Identifying microorganisms using differential staining techniques

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IDENTIFYING MICROORGANISM’S USING DIFFERENTIAL STAINING TECHNIQUES

Introduction:

The purpose of this lab is to experience preparing slides and drawing cells that involve the process of differential staining. In this lab we will be using five different microorganisms. In experiment 4 we will use Staphylococcus epidermidis and Escherichia coli. Staphylococcus epidermidis is a gram-positive cocci shaped bacteria that is known to be one of the leading causes of infections, particularly the foreign body infections (Bukhari, 2004). On the other hand, Escherichia coli is a gram-negative rod shaped bacteria that is known to produce and grow in the intestines of animals and humans often from the ingestion of foods or water (Todar, 2008). In experiment 5 we will use Mycobacterium phlei. M. phlei is a gram-positive cocci shaped bacteria that is of the genus Mycobaterium (SteadyHealth.com, 2004). This type of bacteria is one of the hardest to destroy because of the mycolic acids that cover the cell bodies (SteadyHealth.com, 2004). Also, this bacteria is often the cause of human infections but people infected often respond well to anti-mycobacterial therapy (SteadyHealth.com, 2004). In experiment 6 we will use Bacillus megaterium and Clostridium sporogenes. Bacillus megaterium  is a gram-positive rod shaped bacteria that produces spores. This type of bacteria is one of the largest Eubacteria that is found in soil (Kunkel, 2009). Clostridium sporogenes is a gram-positive rod shaped bacteria that also produces spores. It is found often in soil as well as the intestines of animals and humans. This type of organism is often used to conduct studies involving low acid foods (Kunkel, 2007). In this lab we will explore three major types of differential staining techniques. These techniques are gram stain, acid-fast stain and the spore stain. All these staining techniques involve the use of multiple stains. The gram stain technique is used to distinguish between gram positive bacteria and gram negative bacteria of a particular cell (Kelleher, 2004). A primary stain called crystal violet is used first. Then a stain known as gram’s iodine is used as a mordant to fix the primary stain onto the cells (Kelleher, 2004). A de-colorizer stain known as ethyl alcohol is then used to distinguish between gram positive and gram negative cells (Kelleher, 2004). This stain removes the crystal violet colour from the gram negative cells turning them clear and traps the crystal violet colour in the gram positive cells (Kelleher, 2004). Finally, a stain known as safranin is used to stain the walls of the cell. The clear gram negative cells turn pink and the purple gram positive cells overpower the pink colour and remain the same (Kelleher, 2004). In the acid-fast staining technique the use of 3 stains is used instead of 4. A primary stain known as carbolfuchsin which is dark red is applied to the slide (Kelleher, 2004). With the help of heat and the phenol contained in the stain, the stain is able to penetrate the layer of mycolic acids that are present in the cell walls (Kelleher, 2004). Once this stain breaks through the acid from the cell wall a de-colorizer stain called acid-alcohol  is used to distinguish between acid-fast bacteria and non-acid-fast bacteria contained in the cell (Kelleher, 2004). Seeing that the acid-fast bacteria contain strong cell walls they will remain the same pink colour, while the non-acid-fast bacteria become clear because the acid from the decolorizer is able to remove the carbolfuchsin from the weak cell walls (Kelleher, 2004). Finally, a stain called methylene blue is used where the acid-fast cells remain pink and the non-acid-fast cells absorb the stain and turn blue-green (Kelleher, 2004). In the spore staining technique only 2  types of stains are used. A primary stain called malachite green is applied to slide. With the help of heating, the stain is able to enter the endospores and cooling traps the stain into the spores (Kelleher, 2004). The stain also enters the vegetative cells easily (Kelleher, 2004). Once this staining is complete the excess stain is washed off and this is when the endospores of the bacteria remain green, while the vegetative cells become clear (Kelleher, 2004). Lastly, a stain called safranin is applied to the slide to distinguish between endospores and vegetative cells of the bacteria (Kelleher, 2004). The endospores remain green and the vegetative cells become pink (Kelleher, 2004).

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Materials & Methods

This lab was performed exactly as described in the Fall 2011 BIOL 140L lab manual by the Department of Biology without any deviations. The experiments involved in this lab were Experiment 4: The Gram Stain pages 24-27, Experiment 5: The Acid-Fast Stain pages 28-30 and Experiment 6: The Spore Stain pages 31-33.

Discussion:

Based on the results from the gram stain experiment it can be concluded that the two organisms tested are very different in terms of morphology and gram reaction. Staphylococcus ...

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