Gram stain or Gram staining, also called Gram's method, is a method of staining used to distinguish and classify bacterial species into two large groups (Gram-positive and Gram-negative). The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique. Gram staining differentiates bacteria by the chemical and physical properties of their cell walls by detecting peptidoglycan, which is present in the cell wall of Gram-positive bacteria. Gram-negative cells also contain peptidoglycan, but a very small layer of it that is dissolved when the alcohol is added. This is why the cell loses its initial color from the primary stain. Gram-positive bacteria retain the crystal violet dye, and thus are stained violet, while the Gram-negative bacteria do not; after washing, a counterstain is added (commonly safranin or fuchsine) that will stain these Gram-negative bacteria a pink color. Both Gram-positive bacteria and Gram-negative bacteria pick up the counterstain. The counterstain, however, is unseen on Gram-positive bacteria because of the darker crystal violet stain. The Gram stain is almost always the first step in the preliminary identification of a bacterial organism. While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique. This gives rise to Gram-variable and Gram-indeterminate groups.
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