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 **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.