Abstract
The ability of the Ames test to determine which compound: Tobacco or Diet coke, would meet Ames definition of a mutagen was investigated. Six Petri dishes were created for the Spot-Overlay Assay, all of which contained a different strain of Salmonella Typhimurium, the two potential mutagens, as well as Tryptic Sox Agar (TSA), Davis Minimal Agar (DMA), and our positive and negative controls. The Spot-Overlay Assay showed that Diet Coke was more mutagenic. After the Spot-Overlay Assay, the full Ames test was performed. Six Petri dishes were also made, containing just the more mutagenic of the two compounds, Diet coke at different amounts, ranging from 50?µl, 100?µl, 200?µl, and 400?µl, as well as everything that was in the Spot-Overlay Petri dishes. It was concluded that neither compound met Ames definition of a mutagen.
Introduction
The Ames test is a quick and cost-efficient was to detect the mutagenic properties in compounds developed by Dr. Bruce Ames. Today??™s environment is surrounded by mutagens, which are chemicals that cause mutations in DNA by changing the nucleic acid sequence, 90% of which are carcinogens. Keeping this in mind Dr. Ames developed the Ames test (Gabor, Pesthy, and Bosworth 2009). The Ames test is used in genetic testing, environmental monitoring, as well as, testing for potential carcinogens (Abstract 1). Dr. Bruce Ames defines a mutagen as any chemical agent which induces twice the number of mutations that would occur spontaneously. Among scientist, Salmonella most widely used in the Ames test due to its ability to spot mutagenic substances through their ability to revert the histidine auxotroph of the Salmonella Ames bacteria. This particular mutant strain of Salmonella had three distinct characteristics: lacks the ability to produce histidine, lacks the DNA excision repair mechanism and has an easily penetrable membrane. Another important aspect of the experiment was the Spot-overlay Assay, which detected between the two compounds which met Ames Definition of a mutagen, quickly as well as cheaply.
Our experiment contained Tryptic Soy Agar (TSA), Tobacco and Diet Coke. TSA proved to be a staple in both the Spot-Overlay test and the full Ames test. Tryptic Soy Agar is a gelling agent that contained just enough histidine to allow the mutant Salmonella strains to grow for three generations. Tobacco is a known mutagen aside from being a carcinogen. Research shows Tobacco to be carcinogenic to a variety of animal species and tissues (Wynder and Hoffman 1968). Diet Coke contains Saccharin which is an artificial sweetener that has undergone much research to test its carcinogenic properties. During the sixties many carcinogenic bioassays were performed on rats as well as other rodents. The results of these bioassays showed that rats exposed to diets wit 7.5% sodium saccharine from conception to death had an increase in bladder cancers (Arnold 1983).
The Spot-Overlay Assay:
The potential mutagens used in this study were Diet Coke and Cigarette Tobacco, where the tobacco was extracted from the cigarette and brewed into a tea. We tested the mutagenicity of the potential mutagens using a variation of the Ames test called the Spot-Overlay Assay. The Spot-Overlay test required soft sugar tubes (2mL) containing Tryptic Soy Agar (TSA). The tubes containing the TSA were autoclaved for sterilization and maintained at a liquefying temperature of 47?C to keep the Tryptic Soy Agar from gelling prematurely. After the TSA tubes were made, 6 pre-made Petri Dishes each containing Davis Minimal Agar, which contained no histidine, were obtained and divided into four equal quadrants. The sections were labeled potential mutagen 1 (PM1), potential mutagen 2 (PM2), positive control and negative control. Once the Petri dishes were obtained, using a micropipette 50?µl of our positive control (UV+) was added to 2ml of TSA, swirled to mix. Then we withdrew 50?µl of the overlay mixture and quickly discharged the bacteria in the 1st quadrant of our Petri dish. The same thing was done for our negative control (UV-) and then it was discharged in to the 2nd quadrant. There were three strains of mutated Salmonella: TA 1535, which produced the missense mutation, TA 1537, which had a frameshift mutation (deletion of one nucleotide), and TA 1538, which had a different frameshift mutation (addition of one nucleotide). Each of our mutated strains had two trials meaning they were added to two Petri dishes. After the positive and negative controls were added to the Petri dishes, we made the quadrants for our potential mutagens. For quadrant 3, we pipetted 50?µl of tobacco tea into 2ml of TSA, swirled to mix. Next, we pipetted 50?µl of our mutant strain into the TSA/Tobacco mixture. Finally we extracted 50?µl of the overlay mixture to eject into quadrant 3. The same thing was done for diet coke and added to quadrant 4. After our Petri dishes were complete, we taped the Petri dish halves together to avoid spilling the bacteria. The Petri dishes were left alone for 20 minutes to allow the overlay mixtures to gel. After they gelled, the Petri dishes were inserted into a 37 ?C incubated for 24 hours before removing them to the 4 ?C refrigerator to slow colony growth.
Results
Spot-Overlay Assay:
PM 1 tested the mutagenicity of our potential mutagen, tobacco and PM 2 tested the mutagenicity of our potential mutagen, Diet Coke. Neither of the potential mutagen met Ames definition of mutagen, because our negative control produced more colonies than expected. When it came to the different strains of Salmonella, TA 1538 was the only strain that gave us viable results, meaning results that could be quantified.
Figure 1. The number of colonies recorded for each trial of potential mutagens with different mutant strains of Salmonella Typhimurium. The colonies were obtained using the Spot-Overlay assay which had two trials for the potential mutagens. The trials contained 50?µl of each of the three mutant strains of Salmonella: TA 1535, TA 1537, and TA 1538. Strain 1535 produced no visible colonies, TA strain 1537 had to many colonies to count, and TA 1538 was the only strain that allowed for colonies to be recorded.
Figure 2. Number of Colonies that emerged from the different concentrations of Diet Coke. As we added a high concentration of mutagen the colonies increased. The mutagen met Ames definition of a mutagen between 200?µl and 400?µl.
{draw:frame} The concentration with the highest amount of colonies present was 400?µl. From 0 ?µl to 200 ?µl there was a steady increase in the amount of colonies in diet coke and then a much larger increase of colonies from 200?µl to 400?µl.
Discussion
The purpose of the experiment was to use the Spot-Overlay and Ames tests to determine which compound, Tobacco and Diet Coke, presented with different mutant strains of Salmonella Typhimurium met Ames definition of a mutagen. When doing the Spot-Overlay Assay experiment, we expected to find that Tobacco would meet Ames definition of a mutagen considering Tobacco is a known carcinogen among society. Our findings didn??™t support our initial expectations because Diet coke proved to have higher mutagenic properties than Tobacco. Our data supported our Spot-Overlay null hypothesis which stated, the addition of Tobacco will not meet Ames definition of a mutagen, while dispensing our alternative hypothesis which stated that the addition of Tobacco would meet Ames definition of a mutagen. Neither of our compounds met Ames definition of a mutagen because the colonies the amounted from the negative control (UV-) were much higher than expected, so Diet coke just proved to have higher mutagenic properties. When Diet Coke proved to be more mutagenic the full Ames test was done with the expectations that it would support our alternative hypothesis which stated that Diet Coke would meet Ames definition of a mutagen. Our data supported this. Diet coke became a mutagen between 200?µl and 400?µl. Sometime between 200?µl and 400?µl the amount of colonies doubled that of the colonies present in our negative control.
When doing this experiment, we encountered some problems when it came to dispensing the chemical mixtures onto the Petri dishes. When we pipetted some of the Tobacco mixtures air bubbles were present which could have caused less than expected amounts of colonies. A potential to this solution would be to make sure no air bubble are present in any of the mixtures to allow for the best possible results. Another problem encountered came from counting the colonies that presented on the Petri dishes. Many of Petri dished exhibited far too many colonies to count.
To further investigate would yield many interesting possibilities. One experiment could have more trials in order to stabilize any variations in data, as well as, a shorter incubation time, hopefully taking away from the overgrowth of many of the colonies.
Works Cited
Arnold, D. L. “Two-Generation Saccharin Bioassays.” JSTOR. JSTOR, Apr. 1983. Web. 06 Dec. 2009. http://www.jstor.org.libproxy.txstate.edu/stable/3429532 .
Gabor, Caitlin R., Carolyn Pesthy, and Anthony B. Bosworth. Laboratory Exercises for Functional Biology. Stripes, 2009. Print.
Margolin, Barry H., Norman Kaplan, and Errol Zeiger. “Statistical Analysis of the Ames Salmonella/Microsome Test.” JSTOR. JSTOR, June 1981. Web. 06 Dec. 2009. .
Wynder, E. L., and D. Hoffmann. “Experimental Tobacco Carcinogenesis.” JSTOR. JSTOR, 22 Nov. 1968. Web. 06 Dec. 2009. http://www.jstor.org.libproxy.txstate.edu/stable/1725511 .
Heather30 | Heather Scott | Social Media Today
