Which of the following correctly lists the order of reagents used in the Gram stain procedure?

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

Answer: (D)

Verified step by step guidance

1. Understand that the Gram stain procedure 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: 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 (decolorizer) is used to wash away the stain from Gram-negative cells, while Gram-positive cells retain the dye due to their thick peptidoglycan layer.
5. Finally, safranin is applied as a counterstain to color the decolorized Gram-negative cells pink/red, allowing differentiation under the microscope.