Monday, October 14, 2019
Using Kanamycin Resistance Bacteria Essay Example for Free
Using Kanamycin Resistance Bacteria Essay Kanamycin is a common antibacterial that interferes with bacterial growth, by inhibiting protein synthesis, and causing the mistranslation of mRNA. Kanamycin is commonly used in chicken feed to keep harmful bacteria from getting into the eggs and producing healthier chickens. Recently reports of severe gastroenteritis have been linked to eating raw or undercooked eggs. This has led to the FDA to look for possible sources of contamination. Scientists have now isolated bacteria from batches of eggs known to cause the illness, and they found that the bacteria are resistant to kanamycin. The contaminated eggs were found to have come from three different chicken farms, Acme, Big ALÃ¢â¬â¢s, and CluckyÃ¢â¬â¢s chicken farm, that are geographically separate, and are in different states. The scientists also know that there are three different genes responsible for kanamycin resistance, and that these different genes codes for a certain enzyme that alters the kanamycin molecule differently. The enzymes are located between the inner and outer bacterial membranes, and act on the kanamycin after it passes through the outer membrane. The modification of the kanamycin molecule prevents it from being taken up by the inner membrane, preventing it from reaching the ribosomes. Therefore if any bacteria present has one of the three genes for kanamycin resistance, than kanamycin wonÃ¢â¬â¢t prevent bacterial contamination (Hass C. , Woodward D. , and Ward A. , 2010. ). The purpose of this lab was to determine if there was a shared source of contamination for the three chicken farms, and to make recommendations for steps to prevent further outbreaks. The hypothesis is that all the chicken farms shared the same source of contamination. The guiding questions for the lab are what is the concentration of viable bacteria in the original samples from the three chicken farms? And what is the frequency of resistant bacteria in the original samples? Methods and Materials: This lab is broken up into four different sections. To begin section one of this lab you need to make sure that your lab area is sterile so that there is no contamination of the bacteria. Then each group gets a bacteria sample, and the letter represents which chicken farm the sample came from. Next each group should obtain six plates. Three have kanamycin, and are labeled with a K, and three unlabeled plates. Each group should then put the names of the groupsÃ¢â¬â¢ member s, date, lab section number, letter of bacteria sample, and label one of each of the three sets of plates, K versus non K, 10-2, 10-4, and 10-6. Then label three, empty, sterile, microtubules with the dilutions, 10-2, 10-4, and 10-6 that will be made. Next using sterile techniques add 990 microliters of water into each microtubule. Afterward mix the bacterial suspension by gently flicking the microtubule, as shown by your TA. Then for each dilution factor, use 10 microliters of the bacterial suspension, and use this as the starting sample to make three-fold serial dilutions. For each dilution factor make sure to keep the bacteria well suspended by flicking the tube before removing each sample, and make sure that a fresh pipette tip is used for each dilution. Then use sterile glass beads to distribute the bacteria evenly on the agar surface of the 10-6 plate by gently swirling the beads in a circular motion. Then using the same set of beads for each plate transfer the beads from 10-6 to 10-4, then 10-2. Each group should then flip the dishes upside down and stack the three dishes together. Lastly tape the stacks together, and label the tape with your group member names, and section number. The plates should be incubated for approximately 24 hours, and then placed in a cold storage room until you are ready to count the colonies (Hass C. , Woodward D. , and Ward A. , 2010. ) For section two of this lab each group will be working as one group with the other groups at your lab bench. To begin you will collect the petri dishes that you prepared before. Remove the tape from the stacks and examine your plates for colonies. Each lab bench will have six tubes containing PCR mix. The orange, blue, and yellow tubes will have primers only, and will have some colonies added to them. The red, green, and pink tubes will have primers with the control plasmid so no colonies will be added to these tubes, as they will be used as positive controls. Second identify and number the antibiotic resistant plates labeled Ã¢â¬Å"KÃ¢â¬ which have colonies growing on them. Third, use a white pipette tip and dip it into a colony on the plate labeled number one, and dip that into the orange tube, and close the cap. In turn repeat this step using a new pipette tip each time for colonies two and three, in the blue tube, and the yellow tube respectively. Finally load all six tubes into the PCR machine, and you TA will help you run them. While the PCR machine is running each group can begin working on section three of the lab. To begin with each group will look at the bacteria plates, and count the number of colonies. If the colonies are distributed evenly in the plate then you can divide the plate into four quadrants and just count one quadrant and multiply that number by four. However if they are not, you must count all of the colonies. If there is more than 800 colonies on a plate record the number as lawn growth. Finally record the number of colonies for each plate and use these numbers to calculate the concentration of viable bacteria in the original sample, and the frequency of antibiotic resistant bacteria in the sample. In the last section for the lab each group will be using gel electrophoresis to run their bacteria DNA. Each lab bench will make, and run one gel electrophoresis per table. Once the gel is ready to be loaded, load five microliters of PCR DNA ladder into the first well, as a standard. This should be found in a tube in and ice bucket. Next add two microliters of 6x loading dye into the six sample tubes. The dye should be mixed in thoroughly by gently pipetting up and down after adding the dye. Following that you should load fifteen microliters of each sample into the following six wells. Since lane one will have the DNA ladder lane two starts the samples using the orange tube, then the blue, yellow, red, green, and pink tubes go into lanes three, four, five, six, and seven respectively. Once all the samples are loaded turn on the electrophoresis machine, and wait until the bromophenol blue tracking dye has migrated at least half the length of the gel. Lastly using gloves carefully remove the gel and carry it to the UV light box to view, and photograph the gel (Hass C. , Woodward D. , and Ward A. , 2010. ). Results: The results of this experiment show that the farms do not share the same plasmid that carries the antibiotic resistance gene. Table one shows the individual group data for the concentration and frequency of the antibiotic resistant bacteria. Table two shows the overall frequency of antibiotic resistant bacteria for code A which was taken from Acme Farm, for the section. Table three shows the section data for the overall frequency of antibiotic resistant bacteria, for all three farms, and which plasmid corresponds to that bacteria code. The results showed that for code A which was Acme farm, their resistant bacteria carried plasmid A. For code B, Big AlÃ¢â¬â¢s, and code C CluckyÃ¢â¬â¢s chicken farm, their resistant bacteria carried plasmids B, and C respectively. Figure A shows the gel electrophoresis picture for the bacteria code A. This figure shows that code A does in fact carry the plasmid A. Discussion:Ã Based on our data we can conclude that the three farms had different sources of contamination because the three farms all had different strands of resistant bacteria, as shown by the gel electrophoresis pictures from each farm. Figure one shows the plasmid that correlates to bacteria code A which came from Acme Farm. Based on the results shown in table 3 we learn that our hypothesis that all three farms shared a contamination source was wrong. The three farms each carry a different plasmid that is resistant to the antibiotic so their contamination sources must be different. The overall trends from this data are that there was an overwhelming amount of bacteria in almost every case for the 10-2 dilution factor, and the frequencies of viable resistant bacteria were low so that means there was not a lot of resistant bacteria. Some possible sources of error were the DNA samples were not placed properly in the gel so the electrophoresis was not as reliable, or a fresh pipette tip was not used for each dilution which would have messed up the dilutions. Additional experiments that can be done are use three different farms from the previous experiment and see if the same results are obtained. Our research was significant because it showed that there was not a common source of bacteria for the farms, and that bacteria can have multiple strands of DNA that could be resistant to an antibiotic. The significance of the guiding questions was to give practice calculating the concentrations and frequencies of bacteria. Doing these calculations also gave us an indication of how reliable or data could be based on the amount of viable specimen. Recommendations for the farms would be to figure out where the bacteria is coming from and find a way to keep it from the chickens, or to use a different antibiotic that has less resistant strands.