Question

Arrange the following steps involved in Gram staining technique in correct sequence:
A. Preparation of smear and fixation,
B. Decolourization with alcohol,
C. Addition of iodine,
D. Counter staining, E. Differential feature becomes apparent. Choose the correct answer from the options given below:

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

Hint:

In Gram staining, decolourization is the most critical step for differentiating Gram-positive and Gram-negative bacteria.

Answer 1

The Correct Option is A

Concept:
Gram staining is a differential staining method used to separate bacteria into Gram-positive and Gram-negative groups.

Step 1: Smear preparation and fixation.
First, a thin smear is prepared and fixed on the slide. \[ A=\text{Preparation of smear and fixation} \]

Step 2: Iodine treatment.
Iodine acts as a mordant and forms a complex with the primary stain. \[ C=\text{Addition of iodine} \]

Step 3: Decolourization.
Alcohol is used to remove stain from Gram-negative bacteria. \[ B=\text{Decolourization with alcohol} \]

Step 4: Differential feature becomes visible.
After decolourization, Gram-positive and Gram-negative bacteria start showing differential behaviour. \[ E=\text{Differential feature becomes apparent} \]

Step 5: Counter staining.
Finally, counter stain is applied so that Gram-negative bacteria become visible. \[ D=\text{Counter staining} \] Thus, the correct sequence is: \[ A,C,B,E,D \] \[ \therefore \text{Correct Answer is (A)} \]