VPP 322: Aquatic Animal Diseases, Health Care and Management (1+1)

Preparation of Histopathological Sections of fish Tissue

PROTOCOL

Fish tissues

• Dissect out the tissue from the fish. Liver, kidney, spleen, gill, intestine, heart, brain, muscle or skin depending on the pathology. Limit the size of tissue to 1 cm³ beyond which fixing of the tissue may not be proper.
• Fixation: Preserve the tissue in buffered formalin for 24 hours.
  • Composition of buffered formalin
    • 40% formalin - 100 ml
    • NaH₂PO₄H₂O - 4 g
    • NaHPO₄ - 6 g
    • Tap water - 900 ml
    • Use approximately 25 volumes of fixative for preservation. In case of large tissues or small whole fish, the fixative can be replaced after 12 hours with fresh fixative.
• Wash the tissue in tap water.
• Dehydrate the tissue in a series of graded alcohol and block it after impregnating with molten wax.
  • Dehydration: The tissues, which are fixed, will be dehydrated in this step.
    • 30% - ½ hour
    • 50% - ½ hour 
    • 70% - ½ hour to 45 min)
    • 90% - ½ hour
    • 95% - ½ hour
    • Absolute alcohol - ½ hour
    • Absolute alcohol - ½ hour
  • Clearing
    • Xylene - ½ hour
    • Xylene - ½ hour
  • Impregnation
    • Xylene :wax (2:1) - ½ hour
    • Xylene :wax (1:2) - 1 hour
    • Molten : wax - 2 hour
  • Blocking
    • Take suitable container (boat) for blocking the tissue and embed the tissues in blocks of wax. The orientation of the tissue has to be looked in to at this stage. Care has to be taken to prevent trapping of air bubbles in the blocks.
  • Trimming and sectioning
    • Cut the blocks to required cube shape and fix on the chuck and cut using microtome. The sections are then floated on the water bath maintained at 60°C and collected on clean slides and dried on a slide warmer.
• Staining
  • Put the slides with sections in 60°C oven melting off the excess wax
  • Put in xylene I to dewax – 5 min
  • Change to xylene II for complete dewaxing - 2 min
  • Change to alcohol I 2 – min
  • 90% alcohol – 1 min
  • 70% alcohol – 1 min
  • 50% alcohol – 1 min
  • Wash in running tap water – 2 min
  • Hematoxylin – 5- 10 min.
  • Wash in tap water
  • Eosin – 2 min
  • 30% alcohol – 1 min
  • 50% alcohol – 1 min
  • 70% alcohol – 2 min
  • 90% alcohol – 2 min
  • Absolute alcohol - 2 hour
  • Absolute alcohol - 2hour
  • Xylene I – 3min
  • Xylene – until cover –slipped (Don’t allow to dry the slides)
  • Mount using DPX
• Duration of the staining in hematoxylin and eosin has to be standardized by observing in the microscope after dip in both sides. For shrimp tissues, the fixative used is Davidson’s fixative which is alcohol based. Hence the steps in dehydration from 70% alcohol to be continued
  • Davidson’s fixative
    • Ethyl alcohol - 330 ml
    • Formalin - 220 ml
    • Glacial acetic acid - 115 ml
    • Tap water/Distilled water - 335 ml