Tissue Processing

PROCESSING OF TISSUES FOR LIGHT MICROSCOPY :
(PARAFFIN WAX EMBEDDING):

_ Tissues are processed by the following procedure to obtain thin translucent sections so that they can be examined under microscope by transillumination.


Fixation and Fixatives:

_ Chemical substances like formalin, mercuric chloride, acetic acid, picric acid and glutaraldehyde are used as fi xatives to preserve tissues.
_ All fi xatives have both desirable and undesirable effects.
_ A combination of these fi xatives is often prepared to get the maximum desirable effect. Such combinations commonly used are:
1. Bouin’s fl uid (formalin, acetic acid and picric acid)
2. Formal sublimate (formalin and mercuric chloride)
3. Helly’s fl uid (formalin, mercuric chloride and potassium dichromate)
4. Zenker’s fl uid (acetic acid, mercuric chloride and potassium dichromate)
_ Small pieces of fresh tissues are placed in common fi xatives like 10% neutral formal saline for 24 hours.
_ The purpose of fi xation is
– to preserve the morphology and chemical composition of the tissue,
– to prevent autolysis and putrefaction,
– to harden the tissue for easy manipulation,
– to solidify colloidal material, and
– to infl uence staining.
After fi xation, some hard tissues like bone and tooth, which contain large amount of calcium salts, require an additional step called decalcification before they are subjected for dehydration. Decalcifi cation makes the hard tissues soft, enabling them to be cut with microtome. For decalcifi cation, several decalcifying agents are used, namely 10% nitric acid, 5% trichloroacetic acid and ethylene diamine tetra acetic acid (EDTA).