2027 L3 Principles of Staining

Chromophore

The part of a dye responsible for its color.

Auxochrome

A chemical group that attaches to a chromophore, allowing the dye to bind to tissue.

Metachromasia

A staining phenomenon where a dye changes color upon binding to specific tissue components.

Metachromasia - Normal staining

In tissues without metachromasia, a dye like Toluidine Blue stains the tissue its typical blue colour.

Used to stain mast cell granules, cartilage matrix, and others.

Metachromasia - Metachromatic Staining

When metachromastic substance is present, the dye aggregates and shift colour to purple or red. Colour shift is due to the charge and density of tissue elements.

Purpose of staining in microscopy

• Add contrast to the image

• Locate cells or organelles

• Identify chemical components of interest

Basic dyes

Dyes that bind to acidic tissue components (e.g Haematoxylin)

Acidic dyes

Dyes that bind to basic tissue components (e.g. Eosin)

Neutral dyes

Stain both acidic and basic structures.

Amphoteric dyes

React depending on pH and can bind to both acidic and basic tissue components.

Dye-tissue interactions

• Covalent bonds

• Ionic bonds

• Hydrophobic interactions

• Hydrogen bonds

• Van der Waals forces

Mordant

A non-dyeing compound that enhances dye binding to tissues by acting as a bridge (e.g. iron or aluminium in haematoxylin staining.

Trapping agents

Substances that create large aggregates with the dye, causing it to precipitate in cells or tissues, larger precipitate is harder to remove (e.g. Iodine in Gram staining).

Tissue prep for staining

Most dye staining solutions are aqueous, so to stain the sections, the wax has to be dissolved and the tissue rehydrated.

De-wax (Xylene) —→ Rehydrate (Alcohol) —→ Water —→ Stain

If the section is frozen, ignore this step as the section is already in water.

Hematoxylin and Eosin (H&E)

A common staining technique used to provide diagnostic information in pathology.

Argentaffin reaction

A tissue-driven reduction process resulting in silver deposits (e.g. Enterochromaffin cells in the stomach).

Argyrophil reaction

Requires external reducer/ developer (Formaldehyde as a reducing solution).

Silver staining / Metallic impregnation

A technique used to visualize structures by plating them with silver, as silver is easily reduced and is autocatalytic (self-amplifying reduction).

Silver staining techniques

Gordon and Sweets: Visualizes reticular fibres

Warthin-Starry: Detects thin-spiralled bacteria like Helicobacter pylor

Silver staining pros

• Stable, non-fading

• Offer dense black contrasts ideal for photography

• Detect materials challenging for dyes and plate slender objects, enhancing visibility

Silver staining cons

• Techniques can be inconsistent.

• Solutions are typically very alkaline.

• Sensitivity can lead to nonspecific deposits.

• Stains almost everything it touches.

• Solutions can be explosive.

• Silver is costly.

• Can't be discarded in drains

Mounting media

Substances used to preserve stained specimens; can be permanent (resinous) or temporary (water-based).

Hydrophobic interactions

Weak interactions that occur between dyes and lipophilic tissue components.

Ion-exchange reactions

Reactions used to detect specific minerals, such as bone mineralization (Von Kossa technique).

Silver salts darken under UV light after interacting with bone minerals.

Gram staining

A method that uses iodine as a trapping agent during the staining process.

Special stains

Techniques used to stain specific types of cells or materials not visible with routine stains.