Which of the following is the correct sequence of reagents given below used in the Gram staining process?
(A) Iodine (B) Crystal violet (C) Safranin (D) Alcohol
Choose the correct answer from the options given below:

Correct option is B

Correct Answer: B — (B), (A), (D), (C)

That is: Crystal violet → Iodine → Alcohol → Safranin

Gram Staining Process-

Step 1: Crystal violet (B) – Primary stain
Crystal violet is the first dye applied to the bacterial smear.
It stains all bacteria (both Gram-positive and Gram-negative) purple initially.

Step 2: Iodine (A) – Mordant
Iodine acts as a mordant and forms a crystal violet–iodine (CV–I) complex.
This complex is strongly retained in the thick peptidoglycan layer of Gram-positive bacteria.

Step 3: Alcohol (D) – Decolorizer
Alcohol removes lipids from the outer membrane of Gram-negative bacteria.
As a result, Gram-negative cells lose the CV–I complex, while Gram-positive cells retain it.

Step 4: Safranin (C) – Counterstain
Safranin stains the decolorized Gram-negative bacteria pink/red.
Gram-positive bacteria remain purple because the darker crystal violet masks safranin.

Why other options are incorrect

Option A: (A), (B), (C), (D)
Iodine cannot be used before crystal violet because it acts only as a mordant.
Safranin cannot be applied before decolorization.

Option C: (B), (A), (C), (D)
Safranin is incorrectly placed before alcohol.
Counterstain is always applied after decolorization.

Option D: (C), (B), (D), (A)
Safranin cannot be the first stain in Gram staining.
Iodine must immediately follow crystal violet, not come at the end.