Learning Objectives

• Describe the purpose of staining in the histology lab.
• Learn about dyes and terminology related to dyes.
• Classify dyes based on reaction with tissue, the chemical bond formed, physical and chemical interactions, and methods.
• Learn about the classification systems typically used in histological staining.

Why Do We Stain?

Staining is one of the main testing areas in the histology laboratory. The main purposes of staining are:

• Contrast between and identify tissue elements and their components:
  • Achieved by several methods:
  • Attaching dyes to the tissue.
  • Reacting the tissue with organic reagents to produce colored products.
  • Depositing metals on tissue components to create visible, insoluble black products.

What Are Dyes?

Dyes are organic compounds, primarily derived from natural sources or from coal tar or benzene. They can attach to a substance via chemical bonding.

Dye Terminology

Terminology is crucial for clear communication in histotechnology regarding dyes. Key terms include:

• Chromophore:
  • Definition: A color-emitting part of a molecule that absorbs UV-visible light and reflects the unabsorbed light.
  • Responsible for the color of a molecule.
  • Types of chromophore groups include:
  • Nitro: -NO2
  • Azo: -N=N-
  • Carbonyl: >C=O
  • Ethylene/Acetylene: C=C or C=C
• Auxochrome:
  • Definition: A group of atoms attached to a chromophore that intensifies the color emission but does not itself emit color.
  • Modifies the chromophore by shifting it to a longer wavelength.
  • Works as a color helper.
  • Types of auxochrome groups include:
  • Hydroxyl: -OH
  • Amino: -NH2
  • Alkoxy: -OR
  • Halogens: -Cl, -Br
• Chromogen:
  • Definition: Precursors to colored compounds. They are converted into chromophores through chemical reactions (e.g., oxidation).
  • Reaction: Chromogen + reactive compound = chromophore.

Classification of Dyes

Dyes can be classified based on:

• Chemical Properties: How the dyes work.
• Application: What they are used for.
• Origin: Where they come from.
• Method: Progressive vs. regressive.
• Systems of Classification:
  • Colour Index Number (CIN)
  • Conn's Biological Stains

Chemical Classification of Dyes

Dyes can be classified by their reaction with tissues. Examples include:

• Cationic Dyes:

  • Characteristics: Attach to anionic tissue groups; basic and acidophilic.
  • Examples: Used to stain nuclei, basophil/mast cell granules, cartilage matrix, and cytoplasmic anionic groups (e.g., RNA).
  • Example dye: Hematoxylin.

• Anionic Dyes:

  • Characteristics: Attach to cationic tissue groups; acidic and basophilic.
  • Examples: Used to stain cytoplasm and extracellular structures.
  • Example dye: Eosin.

• Mordant Dyes:

  • Definition: Dyes containing a metallic salt (mordant) that bridges them to structures, forming an insoluble "dye lake."
  • Characteristics: Weak auxochromes, highly basic; act as cationic dyes.
  • Function: Do not stain directly; act as glue.
  • Example dyes: Iron (Weigert's) and aluminum (Harris) hematoxylin.

• Solvent (Lysochrome) Dyes:

  • Characteristics: Lack an auxochrome, hydrophobic and fat-soluble.
  • Mechanism: Work via preferential or selective solubility.
  • Example dyes: Oil Red O and Sudan Black.

• Polychromatic Dyes:

  • Characteristics: Mixture of dye components (both anionic and cationic) forming various colors when applied.
  • Application: Used in rapid diagnosis of frozen sections.
  • Example dye: Methylene Blue.

• Metachromatic Dyes:

  • Definition: Stain certain tissue components (chromotropes) a different color than the original dye, resulting in metachromasia.
  • Mechanism: Due to polymerization and requires water for the reaction.
  • Application: Typically used for staining acid mucins and cartilage.
  • Example dyes: Toluidine Blue, Azure A, Crystal Violet.

• Neutral (Romanowsky) Dyes:

  • Definition: Solutions made by combining acidic and basic dyes.
  • Application: Commonly used in hematology to stain both basic nuclear components and acidic cytoplasmic components.
  • Example dye: Wright's stain.

• Amphoteric Dyes:

  • Definition: Contain both acidic and basic auxochrome groups, allowing for ionization and binding to oppositely charged tissue components (zwitterions).
  • Feature: pH-dependent.
  • Example dye: Acid Fuchsin.

• Leuco Dyes:

  • Definition: Dyes that switch between colorless and colorful forms due to easily reduced and reversible chromophores.
  • Triggers: Changes in temperature, pH, or UV light exposure.
  • Example: Schiff's reagent in Periodic Acid Schiff (PAS) stain.

• Metallic Impregnation:

  • Definition: Using alkaline, saturated silver solutions to precipitate into the tissue.
  • Types of reactions include:
  • Argentaffin: Reactive groups in the tissue reduce silver salts.
  • Argyrophilic: Reactive tissue groups require an outside reducer to reduce silver salts.
  • Metallic Substitution: Silver in solution exchanges with ions of reactive tissue groups.

Physical and Chemical Interaction with Tissues

Interactions with tissues depend on physical and chemical factors.

Physical Interactions

• Definition: Refers to the absorption of dye into tissue.
• Factors affecting physical interactions:
  • Surface area.
  • Density.
  • Permeability.
  • Size of dye molecule.

Chemical Interactions

• Definition: Refers to the bonds and interactions that dyes have with tissues. Types include:
  • Electrovalent Bonds:
  • Definition: Electrostatic attraction of oppositely charged ions.
  • Also known as ionic, electrostatic, and salt linkage.
  • Disrupted by acids, weak bases, water, alcohol, or high electrolyte concentrations.
  • Example: Most common in many dye-to-tissue interactions.
  • Covalent Bonds:
  • Definition: Formed when two atoms share electrons; strongest bond requiring considerable energy to disrupt.
  • Typical in organic chemicals.
  • Example: Schiff’s reagent and Verhoeff’s stain.
  • Hydrogen Bonds:
  • Definition: Attraction between the lone pair of electrons on O or N and partially exposed nucleus of H.
  • Weaker than covalent or ionic bonds.
  • More significant in non-aqueous solvents.
  • Example: Amyloid stains.
  • Van der Waals Forces:
  • Definition: Electrostatic attractions between electrons of one atom and nucleus of another.
  • Found in all staining systems to varying degrees.
  • Characteristics: Short-range forces, weakest.
  • Example: Staining of elastin by aldehyde fuchsin.
  • Hydrophobic Interaction:
  • Definition: Affinity of non-polar molecules for each other.
  • Requirement: Stained preparations must be mounted in aqueous media; dye must be more soluble in substrate than solvent.
  • Example: Oil Red O.

Application Classification of Dyes

Dyes can be categorized based on their usage.

• Examples include:
  • Food dyes.
  • Textile dyes.
  • Tissue dyes.

Origin Classification of Dyes

Dyes can be classified by their origin:

• Natural Dyes:

  • Definition: Derived from natural sources, including plants, minerals, or animals.
  • Characteristics: Typically less vibrant and less consistent.
  • Examples: Hematoxylin, Orcein, Indigo.

• Synthetic Dyes:

  • Definition: Typically derived from petrochemical sources.
  • Characteristics: More vibrant and consistent compared to natural dyes.
  • Examples: Eosin Y, Alcian Blue, Oil Red O.

Method Classification of Dyes

Dyes can be classified based on their usage methods:

• Progressive Dyes:
  • Description: Stain until the desired effect is achieved.
• Regressive Dyes:
  • Description: Overstain the tissue, then differentiate (remove stain) until desired result is achieved.

Systems of Classification

Two main systems are recognized for categorizing dyes and stains used in histology:

• Colour Index Number (CIN):
  • Description: A standardized system for classifying dyes based on chemical structure and properties.
• Conn's Biological Stains:
  • Description: A reference book that classifies biological stains using various properties, including safety and Colour Index (C.I.) number.