Gram Staining Staphylococcus Aureus and Pseudomonas Fluorescens

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The Gram stain is one of the most useful staining procedures in microbiology . It is one of three differential staining techniques, is used to identify divide bacteria into two groups: Gram-positive bacteria and gram-negative (Madigan, Martinko, Dunlap, Clark (2009). These staining reactions take advantage of the fact that cells or structures within cells display dissimilar staining reactions that can be distinguished by the use of different dyes (Brown 2009). A gram positive bacteria can be identified by retaining a purple color dye within its peptidoglycan exterior. A gram-negative bacteria, which doesn’t retain this purple color due to an outer membrane that covers a much thinner peptidoglycan layer, instead retains a pink counterstain.

The purpose of this exercise is to become familiar with the Gram stain method by testing Staphylococcus aureus and Pseudomonas fluorescens and determine their Gram classifications.

Broth cultures of Staphylococcus aureus and Pseudomonas fluorescenes, a Bunsen burner, one inoculating loop, one clean glass slide, one slide dish, bibulous paper, Gram-staining materials (Crystal Violet, Iodine, Ethyl Alcohol, Safranin, DI water), and a microscope.

Smear Preparation
Obtained a clean dry glass slide, Staphylococcus aureus and Pseudomonas cultures. Added drop of water onto the center of slide.
3. Flame-sterilized inoculating loop, top of culture tube, obtained a very small sample of bacteria and smeared onto the water drop on the slide slide Repeated smear with second bacteria culture tube.

Smear was allowed to air dry and was subsequently heat-fixed onto the slide.

Gram Stain
The S. aureus and P. fluorensens smear was covered with Crystal-Violet for 20 seconds and briefly rinsed with distilled water. The smear was covered by Gram’s iodine (mordant) and stood for one minute. The smear was then rinsed with ethyl alcohol (de-colorizer) until mordant was no longer visible...
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