02/09/2025
📚 Gram Stain (Gram’s Staining)
🔹 Principle
Gram staining is based on the structural differences in the bacterial cell wall:
• Gram Positive bacteria have a thick peptidoglycan layer, which retains the primary stain (crystal violet).
• Gram Negative bacteria have a thin peptidoglycan layer and an outer lipid membrane, so they lose the primary stain and take up the counterstain (safranin).
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🔹 Steps of Gram Staining
1. Primary Stain – Crystal Violet
→ All bacteria appear purple.
2. Mordant – Iodine
→ Forms a crystal violet–iodine complex, fixing the dye.
3. Decolorizer – Alcohol/Acetone
• Gram positive → dye retained (remain purple)
• Gram negative → dye lost (colorless)
4. Counterstain – Safranin (or Fuchsin)
• Gram positive → remain purple/blue
• Gram negative → turn pink/red
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🔹 Results
• Gram Positive bacteria → Purple (e.g., Staphylococcus aureus, Bacillus subtilis)
• Gram Negative bacteria → Pink/Red (e.g., E. coli, Salmonella)
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🔹 Applications
• Differentiation of bacteria (Gram + vs Gram –)
• Important in clinical diagnosis of infections
• Helps in antibiotic selection
