Microbiology Unknown Paper.

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Lahela Correa
Microbiology 140
Matthew Tuthill
Unknown Lab Report

There are many reasons for knowing the identity of microorganisms. The reasons range from knowing the causative agent of a disease in a patient, so as to know how it can be treated, to knowing the correct microorganism to be used for making certain foods or antibiotics. This study was done by applying all of the methods that I have been learned so far in the microbiology laboratory class for the identification of an unknown bacterium. The purpose of this lab was to identify two unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible bacteria based on specific biochemical characteristics. Whether the tests performed identified specific enzymatic reactions or metabolic pathways, each was used in a way to help recognize those specifics and identify the unknown cultures. The differential tests used to identify the unknown cultures were Each person in the class was given a heterogeneous broth mixture of 2 microbes. This mixture was streaked onto the following plates: TSA, BAP, MAC, CNA some of which are selective for gram (+) or gram (-) microbes, while others aren’t specific. On the nonspecific TSA plate a large hazy white colonies was formed. On the other hand, there were also small orange colonies fewer in growth. Isolation onto a BAP would allow for a pure culture of just gram (+) which would be used to carry out the rest of the differential test. Isolation onto TSA/ MAC allowed for the growth of gram (-) and would be used for further test. Procedure:

The catalase test was performed only on gram (+) bacteria, as this test would not help in differentiating the gram (-) bacteria because all of the possible unknown gram (-) bacteria were catalase positive. This test is used to detect the presence of catalase, which helps to breakdown toxic hydrogen peroxide produced from the transport of high-energy electrons directly to oxygen. Catalase is tested for by adding hydrogen peroxide to the culture, and looking for the production of gas bubbles. If gas bubbles appear immediately, the culture is catalase positive. However, if no bubbles are observed, the culture is negative for catalase. Results yielded positive. The citrate test was performed only on the gram (-) bacteria and was used to determine the ability of an organism to use citrate as its sole carbon source. Bacteria that possess citrate-permease are able to do this. The medium used for this test contains bromthymol blue dye, which is green at neutral pH, but blue at a basic pH. Bacteria that is able to survive and utilize the citrate, convert ammonium phosphate to ammonia and ammonium hydroxide, which alkalinize the agar, turning it blue. Thus, the conversion of the medium to blue is a positive citrate test. No color change is negative. However, I believe that the citrate test had failed and should have been positive. There was no growth of bacteria in tube, it just didn’t seem to match up with what results I already had from acquired test. The urease test was performed on the gram (+) and (-) bacteria, testing for the breakdown of urea. Urea hydrolysis to ammonia by urease-positive organisms will overcome the buffer in the medium and change it from orange to fuchsia. Rapid urease-positive bacteria will turn the broth pink within 24 hours. Urease-negative bacteria will produce no color change or turn the broth yellow. The results for both organisim yielded yellow which indicated negative reaction. A bacitracin test was performed on the gram (-) bacteria, testing for resistance and sensitivity towards antibiotic. If there is a zone of inhibition around the disc then that means the bacteria is sensitive to bacitracin. If there is not a clear zone around disc the bacteria is resistant to the antibiotic. After streaking the unknown S on to BAP plate 1...
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