Describe the steps involved in performing a Gram stain on a bacterial smear.

• A. Prepare a smear and heat-fix; apply crystal violet (1 min); rinse; apply iodine (1 min) as mordant; decolorize briefly with alcohol; counterstain with safranin (30–60 sec); rinse and air dry
• B. Stain with safranin first
• C. Decolorize with water only
• D. Heat with flame after staining

Answer: (A)

Gram staining differentiates bacteria by cell wall properties using a sequence of reagents that first colors all cells, then locks color in where the wall structure retains it, and finally provides a contrasting stain for cells that lose the initial color. Start with a prepared smear that’s heat-fixed to fix the cells to the slide. Apply the primary stain, crystal violet, for about a minute to color all cells. Rinse to remove excess. Then add iodine as a mordant for about a minute to form a crystal violet–iodine complex that adheres strongly to the cell wall. Rinse again. Briefly decolorize with alcohol; this step is what creates the difference: thick, tightly cross-linked peptidoglycan in Gram-positive cells traps the violet–iodine complex, while the thinner peptidoglycan layer and outer membrane in Gram-negative cells allow it to be washed away. Immediately rinse to stop decolorization. Finally, counterstain with safranin for about 30–60 seconds so any decolorized Gram-negative cells appear pink, then rinse and air dry.

Staining safranin first would erase the differential result, decolorizing with water wouldn’t reliably distinguish organisms, and heating after staining isn’t part of this protocol and could distort results.