Nov 10, 2017 in Medicine

Diagnosis of an Infected Patient

A blood sample that contains bacterium can undergo various tests to be able to identify the genera from which these bacteria may belong to. In this case, focus is placed on three of these bacteria that are suspected to be contained in a blood sample. These bacteria include; Bacillus, Escherichia and Mycoplasma.

First, Bacillus has the ability to form spores which help them adapt to extreme environmental conditions. However, in the case of Escherichia and Mycoplasma, they produce no spores. Another major difference is on the presence of the cell wall. In both Bacillus and Escherichia there exist a cell wall but in Mycoplasma there is a cytoplasmic membrane. This has enabled Mycoplasma to take various shapes.

Next is the size of the bacteria whereby while Bacillus and Escherichia are almost equal in size, this is different in comparison to Mycoplasma which is about 10% the size of Escherichia. In fact, Mycoplasma is regarded as the smallest living organism in the world. The other difference is in the shapes in which these bacteria exist. As had been noted above, Mycoplasma takes any shape depending on the environmental conditions that it exists within. However, the other two has distinct shapes with Bacillus being rod shaped with an extreme curvature and longer in size than their wideness. Again, Bacillus is divided along the short axis that they have. On its part, Escherichia is rod shaped but with no such divisions as in Bacillus.

Staining protocol tests

For each of the bacteria above, a different staining protocol test will be carried out to confirm their presence or absence. These tests which are unique in testing the bacteria include Acid Fast Bacilli Staining Protocol to test the presence of Bacillus bacteria, Gram Stain Protocol to test for Escherichia bacteria, and Hoechst Dye Nuclear (DNA) Staining to test for Mycoplasma bacteria (2012, p. Graumann).

To begin with is the test for Bacillus presence, whereby Acid Fast Bacilli Staining protocol comes to use. During the test, the following procedure needs to be followed. First thing, put your working solution in a Coplin Staining jar and place it in a water bath of 58 - 60 ºC for 10 minutes. Secondly, carry out paraffin removal process to obtain water. Thirdly, place a stain in your working solution and maintain it in the water bath for further 15 minutes. Four, remove the Coplin jar from water bath and pour cold water on it for 2 minutes. Five, take out the slides in the Coplin jar and clean them in cold water for about a minute. Six, carry out differentiation in acid alcohol till no color is dashing from the slides. Seven, wash to remove the presence of acid alcohol. Eight, stain it using methylene blue to just 15 to 30 seconds. Finally clean it in water, carry out dehydration and put it in DPX. From this experiment, the following results can be obtained to confirm the presence. For Acid fast bacilli the coloring will be Red, while Nuclei and other tissue elements will be blue.

The next test is of Escherichia using the Gram Stain Protocol. The first step will involve flooding air-dried and heat-fixed smearing of cells for about a minute. Care should be taken to avoid over smearing or under smearing of the cells. Second, clean the slides in a soft stream of running tap water in for seconds. Third, do flood sliding with caustic iodine for a minute. Four, clean the slides in a soft stream of running tap water for about 2 seconds. Five, do the flooding for decolorizing solution. Six, in about 30 seconds to one minute, do flooding with a counter-stain and safranin. Seven, clean the slides in a soft stream of running tap water, till no color appearance and stain dry it with an absorbent paper. Eight, do an observation under an oil immersion by use of a Bright-field microscope. The result will be red or pink for gram-negative bacteria and blue or purple for gram-positive bacteria.

Finally, there is the Hoechst Dye Nuclear (DNA) Staining used to test for Mycoplasma. The staining should be done using a stock solution of Hoechst 33258 at 4°C and place in darkness, 1.2 mg/ml in 0.9% Water or NaCl. The procedure involved is as follows. First, carryout an aspiration process of media from the cells by cleaning the cells two times using PBS (Ryan KJ, 2004). Second, place the cells in a solution of 1-3 percent formaldehyde in PBS at room temperature (RT) for about 10-20 minutes. Third, do aspiration to the fix solution and add cold methanol (–20°C) and leave it at RT for 20 minutes. Four, do aspiration for methanol and clean about three times using PBS. Five, add to the solution in a ratio of 1:10,000 of Hoechst accumulation in PBS. Six, wash again about five times using PBS. Seven, observe using a microscope fitted with a mercury bulb.

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