Staining Techniques in Microscopy

Staining Techniques

• Stain: A substance that adheres to a cell, giving it color. Color provides contrast, making cells more visible under a microscope.
• Dye: Synthetic stains.
• Pigment: Stains extracted from plants or animals.
• Staining Technique: A microscopy method for enhancing contrast in images.

Classification of Stains

• Origin:
  • Animal origin (e.g., carmine from insect bodies).
  • Plant origin (e.g., hematoxylin from the Mexican tree).
  • Synthetic (e.g., eosin).
• Mode of action:
  • Physical staining: The stain physically combines with the structure by absorption. (e.g., Sudan III, stains fats orange-yellow).
  • Chemical staining: The stain chemically reacts with the structure (e.g., various stains for DNA).
• End color of stained content:
  • Orthochromatic: The stain reacts with the cell content, resulting in the same color. (e.g., cytoplasm with methylene blue gives a blue color).
  • Metachromatic: The stain reacts with the cell content and produces a different color. (e.g., starch with iodine solution gives a blue color).
  • Metachromasia: The reaction between the stain and the content results in a color different from the stain's original color.

Classification of Staining Techniques

• Number of stains used:
  • Monochromatic (simple): Uses one dye.
  • Polychromatic (multiple): Uses several dyes. Multiple dyes allow different structures to be stained in different colors (e.g., trichrome stain uses three dyes for color differentiation).
• Nature of stained material:
  • Staining in toto: Staining the entire tissue mass.
  • Section staining: Staining sections of the tissue. This method firstly sections the specimen, and then stains the separated sections.
• Applied method:
  • Regressive: Over-staining followed by removal of excess stain to reach the desired intensity.
  • Progressive: Sections are gradually stained until the desired intensity is reached.
  • Mordant use: Using a mordant to fix the stain better to the tissue. The mordant is a chemical that aids stain fixation and helps in more intense coloring. (e.g., in Gram staining and iodine solution procedure for crystal violet)
  • Special methods: Specialized methods like fluorescent and electron microscopy.

Vital Staining

• Stains that can diffuse through living tissues without harming them.
  • Intravital: Staining living cells inside the body (in vivo).
  • Supravital: Staining living cells outside the body (in vitro).

Other Concepts

• Acidophilic: Substances that have an affinity for acid stains.
• Basophilic: Substances that have an affinity for basic stains.
• Eosin stain: A stain that stains cytoplasm, usually pink.
• Basic fuchsin: A stain that stains DNA, often creating a pink/purple color.
• Neutral Stains: Stains with both acid and base radicals. These stains are utilized in blood cell staining.