Goal

Assess whether the biological sample contains Gram positive or Gram negative bacterial strains.

Materials

  • Gas burner
  • Crystal Violet
  • 95% alcohol
  • Iodine
  • Safranin
  • 3x Bucket or beaker glass (or wash in between stainings)
  • Tap water
  • Blotting paper (alternatively: Tork roll or toilet paper)
  • Clear nail polish
  • Acetone
  • Cotton swab

Composition of the stains

  • Crystal violet: 1 g of dry crystal violet in 10 mL 95% ethanol
  • Iodine solution: 1 g of iodine in 100 mL water
  • Safranin: 0.25 g of safrin in 10 mL 95% ethanol and 90 mL water

Method

  1. Prepare a drop of sample on your microscope slide according to the Method
  2. Leave the sample to air dry for 5 to 10 minutes.
  3. Fixate by moving the slide back and forth through a flame for a few seconds.
  4. Stain the sample by a droplet of crystal violet and let it stain for max 60 seconds.
  5. Remove excess stain by bathing / dipping your glass slide into a beaker glass / bucket with tap water.
  6. Add the iodine solution for 60 seconds.
  7. Remove excess stain as described before.
  8. Decolourise using 95 % ethanol and immediately rinse with tap water. Don’t leave the ethanol on the slide for too long.
  9. Counter stain with safranin for about 60 seconds.
  10. Remove excess stain as described before.
  11. Remove excess water using blotting paper.
  12. Let slide air dry to get rid of the remaining water.
  13. Put 2 drops of clear nail polish onto the sample.
  14. Cover the sample with a cover slide.
  15. Try to get rid of air bubbles by putting light pressure on the cover slide.
  16. Remove excess nail polish with a cotton swab with acetone.
  17. Take a look with the microscope with full light intensity (open diaphragma)

Interpreting the results

  • Gram positive cells appear violet
  • Gram negative cells appear red

Gram Stain

Microscopic image of a Gram stain of mixed Gram-positive cocci (Staphylococcus aureus ATCC 25923, purple) and Gram-negative bacilli (Escherichia coli ATCC 11775, red). Magnification: 1.000x - Picture by Y tambe CC BY SA 3.0 license

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