Veterinary Clinical Biochemistry and Laboratory Diagnosis-II

FIXATION

• The fixation of tissues is necessary immediately after collection. Tissues should be kept in a fixative for 24-48 hours at room temperature.
• The fixative harden the tissues by coagulation of the cell protein.
• Prevents autolysis and putrification.
• Preserves the structure and architecture of the tissue/cells.
• Prevents shrinkage.
• Increases optical differentiation, which enables components to be more easily seen than when unfixed
• The volume of the fixative added is 10 times the volume of the tissues.

Common Fixatives

• 10% Formalin
  • 40% formaldehyde - 100 ml
  • Distilled or tap water - 900 ml
• Formal saline
  • 40% formaldehyde - 100 ml
  • Sodium chloride - 9 g
  • Tap/distilled water - 900 ml
• Neutral Buffered Formaldehyde
  • 40% formaldehyde - 100 ml
  • Distilled water - 900 ml
  • Sodium dihydrogen phosphate monohydrate- 4g
  • Disodium hydrogen phosphate anhydrous -6.5g

Special Fixatives

• These fixatives are required for special type of investigations.
• Tissue already fixed with formalin, deformalinization is required initially followed by secondary fixation by other fixatives like Zenker’s fluid, Helly’s fluid, Bouin’s fluid etc.
  •  Zenker’s Fluid
    • Mercuric chloride - 5g
    • Potassium dichromate - 2.5 g
    • Sodium sulphate - 1.0g
    • Distilled water - 100 ml
    • Glacial acetic acid 5 ml added just before use

    It gives good histological differentiation. Nucleus and connective tissue stain sharply. Fixation time 4 to 14 hours. It makes tissues brittle if left too long and does not keep well. (Remove mercury and subsequently iodine before staining is done).

    • Step I: Treat sections in 0.3 to 0.6% iodine, 70% alcohol for 5 to 10 minutes and rinse in water.
    • Step II: Decolorise in 5% sodium thiosulphate for 1 to 5 minutes and wash in running water before proceeding with haematoxylin staining.
  • Heidenhan’s SUSA Fluid
    • Mercuric chloride - 4.5g
    • Sodium Chloride - 0.5g
    • 40% formaldehyde - 20 ml
    • Glacial acetic acid - 4 ml
    • Trichloro acetic acid - 2 ml
    • Distilled water - 80 ml
    • This is similar to Zenker’s but does not give the mercury precipitate. Fixation 12 hours followed by washing in 95% ethanol.
  • Bouin’s Fluid
    • Saturated aqueous  acid- 75 ml
    • (about 1,2%)
    • 40% formaldehyde - 20 ml
    • Glacial acetic acid - 5 ml
    • Nucleus stains excellently.
    • Rapid penetration. Good for nervous tissue. But it makes tissues brittle if left for more than 3 days.