was first developed by Dr. Bruce Ames in 1971, and gave researchers a faster and less expensive way to get results. “This assay uses a set of histidine-requiring strains of the bacterium Salmonella typhimurium to detect mutations induced by a test agent.” (Bassi, Lopez, L.C., Moretton, J.). The bacteria used in the experimentation were three mutant strains of S. typhimurium that carry a defective mutant gene making them unable to synthesize the amino acid histidine. The TA 1535 strain had a base substitution that produced a missense mutation in the gene coding for the first enzyme of histidine synthesis. The second strain, TA 1537, displayed a frameshift mutation where one nucleotide was deleted. The third and last strain, TA 1538, had a frameshift mutation where one nucleotide was inserted. (Gabor, C.R., Pesthy, C.). These strains also have cell walls containing defective lipopolysaccharide layers, which allow chemicals to seep into the cells easily. (Gabor, C.R., Pesthy, C.) None of the three strains of salmonella are able to produce histidine, an amino acid essential for the bacterium to grow if not provided externally. But, some types of mutations can be reversed, a back mutation, with the gene regaining its function. These revertants are able to grow on a medium lacking histidine. To start off an Ames test, an assay is carried out using strains of bacteria, such as Escherichia coli or Salmonella that already have a single mutation that cannot produce histidine. The experimental cultures are exposed to the agent to be tested while the positive control cultures are exposed to a known mutagen to confirm that there has been no contamination of the strain. If the mutation screened for has in fact occurred, dense spots in the colonies will form. “Heterocyclic amines (HCAs) formed during heating (broiling, frying or grilling) of various proteinaceous foods such as meats and fishes are well known as potent mutagens in the Ames/Salmonella assay.” (Felton & Knize 1991; Eisenbrand & Tang...
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