Fixation in histopathology is a complex series of chemical events which brings about changes in the various chemical constituents of cell like hardening but doesn’t alter or change the cell structural details and morphology.Instead,fixation is means of preservation of the histopathological specimen.
Unless a tissue is fixed soon after the removal from the body,it will undergo degenerative changes due to processes like autolysis and putrefaction which alters the morphology of the cell.
Principle of Fixation
The fixative brings about a crosslinking of protein which denature or coagulate proteins so that the semifluid state is converted into semisolid state so that it maintains everything in vivo in relation to each other.Therefore a semisolid state facilitates easy manipulation of tissues.
Aims and effects of fixation
If a fresh tissue is kept as such at room temperature,it will become liquefied with foul odour mainly due to action of bacteria that is putrefaction and autolysis so the first and foremost aim of fixation is
- Firstly to preserve the tissue in as if like manner as possible
- To prevent postmortem changes like putrefaction and autolysis
- 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 bacteria action often with formation of gas
- Preservation of chemical compounds and microanatomic constituents so that further histochemistry is possible
- Hardening which is the effect of fixatives that allows easy manipulation of soft tissues like intestines,brain etc
- Solidification which converts the normal semifluid consistency of cells (gell) of the tissue to an irreversible semi solid consistency(solid) for easy manipulation
- For optical differentiation.It alters to varying degrees the refractive indices of the various compounds of cells and tissues so that unstained components are more easily visualized than when unfixed
- To intensify the effects of staining.Example like formaldehyde fixative intensifies the staining characteristics of tissues especially with haematoxylin stain
Properties of Ideal fixatives
- It should be able to Coagulation and precipitation of proteins
- It should be able to easily penetrate the tissue faster.Penetration of the fixatives in the tissues which is done by immersing the tissue in fluid containing the fixative.Note that the faster a fixative can penetrate the tissue,the better and fixative penetrating power depends upon their molecular weight.Formalin fixes faster than osmic acid
- It should be soluble in a suitable solvent preferably in water so that adequate concentrations can be prepared
- The concentration of a fixative should be isotonic or hypotonic
- It should be neutral.Most fixatives are acidic which may help in fixation but can affect the staining of the tissue.
Amount of Fixative required
The fixative to be used,should be at least 15-20 times the bulk of the tissue to be fix.
Large specimen can be section to slices with a thickness of 1.5 cm so that fixative can easily penetrate the tissue.
The volume of museum specimens during fixation is usually greater than 50 times that of the specimen
Preparation of Specimen for the process of fixation
- In order to achieve good fixation,it is important that the fixative penetrates the tissue well hence the tissue section should be less than 4 mm thick so that fixation fluid should be able to penetrate from the peripheries to the center of the tissue.
- For fixation of large organs,perfusion method is used.Perfusion method is the injection through the blood vessels into the organ.Hollow viscera fixation is injected into the cavity like urinary bladder,eyeball etc
- The time necessary for fixation is important.Routinely,10% aqueous formalin at room temperature takes 12 hours to fix the tissue.At higher temperature that is 60 to 65 ° C,the time for fixation is reduced to 2 hours
- Ratio of volume of the fixative to specimen should be 1 : 20.
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