Which of the following correctly lists the steps of the Gram stain procedure in the proper order?

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

Answer: (B)

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 correct sequence of the Gram stain procedure: first, apply the primary stain (crystal violet) to color all cells.
3. Next, add the mordant (iodine), which forms a complex with crystal violet, helping it to fix inside the cell wall.
4. Then, apply the decolorizer (alcohol or acetone-alcohol), which removes the stain from Gram-negative cells but not from Gram-positive cells due to their thicker peptidoglycan layer.
5. Finally, apply the counterstain (safranin), which stains the decolorized Gram-negative cells pink/red, while Gram-positive cells remain purple.