Removal of Fixative

REMOVAL OF FIXATIVE

  • Fixative is removed from tissues by keeping them overnight in running tap water or washing briefly in tap water followed by placing in 70% alcohol. Alcohol removes formalin more rapidly.
  • Sometimes formalin may produce dark brown or black precipitates which are seen in sections under microscope.
  • To remove precipitates, deparaffinize the sections to water and then place in following solutions for 1 hour followed by thorough washing in tap water.
    • Ammonium hydroxide (58%) 2 ml
    • Alcohol(70%) 100ml

Decalcification

  • Decalcification is the chemical dissolution of insoluble calcium salts with a suitable acid or chelating agent.
  • The process is meant for softening hard tissues like bone and teeth to facilitate sectioning in ordinary microtome.
  • Decalcification is done after fixing the tissues.

Acid Decalcifier

  • Buffered formic acid, pH 1.5 – 3.5
    • Formic acid (90%) - 250 ml
    • Distilled water - 750 ml
    • Sodium formate (HCOONa) - 35g
  • Chelating Agent
    • EDTA Disodium ethylenediamine tetracetate 5 to 10 g
    • Dissolved in distilled water 100 ml
    • Add 4% NaOH to pH 7 or 8

Procedure

  • Thoroughly wash the tissue in water to remove the fixative
  • Take to decalcifying solution. Volume of decalcifying fluid should be atleast 20 times that of tissue.
  • Change the decalcifying solution
    • If acid mixture, every 3-5 days
    • If EDTA solution, every 3-5 days
  • End – point decalcification
    • Tissue is pliable and can be trimmed with scalpel.
    • Chemical test - 5 ml of decalcifying solution is made alkaline – pH 7 by adding strong ammonia solution.
  • Add 5 ml saturated aqueous solution of ammonium oxalate (approximately 3%) (NH4)2 C204, H2O) and leave to stand for 30 minutes. If no white precipitate has formed after this time the fluid contains no calcium
  • For EDTA solution pH is adjusted to 3.2-3.6 for maximum sensitivity of oxalate test.
  • Radiography for the presence or absence of calcified spots in the tissues.
  • Wash the tissues in water before dehydration.
Last modified: Saturday, 24 September 2011, 6:58 AM