Question

Select the correct order of the steps involved in Gram staining:
A. Counter staining
B. Crystal violet- Iodine (CV-I)
C. Heat Fixation
D. Decolorizing agent

• A. B, D, C, A
• B. A, D, B, C
• C. C, A, D, B
• D. C, B, D, A

Hint:

Gram Staining Sequence:
1. Heat Fix slide (C) $\to$ 2. Crystal Violet + Iodine (B) $\to$ 3. Alcohol Decolorizer (D) $\to$ 4. Safranin Counterstain (A).
Result: Gram-positive = Purple, Gram-negative = Pink/Red.

Answer 1

The Correct Option is D

Step 1: Understanding the Question:
The question asks for the proper sequential order of procedures involved in performing a Gram stain differential staining procedure on bacterial smears.

Step 2: Key Formula or Approach:
Gram staining differentiates Gram-positive (purple) and Gram-negative (pink/red) bacteria based on cell wall peptidoglycan thickness:
Smear Fixation (C) $\rightarrow{}$ Primary Stain + Mordant (B) $\rightarrow{}$ Decolorization (D) $\rightarrow{}$ Counterstaining (A)

Step 3: Detailed Explation:

• 1. Heat Fixation (C): A bacterial smear is air-dried and gently heat-fixed over a flame to adhere cells to the glass slide and kill microbes.

• 2. Crystal Violet and Iodine Mordant (B): Crystal violet primary stain is applied, followed by Gram's iodine (mordant) application to form large, insoluble Crystal Violet-Iodine (CV-I) complexes inside the peptidoglycan wall.

• 3. Decolorization (D): Applying decolorizer (95% ethanol or acetone). Ethanol dehydrates thick peptidoglycan in Gram-positive walls (trapping CV-I, retaining purple dye), but dissolves outer lipid membranes of Gram-negative walls, leaching out the CV-I complex (making them colorless).

• 4. Counterstaining (A): Applying safranin counterstain, which stains decolorized Gram-negative cells pink/red while Gram-positive cells remain purple.

Step 4: Fil Answer:
The correct sequential order of Gram staining steps is C, B, D, A.