In the Gram stain procedure, which reagent serves as the decolorizing agent?

• A. 95% ethanol (or an acetone-alcohol mixture)
• B. Gram's iodine
• C. Safranin
• D. Crystal violet

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. Recognize that Gram's iodine acts as a mordant, forming a complex with crystal violet to help it adhere to the bacterial cell wall.
3. Identify the decolorizing agent, which selectively removes the primary stain from certain bacteria based on their cell wall structure. This agent is typically 95% ethanol or an acetone-alcohol mixture.
4. Note that after decolorization, Gram-positive bacteria retain the crystal violet-iodine complex and remain purple, while Gram-negative bacteria lose the stain and become colorless.
5. Finally, safranin is used as a counterstain to color the now colorless Gram-negative bacteria pink/red, allowing differentiation under the microscope.