staining

• Staining


  The process of applying dyes on the sections
• Purpose of staining

  • To see and study the architectural pattern of the tissue, the physical characteristics of the cells, and the structural relationship of the tissue and their cells
• Affinity of tissue and cells for dyes


  Varying affinity allows differentiation of structures
• Nucleus


  Attracts basic dyes
• Cytoplasm


  Attracts acidic dyes
• Acidic tissue


  Attracts basic dye
• Basic tissue


  Attracts acidic dye
• Classification of staining

  • Histological
  • Histochemical
  • Immunohistochemical
• Methods of staining

  • Direct
  • Indirect
  • Progressive
  • Regressive
  • Metachromatic
  • Counterstaining
  • Microanatomical
  • Metallic Impregnation
  • Vital
  • Intravital
• Direct staining


  Directly stained = no other processes involved
• Indirect staining


  Involves the usage of Mordant in order for the stain to interact or stay on the tissue
• Mordant


  Link or bridge between tissue and dye; added with dye (lake)
• Accentuator


  Accelerate the speed of staining, does not participate in the staining reaction
• Progressive staining


  Follows a definite sequence, concentration is always increasing, does not wash or decolorize
• Regressive staining


  Tissue is first overstained, then decolorized through selective removal of excess stain
• Classes of differentiators

  • Acid
  • Oxidizing
  • Mordant
• Metachromatic staining


  Stains tissue with a color that is different from that of the stain itself
• Counterstaining


  Application of a different color or stain to provide contrast and background staining
• Microanatomical staining


  Stains the general relationship of tissues and cells, cytoplasmic structures, and bacterial morphology
• Metallic impregnation


  Demonstration of tissue elements by the use of metallic salts (colorless solutions)
• Vital staining

  Selective staining of living cells
• Intravital staining


  Done by injecting the dye into any part of animal body
• Supravital staining


  Stains living cells immediately after removing from the living body
• Categories of dyes

  • Natural
  • Synthetic
• Hematoxylin


  Most valuable stain, derived from Hematoxylin campechianium, requires oxidation/ripening process
• Types of hematoxylin

  • Alum
  • Iron
  • Copper
• Cochineal dyes


  Derived from female Coccus cac
• Categories of dyes used to prepare stains

  • Natural Dyes
  • Synthetic Dyes
• Natural Dyes

  Hematoxylin, Cochineal Dyes, Orcein, Saffron
• Synthetic Dyes

  Aniline or Coal Tar dyes
• Hematoxylin


  Derived from Hematoxylin campechianium, from the heartwood of a mexican tree, originally found in Campeche, Mexico, most valuable stain used
• Hematin


  The active coloring agent in hematoxylin (will not be considered as a stain without undergoing an oxidation or ripening process)
• Oxidation/Ripening of hematoxylin

  1. Exposure to air/sunlight (takes 3-4 months)

  2. Oxidizing agents
• Types of Hematoxylin

  • Alum Hematoxylin
  • Iron Hematoxylin
  • Copper Hematoxylin
• Cochineal Dyes


  Derived from female Coccus cacti (Cochineal bug), widely used a powerful chromatin and nuclear stain, when treated with Alum, will be termed as Carmine
• Orcein


  Derived from Lichens; a vegetable dye, usually colorless but expenses a blue or violet color when treated with Ammonia or Exposure to air, for staining Elastic Fibers
• Aniline Dyes


  "Coal Tar Dyes"; derived from Hydrocarbon benzene, must bind to substrate for it to show color, chromophores, chromogens, auxochromes
• Aniline Dyes by location of chromophore

  • Acid dye
  • Basic dye
  • Neutral dye
• Common Hematoxylin Staining Solutions

  • Ehrlich's Hematoxylin
  • Harris Hematoxylin
  • Coles Hematoxylin
  • Mayer's Hematoxylin
• Common Iron Hematoxylin Staining Solutions

  • Weigert's Hematoxylin Solutions
  • Heidehain's Hematoxylin
  • Phosphotungstic Acid Hematoxylin (PTAH)