Gram Staining in Microbiology

Overview

• Gram Staining: A technique used to differentiate between gram-positive and gram-negative bacterial cells under a microscope.

Bacterial Cell Structure

• Two Main Groups:
  • Gram Positive
  • Gram Negative
• Key Structural Differences:
  • Inner Cell Membrane: Composed of phospholipid bilayer in both types.
  • Peptidoglycan Cell Wall:
    • Thicker in gram-positive cells.
    • Thinner in gram-negative cells.
  • Outer Membrane:
    • Present in gram-negative cells.
    • Absent in gram-positive cells.
  • Outer Slime Capsule: Present in both, providing extra covering.

Gram Staining Process

1. Fixing:
   • Sample bacteria from a petri dish applied onto a glass slide.
   • Heat applied to fix bacteria onto the slide.
2. Crystal Violet Application:
   • Diffuses across all outer layers, entering cells.
   • Stains all cells purple initially.
3. Iodine Application:
   • Binds with crystal violet, forming complexes.
   • Purpose: to fix the crystal violet inside the cells.
4. Ethanol/Acetone Application:
   • In Gram-negative Cells:
     • Dissolves outer membrane.
     • Peptidoglycan wall shrinks.
     • Crystal violet complexes washed out, cells become colorless.
   • In Gram-positive Cells:
     • Cell wall shrinks but retains crystal violet.
     • Cells remain purple.
5. Saffronin Application (Counter Stain):
   • Diffuses into both cell types.
   • Gram-positive Cells: Remain purple as crystal violet dominates.
   • Gram-negative Cells: Appear red/pink as saffronin is the only dye present.

Visualization

• Under the Microscope:
  • Gram-positive cells appear purple.
  • Gram-negative cells appear red.

Conclusion

• Summary of Key Steps:
  • Fixing, crystal violet, iodine, ethanol/acetone, saffronin.
• Visual Differences:
  • Clear distinction between gram-positive and gram-negative cells based on color.