Materials and Methods To perform this experiment we collected samples from

Materials and methods to perform this experiment we

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Materials and Methods To perform this experiment we collected samples from Bintou’s nose and Emma’s ear using a sterile swab. The samples were then inoculated into two different 15ml screw cap tube containing 5ml of m-staphylococcus broth, which we made using powder and distilled water. The tubes were incubated at 37°C. We Gram stained the unknown mixed culture by heat fixing the smear on a slide, flooding with crystal violet for one minute, rinsing with distilled water, flooding with iodine for one minute, rinsing with distilled water, decolorizing with alcohol for five seconds and immediately rinsing with distilled water, flooding with safranin as a counterstain for one minute, rinsing with distilled water, and dry with bibulous paper. Another 15ml tube was inoculated with the unknown mixed culture and incubated at 37°C. After one week we streaked each sample onto an MSA plate. One plate was streaked with the m-staphylococcus broth of Bintou’s nose and one plate was streaked with the m-staphylococcus broth of Emma’s ear. These plates were then incubated at 37°C. The next step was to check the colonies and determine which bacterium was able to ferment the mannitol. The bacteria from Bintou’s nose grew colonies that fermented the mannitol on the MSA plate but the bacteria from Emma’s ear did not grow many colonies at all. It can be presumed that the colonies which fermented and the mannitol and turned the MSA plate yellow belonged to the Staphylococcus aureus species of bacteria. This can be confirmed by a coagulase test by inoculating with a Staphylococcous epidermis sample and a Staphylococcus aureus sample to find what results occur.
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Two colonies well-isolated colonies that were not surrounded by yellow on Bintou’s nose plate were chosen and streaked onto a new plate. This plate was then incubated at 37°C. We only inoculated one plate because Emma’s ear colonies did not grow on the initial MSA plate. The colonies chosen from Bintou’s nose plate are not expected to be an organism of Staphylococcus aureus , which means that molecular identification by PCRing is necessary. By PCRing their 16S rRNA genes we were able to obtain the 16S DNA sequence. A tiny bit of a colony from the new MSA plate was chosen as the target DNA. 0.5ml of sterile saline was put into one sterile 1.5ml centrifuge tube. After sterilizing the loop by heating it in the flame of a Bunsen burner, a single colony was taken and put into the tube. To set up 50ul PCR reaction it is important to add the reagents in a particular order. First, 25ul of 2x SSO SYBR supermix was added. Second, 2ul of 10um 8F primer and then 2ul of 10um 1492R primer was added. Then, 17.5ul of distilled water was added and then 2.5ul of DMSO was added. Finally, one bacterial colony in saline was added. We mixed the reaction and the placed it in the thermo cycler. Two minutes at 72°C and 40 cycles would give us 2 40 copies of the DNA. A Microgen Staph Strip was then inoculated using the bacteria from Bintou’s nose colonies. The black circle hole of the staph strip requires an addition of mineral oil prior
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  • Spring '14
  • GinaM.DiGioia
  • Microbiology, Bacteria, Staphylococcus aureus, Gram-positive bacteria

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