Which of the following lists the correct sequence of reagents used in the Gram stain procedure?

• A. Iodine, crystal violet, safranin, alcohol
• B. Crystal violet, iodine, alcohol, safranin
• C. Alcohol, safranin, crystal violet, iodine
• D. Safranin, alcohol, 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 sequence of reagents used in the Gram stain procedure: first, the primary stain crystal violet is applied to stain all cells purple.
3. Next, iodine is added as a mordant to form a crystal violet-iodine complex, which helps fix the dye inside the cells.
4. Then, alcohol (usually ethanol or acetone-alcohol) is used as a decolorizer to wash out the dye from Gram-negative cells but not from Gram-positive cells.
5. Finally, safranin is applied as a counterstain to color the decolorized Gram-negative cells pink/red, allowing differentiation under the microscope.