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)
- Mount and heat fix the specimen (about 6 times through the flame).
- Flood (cover completely) the entire slide with crystal violet.
- Let the crystal violet stand for about 60 seconds.
- Flood your slide with the iodine solution
- Let it stand for 60 seconds.
- Rinse the slide with water for 5 seconds and immediately proceed to next step.
- Rinse the slide with decolourizer for 20-60 seconds.
- Rinse the slide carefully with water for about 5 seconds.
- Apply the counter stain, safranin, by flooding the slide with the dye
- Let it stand for about 10-15 seconds.
- Rinse with water for 5 seconds
- Dry the slide with paper or allow it to air dry
- 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.