Gram's Stain

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Gram's Stain
    Principle:
        The basic dye (methyl violet) is retained even after decolorization with acetone in gram-positive bacteria.
        In gram - negative bacteria on decolorization with aacetone, basic dye comes out.

     Procedure:
  • Spread the material containing bacteria on a clean glass slide and dry it.
  • Pass the slide over the flame once or twice to fix the smear.
  • Cover the smear with Crystal violet for 1 minute.
  • Wash the slide with the water and cover the smear with Gram's Iodine for 1 minute.
  • Drain off Gram's Iodine.
  • Add Acetone over the smear and immediately wash the slide with water.
  • Cover the smear with diluted Carbol Fuchsin (1:20). Keep it up for 1-2 minutes.
  • Wash the slide with water and then dry it and see the smear under microscope (100x).
  • Keep the condenser upward.

    Result:
  • Gram - Positive Bacteria are seen deep violet in color.
  • Gram - Negative Bacteria are seen pink color.



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