The Gram stain uses four reagents.

1. 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.

2. 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).

3. 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.

4. 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.

An overview of the gram stain method

Figure 1. Overview of the Gram stain method