INTRODUCTION TO FIXATION
Fixation is a process by which the constituents of the cells and thereby tissues, are fixed in a physical and partly chemical state, so that they are able to withstand subsequent treatment with various chemicals, with minimum or no significant distortion or decomposition. Most fixations are carried out by denaturing or precipitating proteins which then forms a spongy meshwork that tends to hold other constituents.
WHAT IS THE AIM OF FIXATION?
Fixation is done to maintain the structure of tissues in almost lifelike conditions before they are ready to be examined under the microscope. Fixation also serves the following important functions.
⇒ It prevents the autolysis and bacterial decomposition/ Putrefaction. Autolysis is most rapid in brain and Kidney.
⇒ It coagulates the tissue to prevent the loss of diffusible substances.
⇒ It fortifies the tissue against the deleterious effect of various stages in the preparation of the section, like Dehydration, Clearing and Wax impregnation.
⇒ Fixation improves the optical differentiation of the tissues. This is actually done by altering the refractive index by varying degrees. This is of real importance since the refractive indices of some of the cellular components are so close to that of the surrounding structure which renders them invisible in the living state when examined under the microscope.
⇒ The protoplasm of the living cell is the mixture of colloids of the semi-solid consistency. When the tissue is treated with the fixatives it is rendered solid i.e. the semi-solid colloids are set into the semi-solid gel.
CLASSIFICATION OF FIXATIVES
Fixatives can be functionally classified into two major groups
Simple Fixatives – These fixatives are made up of simple chemical compounds and take more time for the fixation of tissues. For example, Formalin, Picric acid, Mercuric oxide, osmic acid, Osmium tetroxide etc.
Compound Fixatives – These are the mixtures of a number of fixatives in definite proportion and require a lesser amount of time for fixation. For example, Susa fluid, Carnoy’s fluid, Bouin’s Fluid, Formal saline, buffered formalin etc. The compound fixatives can further be classified into three types as follows:
- Microanatomical fixatives
- Cytological fixatives
- Histochemical fixatives
The Micro anatomical fixatives aim at accurately preserving the relationship of tissue layer and large cell aggregation. These fixatives are used for routine work of normal and histopathological study. For example, buffered formalin, Zenker’s fluid, Bouin’s fluid etc.
The Cytological fixatives are intended to preserve the constituents elements of the cells themselves. According to their mode of action, they can be subdivided into two groups:
- Nuclear fixatives – These are the fixatives that primarily fix the nuclear components of the cells. For example, Carnoy’s fluid, Clarke’s fluid etc.
- Cytoplasmic fixatives – These are the fixatives that primarily fix the cytoplasmic components of the cell. For example, Helly’s fluid, Champy’s fluid etc.
The Histochemical fixatives are used for the Histochemical studies of the tissues where the minimum or no changes in the components to be demonstrated are required. for example, Buffered formalin or vapor fixatives include Formaldehyde, Glutaraldehyde, Acrolein etc.
Fixatives can also be broadly classified into two groups as follows:
Precipitant fixatives – These precipitates the colloidal constituents of the cells as a coagulum. for example, Mercuric chloride, Picric acid, and ethyl alcohol.
Non- Precipitant Fixatives – These fixatives fix protein by denaturing them. These make the protein molecules insoluble. For example, Formaldehyde.
Here is the list of commonly used fixative, their Advantages, and Disadvantages:-
S. No. FIXATIVE ADVANTAGES DISADVANTAGES
1.) FORMALDEHYDE i.) It fixes the proteins without precipitation.
ii.) Has no effect on Carbohydrates.
iii.) Preserves Glycogen and Lipids.
i.) It causes little Shrinkage.
ii.) Over hardens the tissue if left for a long time in formaldehyde solution.
2.) GLUTARALDEHYDE i.) It fixes the tissues quickly
ii.) Suitable for Electron microscopy.
iii.) It forms a more rigid and dense product.
i.) Over hardens the tissue if left for a long time in Glutaraldehyde solution.
ii.) It is not ideal for Immunohistochemistry staining.
3.) POTASSIUM DICHROMATE i.) Homogeneously fixes the cytoplasm.
ii.) Has mordanting effect in the staining process.
i.) Mucoproteins are not well preserved.
ii.) Can cause extensive denaturation.
4.) MERCURIC CHLORIDE i.) It precipitates the proteins and hardens the tissue.
ii.) Has beneficial effect on staining.
iii.) Causes neither Shrinkage nor Swelling.
i.) It damages the tissue lipids.
ii.) It is difficult to make frozen sections after fixing with Mercuric Chloride.
5.) OSMIUM TETROXIDE i.) It fixes fats, conjugated lipids and mitochondria.
ii.) It preserves all the details of tissues.
iii.) Excellent fixative for the Electron microscopy.
i.) May produce Black coloration on the tissue.
ii.) It is very expensive.
iii.) Its vapors are irritating and can cause Conjunctivitis.
6.) PICRIC ACID i.) It precipitates & combines with proteins to form picrates.
ii.) Preferred fixative for connective tissues.
iii.) Prevents over hardening of tissue during dehydration.
iv.) Preserves glycogen well & does not shrink the tissues.
i.) It does not fix the carbohydrates.
ii.) Picric acid is Highly explosive.
S. No. FIXATIVES ADVANTAGES DISADVANTAGES
1.) BUFFERED FORMALIN i.) It fixes proteins without precipitation.
ii.) Fats are preserved and can be stained by suitable methods.
Formalin pigment is not formed.
i.) High strength of formalin can causes the shrinkage of tissues.
ii.) Over-exposure may over-hardens the tissue.
2.) HEIDENHAIN SUSA i.) Tissues are fixed quickly.
ii.) Gives Rapid and even penetration with minimum Shrinkage.
i.) Over exposure can bleaches the tissue and over hardens it.
ii.) Tissue requires a treatment with iodine to remove mercury pigments.
3.) CARNOY's FLUID i.) It is one of the most penetrating fixative.
ii.) It rapidly fixes the tissue.
iii.) After fixation the tissues can be directly transferred to 90-100% Alcohol.
i.) It causes lysis of Red blood cells and much shrinkage.
ii.) Some cytoplasmic granules may be preserved.
4.) BOUIN's FLUID i.) It penetrates evenly and rapidly.
ii.) Causes less shrinkage and can be used to demonstrate glycogen.
iii.) Tissues may be left in it for months without any harm.
i.) It is not suitable for tissues containing mucin, since it becomes greatly swollen.
ii.) The cortex of Kidney is badly preserved.
iii.) It is necessary to remove excess picric acid by washing or by alcohol treatment.
5.) ZENKER's FLUID i.) It rapidly and evenly penetrates the tissue.
ii.) It is a good routine fixative.
i.) It is unstable after the addition of Acetic acid, hence acetic acid (or formalin) should be added just before use.
6.) HELLY's FLUID i.) Excellent for micro anatomical structure analysis.
ii.) Excellent fixative for blood containing organs, bone marrow, spleen etc.
i.) It is necessary to remove excess of dichromate and Mercuric pigments for best results.
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