Microbiology Procedure

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Gram Staining Techniques

	Gram Staining Techniques A Danish scholar, Christian Gram in 1884 devised a differential staining procedure, which differentiates between two kinds of bacteria Gram-positive and Gram-negative bacteria. This procedure is called Gram stain technique. Different steps of Gram stain technique are as follows. A thin smear of the bacterium is prepared on the slide. To the smear crystal violet solution is applied for 30 seconds (step I). The slide is then gently rinsed in clean water, and an iodine solution is applied for 30 seconds (step 2). This, in turn, is rinsed off. If the slide is examined, all cells would be deeply stained and appear blue-purple. Then, 95% ethyl alcohol is applied and this is renewed until all but the thickest parts of the smear have ceased to give off the dye (step 3).
This usually takes from 20 seconds to one minute. The differential feature of the stain becomes now apparent. Microscopic examination of the slide will reveal that Gram-positive bacteria retain the violetiodine combination (retaining of blue-purple colour even after alcohol wash), whereas Gram-negative ones loose the blue-purple colour after alcohol wash, and will be of original colour. The species which retain the stain are called Gram positive, whereas those which yield the stain to alcohol are called Gram-negative bacteria. Then, there is applied a counter stain a dye of some contrasting colour (step 4).
The generally used counter stains are, eosin (red), safranin (red), brilliant green or Bismarck brown. Each of these colours the Gram-negative species. The cells become now clearly visible. The reasons why bacteria respond differently to the Gram stain are not completely understood. It has been postulated that since Gram-negative bacteria have relatively a high lipid content in their cell walls, the alcohol dissolves the lipids, that allows the leakage of crystal violet-iodine complex.
The Gram positive bacteria with less lipids in their cell walls are less susceptible to the action of alcohol. Another theory suggests that the peptidoglycans found in high concentration in the cell walls of Gram-positive bacteria perhaps traps the crystal violetiodine complex in its many cross linkage. The Gram negative bacteria with less peptidogylycan and fewer cross linkages loose the stain readily. It is important to note that there are some characteristic differences between most Gram-positive and Gram-negative bacteria. It should be evident that the property of Gram positiveness is related to very fundamental physiological properties of the cell.

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