![]() ![]() ![]() Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram positive cells. A counterstain, such as the weakly water soluble safranin, is added to the sample, staining it red.Conversely, the the outer membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-iodine complex and the color is lost. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria. A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it.This complex is a larger molecule than the original crystal violet stain and iodine and is insoluble in water. Next, a Gram's iodine solution (iodine and potassium iodide) is added to form a complex between the crystal violet and iodine. Cells are stained with crystal violet dye.Due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain during the decolorization process, while Gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process. Gram staining involves three processes: staining with a water-soluble dye called crystal violet, decolorization, and counterstaining, usually with safanin. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. ![]()
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