6.2. Gram staining

Gram-staining is a four part procedure which uses certain dyes to make a bacterial cell stand out against its background. The reagents you will need are:

  • Crystal violet (the primary stain)
  • Iodine solution (the mordant)
  • Decolourizer (ethanol + acetone)
  • Safranin (the counter stain)

Procedure:

  1. Mount and heat fix the specimen (about 6 times through the flame).
  2. Flood (cover completely) the entire slide with crystal violet.
  3. Let the crystal violet stand for about 60 seconds.
  4. Flood your slide with the iodine solution
  5. Let it stand for 60 seconds.
  6. Rinse the slide with water for 5 seconds and immediately proceed to next step.
  7. Rinse the slide with decolourizer for 20-60 seconds.
  8. Rinse the slide carefully with water for about 5 seconds.
  9. Apply the counter stain, safranin, by flooding the slide with the dye
  10. Let it stand for about 10-15 seconds.
  11. Rinse with water for 5 seconds
  12. Dry the slide with paper or allow it to air dry
  13. View under the microscope at 1,000 times magnification (Fig. 6).

 

Fig. 6. Gram staining of Melissococcus plutonius. The coccoid-shaped bacteria forming pairs or even chains are clearly visible. Photo: Lena Lundgren and Karl-Erik Johansson.

Figure 6