Gram Negative Unknown Lab Report Essay

The following tests according to the lab manual were performed: gram stain, fermentation tubes, methyl red, vogues proskauer, sulfur, indole, motility and growing it up on MacConkey agar. The gram stain was performed incorrectly the first time. This is because the decolorizer was not on the bacterium slide for long enough, giving a false outcome.

The Voges-Proskauer test to detect organisms that are able to ferment glucose, but convert the products to acetoin and 2,3-butanediol. This is deduced by the addition of Reagent A and Reagent B, and the observation of the color change thereafter. Reagent A is a solution of -naphthol and alcohol. Reagent A catalyzes the conversion of acetoin to diacetyl. Diacetyl thens react with guanidine-containing compounds from the peptone to form a red color in the presence of -naphthol. Reagent B is a solution of potassium hydroxide and water. It

Purpose: The purpose of this lab is to help you become a little familiar with some of the tests that can be typically performed in a clinical or research lab facility. These tests may help in determining a particular pathogen’s growth needs.

The enzyme urease breaks urea down into NH3 and CO2. An orange broth containing urea is used for this test and needs to be inoculated with the gram negative bacteria. A pink color in the medium indicates a urease-positive organism, an orange or yellow is negative.

In class, we were given the task of identifying an unknown bacterium broth culture. After receiving number 69, I went through several tests to figure out what bacterium I received. First, I created a slide from my broth by putting a small amount of the unknown broth on to a clean slide and letting it dry for ten minutes. After this, I stained the slide by applying four reagents in order; crystal violet, grams iodine, decolorizer and safranin. From the stained slide, I discovered that this bacterium was gram-negative, which would determine the next couple of tests I would do to identify my unknown bacterium. I began by streaking for confluent growth from my broth culture onto a TSA plate. From the TSA plate, I aseptically transferred a loop

After confirming that the bacteria is gram negative, the following test that will need to be done is the oxidase test. This is done by adding a few drops of oxidase reagent to a swab that has collected a single colony from the TSA plate. If the swab changed to a purple color within 5 seconds, this indicated that the organism is oxidase positive. The organism that was tested was positive for oxidase.

The first method is the pyrogallic acid technique in a solid medium. This technique uses streak cultures on nutrient agar slants. A person pushes a cotton plug into the tube until it almost touches the slant. The space above the cotton gets filled with pyrogallic acid crystals and put sodium hydroxide in there as well. Insert the stopper very tightly and then invert and incubate. The chemicals absorb the oxygen which produces an anaerobic environment. The second method is the shake-culture method in a solid medium. This is a molten and cooled nutrient agar which is inoculated with a loopful of an organism. The tube gets shaken, cooled quickly, and then incubated. The position of growth in the tube makes it the index of gaseous requirements for an organism. The third method os the Paraffin plug technique in a broth medium. It is a medium that has reducing substances like cystein or ascorbic acid in it. This medium gets heated to make the oxygen go away then get quickly cooled and inoculated with a loopful of culture. After that it get immediately sealed with melted paraffin and then gets incubated. The last method is fluid thioglycollate in a broth medium. It is present in redox potential indicator like resazurin, which produces a pink color in an oxidized

Different strains of E.coli can be isolated and identified by a variety of biochemical tests. The biochemical tests commonly used to identify E.coli include: lactose, oxidase, indole, and beta-glucuronidase tests. The use of lactose to identify a strain of E.coli is an appropriate method since intestinal bacteria, such as E.coli, typically undergo lactose fermentation. Using MacConkey agar, microbiologist are able to analyze if lactose is fermented and detected by the change of color in the medium. The oxidase test is used to identify if the bacteria synthesizes the enzyme cytochrome c oxidase that is part of the electron transport chain. When the enzyme cytochrome c oxidase is present, the reduced reagent, tetramethyl-p-phenylenediamine will donate its electron to cytochrome c oxidase and a purple color end product will show in the media. The indole test is performed by microbiologist to determine if E.coli is capable of converting tryptophan to indole. Lastly, beta-glucuronidase test is used to determine if an organism produces the enzyme glucuronidase by hydrolysis of nitrophenyl-beta-glucopyranosiduronic acid. In the presence of glucuronidase, the medium develops a yellow color (Trepeta & Edberg,

The first a catalase test uses hydrogen peroxide to see if it can be reduced to oxygen and water by the presence of bubbles. The next test, Bile Esculin, which is an Esculin medium that if the bacteria reacts with the ferric chloride the agar will turn black. The last test being used is a SIM test which is a few tests in one. It tests for sulfur reduction, which would turn black; motility, which shows growth around the incision and indole production, which is the removal of an amino group and its reaction with the Kovacs reagent turning a red color (Allen, 2016). The whole premise of this lab is to take an unknown sample and try and separate it into both Gram-positive and Gram-negative bacteria.

These tests indicate metabolism characteristics that are consistent with the E. coli bacteria. The SIM agar tests for the bacteria 's ability to break down the amino acids cysteine and tryptophan. E. coli does not use cysteine as source of food, but it does use tryptophan as a food source producing indole, which is indicated by adding Kovac 's reagent. If indole is present, a reddish color will appear, if not, no change occurs. If tryptophan is broken down, a metal sulfide results which produces a black color. The SIM agar is also responsible for indicating bacterial motility, which can be observed if the agar becomes cloudy (which means the bacteria has moved outward), rather than

The TSIA medium test checks for hydrogen sulfide production while also fermenting glucose, lactose, or sacchorase. Acid is produced during fermentation which causes the pH indicator to change the color from red to yellow. The SIM test incorporates sulfur reduction, indole production, and motility all in one medium. If hydrogen sulfide is produced, the agar will become black. Indole production will turn a pink color during a positive reaction and motility is determined by the growth of microbes away from the stab line. The last two tests conducted were the Citrate utilization test and Urease test. Citrate utilization is used to determine if the bacterium can live with citrate as its only carbon source. Citrate utilization can be determined

The Catalase tests involves exposing the colony of interest to hydrogen peroxide to check for the presence of the enzyme Catalase. If the bacteria contain the enzyme, exposure to hydrogen peroxide will trigger the breakdown of it by Catalase as a defense mechanism against the harmful substance. Catalase test positive genera include Bacillus, Micrococcaceae, and Staphylococcus, while Clostridium and Streptococcaceae test negative (Holbrook and Leicht, 2015). The second test administered to gram-positive bacteria is the Mannitol Salt Agar (MSA) test. This test involves observing growth of the bacteria on the MSA medium, which contains very high amounts of sodium chloride. The only strain capable of growth is Staphylococcus, which causes the medium to change in color from red to yellow, indicating fermentation of the

I got exact the same results as the previous tests so I thought to myself “maybe I happened to have two bacteria with identical metabolic capabilities”. Based on the initial results I got, I thought my gram negative bacteria was Citrobacter freundii and Klebsiella terrigena. To confirm my bacteria, I did the KIA tests. If they were Citrobacter freundii and Klebsiella terrigena then I was expected to see black precipitation in one of the KIA tests only, but both of the KIA tubes had black precipitate in it. It didn’t make any sense at all. I thought my gram negative pure cultures was old so they weren’t producing the proper results so I started everything from scratch. I grew new pure cultures, did all the tests again and still got the same results. I had no idea what was going on; the morphology of the two bacteria was completely different from one another. They were clearly two different bacteria but how comes they kept producing the same results for all of the tests. Well, it turned out that my U3's pure culture contained U2 bacteria. My U3's

With a glass-marking pen preferably black, mark four sterile test tubes. The test tubes should be marked 1A, 2A, 3A, and 4A. First, using a mortar and pestle to grind together a spatula of sand, a 0.5 cm pieces of apple, and 1 ml of distilled water. Then pour this mixture into test tube 1A. Second, using a mortar and pestle to grind together a spatula of sand, a 0.5 cm pieces of potato, and 1 ml of distilled water. Then pour this mixture into test tube 2A. Third, using a mortar and

To further enhance this method an indicator medium will also be used. These medium changes colour during fermentation, an indication of pH change due to the presence of acid forming species. Durham tubes will then be employed; when inverted they catch gaseous bubble formed during fermentation. The gas produced at 37C act as a strong indicator to the presence of E.coli Reasoner & Geldreich (1985).