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LAB REPORT OF 7 POSSIABLE UNKNOWN BACTERIA (Proteus vulgaris, Staphylococcus epidermis, Bacillus subtilis, Escherichia coli, Streptococcus agalactiae, Moraxella catarrhalis, or Enterobacter aerogenes UNKNOWN #29 HARRY BROWN 6/11/2012 Dr. Ann Sylvia Microbiology-Bi 234 Spring 2012The purpose of this lab was to determine an unknown bacteria by preforming different test to the unknown and other known bacteria as controls for the testing. Determining unknowns is important in the process of treating different genes of bacteria when they invade the body. I choose unknown # 29, which was prepared by our teacher. It was in broth form and from there nutrient agar slants where used to determine what the optimal growth was for the unknown bacteria. After inoculating two agar slants with the unknown bacteria, one was incubated at 25 degrees Celsius, and the other at 37 degrees Celsius. These two agar slants where incubated for 48 hours and then examined to determine morphology of the bacteria. After that, one tube was chosen to be the reserve stock specimen, which was placed in the refrigerator to be examined at a later date for further growth, and the other was used as the working specimen. The reserve stock specimen had no further growth once placed in the refrigerator which shows that the unknown do not grow in psychrophilic conditions. Gram staining was the next test to be preform on the unknown by using the culture that was grown and or the broth that we first were given of our unknown. Gram staining is an essential step in narrowing down the possibilities of the unknown bacteria. Bacteria are either Gram + or Gram –, which once that could be determined; other biochemical test could be performed to further narrow down the possibilities of the unknown. For example:Test used to identify Gram positive bacteriaTest used to identify Gram negative bacteriaCatalase testOxidation testMannitol Salt agarSugar broths with Durham tubesBlood agar platesUrease testStarch hydrolysis testSimmon’s Citrate agarNitrate brothNitrate brothCoagulase testSulfur Indole motility media (SIM)Motility agarMotility agarCAMP testKliger’s Iron agarSpirit blue agarMethyl Red/ Voges-Proskauer (MR/VP)Bile Esculin AgarMacConkey agarAfter gram staining the unknown #29, twice, with cultures from both original broth and nutrient agar slant, it was determined to be a gram – bacteria. With both gram stains performed, the bacteria seemed to resemble both coccus and bacillus morphology. The first assumption was that it was a diplococcus gram negative bacteria, but with further examination the bacteria where similar to shorten bacillus rods which are common in two of the unknown Gram – bacteria (Esherichia coli and Enterobacter aerogenes). At this point multiple tests were performed to determine which of the two possible bacteria that had been selected from the gram staining. Table 1 has a list of the biochemical test that were performed and the significants of each test.TABLE 1 Test Reason for testGram stainingGram +/-, morphology, characteristicsOptimal growthWhat temp. is best for growth of bacteriaO/F glucose testDetermining if bacteria is fermentative and oxidative metabolismFermintation of glucose w/ Durham tubeAcid and gas productionUreaseCapability to hydrolyze enzyme ureaseHemolysisCapability to hemolysis bloodOxidaseIdentify if microorganism contains enzyme cytochrome oxidase which is important in the electron transport chain.Motility (SIM agar slant)Determine if bacteria are capable to migrate.CitrateDetermine if bacteria can use citrate as its sole carbon source.FTMOxygen requirementsMR (Methyl Red test)Capability to produce organic acidsVP (Voges-Proskauer test)Fermentation of glucose to produce 2,3 fermentation, which is a more neutral end product organic acidGelatin deepProduction of proteases, enzymes that degrade proteins.Nitrate reductionOrganisms ability to reduce Nitrate to NitriteCatalaseAbility of bacteria to produce enzyme, catalase, which detoxifies hydrogen peroxide by breaking it down into water and oxygen gas.The results of these tests are listed on Unknown chart 2 on the next pageConclusion of unknown #29Multiple tests were performed on the unknown bacteria using multiple media to determine growth of the bacteria. Grams stain was perform twice on the unknown bacteria due to the fact that the first gram stain that was preformed looked more like Gram- coccus or coccibacillis. When the first few test that were ran did not have the correct reactions for Moraxella catarrhia which is a gram- coccibacillis bacteria, a second gram stain was performed to verify the correct reaction to the gram stain. The unknown was definitely gram- but the bacteria looked more like shorten rods. With this and the prior results, tests were performed in the direction of two known bacteria that are from the same family and that have similar reactions such as optimal growth, fermentation of glucose, motility, catalase just to name a few. The two bacteria being Escherichia coli and Enterobacter aerogenes do have several significant differences for example; E. aerogenes has a positive test for Voges-Proskauer which means it carries out 2,3-butanediol fermentation were as E. coli does not, E. coli is positive for methyl red test were as E. aerogenes is usually, but not always, negative, E. aerogenes grows on a Simmon’s citrate test were as E . coli does not. When the MR/ VP test were performed, the unknown #29 bacteria had the same results as E. coli, but when the citrate test was performed the unknown #29 bacteria had a positive reaction like the reaction that E. aerogenes has. There was one test that could be performed which was a very reliable test to determine E. aerogenes vs. E. coli which is called the indole test. This test determine if the bacteria can breakdown the amino acid tryptophan into indole by using the SIM’s media which was used when determining motility of the bacteria. E. coli is a positive test, were as E. aerogenes is not. This test could not be performed due to not having Kovac’s reagent available for the testing. Further research has shown that E. coli does have natural variants that cause a positive reaction to citrate, although it is not common. The test performed with the unknown had a citrate agar tube that was half and half. The top half of the tube was bromthymol blue and the other half stayed a green color even after multiple days of incubation. For this reason and the correlation of tests performed on the unknown bacteria #29, I conclude that this unknown bacteria is Escherichia coli. ................
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