8 Staining

• Staining is the process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cells.

  • Physical characteristics and structural relationship of tissues and their cells are thereby studied and evaluated.
• Routine glass slide used in staining → 76 x 22 mm w/ 1-2 mm thick (standard size)
• Nucleus has greater affinity to basic dyes → e.g., hematoxylin
• Cytoplasm has greater affinity to acid dyes → e.g., eosin
• Silver nitrate is the most commonly used agent for impregnation, ca also function as a staining agent
• In histological staining, tissue contents are demonstrated in sections by direct interaction with dye.

  • Also called Micro-anatomical staining
  • Bacterial stains, Specific tissue stains
• In histochemical staining, various constituents of the tissues are studied through chemical reactions.

  • Examples:
  • Perl’s Prussian blue → Hemoglobin
  • Periodic Acid Schiff → Carbohydrates
• Immunohistochemical staining is a combination of immunologic and histochemical techniques.

  • Use of wide range of polyclonal or monoclonal, fluorescent labeled or enzyme-labeled antibodies.
  • Tumor Marker (ck 20, ck 20, ERPR)
• In Direct Staining, sections are stained with simple of aqueous or alcoholic solution of the dye.

  • Example: Methylene blue, Eosin (Blood Smear)
• Indirect Staining requires mordant and accentuator for staining.

  • Example of mordants:
  • Potassium alum with hematoxylin in Erlich’s hematoxylin
  • Iron in Weigert’s hematoxylin.
  • Example of Accentuator
  • Potassium hydroxide in Loeffler’s methylene blue and Phenol in carbol thionine and carbol fuschin
• Mordant is the link/bridge between dye and tissue
• Accentuator speeds up staining reaction by increasing staining power and selectivity
• In Progressive Staining, tissues are stained in a definite sequence and the staining solution is applied for specific periods of time or until the desired intensity of coloring of the different tissue elements is attained.

  • Not washed or decolorized
  • PAP smear, Cytology, Frozen sections staining
• In Regressive Staining, tissues are first overstained to obliterate the cellular details.

  • Excess stain is removed or decolorized until the desired intensity of color is obtained
  • H&E staining
• Differentiation is the process of selective removal of stain during regressive.

  • Usually done using acids, alcohol/ oxidizing agent
• Metachromic staining rntails the use of a specific dyes which differentiate particular substances by staining them with a color that is different from that of stain itself

  • Used for cartilage, connective tissues, epithelial muscles, mast cell granules, amyloid
• Water is necessary for most metachromatic staining and metachromasia → lost if the sections dehydrated w/ alcohol after staining
• Metachromatic dyes are basic dyes belonging to the Thizine and Tripenylmethane group

  
• Counterstaining is the application of a different stain to provide contrast and background to the staining of the structural components to be demonstrated

  
• Microanatomical staining demonstrates the general relationship of tissues with general differentiation of nucleus and cytoplasm without necessarily emphasizing the inclusion bodies

  • Cytoplasmic staining
  • Negative staining
• In Metallic impregnation, specific tissue elements are demonstrated by colorless solutions of metallic salts which are reduced by tissues producing an opaque black deposit on the surface of the tissue or bacteria.

  • Gold (gold chloride)
  • Silver (silver nitrate)
  • Amonniacal silver solution → very explosive
• In using Amonniacal silver solution, clean the container before use and silver glassware should be avoided.

  • It reduces argentaffin cells in melanin and intestinal gland forming black deposit
• Vital stains are stain that can be applied on living cells without killing them

  • Example:
  • Trypan-blue → reticulo-endothelial
  • Janus green → mitochondri
• Intravital stains are non-toxic dye injected (intravenous, peritoneal, subcutaneous) into the body to selectively stain certain cells or tissues.

  • Example:
  • Lithium
  • Carmine
  • India ink
• Supravital staining stains living cells immediately after removal from the living body.

  • Common dyes:
  • Neutral red → best vital stain
  • Janus green → for mitochondria
  • Trypan blue → toxic to the cell → allow the suspension to stand > 1 hour.
  • 1 gram of dye dissolved in 100 ml of sterile distilled water
  • use immediately!
  • Nite blue
  • Thionine
  • Toluidine blue
• Coplin jar are slotted jar holding from 5-9 slide
• Slotted staining dishes holds from 5-19 slides, over which different solutions are poured.
• Metal/Glass Staining racks or Carriers holds from 10 – 30 slides upright
• A) Hematoxylin campechianum

  
• A) Coccus cacti

  
• A) Lichen

  
• A) Crocus sativus

  
• Properties of dye:

  • Chromophore → coloring property or “color bearer”; “Coal Tar dyes”
  • Auxochrome → dyeing property; the “ increasers”
• Types of stain according to source

  
• Types of stain according to Effect:

  • Adjective – acts on tissue but first treated with another substance
  • Substansive – acts immediately, directly on the object
  • Amphoteric – acid and basic grouping
• H&E staining is the most common method (microanatomical studies)

  • Routine
  • Using regressive staining (1% acid alcohol)
  • Consists of overstaining the nuclei, removal of superfluous and excessive color of the tissue constituent by acid diffrentiation
• H&E staining of Frozen section for rapid diagnosis

  • Progressive staining
  • Takes 5-10 minutes

  
• Routine H&E staining in paraffin-embedded sections

  • Regressive staining
  • Most fixative can be used except OSMIC ACID which inhibit hematoxylin
• H&E staining results

  • Nuclei → Blue to blue black
  • Karyosome → Dark blue
  • Cytoplasm, Edema fluid proteins → pale Pink
  • RBCs, Eosinophilic granules, Keratin → Bright orange red
  • Basophil, Cytoplasm, Plasma cells, Osteoblast → Purplish pink
  • Cartilage → pink or light blue to dark blue,
  • Calcium & Calcified bone → Purplish blue
  • Decalcified bone matrix, Collagen, Osteoid → Pink
  • Muscle fibers → deep pink
• Mercuric Chloride increases time of staining in hematoxylin, decreases staining of eosin.