Ames Spot Overlay Test Essay Example
Ames Spot Overlay Test Essay Example

Ames Spot Overlay Test Essay Example

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  • Published: October 1, 2017
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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. hyphimurium 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 d

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ifferent 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. INTRODUCTIONThe 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

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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 ynthesize 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 1993; Stavric 1994) This experiment demonstrates the positive associations between higher consumption of well-done red meat and risk of colon cancer (Probst-Hensch et al. 1997; Sihna et al. 1999 and 2001), breast cancer (Zheng et al.

1998 Sihna et al. 2000) lung cancer (Sihna et al. 1998) and gastric cancer Ward et al. 1997; De Stefani et al. 1998) Similarily, in this paper, we investigated the presence of Salmonella and common mutagens such as Diet Coke, and ThermaFlu to see which of the strains of salmonella used in lab could possibly revert the bacteria back into its wild-type.

One hypothesis was that the potential mutagens, Diet Coke and ThermaFlu, were in fact mutagens. With that in mind, the reason for carrying on this experiment was to test and see if our hypothesis was correct. As the experimentation proceeded, data showed that Diet Coke was the best potential mutagen, and was chosen to continue testing. With this substance being the focal point of the second experiment, the goal was to

test different amounts of Diet Coke with the TA 1535 strain of S. typhimurium.

Hypothesis was that as the concentration of Diet Coke was increased, colony growth would raise as well.Besides testing all of the substances, the experimentation would help build a better understanding of how and why it is that the bacterium Salmonella converts back into its wild-type, and how it is that amino acids carry out this job. MATERIALS & METHODS To commence the experimentation for the first week, some of the materials that will be needed are the two potential mutagens, Diet Coke (100% concentration), and ThermaFlu (100% concentration), as well as Sodium Azide and the three strains of S. typhimurium TA 1535, TA 1537 and TA 1538. Secondly, these substances shall be placed into a DMA plate containing 1mL of agar mixed with histidine.

The DMA plates have a limited amount of nutrients so when salmonella is reverted back into its wild type strain there won’t be a lot of excess growth. The plates should be kept upside down till the experimentation is ready to begin in order to keep the moisture from fogging up the entire plate. The DMA plate should be divided and labeled into four sections, pie-shaped, with the name that corresponds to each chemical being tested. The tubes containing the soft agar are to be held in a water bath of 55 degrees Celsius to keep them liquified.

Table 1. shows the amounts of substances placed into each overlay tube.Table 1. Represents the amounts and volumes of each substance mixed into an overlay tube for the first week. Section 1Section 2Section 3Section 4 Chemical:N/aSodium AzideDiet CokeTherma Flu Chemical (ul)N/a202020 Salmonella

typhimurium (ul)100100100100 DMA Overlay (ml)1111 After the correct amounts of the substances are mixed, 50 ul of each mixture is to be placed into the corresponding sections of the DMA plate, creating an aliquot, or puddle. The plates should then be placed into in a 37-degree Celsius incubator for 72 hours.

Once the incubation time is complete, the lates should be observed and bacterial growth should be recorded. Any growth happening in the negative control will be spontaneous, and should be minimal. The potential mutagens will be considered mutagens if they have double as much growth as the negative control. On the other hand, the positive control should, and will have the most growth because that is a guaranteed true mutagen.

Once the results for the first week have been obtained, they will show whether or not the potential mutagens were in fact mutagenic. After the data has been collected, the best potential mutagen should be tested to see if more growth would happen.In this lab’s case, different amounts of Diet Coke were tested with the TA 1535 strain of salmonella. Strain TA 1535 was used because it showed the best results and clear amounts of bacterial growth from the first week’s experiment.

To carry on testing for the second week, new DMA plates would be needed to cultivate the bacteria with Diet Coke. Table 2. shows the amounts of substances placed into the overlay tubes. The different amounts of Diet Coke, agar, and TA 1535 strain were mixed in the overlay tube before all of the substance was poured onto the DMA plate.

The DMA plates were then placed into the incubator for 72 hours at 37 degrees

Celsius. Table 2. Represents the amounts and volumes that were placed into the overlay tubes for the second week of experimentation. DMA Plate123456 ChemicalNegative ControlPositive ControlDiet CokeDiet CokeDiet CokeDiet Coke Chemical Amount (ul)05050100250500 TA 1535 (ul)250250250250250250 DMA mix (ml)555555 RESULTS Table 3.

Shows the amount of colonies (bacteria) grown in the DMA plates for the first week. Salmonella StrainNegative ControlPositive ControlThermaFluDiet Coke 153512 32 TMTC0 840 13 1537TMTC TMTCTMTC TMTCTMTC TMTCTMTC TMTC 1538192 85148 475 1001 224 As the potential mutagens were left to grow in the Petri dishes over a span of 3 days, results showed that the potential mutagens, Diet Coke and Thermaflu, did in fact turn out to be mutagenic. This was determined by looking at the growth of bacterial colonies. If the potential mutagens had more than twice the growth than the negative control then they would be considered mutagenic The results shown in Table 3. represent the number of colonial growth for each substance for the first week.In strain TA 1535 ThermaFlu had more than twice the growth of colonies than the negative control.

For another group dealing with the same strain of Salmonella, ThermaFlu did not have any bacterial growth. In the same strain, Diet Coke had more than double the growth of the negative control for both groups. Next, the bacterial strain TA 1537 had “TMTC” for each potential mutagen. “TMTC” simply meant there was too many to count. Lastly, both potential mutagens did not have more than double the growth of the negative control in the bacterial strain 1538.

The one exception was the number of colonial growth for one group testing Diet Coke. This was the only time where

growth surpassed that of the negative control under this strain. Table 4. Represents the amount of colonial growth in the DMA plates with the different amounts of Diet Coke for the second week.

DMA PlatesNegative ControlPositive ControlDiet Coke (50 ul)Diet Coke (100 ul)Diet Coke (250 ul)Diet Coke (500 ul) Number of Colonies 1 14 6 100 1 1 Table 4. represents the colony growth of the different levels of Diet Coke and the strain TA 1535 used in the second week of the experiment.In particular, the DMA plate containing 100ul of Diet Coke had the most number of colonial growth compared to the rest of the amounts, as well as the positive and negative controls. Finally, the DMA plates containing the most amount of Diet Coke had the same amount of colonial growth as the negative control. At these volumes of Diet Coke colonial growth came to a plateau. Graph 1.

Colonial Growth vs. Concentrations of Diet Coke In graph 1. the amount of S. typhimurium colonies grown under each concentration of Diet Coke are represented by the dots on the line.DISCUSSION As the results showed, Diet Coke and ThermaFlu proved to be mutagenic based on data collected from the first weeks experiment.

After this experimentation, strain TA 1535 was chosen to continue further testing on one of the potential mutagens. This particular strain of the bacteria was chosen because it was the only strain that showed precise amounts of colonial growth for the given substances. While TA 1535 showed accurate amounts of bacterial growth, table 3 shows that the bacteria strain TA 1537 had “TMTC” which means, too many to count.Moreover, this strain of salmonella could be

said to have reverted back into the wild-type strain quite fast.

A possible reason this strain showed “TMTC” for colony growth was because it was left in the incubator for an extra day. It could have been possible to use this strain if the DMA plates were left in the incubator for the correct amount of time. Next, when it comes to the salmonella bacteria strain 1538, Diet Coke was tested to be a bigger mutagen with more visible colonies than all other strains. ThermaFlu did not prove to be quite as effective when it came to reverting the bacteria back into its wild-type.

However, a lot of the data collected from the colonial growth didn’t make sense, so that automatically voided that strain. Again, both of the potential mutagens, Diet Coke and ThermaFlu, tested to be actual mutagens. These results came in agreement with the first hypothesis because both substances showed more than twice the amount of colony growth as the negative control. In the first experiment sections that were cultivating Diet Coke showed more than twice the growth of than the sections with the negative control in 75% of all DMA plates.

This information was gathered by table 3. In the same way, ThermaFlu was detected to also be a mutagen because it too, had double or more the amount of colony growth than the negative control. An exception to this would be in TA strain 1537 where ThermaFlu had “TMTC. ” In continuation with the experimentation, it was also hypothesized that if the concentrations of the potential mutagens were increased, colony growth would raise as well. Results from the second week also showed that this

hypothesis was correct.

In detail, the TA 1535 strain and Diet Coke were mixed together showing that colony growth steadily increased as the concentration of Diet Coke did, and then came to a steady plateau. One reason for this could be because the bacterium strain might have been affected by the high levels of aspartame found in Diet Coke. Some of the problems encountered throughout both weeks of the experiment were air bubbles when pipetting, and pouring the substances onto the DMA plates. This might have led to contamination of the chemicals from pollutants in the air.A solution to this particular problem is to carefully watch where the substance is being poured onto, and to get it done fast and effectively.

Covering the DMA plates in a quick timely manner will also eliminate the chances of contamination. Another problem encountered was the excessive growth of colonies in the TA 1537 strain of salmonella. This led to an unclear measure of colonies and inaccurate data. This was mainly due to the fact that the DMA plates were left in the incubator for an extra day. Nonetheless, if the DMA plates would have been incubated for the actual correct span of time, the results could have been usable.

An issue that was specifically encountered in the second experiment was the death of bacterium strains due to the large concentrations of aspartame. A way to fix this problem could have been by increasing the amount of Diet Coke in smaller increments, and showing the effect on colony growth with low concentration levels as well. In regards to future experimentation, different strains of Salmonella typhimurium could be used to get a more

varied amount of statistics. Also, Diet Coke could have been studied in a more careful manner that might have shown other factors that contribute to the growth of salmonella.

RESULTS 1. H. Kataoka, S. Nishioka, M. Kobayashi, T. Hanaoka, S.

Tsugane. © 2002 Springer-Verlag New York Inc. Analysis of Mutagenic Heterocyclic Amines in Cooked Food Samples by Gas Chromatography with Nitrogen-Phosphorus Detector. 2. E.

O ksu zog lu, N. Diril, M. Durusoy. © 2000 Springer-Verlag New York Inc. Mutagenic Effects of Plant Growth Hormones with the Salmonella/Microsome Test and the SOS Chromotest.

3. L. C. Lopez, M. D.

Bassi, J. Moretton. © 1999 Springer-Verlag New York Inc. Influence of River Water in the Detection of Cr(VI) Mutagenicity by the Ames Test.

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