Reagents used in the gram stain

The Gram stain uses four reagents.

1. Crystal Violet stains both Gram-positive and Gram-negative bacteria purple by binding to negatively charged cell wall structures. The CV molecules are positively charged and bind to negatively charged bacterial cell wall structures.

2. Iodine is a dye fixer (mordant). This fixes the stain via electrostatic forces, forming a large, insoluble Crystal Violet - Iodine complex (CV-I complex).

3. 95% Ethyl Alcohol decolorizes Gram-negative bacteria but not Gram-positive due to the thicker peptidoglycan layer.

4. Safranin counterstains the Gram-negative bacteria pink, while Gram-positive bacteria remain purple. Like crystal violet, it is a positively charged molecule that binds to negative bacterial cell wall structures.

Steps in the gram staining method. Crystal violet and iodine are added to a bacterial sample and left to react for 60 seconds each, staining all bacteria purple. 95% ethyl alcohol is then added and left to react for 5-10 seconds, removing the purple stain from gram-negative bacteria. Finally, safranin is added and left to react for 45 seconds, staining gram-negative bacteria pink.

Figure 1. Overview of the Gram stain method