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	Possible Errors
Bring slides to dH2O
Flood with filtered crystal violet. Drain slides (do not rinse).	2 min	Stain not filtered: stain deposits obscure tissue Too short time: inadequate staining (pale purple)
Flood with Lugol's iodine	3 min	Skipped step: no crystal violet-iodine complex is formed, so dye is not trapped in gram positive (everything appears gram negative)
Rinse with dH2O. Drain and blot dry.	2 min
Decolourize with acetone alcohol	5-10 dips	Under-decolourized: No differentiation, everything appears gram positive Over-decolourized: Gram positives decolourized, everything appears gram negative
Rinse with dH2O. Check for stain deposits
Flood slides with 5% Neutral Red	5 min	Under-stained: Background pale/colourless, reducing contrast Over-stained: Stain leaches crystal violet-iodine complex, making everything appear gram negative
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