What is Fixation and Various Fixative
FIXATION
When a tissue is removed from the body it begins to decompose. To preserve the natural state (as nearly as possible) the tissue is immediately placed into a solution called fixative and this process is called fixing or fixation. The common cause of poor cellular details and histological preparation are due to autolysis and putrefaction. Autolysis is the lysis of cells by the enzyme liberated as a result of rupture of the lysosomes. The enzymes are known as cathepsin. Cathepsins, some of which are proteinases. Putrefaction means the breakdown of tissue by bacterial action, often with the formation of gas. Both the autolysis and petrification follow rapid upon death of the cells and after the removal of the specimen from the body. Best preservation results are obtained by putting tissue into fixative as soon as possible. A fixative plays important role in :
- Reservation of cell and tissue constituents.
- Hardening of soft tissue.
- Conversion of semifluid consistency of cells to an irreversible semisolid consistency.
- Alteration of reflective indices to varying degrees, which enables unstained components to be more easily seen.
FACTORS AFFECTING FIXATION PROCESSES
- Buffer pH: This is necessary for appropriate chemical reaction. pH is adjust to physiological range 6 to 8.
- Osmolarity : Best results are obtained by using appropriate osmolarity of the fixative. Hypertonic solution give rise to sales swelling and poor fixation. Hypertonic solutions result in cell shrinkage.
- Temperature : Increase in temperature will accelerate the fixation process. However, selection of temperature should be done according to the specification of the fixation procedure. Routinely tissues are submitted for fixation at room temperature. For electron microscopy, the temperature range selected is 0-4 degree Celsius.
- Penetration capacity of fixatives : Thickness of the section should be 1-4 mm thick. Penetration of fixative depend on the diffusion and rate at which fixative reacts with tissue.
- Duration : 12 to 24 hours are required for fixation. Prolonged fixation can cause shrinkage and hardening of tissue. Prolonged fixation also may affect antigenicity of the specimen.
- Agitation : Appropriate agitation improves speed of penetration, resulting in better infiltration and information of the tissue.
- Concentration : Optimum concentration of fixative is required for satisfactory result.
VARIOUS TYPES OF FIXATIVES
The fixatives can be divided into following 3 main groups :
- Microanatomical fixative,
- Cytological fixatives
- Histochemical fixatives.
MICROANTOMICAL FIXATIVES
Microanatomical fixative or mainly used for the routine use. These fixatives are used to preserve the anatomy of the tissue, with the correct relationship of tissue layers and large aggregate of cells. Following. are various examples of microanatomical fixatives :
- 10% (v/v) formalin in 0.9% sodium chloride (normal saline) : This has been the routine fixative of choice for many years, but it has now been largely supplanted by buffered formalin or by formal calcium acetate.
- Buffered formalin
| Formalin | 10 ml |
| Acid sodium phosphate monohydrate | 0.4 g |
| Anhydrous disodium phosphate | 0.65 g |
| Distilled water | 100 ml |
- Formal calcium.
| Formalin | 10 ml |
| Calcium acetate | 2.0 g |
| Distilled Water | 100 ml |
- Buffered formal sucrose.
| Formalin | 10 ml |
| Sucrose | 7.5 g |
| M/15, phosphate buffer ( pH 7.4) | 100 ml |
- Heidenhain susa.
| Mercuric chloride | 4.5 g |
| Sodium chloride | 0.5 g |
| Trichloroacetic acid | 2.0 g |
| Acetic acid | 4.0 g |
| Distilled water | 100 ml |
- Zenker’s fluid.
| Mercuric chloride | 5.0 g |
| Potassium dichromate | 2.5 g |
| Sodium sulfate | 1.0 g |
| Distilled water | 100 ml |
| Glacial acetic acid (add immediately before use) | 5.0 ml |
- Zenkers formal (Helly’s fluid).
| Mercuric chloride | 5.0 g |
| Potassium dichromate | 2.5 g |
| Sodium sulfate | 1.0 g |
| Distilled water | 100 ml |
| Formalin (add immediately before use) | 5.0 ml |
- Bouins fluid.
| Saturated aqueous picric acid | 75 ml |
| Formalin | 25 ml |
| Glacial acetic acid | 5.0 ml |
- Gander’s fluid.
| Saturated picric acid in 95% (v/v) alcohol | 80 ml |
| Formalin | 15 ml |
| Glacial acetic acid | 5.0 ml |
What is a Microtome? and 7 Types of Microtome
CYTOLOGICAL FIXATIVES
Cytological fixative are used when the preservation of intracellular structure or inclusions is very important. These fixatives can be subdivided into :
- Nuclear fixative
- Cytoplasmic fixative.
Nuclear fixative
- Carnoys fluid
| Absolute alcohol | 60 ml |
| Chloroform | 30 ml |
| Glacial acetic acid | 10 ml |
- Clarke’s fluid
| Absolute alcohol | 75 ml |
| Glacial acetic acid | 25 ml |
- Newcomers fluid
| Isopropanol | 60 ml |
| Propionic acid | 40 ml |
| Petroleum ether | 10 ml |
| Acetone | 10 ml |
| Dioxane | 10 ml |
Cytoplasmic fixative
- Champys fluid
| 3 g/dl potassium dichromate | 7.0 ml |
| 1% (v/v) chromic acid | 7.0 ml |
| 2 g/dl osmium tetroxide | 4.0 ml |
HISTOCHEMICAL FIXATIVES
The fixatives used for histochemical studies should produce minimum changes in the elements that is to be demonstrated. Freeze drying technique is ideal for this purpose. The various function of a good histochemical executive or as follows.
- Preservation of the tissue constituents.
- Preservation of. Morphological relationship.
- Preservation of the specific tissue constituents.
- No interference with the reagent to be used in the process of visualization.
For the majority of histochemical methods, cryostat cut sections of rapidly frozen tissue or sections of frozen dried tissue are preferred. These sanctions are used unfixed or fixed by a vapor fixative.
- Buffered formalin is the most common fixative used for histochemical purpose. Immersion is acetone, 0-4 degrees Celsius is widely used for the fixation of tissues in which it is intended to study enzyme (particularly the phosphatase). fixation of section cut from freeze dried material may be affected by immersion in absolute alcohol for 24 hours.
- Vapors fixative : These are used to fix a cryostat cut section of fresh tissue and sections of block of frozen dried tissue. These fixative are used inside an airtight glass container with controlled heat and humidity to fix, cryostat cut sections of fresh tissue and sections or block of frozen dried tissue. Following are various vapour fixative used in histopathology laboratory.
- Formaldehyde : Paraformaldehyde in heated 50-80 degree Celsius to obtain vapors for fixation. Block of tissue require 3 to 5 hours and section required. 1/2 – 1 hour at 5250 degrees Celsius.
- Acetaldehyde : It is used by heating at 80 degree Celsius for 1-4 hours.
- Glutaraldehyde : A 50% (v/v) aqueous solution of glutaraldehyde can be used at 80 degrees Celsius for 2 minutes to 4 hours or at 60 degree Celsius for 7 hours.
- Acrolein or Chromyl chloride : This reagent is used in liquid form at 37 degree Celsius for 1-2 hours.
