Histology Staining Techniques

The Theory of Staining

• Tissues are transparent and colorless, making it difficult to distinguish different structures when examined with a light microscope.
• Histological techniques are used to distinguish tissue components, which can cause changes in the tissue through alteration of contrast or color.

Alteration of Contrast

• Phase contrast and polarized microscope can be used to make parts of the tissue appear grey or black.
• Impregnation methods can also be used to alter the contrast of tissue components.

Alteration in Color (Histological Staining)

• Histological staining involves the production of colors in tissue through dye-staining.
• The coloration is due to the absorption of light by the dye.
• A satisfactory staining method should combine high sensitivity and specificity.
• Sensitivity refers to the capacity of the stain to demonstrate a tissue substance in low concentration.
• Specificity or selectivity refers to the ability to discriminate between individual tissue components.

Staining Processes

• Vital Staining: Living cells can be stained by dissociation in the staining fluid (supra-vital) or by injection of dye into the living organism (intra-vital).
• Elective Solubility: Based on the solubility of the stain in tissue fluids, substances that dissolve in tissue are known as lysochromes.
• Chemical Production of Colored Substance in Tissue: Using a pale or colorless solution that reacts with tissue components to produce colored substances.
• Metallic Impregnation: Metallic compounds are reduced by tissues to produce opaque, black deposits.
• Staining with Dyes: Most staining techniques belong to this group, involving the use of natural or synthetic dyes.

Staining with Dyes

• Types and Structure of Dyes: There are two main types of dyes: natural dyes (e.g., carmine and haematoxylin) and synthetic dyes.
• Chromophores: Are the color-bearing chemical groups in the compound, which are classified into three main types: quinonoid, azo-coupling, and nitro group.
• Auxochromes: Are the ionizing groups that increase the intensity of the color, which are either acidic or basic.
• Dye Characteristics: Dyes can be basic, acidic, neutral, or amphoteric, depending on the number of basic or acidic auxochromes they contain.
• Modifiers: Additional chemical groups that alter the color of the dye or make it deeper.

Mode of Action of Dye Staining

• Chemical Dye Staining: Dye and tissues react together at the ordinary pH range, resulting in electropositive stain units combining with electronegative tissues and vice versa.
• Physical Dye Staining: Dye combines with tissues by adsorption, which is affected by physical factors such as density and permeability.
• Staining Techniques: There are four main staining techniques: progressive, regressive, direct, and indirect staining.

General Factors in Tissue Staining

• Effect of Fixation: Fixation can assist or hinder tissue-dye interaction, and may extract some tissue substances making subsequent staining ineffective.
• Accentuators: Substances used to increase the staining power and selectivity of dyes, but differ from mordants.
• Accelerators: Act like accentuators, used in metallic impregnation for the nervous system.
• Trapping Agents: Hold dyes in combination with tissues or bacteria, preventing them from escaping during differentiation.
• Differentiation: De-staining of over-stained tissue in regressive technique, brought about by washing, oxidizing agents, mordants, or dyes acting as differentiators.