Introduction:-
It is a process by which the cells or tissues are fixed in chemical and partly physical state so that they can withstand subsequent treatment with various reagents, with minimal distortion of morphology and no decomposition
OBJECTIVES:-
After reading this lesson, you will be able to:
*state the aims of fixation *explain the principle of fixation *describe the properties and factors affecting fixation *explain types of fixation.
AIMS OF FIXATION:-
(a) To preserve the tissues as close to their living state as possible (b) To prevent autolysis and bacterial attack
(c) To prevent tissues from changing their shape and size during processing (d) To harden the tissues (e) To allow clear staining of sections subsequently (f) To improve the optical differentiation of cells & tissue.
(c) To prevent tissues from changing their shape and size during processing (d) To harden the tissues (e) To allow clear staining of sections subsequently (f) To improve the optical differentiation of cells & tissue.
PRINCIPLE OF FIXATION:-
Fixation results in denaturation and coagulation of protein in the tissues. The
fixatives have a property of forming cross links between proteins, thereby forming a gel, keeping everything in their in vivo relation to each other.
fixatives have a property of forming cross links between proteins, thereby forming a gel, keeping everything in their in vivo relation to each other.
PROPERTIES OF FIXATIVES AND FACTORS
AFFECTING FIXATION:-
AFFECTING FIXATION:-
1. Coagulation and precipitation of proteins in tissues. 2. Penetration rate differs with different fixatives depending on the molecular
weight of the fixative 3. pH of fixatives – Satisfactory fixation occurs between pH 6 and 8. Outside
this range, alteration in structure of cell may take place. 4. Temperature – Room temperature is alright for fixation. At high temperature
there may be distortion of tissues. 5. Volume changes – Cell volume changes because of the membrane
permeability and inhibition of respiration. 6. An ideal fixative should be cheap, nontoxic and non-inflammable. The
tissues may be kept in the fixative for a long time.
weight of the fixative 3. pH of fixatives – Satisfactory fixation occurs between pH 6 and 8. Outside
this range, alteration in structure of cell may take place. 4. Temperature – Room temperature is alright for fixation. At high temperature
there may be distortion of tissues. 5. Volume changes – Cell volume changes because of the membrane
permeability and inhibition of respiration. 6. An ideal fixative should be cheap, nontoxic and non-inflammable. The
tissues may be kept in the fixative for a long time.
TYPE OF FIXATION:-
*Immersion fixation
*Perfusion fixation
*Vapour fixation
*Coating/Spray fixation
*Freeze drying
*Microwave fixation/Stabilization
*Perfusion fixation
*Vapour fixation
*Coating/Spray fixation
*Freeze drying
*Microwave fixation/Stabilization
The most commonly used technique is simple immersion of tissues/smears in
an excess of fixative. For all practical purposes immersion fixatives are most
useful. These may be divided into routine and special.
SIMPLE FIXATIVES:-
an excess of fixative. For all practical purposes immersion fixatives are most
useful. These may be divided into routine and special.
SIMPLE FIXATIVES:-
1. Formaldehyde: Commercially available solution contains 35%-40% gas by
weight, called as formalin. Formaldehyde is commonly used as 4% solution,
giving 10% formalin for tissue fixation. Formalin is most commonly used
fixative. It is cheap, penetrates rapidly and does not over- harden the tissues.
The primary action of formalin is to form additive compounds with proteins
without precipitation. Formalin brings about fixation by converting the free
amine groups to methylene derivatives
If formalin is kept standing for a long time, a large amount of formic acid
is formed due to oxidation of formaldehyde and this tends to form artefact which is seen as brown pigment in the tissues. To avoid this buffered
formalin is used.
2. Absolute alcohol – it may be used as a fixative as it coagulates protein.
Due to its dehydrating property it removes water too fast from the tissues
and produces shrinkage of cells and distortion of morphology. It penetrates
slowly and over-hardens the tissues.
weight, called as formalin. Formaldehyde is commonly used as 4% solution,
giving 10% formalin for tissue fixation. Formalin is most commonly used
fixative. It is cheap, penetrates rapidly and does not over- harden the tissues.
The primary action of formalin is to form additive compounds with proteins
without precipitation. Formalin brings about fixation by converting the free
amine groups to methylene derivatives
If formalin is kept standing for a long time, a large amount of formic acid
is formed due to oxidation of formaldehyde and this tends to form artefact which is seen as brown pigment in the tissues. To avoid this buffered
formalin is used.
2. Absolute alcohol – it may be used as a fixative as it coagulates protein.
Due to its dehydrating property it removes water too fast from the tissues
and produces shrinkage of cells and distortion of morphology. It penetrates
slowly and over-hardens the tissues.
3. Acetone:-
Sometimes it is used for the study of enzymes especially phosphatases and lipases. Disadvantages are the same as of alcohol.
4. Mercuric chloride:-
It is a protein precipitant. However it causes great shrinkage of tissues hence seldom used alone. It gives brown colour to the tissues which needs to be removed by treatment with Iodine during
dehydration.
5. Potassium dichromate:-
4. Mercuric chloride:-
It is a protein precipitant. However it causes great shrinkage of tissues hence seldom used alone. It gives brown colour to the tissues which needs to be removed by treatment with Iodine during
dehydration.
5. Potassium dichromate:-
It has a binding effect on protein similar to that of formalin. Following fixation with Potassium dichromate tissue must be well washed in running water before dehydration.
6. Osmic acid :-
It is used for fixation of fatty tissues and nerves.
6. Osmic acid :-
It is used for fixation of fatty tissues and nerves.
7. Chromic acid:-
It precipitates all proteins and preserves carbohydrates. Tissues fixed in chromic acid also require thorough washing with water before dehydration.
8. Osmium tetraoxide:-
8. Osmium tetraoxide:-
It gives excellent preservation of cellular details, hence used for electron-microscopy.
9. Picric acid –
9. Picric acid –
It precipitates proteins and combines with them to form picrates. Owing to its explosive nature when dry; it must be kept under a layer of water. Tissue fixed in picric acid also require thorough washing with water to remove colour. Tissue can not be kept in picric acid more than 24 hrs.
COMPOUND FIXATIVES:-
1. Formal saline:-
It is most widely used fixative. Tissue can be left in this for long period without excessive hardening or damage. Tissues fixed for a long time occasionally contain a pigment (formalin pigment). This may
be removed in sections before staining by treatment with picric alcohol or
10% alcoholic solution of sodium hydroxide. The formation of this pigment
can be prevented by neutralizing or buffering the formal saline.
be removed in sections before staining by treatment with picric alcohol or
10% alcoholic solution of sodium hydroxide. The formation of this pigment
can be prevented by neutralizing or buffering the formal saline.
Fixation time – 24 hours at room temprature
2. Formal calcium:-
Useful for demonstration of phospholipids. Fixation time-24 hours at room temperature
3. Zenker’s fluid:-
It contains mercuric chloride, potassium-di-chromate, sodium sulphate and glacial acetic acid.
Advantages :-even penetration, rapid fixation
Disadvantages –
After fixation the tissue must be washed in running water to remove excess dichromate. Mercury pigment must be removed with Lugol’s iodine.
4. Zenker’s formal (Helly’s fluid):-
In stock Zenker’s fluid, formalin is added
instead of acetic acid.
instead of acetic acid.
Advantages :- excellent microanatomical fixative especially for bone
marrow, spleen & kidney.
marrow, spleen & kidney.
5. Bouins fluid :-
It contains picric acid, glacial acetic acid and 40% formaldehyde.
Advantages :-(a) Rapid and even penetration without any shrinkage. (b)Brilliant staining by trichrome method. It is routinely used for preservation
of testicular biopsies.
of testicular biopsies.
Points to Remember:-
1. 10% buffered formalin is the commonest fixative. 2. Tissues may be kept in 10% buffered formalin for long duration. 3. Volume of the fixative should be atleast ten times of the volume of the
specimen. The specimen should be completely submerged. 4. Special fixatives are used for preserving particular tissues. 5. Formalin vapours cause throat/ eye irritation hence mask/ eye glasses and
gloves should be used. 6. Tissues should be well fixed before dehydration. 7. Penetration of fixatives takes some time. It is necessary that the bigger
specimen should be given cuts so that the central part does not remain
unfixed.
8. Mercury pigment must be removed with Lugol’s iodine. 9. Biopsies cannot be kept for more than 24 hours in bouin’s fluid without
changing the alcohol. 10. Glutaraldehyde and osmion tetraoxide are used as fixatives for electron microscopy.
specimen. The specimen should be completely submerged. 4. Special fixatives are used for preserving particular tissues. 5. Formalin vapours cause throat/ eye irritation hence mask/ eye glasses and
gloves should be used. 6. Tissues should be well fixed before dehydration. 7. Penetration of fixatives takes some time. It is necessary that the bigger
specimen should be given cuts so that the central part does not remain
unfixed.
8. Mercury pigment must be removed with Lugol’s iodine. 9. Biopsies cannot be kept for more than 24 hours in bouin’s fluid without
changing the alcohol. 10. Glutaraldehyde and osmion tetraoxide are used as fixatives for electron microscopy.
Most Commonly used Fixatives in the Laboratory are 10% Formalin:-
Formaldehyde (40%) 10ml
Distilled water 90 ml