In Gram staining, which of the following is the mordant that forms a complex with crystal violet and allows retention of the dye in Gram-positive bacteria?

Question

Accepted Answer

B. Gram's iodine. ## Gram Staining Mechanism

**Key Point:** Gram's iodine is the mordant in Gram staining. It forms a large crystal violet–iodine complex that is retained in the thick peptidoglycan layer of Gram-positive bacteria but is washed out of Gram-negative bacteria during the decolorization step.

## Step-by-Step Gram Staining Process

1. **Crystal violet application** — Primary stain; binds to all bacterial cells (purple)
2. **Gram's iodine application** — Mordant; forms crystal violet–iodine complex, increasing dye size and affinity
3. **Acetone-alcohol application** — Decolorizer; removes the complex from Gram-negative bacteria (thin peptidoglycan) but not from Gram-positive bacteria (thick peptidoglycan)
4. **Safranin application** — Counterstain; colors decolorized Gram-negative bacteria pink/red

## Gram-Positive vs. Gram-Negative Staining

| Feature | Gram-Positive | Gram-Negative |
| --- | --- | --- |
| Peptidoglycan thickness | Thick (20–80 nm) | Thin (5–10 nm) |
| Teichoic acids | Present | Absent |
| Outer membrane | Absent | Present |
| Crystal violet–iodine retention | Yes (purple) | No (decolorized) |
| Final color after safranin | Purple | Pink/red |

**High-Yield:** The mordant (Gram's iodine) is essential for the differential staining; without it, both Gram-positive and Gram-negative bacteria would be colored by crystal violet alone and could not be differentiated.

**Mnemonic:** **CIG-SA** = Crystal violet, Iodine (Gram's), decolorizer (acetone-alcohol), Safranin (counterstain) — the four key reagents in order of application.

Answer

A. Methylene blue

Answer

C. Safranin

Answer

D. Acetone-alcohol

In Gram staining, which of the following is the mordant that forms a complex with crystal violet and allows retention of the dye in Gram-positive bacteria?

Explanation

Gram Staining Mechanism

Step-by-Step Gram Staining Process

Gram-Positive vs. Gram-Negative Staining

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Feature	Gram-Positive	Gram-Negative
Peptidoglycan thickness	Thick (20–80 nm)	Thin (5–10 nm)
Teichoic acids	Present	Absent
Outer membrane	Absent	Present
Crystal violet–iodine retention	Yes (purple)	No (decolorized)
Final color after safranin	Purple	Pink/red