Salome, Ozioko 12/17/12 BS:203 J.

Berton

Unknown Lab Report #1 INTRODUCTION: This study was aimed to identify unknown bacteria. Identification of unknown bacteria yields multiple benefits in many different areas in the research of microorganisms. There were many reasons for determining the identity of this microorganism. The first reason can be to study the morphological characteristics of the bacteria. Other reason might range from knowing the causative agent of a disease in a patient; this will help to know how this disease can be treated. Identifying the correct bacteria can be also important in knowing the correct microorganism to be used for making certain foods or antibiotics. This experiment was done by applying all the methods that have been learned so far in the microbiology laboratory class for the identification of an unknown bacterium. This study was also done by completing many different tests on the chart given out by the instructor. The identification of the unknown culture was accomplished by separating and differentiating possible bacteria based on specific biochemical characteristics whether the tests performed identified the bacterias shape, color, or its reaction to different materials, each was used in a way to help recognize those species and to identify the unknown bacteria. In this experiment performed many different test deals with things such as the presence of enzymes, fermentation abilities and different chemical reactions. The different test used to

identify the unknown bacteria was a cultured TSA plate (trypticase soy Agar), Simmonss slant, gram stain, oxidase test, nitrate test and other biochemical tests performed. Each test performed, provided us with some key information about the unknown microbes in question and how the bacteria function.

MATERIALS AND METHODS: The first procedure that needed to be done was to streak the unknown out on a Trypticase Soy Agar plate, using the T streak method described in the lab manual. This needed to be done in order to test the purity of the unknown # 1. The streak was done by obtaining a sample with a sterile loop and dragging the loop back and forth across the agar surface and incubates the streaked plate in an upturned position for hours. After the plates were incubated and grown, the morphology was observed and recorded and a Gram stain was performed. Quality control bacteria were Gram stained along with the unknown to make sure that the Gram stain reaction was done correctly. After determining the Gram reaction, specific biochemical tests were performed. The biochemical tests were chosen from the unknown identification tables that were given by the instructor. Since unknown was determined to be a Gram negative rod, an oxidase and nitrate test was performed and the reactions were observed to narrow down the unknown. Next, a PR Lactose, PR Dextrose and PR Manitol test were done. A Citrate test was run on the organisms to determine if the bacterium could use citrate as its sole carbon and energy source. A Methyl Red test and then aVoges Proskauer test were also performed to see what end products resulted when the test organisms degraded glucose. A confirming Indole test was also run on the organism. All of these tests were performed by the methods outlined in the laboratory manual by Alfred E. Brown.

MATERIALS: Carbohydrates broths with Durham tubes and phenol red indicator with the following sugars: 1 glucose tube 1 lactose tube 1 mannitol tube 2 MR-VP broth tubes 1 simmons citrate tube 1 trypticase soy agar (TSA) plate 1 Nitrate broth tube 1oxidase tube

RESULT: The Unknown # 1 bacterium had the following morphology on a TSA plate: medium sized opaque cream colored colony. After determining that it was a Gram negative rod, an oxidase test was performed. The Table below lists all the physiological and biochemical tests done, the observation and results: Bacteria Unknown #1 Test Gram stain Oxidase test PR Lactose test PR Dextrose test PR mannitol test Nitrate test Citrate observation Pink Rod Blue color change Yellow color change Yellow color change Yellow color change Pink color change Color change from green to blue Methyl Red test VP A& B Indole test Motility test Detect motility Color change to red inconclusive Positive Inconclusive Negative results Gram negative rod Positive oxidase test Positive Positive Positive Positive Nitrate test Positive Nitrate test

Fermentation test

Oxidase/Nitrate test result on a colony

Citrate test result

Discussion: After several differential tests, it was concluded that the unknown #1 was K. pneumonia. After performing the Gram stain to determine that the unknown was a Gram negative rod, the organism was grown on a TSA slant for use in inoculating the rest of the biochemical tests. At first, my group was thinking that the unknown #1 is E.coli due to the result that was observed from the gram stain, Oxidase and nitrate test done. All of the biochemical tests worked well for this assumption except for the citrate test. After the citrate Simmons slant test was done, it was observed that the color changed to blue which is a positive result and it indicates use of citrate bacteria produced on top. This positive result was inconsistent with the rest of the results since E. coli supposed to have a negative citrate test result and positive result was observed in the organism. For this reason, my group now leans towards k. pneumonia. It was suggested that indole test, and MR-VP test should be run on the organism before it can come to conclusion. After running these tests, the indole test showed negative the MR test was positive and the VP test was inconclusive. The negative result observed in the indole test performed and the MR-VP results were consistent with the other data. Therefore it was concluded that the unknown #1 was Klebsiella pneumonia. h

Work Cited
Brown, Alfred E. Benson's Microbiological Applications: Laboratory Manual in General Microbiology. 12th edition. New York: McGraw-Hill, 2012. Print.