What is the correct sequence of steps in the basic Gram stain procedure?

• A. Apply crystal violet, decolorize briefly with alcohol, add iodine, counterstain with safranin.
• B. Apply crystal violet, add iodine, decolorize briefly with alcohol, counterstain with safranin.
• C. Add iodine, apply crystal violet, decolorize, counterstain with safranin.
• D. Counterstain with safranin, apply crystal violet, add iodine, decolorize.

Answer: (B)

Differentiating Gram-positive and Gram-negative bacteria relies on how the cell wall interacts with the dye sequence used. Start by applying crystal violet, the primary stain that colors all cells. Next, add iodine, which acts as a mordant to form a larger crystal violet–iodine complex inside the cells, helping the stain stay bound in cells with thick peptidoglycan. Then briefly decolorize with alcohol; this step removes the dye from cells with thinner cell walls, making Gram-negative cells lose the color while Gram-positive cells keep it. Finally, counterstain with safranin to color the decolorized cells pink, so Gram-negative bacteria appear pink and Gram-positive remain purple. This specific order is essential because altering it prevents proper formation of the dye complex or proper removal of color, leading to incorrect results.