Which sequence correctly describes the key steps in performing a Gram stain?

Which sequence correctly describes the key steps in performing a Gram stain?

• A. Place slide on a staining rack; STAIN the slide with crystal violet for 1-2 min; Pour off the stain; Flood slide with Gram's iodine for 1-2 min; Pour off the iodine; Decolorize by washing briefly with acetone (2-3 seconds); Wash slide thoroughly with water; Flood slide with safranin counterstain for 2 min; Wash with water; Blot excess water and dry in hand over bunsen flame.
• B. Stain with crystal violet; Flood with iodine; Decolorize with acetone; Counterstain with safranin; Place slide on rack; Wash.
• C. Place slide on a staining rack; Flood slide with Gram's iodine 1-2 min; Stain with crystal violet 1-2 min; Decolorize with acetone; Wash; Counterstain.
• D. Place slide on a staining rack; Stain with safranin; Then crystal violet; Then iodine; Then decolorize.

Answer: (C)

The main idea being tested is the correct sequence of steps in the Gram stain, which relies on the order of applying a primary stain, a mordant, a decolorizer, and a counterstain to differentiate cell wall types. The correct sequence starts with crystal violet as the primary stain, then Gram’s iodine as the mordant to fix the dye, followed by a brief decolorization with acetone or alcohol, and finally a safranin counterstain. This order matters because Gram-positive cells have a thick peptidoglycan layer that traps the crystal violet–iodine complex, so they stay purple, while Gram-negative cells lose the color during decolorization and take up the pink counterstain. In the proper procedure you’d place the slide on a rack, stain with crystal violet for about 1–2 minutes, rinse; flood with Gram’s iodine for about 1–2 minutes, rinse; decolorize briefly with acetone (2–3 seconds), rinse; then counterstain with safranin for about 2 minutes, rinse, and blot dry. This exact order is what yields the correct differential result. The option that lists this sequence is the one that aligns with the correct protocol. The other options mix up the order—such as applying the iodine before the primary stain or introducing the counterstain at the wrong time or skipping the proper sequence—so they wouldn’t produce the correct differentiation between Gram-positive and Gram-negative cells.

The main idea being tested is the correct sequence of steps in the Gram stain, which relies on the order of applying a primary stain, a mordant, a decolorizer, and a counterstain to differentiate cell wall types. The correct sequence starts with crystal violet as the primary stain, then Gram’s iodine as the mordant to fix the dye, followed by a brief decolorization with acetone or alcohol, and finally a safranin counterstain. This order matters because Gram-positive cells have a thick peptidoglycan layer that traps the crystal violet–iodine complex, so they stay purple, while Gram-negative cells lose the color during decolorization and take up the pink counterstain.

In the proper procedure you’d place the slide on a rack, stain with crystal violet for about 1–2 minutes, rinse; flood with Gram’s iodine for about 1–2 minutes, rinse; decolorize briefly with acetone (2–3 seconds), rinse; then counterstain with safranin for about 2 minutes, rinse, and blot dry. This exact order is what yields the correct differential result.

The option that lists this sequence is the one that aligns with the correct protocol. The other options mix up the order—such as applying the iodine before the primary stain or introducing the counterstain at the wrong time or skipping the proper sequence—so they wouldn’t produce the correct differentiation between Gram-positive and Gram-negative cells.