Gram Staining in Microbiology

Overview

• The Gram stain is the most widely used method in microbiology for staining bacteria.
• It is a differential stain that classifies bacteria into two groups: gram-positive and gram-negative.

Procedure Steps

1. Preparation of the Slide

• Transfer cells from a fresh culture to a clean slide and allow to dry.
• If using cells from an agar plate, first transfer to a liquid medium for dilution.
• Aim for a thin, barely visible film on the slide.
• Fresh cultures are essential; aged cells may lose staining capability.

2. Fixing the Cells

• Fix cells to the slide by passing it slightly above a Bunsen burner flame.
• The slide should feel warm to the touch, but not too hot.

3. Staining with Crystal Violet

• Stain fixed cells with the basic dye crystal violet for 30-40 seconds.
• Rinse with water to remove excess stain.
• All cells appear purple at this stage.

4. Application of Gram's Iodine

• Add Gram's iodine solution and retain for about one minute.
• Iodine forms a dye-iodine complex with crystal violet, decreasing solubility in cells.
• Cells still appear purple after this step.

5. Decolorization

• Decolorize with ethanol or acetone (this is the differential step).
• Gram-positive bacteria retain crystal violet, while gram-negative bacteria do not.
• Apply ethanol dropwise, tilting the slide until effluent starts becoming colorless.
• Note: Excessive decolorization can cause even gram-positive cells to lose the dye complex.
• Rinse off excess ethanol with water.

6. Counterstaining with Safranin

• Cover cells with the counterstain safranin for 20-30 seconds.
• Rinse with water and dry the slide with filter paper.
• Under the microscope:
  • Gram-positive bacteria appear purple
  • Gram-negative bacteria appear pink.

Cell Wall Structure Correlation

• The Gram stain correlates with the structure of the bacterial cell wall:
  • Gram-positive bacteria:
    • Have a thick peptidoglycan layer that acts as a permeability barrier, retaining the dye.
    • Ethanol shrinks the peptidoglycan, helping to retain the crystal violet iodine complex.
  • Gram-negative bacteria:
    • Have a thin peptidoglycan layer with large pores.
    • Ethanol extracts lipids, increasing porosity and removing the crystal violet iodine complex.