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Microbiology Gram Stain Procedure
Created by:
Paul J. Deziel, RPA-C, MHA
Editor-in-Chief/Publisher
All inquiries: deziel@charter.net
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Copyright © 1996-2005
All Rights Reserved
Rochester, MN
U.S.A
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Procedure for performing the gram stain:
Gram stain
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1. make a thin smear on a clean glass slide.
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2. air-dry and gently heat-fix the smear by passing the slide through a flame 1 or 2 times. Do not make the slide too hot to touch, as this may cause artifacts
and alter cell morphology.
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3. after cooling the slide, flood with crystal or gentian violet for 10 seconds and then rinse gently with tap water.
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4. flood with gram's iodine for 10 seconds and then rinse again.
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5. decolorize with 95% ethanol or an acetone-ethanol mixture and repeat the tap-water rinse. dcolorization is the critical step in the staining procedure.
decolorization is complete when the blue stain is no longer visible and when the solvent is colorless as it runs from the slide.
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6. counterstain with safranin for 10 seconds and repeat the tap-water rinse.
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7. air-dry or blot gently with clean, absorbent paper.
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