Q.46 Arrange the steps involved in Gram-stain procedure-
(A). Staining with Crystal Violet
(B). Staining with Gram’s lodine
(C). Decolorization
(D). Staining with Safranine
1. (A), (B), (C), (D)
2. (A), (B), (D), (C)
3. (B), (A), (D), (C)
4. (C), (B), (D), (A)
The Gram stain differentiates bacteria by cell wall properties through a precise four-step sequence.
Correct Answer
1. (A), (B), (C), (D) – Crystal Violet → Gram’s Iodine → Decolorization → Safranin.
Why This Order Works
Gram-positive bacteria retain the purple Crystal Violet-Iodine complex after decolorization, appearing violet. Gram-negative bacteria lose it and take up red Safranin.
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(A) Crystal Violet: Primary stain binds all bacteria purple (30-60s).
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(B) Gram’s Iodine: Mordant fixes dye into insoluble complex (1 min).
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(C) Decolorization: Alcohol/acetone removes complex from gram-negatives (10-20s).
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(D) Safranin: Counterstain colors decolorized gram-negatives red (30-60s).
All Options Explained
1. (A), (B), (C), (D)
Standard protocol: Primary stain → mordant → decolorize → counterstain. Ensures differential coloring.
✅ Correct: Universal lab sequence.
2. (A), (B), (D), (C)
Safranin before decolorization traps counterstain in all cells, masking differentiation—no color contrast.
❌ Incorrect: Decolorization must precede counterstain.
3. (B), (A), (D), (C)
Iodine before Crystal Violet lacks dye to fix; ineffective staining from start.
❌ Incorrect: Primary stain always first.
4. (C), (B), (D), (A)
Decolorization first on unstained cells does nothing; skips essential primary staining.
❌ Incorrect: No logical sequence.
| Option | Sequence | Outcome | Valid? |
|---|---|---|---|
| 1 | A→B→C→D | Proper gram+ purple, gram- pink | ✅ Yes |
| 2 | A→B→D→C | All cells mixed colors | ❌ No |
| 3 | B→A→D→C | Weak/no staining | ❌ No |
| 4 | C→B→D→A | Unstained cells | ❌ No |
Key Exam Tip: Memorize A-B-C-D for Gram staining success.
