Using Kanamycin Resistance Bacteria to find the Sources of Contamination of Three Chicken Farms Introduction:
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 AL’s, and Clucky’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 won’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 groups’ 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...
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