Unknown Project 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 Methyl Red test is a differential test for bacterial respiration used to differentiate strains of coliform bacteria capable of performing mixed acid fermentation that will lower the pH despite the phosphate buffer (http://faculty.deanza.fhda.edu). Mixed acid fermentation is confirmed by using methyl red as an indicator. It is red ant pH 4.4 and below, yellow at pH 6.2 and above, and orange in between. Red is a positive result reported as (+), yellow is a negative result reported as (-), and orange is negative or inconclusive.

The null hypothesis will be that the test tubes with an increase in temperature, pH values, enzyme concentrations, and substrate concentration will have a very small color change or no color change at all. The alternate hypothesis is that the test tubes containing an increase in temperature, pH values, enzyme concentrations, and substrate concentration will all have an intense color change; the more the change, the more intense the color change will be.

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.

For the Urease test, I incoluated my Urea test tube with my unkown bacteria from a TSA plate using and inoculating loop. The Urea tube was then incubated at 37 degrees Celsius for 8 days to observe for a color change. The Urea tests for the ability of a bacteria grown in urea broth produces urease. This medium contains the pH indicator phenol red. If urease is produced the pH of the media will raise thus causing the phenol red to change from yellow to a pink color.

The medium contains sodium citrate that serves as sole carbon source and ammonium phosphate as the sole nitrogen source. These organisms produce the enzyme citrate-permease that can transport the citrate into the cell and produce pyruvate from it. Bromthymol blue dye is used as an indicator that is green at pH 6.9 and blue at pH 7.6 and above (Leboffe and Pierce). A positive result is obtained if the color is blue meaning that bacteria utilize the citrate and convert the ammonium phosphate into ammonia and ammonium hydroxide.

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Ensure that you note any requirements that are out of scope to achieve absolute clarity about what is and is not covered by this project, and to avoid the potential for problems later on.

One MR-VP broth was the control tube, which was not inoculated at all. The other MR-VP broth was the treatment broth that was inoculated with the colony 2 stock culture at 30 degrees Celsius for five days. The reason for completing this test first after gram staining was because it was a 5-day test that could be left over the weekend. There were no variables, it was either negative or positive, and there would be no misleading guesses. The plan I had going into this was to perform all the tests possible with no variables or not determined results to reduce the potential for errors. On Monday I will get the control and treatment inoculated test tubes back to determine wither the unknown organism is positive or negative based off of the color in which the broth will

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Purpose: The MSA streak plate test is used to differentiate between different microbes by their reaction on the specific media. The remaining microbes are Staphylococcus aureus and Staphylococcus epidermidis. On the MSA plate, Staphylococcus aureus turns the MSA plate yellow. Staphylococcus epidermidis turns the MSA plate a darker red (Harley, 2014).

The gram stain result showed that my unknown was a pink long individual rods this means that it is a gram negative bacilli. The growth on the agar of unknown appear off-white with straight growth and the broth growth was uniform fine turbidity. Uniform fine turbidity means there is cloudy growth throughout the test tube. As time progressed the broth appeared clear with some sediment. This could suggest that the bacterium is a slow grower or that the cells are older and dying. This can affect the results of testing. We did to place an agar slant and broth tube in the incubator for 48 hours at 25 degrees Celsius to see if the temperature affects the growth. The agar slant looked the same as agar slant that was grown at 37 degrees Celsius. The broth that was incubated for 48 hours at 25 degrees Celsius did suggest that it was able to grow better at this temperature being the growth in tube with the thick white clunks floating throughout and the uniform fine turbidity.

The Indole portion of the test is performed by adding Kovac's reagent to the inoculated SIM medium. The Kovac's reagent reacts with the indole (if indole is present) to produce a pinkish-red or reddish-purple ring around the top of the test tube. If indole isn't present, there will be no color change. The presence of indole indicates that the bacteria produces tryptophanase, an enzyme which breaks down tryptophan into smaller components, one of which being indole.

The decolorized Gram negative cells are stained pink. With the results from the Gram stain I was able to follow the “Unknown Identification Flowchart” to the next step, which was to prepare for the Starch Hydrolysis Test by inoculating a starch plate.

Each test performed in the lab on theses unknown bacteria have a very specific significance. With each test performed correctly the lab officer is able to move closer towards a proper identification of the unknown bacteria. After performing Gram staining and deciphering if the unknown was gram positive or negative the lab officer was then able to proceed to the next step of identification. The gram positive unknown’s reaction to the catalase test informs the tester of the Genus theyre working with. This indicates which tests to perform next. The MacConkey agar is a selective medium that only allows the growth of gram positive bacteria confirming the results received from the gram staining procedure. The NaCl growth test indicates whether or not the organism is able to