Staining in Histology

Staining

• Staining is a process that renders different tissue components more visible through variation in color, promoting easier optical differentiation and identification of cell and tissue components.
• It involves treating tissue or cells with a series of reagents to acquire a color, making it transparent and facilitating visualization under the microscope.

Purpose of Staining

• Outlines tissue and cellular components
• Enables identification of tissues
• Establishes the presence or absence of disease processes in histology and histopathology

Common Staining Methods

• H&E Staining (Histopathology)
• Gram's Stain & Ziel-Neelsen Staining (Microbiology)
• Romanowsky Staining (Hematology)
• Giemsa Staining (Parasitology)
• Papanicolaou Staining (Cytology)

General Procedure

• Begins with complete removal of paraffin in fixed sections of slides through dewaxing
• Dewaxing involves heating the slides in 60°C for at least 30 minutes to soften the wax, followed by immersion in xylene and descending concentrations of alcohol to distilled water prior to primary stain

Staining Mechanism

• Principles of histological technique for distinction of tissue components are based on:
  • Alteration of contrast
  • Alteration of color
  • Alteration of contrast between the parts of the tissue

Nuclear Staining

• Nuclei are acidic, anionic, or negatively charged
• Stain with basic dye (blue) under the microscope, which is cationic or positively charged
• Reaction results in a basophilic reaction, forming dye-salt unions, producing a bluish to purple color

Cytoplasmic Staining

• Cytoplasm is amphoteric due to terminal amino and carboxyl groups
• May appear basophilic or acidophilic, depending on the substance present in the cytoplasm
• Net charge of cytoplasmic proteins:
  • Below pH 6: positive (+) with high affinity to negative (-) charged dye, resulting in reddish to orange color
  • Above pH 6: negative (-) with high affinity to positive charged (+) or cationic dyes, resulting in purplish to bluish color

Dyes/Stains

• Chemical or reagents with aromatic benzene ring compounds (or derivatives) that possess the twin properties of:
  • Color band
  • Ability to bind tissues
• All dyes are organic compounds, mostly derivatives of coal tar or benzene
• Now, there are synthetic dyes

Integral Components of Dyes

• Chromophores
  • The group on the benzene ring that confers color (C=C, C=O, C=S, N=N, N=O, and NO2)
  • Alters the light resonance properties of the compound, resulting in unequal absorption when white light is passed through

Learn about the process of staining in histology, which enhances the visibility of tissue components by introducing color variations, allowing for easier identification and differentiation of cell and tissue components.