FIXATION

• Fixation


  The process of killing, hardening and preserving constituents of tissues with the least alteration from the living state
• Fixatives


  Solutions responsible for killing, hardening, and preserving the different constituents found in tissues (carbohydrates, lipids, fats, and other metabolic processes)
• Fixation is the most crucial step in the laboratory because if we fail to preserve the tissue, the consequent processes will not be determined or will not be useful
• Main factors involved in fixation

  • Hydrogen ion concentration (pH 6-8)
  • Temperature (room temperature for surgical specimens, 0-4C for electron microscopy and some histochemistry)
  • Thickness of section (no more than 4mm thick)
  • Osmolality (slightly hypertonic solution, 400-450mOsm)
  • Concentration (10% formaldehyde)
  • Duration of fixation (2-6 hours)
• The volume of the fixative should be 10-25 times the volume of the tissue and must at least 3/4 fully submerge the tissue
• Effects of fixatives in general

  • Harden and soft friable tissues
  • Make the cells resistant to damage and distortion
  • Inhibit bacterial decomposition
  • Increase optical differentiation of cells and tissues
  • Act as mordants and accentuators
  • Reduce risk of infections
• Formaldehyde


  A gas produced by the oxidation of methyl alcohol, commonly used at 10% concentration, buffered to pH7 with phosphate buffer
• 10% Neutral Buffered Formalin


  Phosphate-Buffered Formalin, best fixative for tissues containing iron pigments, recommended for preservation and storage of surgical, post-mortem and research specimens
• 10% Neutral Buffered Formalin formula: Sodium dihydrogen phosphate (anhydrous) 3.5 grams, Disodium hydrogen phosphate (anhydrous) 6.5 grams, 40% Formaldehyde 100mL, Distilled water 900mL
• Fixation time for 10% Neutral Buffered Formalin is 4-24 hours
• Disadvantages of 10% Neutral Buffered Formalin

  • Longer to prepare
  • Positivity of mucin to PAS is reduced
  • Produce gradual loss in basophilic staining of cells
  • Reactivity of myelin to Weigert's iron hematoxylin stain is reduced
  • Is inert towards lipids, especially neutral fats and phospholipids
• Factors that retard fixation

  • Size and thickness of the tissue specimen
  • Presence of mucus
  • Presence of fat
  • Presence of blood
  • Cold temperature
• Factors that enhance fixation

  • Size and thickness of tissues
  • Agitation
• Difficulties encountered with fixation

  • Failure to arrest early autolysis of cells
  • Removal of substances soluble in fixing agent
  • Presence of artifact pigments on tissue sections
  • Tissues are soft and feather-like in consistency
  • Loss or inactivation of enzymes needed for study
  • Shrinkage and swelling of cells and tissue structure
  • Tissue blocks are brittle and hard
• Characteristics of a good fixative

  • It must be cheap
  • Stable
  • Safe to handle
  • It must kill the cell thereby producing minimum distortion of cell constituents
  • It must inhibit bacterial decomposition and autolysis
  • It must produce minimum shrinkage of tissue
  • It must permit rapid and even penetration of tissues
  • It must harden tissues thereby making the cutting of sections easier
  • It must be isotonic, causing minimal physical and chemical alteration of the cells and their constituents
  • It must make the cellular components insoluble to hypotonic solutions and render them insensitive to subsequent processing
  • It must permit the subsequent application of many staining procedures to facilitate easier and more profitable examination