Fixation and Fixatives in Histopathology
Fixation is a crucial step in histopathology, preserving tissue samples in their most natural state for microscopic examination.
Fixatives are chemical substances used to achieve this preservation by stabilizing cellular structures and preventing decomposition.
This article explains the purpose, types, and common fixatives used in histopathology.
What Is Fixation?
Fixation is the process of preserving tissue by:
- Preventing autolysis (self-digestion) and putrefaction (decay).
- Maintaining the natural structure of cells and tissues.
- Enhancing tissue’s ability to withstand subsequent processing steps like sectioning and staining.
What Are Fixatives?
Fixatives are chemicals that preserve tissue by denaturing proteins or crosslinking cellular components.
They prevent enzymatic degradation and bacterial growth.
Types of Fixatives
Fixatives are broadly classified based on their mechanism of action and composition:
1. Aldehyde Fixatives
- Examples: Formaldehyde, Glutaraldehyde
- Action: Crosslink proteins by forming covalent bonds, preserving cell structures.
- Usage: Formalin (10% neutral buffered formalin) is the most common fixative in routine histopathology.
2. Oxidizing Fixatives
- Examples: Potassium dichromate, Osmium tetroxide
- Action: Oxidize cellular components, particularly lipids.
- Usage: Primarily for electron microscopy.
3. Coagulant Fixatives
- Examples: Ethanol, Acetic acid
- Action: Precipitate proteins, causing coagulation and hardening of tissue.
- Usage: Ideal for cytological preparations and nucleic acid preservation.
4. Compound Fixatives
- Examples: Bouin’s solution, Zenker’s fluid
- Action: Combine different chemical fixatives to optimize preservation for specific tissue types.
- Usage: Special staining techniques and research purposes.
Factors Influencing Fixation
| Factor | Effect |
|---|---|
| Fixative Type | Determines the chemical reaction and preservation efficiency. |
| Temperature | Higher temperatures speed up fixation but may cause artifacts. |
| Fixative Volume | Should be at least 10 times the tissue volume for effective fixation. |
| Duration | Over-fixation can harden tissue; under-fixation may cause degradation. |
Common Fixatives and Their Applications
| Fixative | Composition | Applications |
|---|---|---|
| 10% Formalin | 10% formaldehyde in water with buffer | Routine histopathology |
| Glutaraldehyde | 2.5–4% aqueous solution | Electron microscopy |
| Bouin’s Solution | Picric acid, formaldehyde, acetic acid | Soft tissues and special staining |
| Ethanol | 70–100% alcohol | Cytology and nucleic acid studies |
| Osmium Tetroxide | Aqueous solution of osmium tetroxide | Lipid preservation in electron microscopy |
Conclusion
Fixation and fixatives are essential in histopathology for preserving tissue morphology and enabling accurate microscopic examination. The choice of fixative depends on the tissue type and the purpose of the study, ensuring reliable diagnostic and research outcomes.