In the Gram stain procedure, what is the correct sequence of reagents applied to a heat-fixed bacterial smear (excluding the initial water rinse)?

• A. Iodine (mordant), crystal violet, safranin, decolorizer (alcohol/acetone)
• B. Crystal violet, decolorizer (alcohol/acetone), iodine (mordant), safranin
• C. Crystal violet, iodine (mordant), decolorizer (alcohol/acetone), safranin
• D. Safranin, crystal violet, iodine (mordant), decolorizer (alcohol/acetone)

Answer: (C)

Verified step by step guidance

1. Step 1: Understand the purpose of each reagent in the Gram stain procedure. Crystal violet is the primary stain that initially colors all the bacteria purple.
2. Step 2: Apply iodine, which acts as a mordant. The iodine forms a complex with the crystal violet inside the bacterial cells, making the dye less soluble and more firmly attached.
3. Step 3: Use the decolorizer (usually alcohol or acetone). This step differentiates bacteria by washing out the crystal violet-iodine complex from Gram-negative cells, while Gram-positive cells retain the complex due to their thicker peptidoglycan layer.
4. Step 4: Apply safranin, the counterstain. This stains the decolorized Gram-negative bacteria pink/red, allowing differentiation from the purple Gram-positive bacteria.
5. Step 5: Summarize the correct sequence of reagents applied to the heat-fixed smear as: crystal violet, iodine (mordant), decolorizer (alcohol/acetone), and finally safranin.