C453task2May

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Introduction When performing microbial experiments, it is imperative to reduce/eliminate any cross contamination of other environmental organisms that may affect your outcome. The first step in ensuring aseptic techniques is through a clean working environment. Using a halogen-based cleaner to remove organisms is imperative. Next, you need to ensure that there is no human contamination through droplet sources. This can be achieved using a simple mask. Gloves will also be needed to ensure the naturally occurring flora that lives on the skin does not get introduced into sample. Once you have a disinfected area and have proper protection, it is time to prepare your samples. Using a modified Bunsen Burner, you can remove the tops of the specimen vials one at a time and gently expose the neck of the tube to heat. This will prevent any unwanted organisms from entering the test tube. During the transfer of the bacterium into the broth, ensure the cap is held in a fashion that prevents surfaces from coming into contact that could later be trapped in your vial. If contaminates get introduced into the specimen, it can alter lab results and cause unnecessary treatments and repeat tests. Methods Methyl Red, Voges-Proskauer (MRVP) was performed through utilization of Staphylococcus Epidermidis and Escherichia coli that had previously been growing in a nutrient broth. Using two MRVP broths, four drops of the bacteria were transferred into new tubes using the aseptic technique mentioned above. Once each MRVP broth had the bacteria transferred, they were left at 24-degrees Celsius for 48-hours. Next, two empty test tubes were labeled, one for Staph. Epidermidis and one for E. Coli. Half of the Staph. Epidermidis was placed in its corresponding test tube and the same action was

completed for E. Coli. Now, we have two tubes of each bacteria. For the Methyl Red testing, seven drops of the reagent were added to one vial of Staph. Epidermidis, and seven drops added to one of the vials containing E. Coli. The VP testing was completed using the remaining two vials of bacteria. However, reagents Barritt’s A and B were added. Twelve drops of Barritt’s A and four drops of Barritt’s B were added to each vial. Carbohydrate Fermentation involved the use of Phenol Red Broth. Six Phenol Red tubes were labeled; three with yeast and three with Staph. Epidermidis. Then they were labeled with their reagent fructose, glucose, or mannitol. Once clearly labeled, the reagents were added to their designated test tube using estimation to divide the reagents. Afterwards, a Durham tube was placed inverted into each test vial and inspected to verify no trapped air was within the Durham tube. All six were then left in a 24-degree Celsius environment for 24-hours. Motility testing involved the use of previously grown bacteria Staph. Epidermidis and E. Coli. on a nutrient rich agar. Using two Motility agars, each one inspected for viscosity, were then inoculated with one of the bacteria. Careful attention to directly inserting the bacterium into the agar and pulling in straight out. The vials were then allowed to incubate for 24-hours at 24- degrees Celsius. Catalase testing was performed on Staph. Epidermidis and E. Coli. Two slides were labeled with the bacteria and a circle was drawn onto the slide. Using the previously grown bacteria plates, a small colony of each bacteria was placed into the designated circle. Next, one drop of hydrogen peroxide reagent was placed on top of each bacterial slide to facilitate a catalase reaction.

Results E. Coli. underwent a MRVP test. The results indicate E. Coli. does produce and an alternate pathway through the release of acetoin; it does not produce an alternate pathway for butanediol. The catalase test showed no reaction to the hydrogen peroxide reagant. The motility test was positive. Staphylococcus epidermidis tested negative for fermentation glucose, fructose, and mannitol. An MRVP test concluded Staph. Epidermidis does produce a mixed acid through the release of acetoin; it does not produce an alternate pathway butanediol. The catalase results were positive for the conversion of hydrogen peroxide to hydrogen and water. The motility test was negative.

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Saccharomyces cerevisiae tested positive fermentation with glucose, fructose, and mannitol. Discussion Escherichia coli is motile, motile bacteria that use flagella to chemotaxis. The Methyl-Red test shows the acidic release of acetoin. The VP did not show use of an alternate pathway because it did not release butanediol. One positive still allows the determination of E. Coli. to be an anerobic organism. Staphylococcus epidermidis is a non-motile bacterium which means they lack the flagella needed to move. The Methyl-Red test was positive meaning it does release acetoin but does not release butanediol. Again, one positive allows determination that Staph. Epidermidis is an anaerobic bacterium. Saccharomyces cerevisiae created an anaerobic reaction when released gas during fermentation. This caused the phenol red to turn yellow due to a pH chemical indicator. Results identify Saccharomyces cerevisiae as anaerobic eukaryotic organism.

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