Gram Staining (Histology)
The gram stain is a microorganism stain that primarily targets gram positive and game negative bacteria (although it can be used to stain other organisms like yeast).
- Classification: Microorganism stain
- Target Tissue: Gram positive and gram negative microorganisms
- Staining Principle: Differential staining
- Staining Mechanism: Ionic bonding
- Controls:
- Positive: appendix (appendicitis tissues), tissues containing microorganisms
Staining Mechanism
Primary: Basic (+) stains are applied to the tissue. The dye diffuses through the cell wall and forms ionic bonds with negatively charged phosphate groups present in the bacterial nucleic acids.
Staining Procedure
| Procedure | Time | Rationale | Possible Errors | Troubleshooting |
|---|---|---|---|---|
| Bring slides to dH2O
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| Flood with filtered crystal violet.
Drain slides (do not rinse). |
2 min |
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| Flood with Lugol's iodine | 3 min |
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| Rinse with dH2O. Drain and blot dry. | 2 min | |||
| Decolourize with acetone alcohol | 5-10 dips |
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| Rinse with dH2O. Check for stain deposits | ||||
| Flood slides with 5% Neutral Red | 5 min |
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| Rinse in RTW | ||||
| DCM | ||||
Other Modifications
Modifications of the gram staining procedure can be used to increase contrast by making the background tissue stain more distinctly from gram negative bacteria.This is done by adding an acid dye to the counterstain, which stains other tissue components.
- Brown-Hopps:
- Basic fuchsin: GN bacteria - red
- Gallego: background tissue - yellow
- Brown and Brenn
- Safranin: GN bacteria - pink
- Picric acid: background tissue - yellow
- Gram Twort
- Neutral red: GN bacteria - red/pink
- Fast Green FCF (Twort): background - green