Goal

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

Materials

  • Gas burner
  • Crystal Violet
  • 95% alcohol
  • Iodine
  • Safranin
  • Tap water
  • Blotting paper

Method

  1. Prepare a drop of sample on your microscope slide according to our 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. Wash the crystal violet by tap water.
  6. Add the iodine solution for 60 seconds.
  7. Wash with tap water.
  8. Decolorize using 95 % ethanol and immediately rinse with tap water. Don’t let it on the slide for too long.
  9. Counter stain with safranin for about 60 seconds.
  10. Wash of the stain with tap water.
  11. Remove excess water using the blotting paper.
  12. Cover the sample with a cover slide.
  13. 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

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

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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