Which of the following describes the correct sequence of the Gram stain procedure?

• A. Crystal violet, iodine, alcohol, safranin
• B. Iodine, crystal violet, safranin, alcohol
• C. Safranin, alcohol, iodine, crystal violet
• D. Alcohol, crystal violet, safranin, iodine

Answer: (A)

Verified step by step guidance

1. Understand that the Gram stain is a differential staining technique used to classify bacteria into Gram-positive and Gram-negative based on their cell wall properties.
2. Recall the sequence of reagents used in the Gram stain procedure: first, the primary stain (crystal violet) is applied to stain all cells.
3. Next, the mordant (iodine) is added, which forms a complex with crystal violet, helping it to fix inside the cell wall.
4. Then, the decolorizer (alcohol or acetone-alcohol) is applied to wash out the stain from Gram-negative cells but not from Gram-positive cells due to their thicker peptidoglycan layer.
5. Finally, the counterstain (safranin) is applied to stain the decolorized Gram-negative cells pink/red, while Gram-positive cells remain purple.