Micro Unknown Lab Report

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Gram Negative Unknown|
Biology 3444-006|
Lena Wallace|


The purpose of this lab was to identify an unknown bacteria culture using differential tests. The identification of the unknown culture was accomplished by identifying the bacteria based on its specific metabolic characteristics and morphology. It is suggested that culture 11 is a sample of Enterobacter aerogenes. Introduction:

This experiment was centered on metabolic and biochemical testing procedures. The rationale of performing these tests was to distinguish six different microbes from one another and to compare how their metabolic and biochemical processes differ from species to species to determine the unknown sample. The tests included: Triple sugar iron agar (TSAI), the Sulfide Indole Mobility (SIM) test, Glucose fermentation, the Methyl Red test, the Voges-Proskauer test, Citrate test, the Urease Test, and finally the Gelatin test. The microbes that were tested during this lab were: Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, and Salmonella typhimurium. The sample labeled #11 could have been any of the six microbes. A gram stain was performed to assess the shape and other characteristics of the bacteria, and to ensure that there was no gram positive contamination. Gram positive cells have a thick outer peptidoglycan layer that traps the crystal violet-iodine complex more than gram negative cells. As a result, they are less vulnerable to the de-colorization step with alcohol making them appear purple in color, while the gram bacteria negative appear pink. Triple sugar iron agar slant tests for multiple things: sugar fermentation of glucose, lactose, and sucrose, and the production carbon dioxide and hydrogen sulfide. The gases are easy to identify. If any carbon dioxide is produced cracks or bubbles appear inside of the medium, and sometimes enough CO2 is produced to push the slant up towards the top, this will be reported as +g. The H2S is identified by how the gas reacts with an iron compound and makes the agar turn black. There are two possible types of sugar reactions that take place in the areas of the butt and the slant of the medium. The outcome of sugar metabolism will be acid production, so the pH indicator phenol red will turn yellow, and be reported as A. if there is no sugar metabolism, or alkaline by-products are made, will cause the indicator to stay the same color red, and reported as a K. TSIA medium is prepared as a shallow agar with a deep butt, providing for both an aerobic and anaerobic environment. A TSIA medium must be checked within about 12 hours to see if it ferments glucose, and again after 24 hours to see if it ferments lactose and sucrose. If the slant returns to being red and the butt is still yellow after this time period, the organism ferments glucose but not the other sugars. If it is completely yellow after the time interval, this indicates that the organism ferments all three sugars. SIM Medium is used as differential test of microorganisms on the basis of hydrogen sulfide production, indole production, and motility. The Sulfur reduction test is useful in differentiating enteric organisms, the Indole test is used for differentiating the Enterobacteriaceae, and the Motility test is useful for testing a wide variety of organisms (condalab.com). Casein is rich in tryptophan which is reduced and produces indole by the enzyme tryptophanase. Ferric ammonium sulfate is the indicator for H2S production. Once the medium was done incubating Kovacs’ reagent was added to the tube. If the sample was positive the reagent would have a color change to red, if the reagent remained clear, a negative result was reported. Glucose fermentation uses Phenol Red Broth as differential test medium typically used to differentiate based on the color change of the pH indicator. Phenol red turns yellow below a pH of 6.8, pink...
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