Gram Staining

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The Gram staining method is named after the Danish bacteriologist who originally developed it in 1882, Hans Christian Gram. The Gram staining process is one of the most important staining techniques in microbiology. It is almost always the first test performed for the identification of bacteria. The primary stain of the Gram's method is either crystal violet or methylene blue, each almost equally effective. The microorganisms that retain the crystal violet and iodine mixture appear purplish brown under microscopic examination. These microorganisms that are stained by the Gram's method are classified as Gram positive. Others that are not stained by crystal violet are referred to as Gram negative, and appear in shades of red. Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye or methylene blue. The cell walls for Gram positive microorganisms have a higher peptidoglycan and lower lipid content than gram negative bacteria. Bacteria cell walls are stained by the crystal violet. Iodine is then added as a mordant to form the crystal violet and iodine complex so that the dye cannot be removed easily. This step is usually called fixing the dye. However, the next treatment with a decolorizer, which is a mixed solvent of ethanol and acetone, dissolves the lipid layer from the gram negative cells. The removal of the lipid layer increases the discharge of the primary stain from the cells into the surrounding solvent. On the other hand, the solvent dries out the thicker Grampositive cell walls, which closes the pores as the cell wall shrinks during the drying out. As a result, the diffusion of the crystal violet and iodine complex is blocked, and the bacteria remain stained. The length of the decolorization is important in differentiating the gram positive bacteria from the gram negative bacteria. A prolonged exposure to the decolorizing agent will remove all the stain from both types of bacteria. Some Grampositive bacteria may...
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