The Gram stain uses four reagents.
Crystal Violet (CV) is the Primary stain, and is used to color both Gram-positive and Gram-negative bacteria purple.
The CV molecules are positively charged and bind to negatively charged bacterial cell wall structures.
Iodine (I) acts as a dye-fixator (mordant) that fixes the purple stain in both Gram-positive and Gram-negative bacteria.
The Iodine molecules bind to Crystal Violet molecules via electrostatic forces, forming a large, insoluble Crystal Violet - Iodine complex (CV-I complex).
95% Ethyl Alcohol (Ethanol) is used to decolorize the Gram-negative bacteria. The alcohol dissolves the outer membrane and disrupts the thin peptidoglycan,
allowing the purple CV-I complexes to escape the Gram-negative cell wall. Gram-positive bacteria remain purple after the alcohol wash,
due to their thick and robust peptidoglycan which withstand the alcohol and retain the CV-I complexes.
Safranin is used as a counterstain, staining the Gram-negative bacteria, pink. Like crystal violet, it is a positively charged molecule that binds to negative bacterial cell wall structures.
The Gram-positive bacteria are already colored purple by the darker Crystal Violet and remain purple despite the addition of the pink safranin.
Figure 1. Overview of the Gram stain method