It is a complex series of chemical events which brings about changes in the various chemical constituents of cell like hardening, however the cell morphology and structural detail is preserved. Unless a tissue is fixed soon after the removal from the body it will undergo degenerative changes due to autolysis and putrefaction so that the morphology of the individual cell will be lost.
Principle of Fixation
The fixative brings about cross linking of proteins which produces denaturation or coagulation of proteins so that the semi fluid state is converted into semisolid state; so that it maintains everything in vivo in relation to each other. Thus semisolid state facilitates easy manipulation of tissue.
Aims and Effects of fixation
If a fresh tissue in kept as such at room, temperature it will become liquefied with a foul odour mainly due to action of bacteria i.e. putrefaction and autolysis so the first and fore most aim of fixation is:
- To preserve the tissue in as if like a manner as possible.
- To prevent postmortem changes like autolysis and putrefaction. Autolysis is the lysis or dissolution of cells by enzymatic action probably as a result of rupture of lysosomes. Putrefaction is the breakdown of tissue by bacterial action often with formation of gas.
- Preservation of chemical compounds and microanatomic constituents so that further histochemistry is possible.
- Hardening: The hardening effect of fixatives allows easy manipulation of soft tissues like brain, intestines etc.
- Solidification: Converts the normal semi-fluid consistency of cells (gel) to an irreversible semisolid consistency (solid).
- Optical differentiation: It alters to varying degrees the refractive indices of the various components of cells and tissues so that unstained components are more easily visualized that when unfixed.
- Effects of staining: Certain fixatives like formaldehyde intensify the staining character of tissue especially with haematoxylin.
Properties of fixatives
- Coagulation and precipitation as described above.
- Penetration Fixation is done by immersing the tissue in fluid containing the fixative. Faster fixative can penetrate the tissue better it is penetration power depends upon the molecular weight. e.g Formalin fixes faster than Osimic Acid.
- Solubility of fixatives: All fixatives should be soluble in a suitable solvent, preferably in water so that adequate concentrations can be prepared.
- Concentration: it is important that the concentration of fixative is isotonic or hypotonic.
- Reaction: Most fixatives are acidic. It may help in fixation but can affect staining so has to be neutralized. e.g. Formalin is neutralized by adding of calcium carbonate.
Amount of Fixative:
The fixative should be at least 15-20 times the bulk of tissue. For museum specimens the volume of fixative is > 50 times.
Note: If the specimen is large then see that the sections are made to make slices which have a thickness of 1.5 cm so that fixative can penetrate the tissue easily.