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Due to multiple complications with the PC, a new hypothesis oldest be formulated. Methods: In this experiment, a 1 gram sample of contaminated dirt was taken and mixed with 100 ml of sterile water then shaken. After allowing the sediment to settle to the bottom, a sample of the water was taken. With the sample, a sterile loop was used to inoculate a TTS Petri plate by doing a quadrant streak, which is aseptically done by making an initial streak, then sterilizing and repeating by streaking out of one portion of the previous streak no more than the initial streak.

This was done a maximum of four times, insuring that the bacteria culture s “thinned” out enough to take a sample from an individual colony. This was repeated one other time and both were refrigerated at 37 degrees for a minimum of 24 hours. Each plate was labeled with names, the date, the class section, and “unknown” (Lowlife, M. , & Pierce, B. (2012)). After retrieving the plates from the fridge, a sample was aseptically taken off an individual colony, from either TTS plate, with a loop and inoculated on a TTS slant. As before label the slant accordingly. The slant was refrigerated as well for 24 hours at 37 degrees.

After retrieving the slant, inoculate a broth tube with a loop full of bacteria then allow the bacteria to grow in the broth (Lowlife, M. , & Pierce, B. (2012)). Staining is the next step in identifying the unknown bacteria. In order to stain the bacterium, a drop of water was taken aseptically and placed on a slide. After flaming the loop, a drop of the bacteria was added to the slide and mixed together along the slide so that it could air dry. Once dry, the slide was passed over an open flame in order to “heat fix” the bacteria to the slide (Lowlife, M. & Pierce, B. (2012)). And now for the actual staining of the bacterium.

The first stain was a simple stain and methyl blue was used. A couple of drops of the methyl blue were added to the slide, making sure it covered all areas where the bacteria was, and was set aside for 1 minute. After it was then rinsed with water and blotted dry. When looked at under a microscope, the shape of the bacteria is made visible (Lowlife, M. , & Pierce, B. (2012)). The gram stain started off the same way as the simple stain, start with a heat iced slide which is where a drop of water is added to a slide then mix a drop of bacteria along the slide so that it could air dry.

Once dry, the slide was passed over an open flame, as previously described, heat fixing the bacteria to the slide. Next, the smear was covered with the crystal violet stain for 1 minute then rinse with distilled water. Then the smear was covered with gram’s iodine as a mordant for 1 minute and rinse with distilled water. Immediately after it was rinsed with 95% ethanol for no longer than 10 seconds to decolonize and again immediately rinsed with water after. If left UN-rinsed, the decentralization process will strip the color from both the gram positive and negative cells.

Lastly, the countersink seafaring was added for 1 minute and rinsed with distilled water and was blotted dry. (The seafaring is used to stain the gram negative cells after the decentralization process. ) When looked at under the microscope, the bacteria should be readily identifiable in regards to being negative (pink) or positive (purple) (Lowlife, M. , & Pierce, B. (2012)). For the acid-fast stain, start with a heat fixed slide as described in the previous taints where a drop of water is added to a slide then mix a drop of bacteria along the slide so that it could air dry.

Once dry, the slide was passed over an open flame “heat fixing’ the bacteria to the slide. For this stain, the Seize-Nelson method was used. In this method, cover the smear with a strip of tissue paper and a couple of drops of carboniferous on top of the tissue paper. The slide was then placed over steam for 5 minutes making sure the paper stayed saturated with the dye then after the time was up, it was rinsed with water. Immediately after, the slide was rinsed with acid-alcohol for no longer than 10 seconds and rinsed with water right after.

A methyl blue countersink was added to the slide for 1 minute then rinsed with water and blotted dry. When looked at under a microscope, the bacteria was then categorized as either an acid-fast stain which is red from the carboniferous and acid-fast negative which is blue from the methyl blue (Lowlife, M. , & Pierce, B. (2012)). For the DNA isolation, Results: The results of these experiments were that the simple stain seemed to be calculus bacteria due to their rod shape seen in the simple stain.

In the Gram negative stain the gram cells were pink due to the seafaring, and in the acid- fast stain the cells were acid-fast negative resulting in the cells retaining the methyl blue. Conclusion: Based on the gathered data, it is hypothesized that the bacteria is of the bacillus family due to the rod shape. Within the bacillus family, there is S. Epidermis, B. Cereus, and M. Kinematics. The acid-fast reaction was negative and the gram stain was negative. The prognosis of the bacteria is still unknown at this mint due to too many variables.

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