Pharmaceutical Microbiology (BP303T): Staining, Sterility & Assay Guide

Chapter 1: Morphological Classification & Gram Staining

Developed in 1884 by Danish physician Christian Gram, the Gram stain differentiates bacteria based on physical and chemical differences in their peptidoglycan cell walls.

Staining Protocol:

1. Primary Stain: Crystal Violet (1 min) → All cells turn violet.
2. Mordant: Gram's Iodine (1 min) → Forms insoluble Crystal Violet-Iodine (CV-I) complex inside cells.
3. Decolorizer: 95% Ethanol or Acetone-Alcohol (15-30 sec) → Washes lipid-rich outer membrane and thins wall in Gram-negative; dehydrates thick peptidoglycan in Gram-positive cells.
4. Counterstain: Safranin (1 min) → Imparts pink/red color to decolorized Gram-negative cells.