Gram Stain


Gram Stain, Gram Positive, Gram Negative

  • Indications
  • Evaluation of Infection
  • Specimens
  1. Infected body fluids
  2. Aspirated abscesses
  3. Exudates
  • Technique
  1. Thin smear of material spread on slide
    1. Allow slide to air dry
  2. Fix material onto the slide
    1. Place side on warmer at 70 degrees Celsius or
    2. Pass slide through Bunsen Burner flame 3-4 times
  3. Overlay the slide with crystal violet for 1 minute
  4. Wash slide thoroughly with water
  5. Overlay slide with Gram Iodine for 1 minute
  6. Wash slide thoroughly with water
  7. Flood slide surface with decolorizer (acetone-Alcohol)
    1. Perform until no violet color washes off slide
    2. May require up to 10 seconds
  8. Wash slide completely with water
  9. Overlay smear with safranin counterstain for 1 minute
  10. Wash slide thoroughly with water
    1. Allow excess water to run off slide
  11. Allow slide to air dry or blot with bibulous paper
  12. Examine under high power (100x) oil immersion
  • Interpretation
  1. Gram Positive (Stains dark blue to purple)
    1. Gram Positive Cocci
  2. Gram Negative (Stains pink to red)
    1. Gram Negative Rod
    2. Gram Negative Coccobacilli