The Gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. Through a series of staining and decolorization steps, organisms in the Domain Bacteria are differentiated according to cell wall composition. Gram-positive bacteria have cell walls that contain thick layers of peptidoglycan (90% of cell wall). These stain purple. Gram-negative bacteria have walls with thin layers of peptidoglycan (10% of wall), and high lipid content. These stain pink. This staining procedure is not used for Archeae or Eukaryotes as both lack peptidoglycan. The performance of the Gram Stain on any sample requires 4 basic steps that include applying a primary stain (crystal violet) to a heat-fixed smear, followed by the addition of a mordant (Gram's Iodine), rapid decolorization with alcohol, acetone, or a mixture of alcohol and acetone and lastly, counterstaining with safranin.
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