In the Gram stain procedure, what is the correct sequence of reagents applied to a heat-fixed bacterial smear?

• A. Iodine (primary stain) → crystal violet (mordant) → safranin (decolorizer) → alcohol/acetone (counterstain)
• B. Safranin (primary stain) → iodine (mordant) → alcohol/acetone (decolorizer) → crystal violet (counterstain)
• C. Crystal violet (primary stain) → iodine (mordant) → alcohol/acetone (decolorizer) → safranin (counterstain)
• D. Crystal violet (primary stain) → alcohol/acetone (mordant) → iodine (decolorizer) → safranin (counterstain)

Answer: (C)

Verified step by step guidance

1. Understand that the Gram stain procedure involves four main reagents applied in a specific order to differentiate bacterial cell walls based on their ability to retain the primary stain.
2. Identify the primary stain, which is the first reagent applied to the heat-fixed bacterial smear. In the Gram stain, this is crystal violet, which stains all cells purple initially.
3. Next, apply the mordant, which is iodine. The iodine forms a complex with crystal violet inside the cells, helping to fix the dye and increase its retention in Gram-positive bacteria.
4. Then, use the decolorizer, typically alcohol or acetone. This step selectively removes the crystal violet-iodine complex from Gram-negative cells due to their thinner peptidoglycan layer, while Gram-positive cells retain the complex.
5. Finally, apply the counterstain, safranin, which stains the decolorized Gram-negative bacteria pink/red, allowing differentiation between Gram-positive (purple) and Gram-negative (pink/red) cells.