In the Gram stain procedure, which reagent is used as the decolorizer to remove the crystal violet–iodine complex from Gram-negative cells?

• A. 95% ethanol (or an acetone-alcohol mixture)
• B. Gram’s iodine (mordant)
• C. Safranin (counterstain)
• D. Crystal violet (primary stain)

Answer: (A)

Verified step by step guidance

1. Understand the purpose of each reagent in the Gram stain procedure: Crystal violet is the primary stain that colors all cells purple initially.
2. Gram's iodine acts as a mordant, forming a crystal violet–iodine complex that helps retain the dye in Gram-positive cells.
3. The decolorizer, typically 95% ethanol or an acetone-alcohol mixture, is applied next to selectively remove the crystal violet–iodine complex from Gram-negative cells due to their thinner peptidoglycan layer.
4. After decolorization, Gram-negative cells become colorless, while Gram-positive cells remain purple because the complex is retained.
5. Finally, safranin is used as a counterstain to color the now colorless Gram-negative cells pink/red, allowing differentiation under the microscope.