Which of the following is NOT part of the Gram stain procedure?

• A. Heat fixation after staining
• B. Application of crystal violet
• C. Counterstaining with safranin
• D. Addition of iodine as a mordant

Answer: (A)

Verified step by step guidance

1. Step 1: Understand the purpose of the Gram stain procedure, which is to differentiate bacterial species into Gram-positive and Gram-negative based on their cell wall properties.
2. Step 2: Recall the main steps of the Gram stain: (a) Application of crystal violet (primary stain), (b) Addition of iodine (mordant) to form a crystal violet-iodine complex, (c) Decolorization with alcohol or acetone, and (d) Counterstaining with safranin to stain Gram-negative bacteria.
3. Step 3: Recognize that heat fixation is a preparatory step done before staining to fix bacteria onto the slide, but it is not performed after staining.
4. Step 4: Compare each option to the known steps: Application of crystal violet, addition of iodine, and counterstaining with safranin are all part of the Gram stain procedure.
5. Step 5: Conclude that 'Heat fixation after staining' is NOT part of the Gram stain procedure, as heat fixation is done before staining, not after.