Veterinary Clinical Biochemistry and Laboratory Diagnosis-II

PROCESSING OF TISSUES

• Most of the fixatives employed are aqueous and water has to be removed in order to embed the tissue in paraffin wax.
• The loaded tissue capsule should be filled in a cotton gauze and is placed under tap water or in a tissue washing tank, which is connected to running water source.
• The water flow should be adjusted to maintain a slow but continuous flow.
• At least six hours flow is essential to wash off the preservative thoroughly from the tissues.

The major steps of tissue processing are

• Dehydration
• Clearing
• Infiltration and Impregnation 
• Embedding

DEHYDRATION

• It is process by which water is removed from fixed tissues, by passing the tissues in ascending grades of alcohol to prevent undue shrinkage of tissues.
  • Ethyl alcohol 50% - 8 hours
  • Ethyl alcohol 70% - 2 hours
  • Ethyl alcohol 90% - 2 hours
  • Absolute alcohol – I - 1 hour
  • Absolute alcohol – II - 1 hour
• Dioxane can be used instead of alcohol. Here shrinkage is minimum and dehydration is quick.

CLEARING

• Removes alcohol from the tissues and prepare them for penetration by paraffin during embedding. Clearing agent commonly used is Xylene.
• • Xylol - I    - 30 min.
  • Xylol – II - 30 min.
•  After clearing, tissues appears transparent
• Xylol is cheap and quick in action – makes tissues transparent – causes shrinkage and hardening if tissues are kept longer.
• Other clearing agents are : Cedar–wood oil, toluene, benzene and chloroform, but are expensive.

INFILTRATION AND IMPREGNATION 

• Usually paraffin  wax  is  used.    It takes place in an oven heated to 54-62 ^◦C, the temperature depends on melting point of wax.
• Tissues are transferred to molten paraffin wax which causes diffusion of xylene into surrounding molten wax (Infiltration).
• The wax diffuses into tissues to replace xylene (Impregnation ). Minimum two changes in wax (paraffin - melting point 50 to 56^◦C) kept in cups replace the clearing agent.
• The tissue is kept for 30 minutes in each cup. Over heated wax can cause cooking of the tissues.
• After cooling, wax provides firmness and support to tissues when these are cut on the microtome.
• Celloidin is also used.

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EMBEDDING

• It is also known as casting or blocking.
• Embedding is transferring of infiltrated tissue into molten wax and solidifies into a block when cools down to room temperature. Leuckhart’s L pieces commonly referred as embedding Ls to prepare tissue block.
• Two L shaped moulds are arranged in the form of a rectangle over a porcelain slab.
• Melted paraffin is poured into the mould and the tissue is so oriented that the cutting surface of the tissue faces the porcelain slab.
• The moulds are removed as soon as paraffin sets and the block is ready for sectioning.

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