Unknown Lab Report Unknown #14 Introduction Throughout the quarter, students in the microbiology lab have done a series of biochemical test that have helped them to differentiate between many different organisms. However, during these test, the identities of the organisms that the test were performed with were given to the students in advance. While this is helpful in a classroom setting, this luxury is not always present in the real world. Being able to determine the identities of microorganisms is a very important task that can help people in many different settings. For example, doctors and medical researchers must be able to determine the identity of microbes present in a sick patient or host in order to determine the correct treatments that need to be applied to cure the ill person. Additionally, researchers in a lab setting must be able to do this in order to further develop their research in whichever ways necessary. Ultimately, the students in class were tested for their aptitude to determine an unknown organism given to them by performing the test that they used throughout the class. Methods and Materials The initial test that was performed to the unknown bacteria was a Gram stain. This test was done in accordance with the procedure that is described on page 200 in the lab manual. First, a drop of culture was aseptically transferred to a slide and dried. After heat fixing the smear on a slide, crystal violet was applied for one minute. After rinsing the dye off with water, iodine was applied to the smear for one minute in order to lock in the dye. Once again rinsing after this step, decolorizer was added to decolorize the cells. Lastly, after another water rinse, safranin was added to the slide for one minute and then rinsed and dried. The slide was then looked at under a microscope. Additionally, a TSA plate was inoculated in a quadrant streak as described on page 43 in the manual. This was done and then the plate was incubated at 35° C. After the plate was incubated for a few days, the next test preformed was an Oxidase test. In line with the parameters of the test as described on page 329, a sterile cotton swab was used to gather a sample from the TSA plate, and then the oxidase reagent was dropped onto the swab to determine any change in color. Furthermore, a Methyl Red/Voges-Proskauer tube was inoculated with the culture of the unknown bacteria. After incubating for the 48 hours at 35° C as indicated by the process outlined on page 313 in the lab manual, the MR/VP broth was separated into two separate tubes and the Methyl Red reagent was added to one tube and the two reagents, containing α-Naphthol, Ethanol, and Potassium Hydroxide, for the Voges-Proskauer test was added to the other tube and left to sit for an hour. Lastly, the final test performed was a MacConkey Agar test. Following the procedure on page 260 in the lab manual, the MacConkey plate, which contains lactose, bile salts, neutral red, and crystal violet, was inoculated with the unknown broth and incubated at 35° C for at least 48 hours. Results Test Gram Stain Purpose To determine the Gram reaction of the organism and identify the morphology Oxidase Test To determine the presence of cytochrome c Methyl Red Test To determine if the organism ferments glucose through mixed acid fermentation Voges-Proskauer To determine if Test the organism performs 2,3butanediol fermentation MacConkey Test To determine if the organism ferments lactose into acid end products Reagents Crystal Violet, Iodine, Alcohol, Safranin Observations Pink Rods Oxidase Reagent No color change Methyl Red reagent No color change Reagent a (αNaphthol and Ethanol) Reagent b (Potassium Hydroxide and Water) Lactose, Bile Salts, Crystal Violet, Neutral Red No color change Pink growth on media Result Gram Negative, rod-shaped bacteria Oxidase test negative (no cytochrome c) Negative Methyl Red test (no mixed acid fermentation) Negative VogesProskauer test (no acetoin production through 2,3butanediol fermentation) Positive MacConkey Test (Lactose fermentation) Discussion After performing all of my test, the results from each test was gathered and interpreted. After performing the gram stain procedure, the possible organisms could be narrowed down to Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Providencia stuartii, Salmonella typhimurium, Proteus vulgaris, and Shigella sonnei. Once an Oxidase test was performed, Pseudomonas aeruginosa could be ruled out because this organism is the only one that is positive for cytochrome c in the group of bacteria. Furthermore, after the results for the Methyl Red test was gathered, the group of possible organisms was further refined to Enterobacter aerogenes, Klebsiella pneumoniae, and Shigella sonnei, for these organisms are both Oxidase and Methyl Red test negative. Additionally, once the Voges-Proskauer test result was observed to be negative, the possible organism list was reduced to only Klebsiella pneumoniae and Shigella sonnei, as these are the organism that meet the criteria that has been observed through the test. Ultimately, after the last test, the MacConkey test, the clear organism that emerged was Klebsiella pneumoniae, for this organism was the only lactose fermenter that was left in the group following the previous test. Flow Chart ID of Unknown Organism After performing a Gram stain, Oxidase test, Methyl Red test, Voges-Proskauer test, and MacConkey test, the organism that was isolated from the group and is the identity of the unknown organism is Klebsiella pneumoniae.